In vitro and in vivo antioxidant activity of a water-soluble polysaccharide from dendrobium denneanum

Article abstract of DOI:10.3390/molecules16021579

The water-soluble crude polysaccharide (DDP) obtained from the aqueous extracts of the stem of Dendrobium denneanum through hot water extraction followed by ethanol precipitation, was found to have an average molecular weight (Mw) of about 484.7 kDa. Monosaccharide analysis revealed that DDP was composed of arabinose, xylose, mannose, glucose and galactose in a molar ratio of 1.00:2.66:8.92:34.20:10.16. The investigation of antioxidant activity both in vitro and in vivo showed that DDP is a potential antioxidant.

Full text of DOI:10.3390/molecules16021579

Molecules 2011, 16, 1579-1592; doi:10.3390/molecules16021579
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
In Vitro and In Vivo Antioxidant Activity of a Water-Soluble
Polysaccharide from Dendrobium denneanum
1
,†
2
1,†,
3
3
4
Aoxue Luo , Zhongfu Ge , Yijun Fan *, Aoshuang Luo , Ze Chun and XingJin He
1
Department of Landscape Plants, Chengdu Campus of Sichuan Agriculture University, Chengdu
11130, China
6
2
United States Geological Survey, Lake Michigan Ecological Research Station, Porter, IN 46304,
USA
3
4
Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
College of Life Sciences, Sichuan University, Chengdu 610064, China
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: Yijfan@yahoo.com.cn.
Received: 12 January 2011; in revised form: 9 February 2011 / Accepted: 11 February 2011 /
Published: 14 February 2011
Abstract: The water-soluble crude polysaccharide (DDP) obtained from the aqueous
extracts of the stem of Dendrobium denneanum through hot water extraction followed by
ethanol precipitation, was found to have an average molecular weight (Mw) of about
4
84.7 kDa. Monosaccharide analysis revealed that DDP was composed of arabinose,
xylose, mannose, glucose and galactose in a molar ratio of 1.00:2.66:8.92:34.20:10.16. The
investigation of antioxidant activity both in vitro and in vivo showed that DDP is a
potential antioxidant.
Keywords: Dendrobium denneanum; polysaccharide; antioxidant activity
1
. Introduction
Dendrobium denneanum (Orchidaceae) is a precious herbal plant highly valued in Traditional
Chinese Medicine and archived in the Pharmacopoeia of the People's Republic of China. Sections of
the stem from the Dendrobium species have been used for the treatment of salivary, ophthalmic

Molecules 2011, 16
1580
disorders, fever and chronic superficial gastritis, or as a tonic for promoting the production of body
fluids and for improving the quality of life [1]. As for Dendrobium species phytochemicals, much
research has been carried out on the low molecular compounds, such as bibenzyl [2], phenanthrene [3],
and alkaloids [4]. Based on previous studies, polysaccharides exist widely in numerous plants and are
identified as essential biomacromolecules in plant life, playing important roles in cell-cell
communication, cell adhesion, and molecular recognition in the immune system [5-8]. In recent years,
some bioactive polysaccharides isolated from natural sources have attracted much attention in the field
of biochemistry and pharmacology [9,10]. It has been proven that polysaccharides are major active
constituents in Dendrobium species. Polysaccharides from some Dendrobium species, such as
Dendrobium nobile Lindl [11,12], Dendrobium huoshanense [13] and Dendrobium chrysotoxum Lindl
[
14], have been isolated and reported to have beneficial effects on antioxidation, immunity stimulation
and antitumor activities. However, the polysaccharides from Dendrobium denneanum have been little
reported [15]. Therefore, the purpose of the present investigation was to elucidate the isolation and
characterization of water-soluble polysaccharide from the stems of Dendrobium denneanum, as well as
to evaluate its antioxidant activities in vitro and in vivo.
2
. Results and Discussion
2
.1. Polysaccharide isolation and purification
The total yield rate of crude polysaccharides was 15.3% by the boiling-water extraction method,
followed by ethanol precipitation. Because of the presence of some colored materials and residual
protein, DDP was a brown water-soluble powder. AB-8 and ADS-7 macroporous resin were used for
further purification of the resulting DDP. After application of these two macroporous adsorption
resins, protein eventually disappeared in the DDP and the polysaccharide DDP had gray-colored
powder appearance. The total sugar content of the polysaccharide was estimated to be 99.7% by the
phenol-sulfuric acid method. Previous research has indicated that the molecular weight of
polysaccharide was an important factor responsible for biological activities [15]. Determining the
molecular weight was therefore the first step for the study of the DDP polysaccharide. The molecular
weight (M
w
) of DDP was calculated to be 484,700 Da based on the calibration curve obtained with
standard dextrans.
2
.2. Monosaccharide composition of DDP
Monosaccharide composition of DDP was analyzed by the trifluoroacetic acid hydrolysis and GC-
MS analysis method. The results shown in Figure 1 indicate that glucose is a major monosaccharide
constructing the backbones of DDP. DDP was found to be composed of arabinose, xylose, mannose,
glucose, and galactose with a molar ratio of 1.00: 2.66: 8.92: 34.20: 10.16.

