Structure and antioxidant activity study of sulfated acetamido- polysaccharide from Radix Hedysari

Article abstract of DOI:10.1016/j.fitote.2013.05.011

A new sulfated acetamido-heteropolysaccharide, HPS4-2A, was obtained by aqueous extraction followed by precipitation with ethanol and fractionation with DEAE column chromatography from Radix Hedysari. It was composed of rhamnose, arabinose, glucose, galactose and 2-acetamido-2-deoxy-D-galactose in the molar ratio of 10.09%:25.90%:25.90%:25.0%:12.30%. Elemental analysis indicated that HPS4-2A was a sulfated polysaccharide containing small amount of sulfate groups (1.87%). Partial acid hydrolysis, GC, GC-MS, 1D and 2D NMR spectroscopy analysis of the HPS4-2A revealed a predominance of glucose, galactose and 2-acetamido-2-deoxy-D-galactose linked in a highly-branched structure. Themolecular weight of HPS4-2Awas determined by HPSEC and HPSEC-MALLS. AFMstudy indicated that HPS4-2A took a highly branched conformation, which in consistent with the result studied by SEC-MALLS. Structural features of HPS4-2A were also investigated by SEM and TEM. Antioxidant assays demonstrated that HPS4-2A possessed of strong DPPH and hydroxyl radicals scavenging activities, suggesting that HPS4-2A could potentially be used as natural antioxidant.

Full text of DOI:10.1016/j.fitote.2013.05.011

Fitoterapia 89 (2013) 20–32
Contents lists available at SciVerse ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Structure and antioxidant activity study of sulfated acetamido-
☆
polysaccharide from Radix Hedysari
a
b
a
a
a
a
a
Zilong Dang , Demei Feng , Xiaohua Liu , Tao Yang , Long Guo , Jin Liang , Jiandi Liang ,
a
a
a,
Fangdi Hu , Fang Cui , Shilan Feng ⁎
School of Pharmacy, Lanzhou University, 199 Donggangxi Road, Lanzhou 730000, PR China
a
b
The Second People's Hospital of Gansu Province, Hezhengxi Road 388, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 January 2013
Accepted in revised form 8 May 2013
A new sulfated acetamido-heteropolysaccharide, HPS4-2A, was obtained by aqueous extraction
followed by precipitation with ethanol and fractionation with DEAE column chromatography
from Radix Hedysari. It was composed of rhamnose, arabinose, glucose, galactose and
2-acetamido-2-deoxy-D-galactose in the molar ratio of 10.09%:25.90%:25.90%:25.0%:12.30%.
Keywords:
Radix Hedysari
Elemental analysis indicated that HPS4-2A was a sulfated polysaccharide containing small
amount of sulfate groups (1.87%). Partial acid hydrolysis, GC, GC–MS, 1D and 2D NMR
spectroscopy analysis of the HPS4-2A revealed a predominance of glucose, galactose and
2D NMR
Structure
Polysaccharide
Atomic force microscopy
Electron microscopy
2-acetamido-2-deoxy-D-galactose linked in a highly-branched structure. The molecular weight of
HPS4-2A was determined by HPSEC and HPSEC-MALLS. AFM study indicated that HPS4-2A took a
highly branched conformation, which in consistent with the result studied by SEC-MALLS.
Structural features of HPS4-2A were also investigated by SEM and TEM. Antioxidant assays
demonstrated that HPS4-2A possessed of strong DPPH and hydroxyl radicals scavenging
activities, suggesting that HPS4-2A could potentially be used as natural antioxidant.
©
2013 The Authors. Published by Elsevier B.V. All rights reserved.
1
. Introduction
group study had shown that representative constituent of
HPS2 (the second fraction of HPS) was mainly composed of
dextran [7]. According to our previous study, the crude
polysaccharide HPS4 (the fourth fraction of HPS) had
potential radioprotective effect on acute radiation injured
mice.
Radix Hedysari was a traditional Chinese medicine, and it
is often used as a substitute for Astragalus membranaceus
Fisch.) bge in the folk. Distributed throughout north China, it
(
was used to strengthen the immune system and treat
diarrhea, diabetes mellitus, chronic nephritic proteinuria,
inflammation and many other diseases [1,2]. The Radix
Hedysari polysaccharides (HPS) have been reported to
exhibit various pharmacological activities include enhance-
ment of the immune system, antitumor, hypoglycemic,
antioxidation and anti-aging properties [3–6]. Our research
The physical and chemical properties of the polysaccharides
(
such as the molecular weight, monosaccharide composition,
degree of branching, substituents and glycosidic linkages) were
related with its biological activities. Elucidation of polysaccha-
ride structure characteristic was necessary for understanding
the mechanism of biological activity and expounding the
structure–activity relationships [8,9].
HPS were purified into four homogeneous fractions
HPS4-1A, HPS4-1B, HPS4-1C and HPS4-2A) by DEAE-52
and Sephadex G-200 column chromatography. In this paper,
the structure of main fraction (HPS4-2A) was elucidated
using partial acid hydrolysis, methylation, GC, GC–MS, 1D
and 2D NMR, and its conformation revealed by SEC-MALLS,
☆
This is an open-access article distributed under the terms of the Creative
(
Commons Attribution License, which permits unrestricted use, distribution,
and reproduction in any medium, provided the original author and source
are credited.
⁎
Corresponding author. Tel./fax: +86 931 8915686.
E-mail address: fengshl@lzu.edu.cn (S. Feng).
0367-326X/$ – see front matter © 2013 The Authors. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.fitote.2013.05.011

Z. Dang et al. / Fitoterapia 89 (2013) 20–32
21
SEM, TEM and AFM. 2D NMR included COSY and TOCSY
70%, V/V final ethanol concentrations) to achieve two crude
polysaccharides accordingly, named as HPS4-1 and HPS4-2.
HPS4-2 was redissolved in distilled water and applied to a
DEAE-52 column (2.7 cm × 70 cm). The column was eluted
stepwise with distilled water, 0.1–1.0 M NaCl aqueous solution
and fractions collected at a flow rate of 0.5 ml/min (each test
tube hold 12 min) and monitored by the phenol-sulfuric acid
method. Then the major fraction, named HPS4-2A (eluted by
1
1
(
homonuclear H/ H correlation spectroscopy), HSQC and
1
3
1
HMBC (heteronulcear C/ H multiple-quantum correlation
spectroscopy). The antioxidant activity of the HPS4-2A was
evaluated in vitro by 1,1-diphenyl-2-picrylhydrazyl (DPPH)
free radical and hydroxyl radical scavenging assay.
2
2
2
. Experimental
1
.0 M NaCl) was concentrated, dialyzed and lyophilized
.1. Materials and general methods
according to the elution curve. The following studies are
aimed at HPS4-2A.
.1.1. Materials
Radix Hedysari was purchased from Wudu County (Gansu
2.3. Elemental analysis
Province, China) and identified by Prof. Zhigang Ma, School of
Pharmacy, Lanzhou University. The roots of Radix Hedysari
were dried in shade and ground into powder. The standard
monosaccharides, including rhamnose, arabinose, xylose,
mannose, glucose, galactose, glucuronic acid and galacturonic
acid were all from National Institute for the Control of
Pharmaceutical and Biological Products (Beijing, China) and
Elemental analysis was carried out by Elementar Vario EL
instrument (Elementar, Germany) to analyze weight per-
centages of carbon (C), hydrogen (H), oxygen (O), sulfur (S)
+
and nitrogen (N). Sulfated group content (\SO
2
O\Na
,
sodium salt) was analyzed using the factor of 3.22 to convert
measured weight percentage of sulfur to sulfate.
2
-acetamido-2-deoxy-D-galactose from Hai Hang Industry
Co., Ltd (Jinan, China). DEAE-52 cellulose was purchased
from Whatman Co. (Maidstone, UK), Sephadex G-200, and
G-100 were purchased from Treechem (Shanghai, China).
Dextran of different molecular weights (668,000, 410,000,
2.4. Molecular weight and homogeneity determination
2.4.1. High performance size exclusion chromatography (HPSEC)
The molecular weight and homogeneity of HPS4-2A was
determined using HPSEC method, which was conducted on a
Waters HPLC, including a pump (Waters 600), a RI detector
(Waters 2414) and a photodiode array detector (Waters 2998).
HPSEC was performed on successively linked of two columns
(Ultrahydrogel™ 1000 and Ultrahydrogel™ 500 columns,
300 × 7.8 mm, waters, Massachusetts, USA) and eluted with
2
73,000, 148,000, 48,600, 23,800, 11,600, and 5200 Da) was
purchased from PSS (USA). All chemicals used were of
analytical grade unless otherwise specified.
2
.1.2. General methods
Total carbohydrate content was determined by phenol-
sulfuric acid method [10], with glucose and galactose as the
standards at 490 nm. Protein content was assayed by
Bradford method with bovine serum albumin as reference
protein [11]. Uronic acid content was determined according
to GC method [12], and m-hydroxydiphenyl method [13] by
measuring the absorbance at 525 nm and using galacturonic
acid and glucuronic acid as standards. GC analysis used a
fused-silica capillary column OV-101 (60 m × 0.25 mm
i.d., × 0.25 μm film thickness), and was carried out using a
Shimadzu GC-2010 (Shimadzu, Japan) with flame ionization
detector.
0.9% (W/V, 0.154 M) NaCl containing 0.02% (W/V) NaN
3
(1.0 ml/min). The temperature of columns and RI detector
was maintained at 35 °C.
Dextran standards were used to establish a standard curve
(retention time was horizontal axis and logarithm of molecular
weight of dextrans was vertical axis). The obtained data were
analyzed by Empower (Version 2.0,Waters) software.
2.4.2. HPSEC-MALLS
The molecular weight and homogeneity of HPS4-2A were
also can be determined by HPSEC-MALLS method. The
HPSEC-MALLS system consisted of
a separation module
2
.2. Isolation and purification of polysaccharide
(Waters 600), a photodiode array detector (Waters 2998) set
at 280 nm, a multi-angle laser light scattering detector (Wyatt
DAWN-EOS) with a laser at 690 nm, along with a refractive
index monitor (Waters 2414). HPS4-2A was dissolved with
mobile phase (5 mg/ml) and filtered (0.45 μm) before injec-
tion (50 μl). Then eluted with 0.9% (W/V, 0.154 M) NaCl
The extraction process was conducted according to the
earlier report [14]. Briefly, the ground dry roots of Radix
Hedysari (4000 g) were extracted 3 times with tap water
(
40,000 ml) at 100 °C for 3 h each time. The combined
aqueous extracts filtered through a cotton cloth bag and
centrifuged (3000 g for 15 min) and then concentrated in a
rotary evaporator at 60 °C. Then the aqueous solution was
precipitated by 95% ethanol at a final concentration of 70%
3
containing 0.02% (W/V) NaN (1.0 ml/min) from two columns
as mentioned in Section 2.4.1. Data for molecular weight
determination were analyzed using ASTRA software (Version
4.73.04, Wyatt Technology Corp.) using a dn/dc of 0.138 ml/g.
(
V/V). The sediment was dissolved in water, and precipitated
by 95% ethanol at a final concentration of 40% (V/V), the
supernatant concentrated to a proper volume under reduced
pressure and lyophilized to get the crude polysaccharide.
Then, the crude polysaccharide from Radix Hedysari, named
HPS4 was obtained after deproteinized according to Sevage
2.5. Compositional analysis
The compositional analysis of HPS4-2A was performed by
the alditol acetate method [16], with minor modifications.
Briefly, HPS4-2A was hydrolyzed by 2 M trifluoroacetic acid at
121 °C for 3.5 h followed by reduction in distilled water with
method [15] and decolored by H
2 2
O . HPS4 was redissolved in
distilled water and precipitated by 95% ethanol (to give 40%,
4
NaBD 1.5 h at room temperature, and subsequent acetylation