Molecules 2011, 16
Figure 1. Monosaccharide composition of DDP by GC-MS analysis.
1581
2
.3. Infrared spectra of DDP
As shown in Figure 2, the sample of DDP exhibited a significant, broad characteristic peak at
−1
−1
around 3,446 cm for the hydroxyl group, as well as a weak C-H band at around 2,927 cm [16]. The
−1
band at 1,638 cm was due to the bound water [17]. The polysaccharide also appears to have a specific
−1
band between 1,200 and 1,000 cm , which is dominated by ring vibrations overlapped with stretching
vibrations of (C-OH) side groups and the (C-O-C) glycosidic band vibration [18]. A characteristic peak
−1
at around 894 cm was also found in DDP, indicating the β-configuration of the sugar units [19].
−1
Positive specific rotation and characteristic absorption at around 869 cm were identified in the IR
spectrum, indicating the α-configuration of the sugar units [20].
Figure 2. FT-IR spectra of the polysaccharide DDP.

Molecules 2011, 16
.4. NMR identification
1582
2
1
13
The H-NMR and C-NMR results for DDP are shown in Table 1 [21-24]. Based on the previous
discussions in the literature, the signals were centered between δ 99.69 and 102.57 ppm, indicating an
1
α anomeric configuration for all monosaccharide residues of DDP (Figure 3A) [25,26]. The H-NMR
spectrum of DDP showed two anomeric protons at 5.30 and 4.86, which were assigned as (1→4)-α-D-
Glcp and (1→6)-α-D-Glcp(Li et al. 2008), respectively (Figure 3B).
1
13
Table 1. H NMR and C NMR chemical shifts for DDP in D O at 27 °C.
2
Sugar residues
H-1
C-1
5.12
H-2
C-2
3.84
69.40
4.08
H-3
C-3
3.87 4.01
70.02 70.02 71.25
3.87 3.74 3.76
71.25 69.40 73.37
73.37 76.58 70.02
3.70 3.56
3.74 3.63
76.58 78.00 76.86
H-4
C-4
H-5
C-5
4.08
H-6
C-6
α-D-Galp-（1→
α-D-Manp-（1→
3.70
60.51
4.00
60.51
65.80
3.66
3.84
62.50
9
9.69
5.12
1
02.57 70.02
→
→
4,6)-α-D-Glcp-（1→
4)-α-D-Glcp-(1→
99.69
.86
5.40
71.25
3.54
3.85
4
4.01
4.01
1
02.57 73.37
1
3
1
Figure 3. NMR analysis of DDP. A for C-NMR analysis of DDP, and B for H-NMR
analysis of DDP.

Molecules 2011, 16
1583
2
.5. Antioxidant activities analysis
2.5.1. Effect of scavenging DPPH radicals
In this experiment, the scavenging ability of the purified polysaccharide DDP on DPPH free radical
were examined in the concentration range of 10–2,000 μg/mL using the DPPH colorimetric assay
(shown in Figure 4A).
Figure 4. Antioxidant activity of DDP in vitro. A: The scavenging effect of DDP on DPPH
radical; B: The scavenging effect of DDP on hydroxyl radical; C: The scavenging effect of
DDP on ABTS radical. Results are presented as means ± standard deviations.
The results indicated that the activities of both samples increased in a concentration dependent
manner. Furthermore, the scavenging activities of DDP increased very significantly with increasing
concentrations. Especially at the high dose (2,000 μg/mL), DDP exhibited very high radical
scavenging activity (92.06%), which was close to that of vitamin C (p < 0.05). However, in the low
doses (10 to 1,000 μg/mL), the radical scavenging activity of DDP was lower than that of vitamin C.
Therefore, it is obvious that the polysaccharide DDP has strong antioxidant activity in the high doses.

Molecules 2011, 16
.5.2. Scavenging effects of polysaccharide on hydroxyl radicals
The results of hydroxyl radical scavenging activities of DDP compared to those of vitamin C are
1584
2
shown in Figure 4B. The samples exhibited obvious hydroxyl radical scavenging activities in a
concentration-dependent manner. The polysaccharide was found to have the ability to scavenge
hydroxyl radicals at concentrations between 500 and 2,000 μg/mL. At 500 μg/mL, DDP revealed an
excellent hydroxyl radical scavenging activity (48.9%), similar to that of vitamin C (50.1%). At the
high dose of 2,000 μg/mL, DDP also exhibited strong activity (79.8%), which was higher than that of
vitamin C. These results clearly showed that DDP has potential hydroxyl radical scavenging
antioxidant ability.
2
.5.3. Scavenging effects of polysaccharide on ABTS
The ABTS radical cation decoloration assay, which employs a specific absorbance (734 nm) at a
wavelength well separated from the visible-light range and requires only a short reaction time, has
been widely applied to evaluating the total antioxidative activity in both lipophilic and hydrophilic
samples [27]. The scavenging ability of DDP on ABTS free radical in the present experiments is
shown in Figure 4C. The scavenging power of all samples correlated well with increasing
concentrations. Moreover, vitamin C exhibited evident scavenging effect especially at high doses
(
from 500 to 2,000 μg/mL). At 2,000 μg/mL, the scavenging ability was 98.16%. In contrast, the
sample DDP revealed insufficient scavenging power against ABTS radical. Even at a high
concentration of 2,000 μg/mL, its scavenging ability was only 25.56%, which was far lower than that
of the control. Therefore, the results indicated that the polysaccharide DDP has no significant effect on
ABTS radical scavenging.
2
.6. Antioxidant activity in vivo
Superoxide dismutase (SOD) activity (U/mL) was tested with the SOD Assay Kit. Superoxide was
generated in xanthine oxidase and hypoxanthine, and the superoxide scavenging effect of serum was
determined according to the method of Oyanagui [28]. SOD activity of the serum was expressed in
U/mL of the sample. As shown in Figure 5A, SOD activities of different dose of DDP exhibited dose-
dependent manner. At 200 mg/kg, particularly, SOD activity of DDP was 132.79 U/mL, which was
close to that of vitamin C. However, SOD activity at low concentrations was much less evident, which
is similar to that of the negative control. The results were therefore an indication of enhancement SOD
activity of DDP for high concentrations.
The concentrations of MDA in blood serum from the mice were determined with an MDA Assay
Kit. The MDA value was estimated according to the thiobarbituric acid (TBA) method [29]. The
samples added with TBA were heated in an acidic environment. The absorbance of the resulting
solution was measured at 532 nm. The results in Figure 5B exhibit a significant pattern of a decreasing
MDA concentration in blood serum with increasing DDP concentration. At 200 mg/kg, the
concentration of MDA was 5.95 nmol/mL, close to that of the positive control. This can be interpreted
as a significant effect of DDP at high concentrations on MDA scavenging in vivo.