22
Z. Dang et al. / Fitoterapia 89 (2013) 20–32
by acetic anhydride with pyridine as the catalyst at 100 °C for
h. The standards, including rhamnose, arabinose, xylose,
JSM-6380LV scanning electron microscopy (JEOL, Tokyo,
Japan), which operated at 5 kV. A working distance of
8 mm and slow scan mode were used for imaging.
1
mannose, glucose, galactose, 2-acetamido-2-deoxy-D-galactose,
treated in parallel with sample. The alditol acetate derivatives
produced were separated by GC on an DB-5MS fused silica
capillary column (50 m × 0.25 mm i.d., 0.25 μm film thick-
ness) with the GC oven programmed from 150 °C to 170 °C at a
rate of 5 °C/min, and then to 225 °C at a rate of 8 °C/min, held
for 1 min and then to 250 °C at a rate of 5 °C/min, hold for
2.9.2. Transmission electron microscope (TEM)
A drop of aqueous solution of HPS4-2A was first placed on
a carbon-coated TEM copper grid (Quantifoil, Germany) and
allowed to air-dry. The sample was then negatively stained
with phosphotungstic acid (Merck, Germany):
a drop
5
min and detected by MS using an Varian 320-MS (320-MS is
phosphotungstic acid aqueous solution (1%, W/W) was
dripped to the carbon-coated TEM grids. The grid was then
air-dried, and put them into the electron microscope. The
samples were viewed at 80 kV, using a JEOL JEM-1230 TEM
(JEOL, Tokyo, Japan). All the micrographs were collected
using Gatan 1.35 K × 1.04 K × 12 bit ES500W CCD camera
at different magnifications from different parts of the
specimens.
a triple quadropole mass spectrometer, Varian, USA). Identifi-
cations were based on peak retention time and by comparison
of electron impact mass spectra with standard spectra, and
further contrast to standard samples. Quantification of each
monosaccharide was carried out by integration of the chro-
matographic peak area.
2
.6. Partial hydrolysis analysis
2.9.3. Atomic force microscope (AFM)
HPS4-2A (50 mg) was hydrolyzed with 4 ml 0.1 M TFA at
00 °C for 1 h. After cooling, TFA was evaporated under
The sample was prepared by spreading of a dilute (1 μg/ml)
1
polymer solution onto a freshly cleaved silicon wafer surface
and successively air-drying under ambient temperature [19].
The atomic force microscopy (MultiMode 8, Bruker, Germany)
was operated in the tapping-mode [20].
reduced pressure. The hydrolysate was dissolved and dia-
lyzed against distilled water (M cut-off 3500 Da) to obtain
w
two fractions, inside dialysis bag fraction (HPS4-2AI) and
outside dialysis bag fraction (HPS4-2AL). HPS4-2AI and
HPS4-2AL were collected. Then HPS4-2AI was applied to
Sephadex G-100 column (2.6 cm × 70 cm), and the major
peak fractions were collected, named HPS4-2AH. HPS4-2AH
and HPS4-2AL were collected for monosaccharide composi-
tion analysis by GC–MS as described in Section 2.5.
2.10. Assay for antioxidant activities of the HPS4-2A
2.10.1. Effect of scavenging 1,1-diphenyl-2-picryl-hydrazyl
(DPPH) radicals
The DPPH free radicals' scavenging activity of HPS4-2A
was analyzed by the method of literature [21–23], with
some modifications. The 0.01 mmol/l solution of DPPH in
2
.7. Methylation analysis
5
0% ethanol was prepared daily before using. One
HPS4-2A (5 mg) was methylated using CH
3
I and NaOH in
milliliter of sample solution (solvent was 50% ethanol)
with different addition quantity (0.5, 1, 2, 4, 8 mg) was
mixed thoroughly with 2 ml of freshly prepared DPPH and
2 ml of 50% ethanol, and then left to stand for 30 min at
room temperature in the dark and the absorbance was
then recorded at 517 nm against a blank. Ascorbic acid
was used as reference compound. The scavenging activity
to the DPPH radical was calculated using the following
equation:
DMSO [17]. The methylated polysaccharides were acetylated to
partially methylated alditol acetates as described by [18]. The
derivatives produced were analyzed by GC–MS as described in
Section 2.5. Quantification of each methylated monosaccharide
was determined by integration of the chromatographic peak
area.
2
2
.8. Spectrum analysis
.8.1. IR spectrum
HPS4-2A were mixed with KBr pellets for FT-IR measure-
Scavenging abilityð%Þ ¼ ½1−ðA −A Þ=A ꢀ ꢁ 100%:
s
e
b
ment and recorded on a Nicolet Nexus 670 spectrometer
(
Nicolet, USA) in the frequency range of 4000–400 cm−1
.
Where A
consisted of 2 ml DPPH solution and 3 ml of 50% ethanol; A
was the absorbance of mixed solution that consisted of 1 ml
sample, 2 ml DPPH solution, 2 ml of 50% ethanol; and A was
the absorbance of mixed solution that consisted of 1 ml
sample and 4 ml of 50% ethanol.
b
was the absorbance of mixed solution that
s
2
.8.2. NMR spectrum
HPS4-2A (30 mg) was exchanged with deuterium by
freeze-drying with D O three times. Then the sample was
dissolved in 0.5 ml D O and H and C spectra were recorded
on a Bruker NMR spectrometer (600 Hz INOVA 600NB) at
e
2
1
13
2
2
2
2
5 °C.
2.10.2. Measurement of hydroxyl radical scavenging activity
The scavenging activity of hydroxyl radicals was investi-
gated based on the method described by Zhang et al. [24] and
with a minor modification. One milliliter test sample (0.5, 1,
2, 4, 8 mg/ml) was mixed with a solution containing 1 ml
phenanthroline (5 mM), 0.4 ml phosphate buffer (50 mM,
.9. Ultrastructure analysis
.9.1. Scanning electron microscope (SEM)
The powder of the HPS4-2A was directly pasted on a
carbon conductive tape, and sputtered with gold (Balzers/
Union FL-9496) for 1 min. The sample was observed in a JEOL
pH 7.4), 0.25 ml deionized water, 0.5 ml FeSO
4
(7.5 mM)
and 0.1 ml H (1%). Then the mixture was incubated at
2 2
O