Molecules 2011, 16
Figure 5. Antioxidant activity of DDP in vivo. A: SOD activity analysis in mice; B:
1585
Determination of MDA contents in blood serum from the mice. Results are presented as
means ± standard deviations.
3. Experimental
3.1. Materials and chemicals
DPPH (1,1-diphenyl-2-picrylhydrazyl) radical and Vitamin C were purchased from Sigma (St.
Louis, MO, USA). Dextrans of different molecular weights were purchased from Pharmacia Co.
Uppsala, Sweden). The standard monosaccharides (glucose, mannose, rhamnose, galactose, xylose
(
and arabinose) were purchased from the Chinese Institute for the Control of Pharmaceutical and
Biological Products (Beijing, China). ABTS (2,2-azinobis-6-(3-ethylbenzothiazoline sulfonic acid)
radical was purchased from Merck (Darmstadt, Germany). AB-8 and ADS-7 were purchased from the
Chemical Plant of Nankai University (Tianjin, China). SOD Assay Kit001 and MDA Assay Kit A003
were purchased from the Institute of Biological Engineering of Nanjing Jianchen (Nanjing, China).
Trifluoroacetic acid (TFA), pyridine, methanol, and acetic acid, ethanol, acetic anhydride and all other
chemicals and reagents were analytical grade.
3
.2. Extraction the crude polysaccharide from Dendrobium denneanum
The stems of Dendrobium denneanum were thoroughly washed with water, dried at 60 °C, and then
powdered with a pulverizer. The powder was extracted with petroleum ether at 70 °C for 2 h to remove

Molecules 2011, 16
1586
lipids. After filtered, the residues were air-dried, refluxed again with 80% ethanol at 90 °C for 2 h, and
then extracted with double-distilled water at 100 °C for 2 h thrice, filtered. The combined filtrate was
precipitated with ethanol (4 times the volume of aqueous extract) at 4 °C for 24 h. After filtration and
centrifugation, the precipitate was dissolved in double-distilled water and deproteinized five times with
the Sevag reagent [30]. It was subsequently dialyzed against deionized water for 72 h and lyophilized,
which finally yielded the desired crude Dendrobium denneanum polysaccharides (DDP).
3.3. Purification of polysaccharide
Two macroporous adsorption resins, AB-8 and ADS-7, were used for purifying the crude
polysaccharides of Dendrobium denneanum. AB-8 was selected for the decoloration of the DDP. The
method was described by Xia et al. [31]. ADS-7 was used to remove the residuary protein according to
the methods of Li et al. [32]. The crude polysaccharides were first dissolved in double-distilled water.
After membrane filtration (0.45 μm, Nucleopore), the filtrate was injected to column (26 × 300 mm) of
AB-8 and ADS-7, respectively. The columns were eluted with distilled water. The carbohydrate
contents of the samples were determined by the phenol-sulfuric acid method using glucose as standard
[
33]. Protein content was determined according to the method of Bradford et al. [34], with bovine
serum albumin as standard.
3
.4. Determination of molecular weight
The molecular weight of DDP was determined by the Gel Permeation Chromatography (GPC)
technique as described by Yamamoto et al. [35] using a Waters HPLC apparatus (Waters 515, Waters
Co. Ltd., USA) equipped with an Ultrahydrogel Linear Column (300 × 7.8 mm) and a model 2410
Refractive index detector (RID). The column was eluted with 0.2 M phosphate buffer (PH 7.0) with a
flow rate of 0.7 mL/min and calibrated with Dextran standards (molecular weights: 2,500, 4,600,
7
,100, 10,000, 21,400, 41,100, 84,400, 133,800, 200,000 Da).
3
.5. Analysis of monosaccharide compositions
DDP (10 mg) was hydrolyzed in a sealed glass tube with 2 M TFA (2 mL) at 100 °C for 8 h [36].
The hydrolysate was dried at a reduced pressure in preparation for acetylation. The acetylation was
carried out using hydroxylamine hydrochloride (10 mg) and pyridine (0.5 mL) heated in a water bath
at 90 °C for 30 min. The tube was afterwards cooled to room temperature before acetic anhydride
(
0.5 mL) was added and mixed thoroughly by vortexing. The tube was sealed and incubated in a water
bath shaker at 90 °C for 30 min. following a cooling process, an aliquot of the acetylation product
3 μL) was loaded onto an Rtx-5SilMS column (30 m × 0.32 mm × 0.25 μm) of a GC-MS (QP2010,
Shimadzu, Japan). This step was carried out under the following conditions: injection temperature:
40 °C; detector temperature: 240 °C; column temperature programmed: 160 °C held for 2 min,
(
2
increased to 240 °C at a rate of 5 °C/min and finally maintained at 240 °C for 5 min. Nitrogen was
used as the carrier gas with a flow rate 1.0 mL/min.