Z. Dang et al. / Fitoterapia 89 (2013) 20–32
23
3
7 °C for 60 min. The ability to scavenge hydroxyl radicals
The explanations of the discrepancy of HPS4-2A's molec-
ular weight measured by HPSEC-MALLS and HPSEC listed as
follows: the HPSEC method used unbranched dextrans as
standards and their monosaccharide composition containing
only glucose, while HPS4-2A was a heteropolysaccharide
composed of five different monosaccharides, and had more
branches (this conclusion in the methylation, and the results
of partial acid hydrolysis and NMR have also been confirmed)
and larger molecular radius, which make a big difference
between the two structure. Although the results are different
and strongly depended on the methods used for analysis, the
authors believe that the result of HPSEC-MALLS is much more
reliable according to the above explanation.
was calculated with following equation:
Scavenging effectð%Þ ¼ ðA −A Þ=ðA −A Þ ꢁ 100%:
1
2
3
2
Where A
10 nm using UV-1700 spectrophotometer (Shimadzu,
Kyoto, Japan), A was the absorbance when sample was
replaced by deionized water, and A was the absorbance
when H and sample were replaced by distilled water.
1
was the absorbance of sample measured at
5
2
3
2 2
O
3
. Results and discussion
3
.1. Physicochemical property of HPS4-2A
3.3. Compositional analysis
The total sugar content of HPS4-2A was 98.98%. The
protein content of HPS4-2A was very low, only 0.26%. The
negative result of m-hydroxydiphenyl reaction and GC
method suggested that uronic acid was absent. Elemental
analysis revealed that the elemental composition of HPS4-2A
was C, H, O, N, S, and its content was 34.51% (mass %), 6.29%,
The monosaccharide composition of HPS4-2A was ana-
lyzed by GC–MS and compared with the monosaccharide
standards. The result showed that HPS4-2A was mainly
composed of rhamnose, arabinose, glucose, galactose and
2-acetamido-2-deoxy-D-galactose in the molar ratio of
10.09%:25.90%:25.90%:25.0%: 12.30%.
5
5.36%, 3.26% and 0.58%, respectively. The molar ratio of C/H/O
was around 1:2:1, which was in line with molar ratio of three
chemical elements in carbohydrate. The total content of
3.4. Partial acid hydrolysis analysis
2
sulfate ester (\SO O\) was 1.87%, which means approxi-
mately one sulfate ester was present per 27–32 sugar units
on average.
The high molecular weight fraction, HPS4-2AH, yields
44.0% of the HPS4-2A, composed of rhamnose (6%), arabi-
nose (0.4%), glucose (40.8%), galactose (33.6%) and 2-
acetamido-2-deoxy-D-galactose (19.2%). The lower molecu-
lar weight fraction, HPS4-2AL, the sugar compositions
showed that arabinose (35.8%) and glucose (41.9%) were
the main constituents and with a relatively small amount of
rhamnose (15.1%) and galactose (6.7%). For HPS4-2AH, the
amount of arabinose and rhamnose decreased considerably
compared with HPS4-2A, whereas the amount of glucose,
2-acetamido-2-deoxy-D-galactose and galactose increased.
According to above data, the main backbone of HPS4-2A was
composed of glucose, galactose and 2-acetamido-2-deoxy-
D-Gal, and substituted by the side chains of mainly arabinosyl
and glucosyl residues.
3
3
.2. Molecular weight and homogeneity determination
.2.1. HPSEC
HPS4-2A showed a single symmetrical peak in HPSEC
profile (mobile phase was deionized water or 0.154 M NaCl),
indicating it was a homogeneous polysaccharide. HPSEC
result showed its molecular weight was more than 388.0 kDa
(
mobile phase was 0.154 M NaCl).
3
.2.2. HPSEC-MALLS
The HPS4-2A exhibits a single peak with good symmetry
detected by differential refraction detector. This suggests it
was a homogeneous polysaccharide in the sodium chloride
aqueous solution. The RMS (root mean square) radius,
3.5. Methylation analysis
w
weight average molecular weight (M ), and polydispersity
index (M /M ) were measured to be 30.5 nm, 272.0 kDa,
w
n
The total ion current peaks from the partially methylated
alditol acetates (PMAA) were identified by their retention time,
characteristic ion fragments in MS spectra libraries and
contrast to referenced literatures [26]. The molar ratio of
methylated monosaccharide residues was calculated based on
the integral area of peaks. The majority of the terminal units
were T-L-Araf (18.25%), with relatively small amount of T-Glcp
(9.76%). The branched sugar residues →4,6) Glcp (1→, →3,6)
Galp (1→, →4,6) Galp (1→, →2,4) Rhap (1→ and →3,4)
2-NAc-Galp (1→ with the molar ratio of 12.30%, 5.90%, 5.68%,
5.12% and 0.86%, respectively. The unsubstituted sugar residues
were shown to be →2) Rhap (1→, →5) Araf (1→, →6) Galp (1
→, →4) Glcp (1→ and →6) Glcp (1→, in percentage of 12.06%,
11.46%, 6.42%, 6.15% and 5.86%, respectively. Although low
values for the proportion of partially methylated amino sugars
were not uncommon, smaller amounts of partially methylated
2-acetamido-2-deoxy-D-galactose (0.86%) were obtained. It
was unclear why a low yield of the acetamino sugar was
and 1.633, respectively. The conformation plot present to be
U shapes, which indicated it was a branched molecule and in
consistent with the result showed by Molar mass-versus-
elution volume and RMS radius-versus-elution volume plots
of HPS4-2A (the figure can be seen in Fig. A.1 of Appendix A).
The plots showed that HPS4-2A had abnormal SEC elution
behavior: after the normal decrease of the molar mass or
RMS radius with increasing elution volume, both plots curved
up.
The abnormal SEC elution behavior was caused by the
“
anchoring effect” [25] of branch structure of HPS4-2A. The
so-called “anchoring effect” is, a branch of the branched
polymer stuck in the gel pores of the gel column, which leads
to extending retention time of the whole branched molecule
in the gel column. Therefore, the region of high elution
volumes was a mixture of small molecules and large branched
molecules.

24
Z. Dang et al. / Fitoterapia 89 (2013) 20–32
1
obtained in the methylation analysis. In addition, there was
reference to the published data for similar molecules. The H
trace amount of →2,3,4) Galp (1→ (0.18%) and T-Rhap
NMR spectrum (Figure not given) showed more than six
peaks in the anomeric region, the dominant peaks were
centered on δ 5.00 and 4.40 ppm. These overlapping peaks
can be clearly observed in the anomeric area of the HSQC
(Fig. 1a). For example, from δ 4.85 ppm to δ 5.4 ppm, eight
(
trace) existed, this may be caused by side reactions in the
methylation process. According to the data of the methyla-
tion, and then combined with the results of the partial acid
hydrolysis, we can tentatively draw the following conclu-
sions: HPS4-2A was mainly composed of →4,6) Glcp (1→,
cross peaks were observed, with the corresponding 13
C
→
4,6) Galp (1→, →3,6) Galp (1→, →3,4) 2-NAc-Galp (1→ as
chemical shifts identified as δ 108.0, 107.5, 102.0, 101.4,
101.0, 100.0, 99.5 and 97.5 ppm respectively. In the region of
δ 4.3 ppm to δ 4.8 ppm, there were four major cross peaks
with their 13C chemical shifts identified at δ 104.6, 104.0,
the backbone with some →5) Araf (1→, →2) Rhap (1→, →4)
Glcp (1→ and →6) Glcp (1→, which maybe exist in the side
chains.
1
04.0 and 102.8 ppm, respectively. The signal at δ 19.8 ppm
13
3
3
.6. Spectrum analysis
belongs to the methyl group of rhamnose in
C NMR
spectrum; the resonances at δ 1.10 ppm and 1.20 ppm in
the HSQC spectrum showed that the rhamnose was in two
different chemical environments. The 13C signal at δ 22.9,
.6.1. IR spectrum
The evidence of sulfate ester was confirmed by the
appearance of the peaks at 817.8 (very weak) and
179.3 ppm and an upfield proton chemical shift at
2.04 ppm and downfield proton chemical shift at
δ
δ
−
1
1
240.71 cm (Figure not given), which was originated from
a
the bending vibration of C\O\S of sulfate in axial position and
stretching vibration of S\O, respectively. The absorption in the
8.20 ppm indicated that a NAc group was present. HSQC
spectrum showed twelve cross peaks in the anomeric region,
which indicated that around twelve spin systems were
present in HPS4-2A. The anomeric chemical shifts of sugar
residues were designated as follows: δ 5.06, 107.5 for residue
A, δ 5.24, 108.0 for residue B, δ 4.76, 104.0 for residue C … and
δ 4.42, 102.8 for residue L (Table 1).
−
1
range of 1200–1000 cm
that appeared in IR spectrum
indicated that the monosaccharide in HPS4-2A had a pyranose
−
1
ring. The strong absorption peak at 1615.08 cm
weak one at 1414.40 cm
and the
also demonstrated the character-
istic IR absorption of polysaccharide. The strong signal at
−
1
−
1
3
407.27 cm
O\H. Unfortunately, the signal of carbonyl stretching, νC_O
amide I), and the NH bending, δ NH (amide II), was not
observed.
was from stretching vibration of N\H and
3.6.2.1. Assignment of residue D (→2,4) α-L-Rhap (1→). The
H-1 signal of residue D at δ 5.38 ppm indicated that it was an
α-linked residue. Residue D was pyran rhamnose due to the
(
signal for an exocyclic-CH
3
group (H-6, δ 1.20 ppm).
According to the COSY spectrum, the chemical shift of the
H-5 (δ 3.64 ppm) could be assigned from its correlation with
H-6. Base on the connectives between H-1 and H-2, H-2 and
H-3, H-3 and H-4, H-2 to H-4 were assigned and their cross
peaks were marked in Figs. 2 and 3a. The 13C chemical shifts
3
.6.2. NMR spectrum
The HPS4-2A was further analyzed by 1D and 2D NMR
spectroscopy. The C and 1H assignments for the HPS4-2A
were reported in Table 1 and were assigned on the basis of
2
13
D COSY, TOCSY, HSQC, and HMBC experiments, along with
Table 1
1
3C and 1H NMR assignments for HPS4-2A.
Sugar residues and sugar type
C-1/H-1
107.5
C-2/H-2
C-3/H-3
C-4/H-4
C-5/H-5
C-6/H-6
A →5) α-L-Araf (1→
82.2
4.15
81.3
4.18
72.4
3.77
77.3
4.18
76.2
4.08
72.4
3.76
73.9
3.44
73.3
3.82
72.0
3.44
72.3
3.55
74.2
3.60
52.4
4.04
76.0
4.00
76.0
3.96
82.6
3.86
73.2
3.97
71.8
3.81
73.4
3.94
76.0
3.95
75.3
3.62
73.0
3.66
75.0
3.66
75.7
3.65
82.0
4.00
81.5
4.20
85.0
4.10
69.5
4.20
82.0
3.81
74.2
3.48
74.0
3.66
79.6
3.77
71.8
3.74
71.0
3.91
78.5
4.14
76.2
3.42
74.3
4.20
68.2
3.79/3.91
62.4
3.71/3.66
73.9
3.79
71.5
3.64
70.2
3.70
73.9
3.54
71.5
3.68
70.6
3.93
74.6
3.88
75.3
3.88
73.2
3.68
74.0
3.76
–
5
.06
108.0
.24
104.0
.76
100.0
.38
101.0
.24
99.5
.87
102.0
.20
97.5
.98
104.6
.57
104.0
.38
101.4
.96
102.8
.42
B α-L-Araf (1→
–
5
C →3,6) β-D-Galp (1→
D →2,4) α-L-Rhap (1→
E →2) α-L-Rhap (1→
F α-D-Glcp (1→
70.8
3.87/3.78
19.8
1.20
19.8
1.10
63.8
3.90
63.0
3.56/3.42
69.2
4.19/3.88
69.6
3.98
71.3
3.72
76.1
4.01
62.0
4
5
5
4
G →4) α-D-Glcp (1→
H →6) α-D-Glcp (1→
I →6) β-D-Galp (1→
J →4,6) β-D-Galp (1→
K →4,6) α-D-Glcp (1→
L →3,4)2-NAc-β-D-Galp (1→
5
4
4
4
4
4
3.80

Z. Dang et al. / Fitoterapia 89 (2013) 20–32
25
2
Fig. 1. 2D HSQC (a) and HMBC (b) nuclear magnetic resonance spectrum of HPS4-2A in D O at 25 °C.
were assigned according to the HSQC spectrum. The 13C
chemical and H shifts of the →2,4) α-L-Rhap (1→ residue
corresponding to the literature values [9,27,28].
spectrum (Figure not given), we could observe three cross
peaks (δ 3.81 ppm, δ 3.70 ppm, δ 3.48 ppm) were correlated to
H-1 (δ 5.24 ppm) of residue E. Hence residue E was tentatively
assigned to be → 2) α-L-Rhap (1 → by comparison with
literature values [9,26]. Then the correlation between H-6 (δ
1.10 ppm) and H-5 (δ 3.70 ppm) was observed when we came
back to the COSY spectrum. After the protons from H-1 to H-6
had been identified, the chemical shifts of 13C were assigned
(Table 1) from the HSQC spectrum and they were matched well
with literature [9,26].
1
3
.6.2.2. Assignment of residue E (→2) α-L-Rhap (1→). The
anomeric cross peak signals of residue E (δ 5.24 ppm) were
shown in the HSQC spectrum. The connection between H-1 and
H-2 could obtain from COSY spectrum (Fig. 2), but it was
difficult to recognize the connectives between H-3 and H-4 due
to overlapping in the region 3.40–3.80 ppm. In the TOCSY

26
Z. Dang et al. / Fitoterapia 89 (2013) 20–32
Fig. 2. COSY spectrum of HPS4-2A, the dotted lines is the connective between H-1 and H-2 of all residues.
3
.6.2.3. Assignment of residue F (α-D-Glcp(1→)). Anomeric
shifts assigned above, all in consistent with the previous
proton signal of residue F at δ 4.87 ppm indicated that it was
an α-linked residue, which was confirmed by C-1 signal at δ
literature of →4) α-D-Glcp (1→ [7,30–33].
9
9.5 ppm according to HSQC spectrum. In Figs. 2 and 3b, we
3.6.2.5. Assignment of residue K (→4, 6) α-D-Glcp (1→). We
could know from the results of the methylation, the content
of →4, 6) α-D-Glcp (1→ was 12.30% in HPS4-2A. In the HSQC
could observe stepwise connectivities from the H-1 signal at
δ 4.87 to H-2 at δ 3.76, H-3 at δ 3.94, H-4 at δ 3.66, H-5 at δ
1
3
.54, and H-6 at δ 3.90. From TOCSY spectrum, we also could
spectrum, residue K had a strong cross peak between H-1 (δ
found the relation between H-1 (δ 4.87 ppm), and H-2 (δ
4.96 ppm) and 13C-1 (δ 101.4 ppm), indicating that it was
α-linked. It was determined as →4,6) α-D-Glcp (1→. The
chemical shifts from H-1 to H-4 were assigned from 2D COSY
(Figs. 2 and 3d). Although the cross peak of H-5, 6 was
difficult to assign due to overlapping in the region δ 3.50–
4.10 ppm, when referring to the chemical shifts of →4,6)
α-D-Glcp (1→ in the literature [30,32], H-5 and H-6 can be
assigned as δ 3.68 and δ 4.01 ppm, respectively. The chemical
shifts of H-6 (δ 4.01 ppm) obtained by TOCSY spectrum,
13
3
(
.76 ppm), and H-3 (δ 3.94 ppm). The C chemical shifts
Table 1) of residue F were obtained from HSQC spectrum,
and they were all in consistent with the previous information
7,29–31]. From the results of methylation analysis and NMR
[
experiments, it could be concluded that residue F was
terminal α-D-glucose.
3
.6.2.4. Assignment of residue G (→4) α-D-Glcp (1→). Residue
13
G had a downfield anomeric proton signal at δ 5.20 ppm
indicated that it was an α-linked residue. The chemical shifts
of H-2, H-3, H-4, H-5, H-6 and H-6′ were obtained from COSY
further conformed data obtained from literature. Their
C
chemical shifts corresponding to 1H were assigned from the
HSQC spectrum. All the chemical shifts assigned were
matched well with literature [30,32].
spectrum (Figs.
2 and 3c) according to the stepwise
connectives from H-1. After the protons had been identified,
the chemical shifts of their corresponding carbons (Table 1)
were determined from the HSQC spectrum. The chemical
3.6.2.6. Assignment of residue L (→3,4) 2-NAc-β-D-Galp (1→).
In the HSQC spectrum, we could observe residue L had a cross

Z. Dang et al. / Fitoterapia 89 (2013) 20–32
27
Fig. 3. Enlargement of COSY spectrum of HPS4-2A, the dotted lines are the correlations of protons of→2, 4) α-L-Rhap (1→ (a), α-D-Glcp (1→(b), →4) α-D-Glcp (1
(c) and →4,6) α-D-Glcp (1→ (d), respectively.
→
peak between 1H-1 (δ 4.42 ppm) and 13C-1 (δ 102.8 ppm),
indicating that it was a β-linked residue. The assignment of
H-2 (δ 4.04 ppm) was conducted by its correlation with H-1
signal at δ 4.42 ppm in the COSY spectrum (Fig. 2). The
assignment of H-3 (δ 4.00 ppm), H-4 (δ 4.20 ppm) and H-5
respectively. Because of weak and overlapping signals in the
spectrum, they could only be partially assigned. The con-
nective between H-1 and H-2 of the residues could obtain
from COSY spectrum (Fig. 2) and the anomeric carbon
chemical shifts could obtain from HSQC spectrum. Based on
the above information, as well as the previous literature
values [9,27,31,37–40], the residues A, B, C, H, I and J were
assigned to be →5) α-L-Araf (1→, α-L-Araf (1→, →3,6)
β-D-Galp (1→, →6) α-D-Glcp (1→, →6) β-D-Galp (1→ and
→4,6) β-D-Galp (1→, respectively, and their chemical shifts
were all summarized in Table 1. Proton signals of H-2 (δ
4.15 ppm) of residue A and H-2 (δ 3.44 ppm), H-3 (δ 3.66 ppm)
and H-5 (δ 3.88 ppm) of residue I were further confirmed by
TOCSY spectrum.
(
δ 3.76 ppm) according to data from TOCSY spectrum and the
literature values [34–36]. The allocation of H-6 resonance of
residue L was supported by comparison with previous
1
3
reports [34–36]. The C chemical shifts of residue L were
obtained from HSQC, and they were all in consistent with the
13
previous information [34–36]. The
C signal at δ 22.9,
1
2
8
79.3 ppm and an upfield proton chemical shift at
.04 ppm and downfield proton chemical shift at
.20 ppm indicated that an NAc group was present. The
δ
δ
a
correlation (Fig. 1a) of the resonance between H-2 (δ
It was worth noting that near residue I had an anomeric
proton and carbon chemical shift at δ 4.56 ppm, δ 103.8 ppm
respectively in HSQC spectrum, suggesting that there was
doublets coexist with residue I. This indicated that the
residue I might have two chemical environment. Indeed,
lowfield chemical shift values for C-6 (69.6) and H-6
(3.98 ppm) of residue I [41] suggest glycosylation or an
electron withdrawing group exists in this position.
4
2
.04 ppm) and C-2 (δ 52.4 ppm) indicated residue L was a
-acetamido-2-deoxyglycose. Based on above results, we
confirmed that residue L was →3, 4) 2-NAc-β-D-Galp (1→.
3
.6.2.7. Assignment of remaining residues. In the HSQC
spectrum, residues A, B, C, H, I and J had anomeric proton
chemical shifts at δ 5.06, 5.24, 4.76, 4.98, 4.57 and 4.38 ppm,

28
Z. Dang et al. / Fitoterapia 89 (2013) 20–32
Table 2
determined and inter-residual connectivity was observed in
The significant 3JH,
connectivities observed in an HMBC spectrum for the
C
HMBC spectrum. For example, cross peaks were found
between C-1 (δ 101.4 ppm) of residue K and H-4 (δ
3.42 ppm) of residue K (K C-1, K H-4), H-1 (δ 4.96 ppm) of
residue K and C-3 (δ 82.0 ppm) of residue L (K H-1, LC-3).
Cross peaks between C-1 (δ 99.5 ppm) of residue F and H-2
anomeric protons/carbons of the sugar residues of HPS4-2A.
Sugar residues and sugar linkages H-1/C-1 Observed connectivities
δ H/δ C δ H/δ C Residue Atom
A →5) α-L-Araf (1→
5.06
82.0
4.2
D
E
C-4
C-2
(
δ 4.18 ppm, 4.08 ppm) of residue D and E (F C-1, D H-2 and
7
F C-1, E H-2) were observed in the same way, indicating that
some of the terminal glucopyranosyl residues were linked
with →2) α-L-Rhap (1→ by 1,2-O-glycosidic bonds. With
the same method, the cross peaks of other residues were
found and summarized in Table 2.
Unfortunately, the position of sulfate group in HPS4-2A
can't be determined by NMR spectra. But according to the
data obtained, we speculate that the most likely connection
position of sulfate group at O-6 of residue I, and we will
further study the location of the sulfate group of HPS4-2A in
the subsequent research.
1
5.24
1
4.76
07.5
B α-L-Araf (1→
68.2
78.5
A
J
C-5
C-4
08.0
C →3,6) β-D-Galp (1→
D →2,4) α-L-Rhap (1→
E →2) α-L-Rhap (1→
1
04.0
5.38
1
5.24
00.0
3.77
71.3
G
J
L
H-4
C-6
C-4
7
4.3
1
4.87
01.0
F α-D-Glcp (1→
69.2
9.6
4.18
.08
H
I
D
E
K
C-6
C-6
H-2
H-2
C-6
6
9
9.5
4
As a result of all the structural information described, a
possible molecular structure of HPS4-2A was proposed as in
Fig. 4.
G →4) α-D-Glcp (1→
H →6) α-D-Glcp (1→
I →6) β-D-Galp (1→
J →4,6) β-D-Galp (1→
K →4,6) α-D-Glcp (1→
5.20
76.1
76.1
82.6
1
02.0
4.98
9
4.57
K
C
C-6
C-3
7.5
3.7. Ultrastructure analysis
1
04.6
4.38
To provide direct evidence of the chain conformation of the
polysaccharides, SEM, TEM and AFM were used to observe their
morphology.
1
04.0
3.42
82.0
3.42
K
L
K
H-4
C-3
H-4
4.96
1
01.4
3.7.1. Scanning electron microscope
3
.6.3. Linkage sites and sequence among residues
A long-range HMBC experiment performed on the HPS4-2A
Morphological of HPS4-2A was observed using SEM after
lyophilized. The SEM images showed thin lamellar structure
that is probably due to shrinkage occurring during the water
evaporation of polysaccharide solution while drying. This
phenomenon is more evident for the freeze-dried samples
[42]. The high magnification image of HPS4-2A showed
smooth surface appearance. SEM images of HPS4-2A can be
seen in Fig. A.2(a and b) of Appendix A.
enable us to identify linkage patterns between sugar residues, as
shown in Fig. 1b, and summarized in Table 2. In this experiment,
the correlations of the resonances between protons and carbon
atoms help us define what kind of glycoside linkage that
connects two sugar residues. The cross peaks of anomeric
protons and carbons of each sugar residue of HPS4-2A were
Fig. 4. Proposed structure of HPS4-2A.

Z. Dang et al. / Fitoterapia 89 (2013) 20–32
29
3
.7.2. Transmission electron microscope
Usually, TEM is used to observe the morphology and
single polysaccharide chain (about 0.1–2 nm), suggesting
that inter- and/or intra-molecular aggregation was involved.
However, it was possible that the drying process could have
some influence over the conformation of the molecules [44].
The results obtained in the present study were consistent
with results obtained by SEM and SEC-MALLS.
The morphology observed under SEM has a clear distinc-
tion with the morphology observed by AFM. The reason was
that the SEM used the lyophilized sample as observation
object, and in freeze-drying process, water evaporating faster
than natural-drying, therefore the polysaccharide molecules
cannot stretched and stacked together to form a thin lamellar
structure. While the atomic force microscope observation
was carried out on the sample dried in the natural state,
although the aggregation occurs in the drying process, still
part of the polysaccharide molecules existed as stretched
state.
dimension of the particles and macromolecules [43]. TEM
images of HPS4-2A can be seen in Fig. A.2(c and d) of
Appendix A. The observed polysaccharide exhibited spherical
features arranged disorderedly. The diameters of the particle
observed by TEM were in the range of 100–800 nm which
weren't in agreement with that determined by MALLS.
Apparently, the polysaccharide molecules were aggregated
in water. There are two reasons for this phenomenon, one is
the highly branched structure of HPS4-2A can entangled with
each other in aqueous solution, which lead to aggregation,
the other one is negative staining reagent changed the
sample pH and ionic strength, which make aggregation is
more obvious.
3
.7.3. Atomic force microscope
The topographical AFM planar image of HPS4-2A depos-
ited from a 1 μg/ml water solution was shown in Fig. 5.
Pictures shown in Fig. 5 represent dilute regions within
which individual molecules can be seen. Branched (arrows
A), beadlike shape (arrows B), and irregular and spherical
lumps (arrows C, arrows D) can be seen in Fig. 5a and b. The
diameter and height of the lumps ranged from 10 to 100 nm
and 5 to 50 nm, respectively. The heights of the spherical
structures in Fig. 5c and d were much higher than that of a
3.8. Antioxidant activity
3.8.1. Effect of scavenging DPPH radicals
The ability of the HPS4-2A to scavenge DPPH radicals was
shown in Fig. 6a and compared with that of ascorbic acid. DPPH
radicals' scavenging rates of the HPS4-2A were dependent on
its concentration; the scavenging rate at concentration of
8 mg/ml was 85.28% while the rate of ascorbic acid at the same
Fig. 5. Atomic force microscopy (AFM) planar (a and b) and cubic (c and d) images of HPS4-2A.

30
Z. Dang et al. / Fitoterapia 89 (2013) 20–32
Fig. 6. Free-radical scavenging of HPS4-2A. DPPH radicals (a) and hydroxyl radicals (b).
concentration was 95.26%, suggesting good DPPH radicals'
scavenging ability of HPS4-2A.
only a possible mechanism according to the speculation from
the literatures, and the anti-oxidation mechanism of HPS4-2A
is under in-depth studying now.
3
.8.2. Measurement of hydroxyl radical scavenging activity
The result of hydroxyl free radical-scavenging ability of
the HPS4-2A was shown in Fig. 6b and the ascorbic acid was
regarded as positive control. The hydroxyl radical scavenging
rate of HPS4-2A and ascorbic acid at 8.0 mg/ml was 66.93%
and 87.01%, respectively, and in a concentration-dependent
manner. This result proved that HPS4-2A had significant
effect on scavenging hydroxyl radicals.
4. Conclusion
HPS4-2A, a heteropolysaccharide, which highly branched
structure has been elucidated by SEC-MALLS, methylation
analysis combined with GC–MS and 1D, 2D NMR spectro-
scopic technique. Its branched and spherical conformation
has been studied by SEM, TEM and AFM. In summary, the
backbone was composed of →4,6) α-D-Glcp (1→, →4,6)
β-D-Galp (1→, →3,6) β-D-Galp (1→ and →3,4) 2-NAc-β-D-
Galp (1→ (not common in plant polysaccharides) residues
substituted at O-6, O-6, O-3 and O-4 positions, respectively.
The antioxidant mechanism of Vitamin C and HPS4-2A is
not identical. Vitamin C has antioxidant activity due to its
enol group, which had strong reducing action. The antioxi-
dant effect of HPS4-2A may be a result of combination of
multiple mechanisms. According to the literatures [45–47],
we can speculate the DPPH free radical scavenging mecha-
nism of HPS4-2A may be due to its hydrogen-donating
ability. DPPH radical was scavenged by HPS4-2A through
donation of hydrogen to form a stable DPPH molecule [48].
Sulfate group act as an electrophile which promotes the
intramolecular hydrogen donation [46,49–52], and resulting
in enhancing DPPH radical scavenging capacity of HPS4-2A.
Both of the two mechanisms might be responsible for the
hydroxyl radical scavenging ability of HPS4-2A as the earlier
researchers suggested [52]: one suppresses the generation of
the hydroxyl radical, and the other scavenges the hydroxyl
radicals generated. HPS4-2A may ligate to the metal ions which
react with hydrogen peroxide to give the metal complexes. The
metal complexes thus formed could not further react with
hydrogen peroxide to give hydroxyl radicals. As reported, in
the absence of transition metal ions, hydrogen peroxide was
fairly stable [53]. Radical scavenging ability of HPS4-2A was
Side chains are terminated by arabinofuranosyl (Araf),
glucopyranosyl (Glcp) residues and occasionally by SO
−
3
(Shi also had similar findings in literature [7]. HPS4-2A
exhibit strong antioxidant activities and further investiga-
tion on its antioxidant property in vivo and the others
biological activities, especially radioprotective activity of
HPS4-2A is in progress.
Acknowledgment
The authors would like to acknowledge the financial
assistance provided by National “Twelfth Five-Year” Plan for
Science
& Technology Support (No. 2011BAI05B02) for
supporting this research. The authors would like to thank
Professor Wei Zhou, for providing instrumental facilities of GC–
MS from Gansu entry–exit inspection and quarantine bureau.
The authors would also like to express gratitude to College of
chemistry and chemical engineering of Lanzhou University for
the NMR spectra and elemental analysis. The authors would
also like to express their appreciation to Lanzhou Institute of
Chinese Physics, Chinese Academy of Sciences, for providing
AFM, TEM, and SEM analyses.
also related to the number of acetamido groups in
a
polysaccharide molecule, the existing of amino can enhance
the radical scavenging ability, as Guo [54] reported. In addition,
it has been reported that the antioxidant ability of the
polysaccharide was bound up with monosaccharide composi-
tion [55] and molecular weight [56]. What described above was

Z. Dang et al. / Fitoterapia 89 (2013) 20–32
31
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25] Podzimek S. Light scattering, size exclusion chromatography and
asymmetric flow field flow fractionation.New Jersey: John Wiley &
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
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