Molecules 2011, 16
.6. Infrared spectra of DDP
The purified polysaccharide was ground with KBr powder and then pressed into pellets for the
1587
3
−1
estimation of the infrared (IR) spectra of DDP in the 4,000–500 cm frequency range. Spectra were
recorded on a Perkin-Elmer Fourier Transform IR spectrophotometer (Perkin-Elmer Corp., USA) [37].
3.7. NMR Identification
2
Twenty milligrams of DDP was dissolved in D O (0.5 mL, 99.9%), freeze-dried, and redissolved in
1
13
D
2
O (0.5 mL). The H-NMR and C-NMR spectra were measured in an NMR tube (5-mm diameter)
at 27 °C with a Bruker Avance 600 spectrometer. The chemical shift was expressed in parts per million
ppm).
(
3
.8. Assays for antioxidant activities
3
.8.1. DPPH radicals scavenging assay
DPPH radical is a stable free radical that shows a maximum absorption at 517 nm, and is widely
used to evaluate the free radical scavenging ability of natural compounds. In the DPPH assay, the
antioxidants were able to reduce the stable radical DPPH to the yellow coloured diphenyl-
picrylhydrazine. Therefore, the antioxidant activities of a sample can be expressed as its ability in
scavenging the DPPH radical. In this study, the antioxidant activities were determined by the DPPH
scavenging activities of the purified polysaccharides as described by Shimada et al. [38] with some
modifications. Vitamin C was used as reference material. Briefly, 0.1-mM solution of DPPH in
methanol was prepared, 1.0 mL of which was then mixed with purified polysaccharide solutions
(
3.0 mL) of various concentrations (10–2,000 μg/mL). The solution was kept at room temperature for
0 min before the absorbance at 517 nm (A517) was measured. The DPPH scavenging effect was
3
calculated as follows: DPPH scavenging effect (%) = [A −(A−A )] / A × 100 (A : the A of DPPH
0
b
0
0
517
b
without the sample; A: the A517 of the sample and DPPH; A : the A517 of the sample without DPPH).
3.8.2. Hydroxyl radical scavenging assay
The antioxidant activity was determined by the scavenging effect of hydroxyl radical as described
by Smirnoff and Cumbes [39], with some modifications. Briefly, vitamin C was used as reference
material. DDP samples of different concentrations (10–2,000 μg/mL) were incubated with 2 mM
EDTA-Fe (0.5 mL), 3% H
2
O
2
(1 mL), and 360 μg/mL crocus in a 4.5-mL sodium phosphate buffer
(150 mM, pH 7.4) at 37 °C for 30 min. Hydroxyl radical was detected by monitoring the absorbance at
5
C S C S
20 nm. The hydroxyl radical scavenging effect (%) was calculated as [(A −A ) / A ] × 100, where A
and A are the A of the samples and the control group, respectively. The control group used distilled
C
520
water and a sodium phosphate buffer in place of H
2 2
O .
3
.8.3. ABTS radicals scavenging assay
The ABTS radical cation scavenging activity of DDP was identified using the method given by
Re et al. [40] with some modifications. ABTS was produced from the reaction of 7 mM of ABTS

Molecules 2011, 16
1588
solution with 2.45 mM of potassium persulphate. The mixture was then kept in the dark at room
temperature for 16 h. At the time of use, the ABTS solution was diluted with ethanol to yield an
absorbance of 0.70 ± 0.02 at 734 nm. Samples (0.2 mL) of various concentrations (10–2,000 μg/mL)
were mixed with ABTS solutions (2 mL). After reaction at room temperature for 6 min, the absorbance
at 734 nm was recorded. The ABTS scavenging effect was calculated based on the following formula:
ABTS scavenging effect (%) = [A −(As−Ab) ]/A ×100, where A , As, and A are the A of the
0
0
0
b
734
ABTS without the sample, of the sample and ABTS, and of the sample without ABTS, respectively.
3
.8.4. Antioxidant activity in vivo
Kunming mice (eight weeks old of age, 20 ± 2 g of weight) were provided by Sichuan Academy of
Medical Science, China. The mice were kept in separated cages at a temperature of 21 ± 1 °C and a
0–60% of relative humidity. They underwent 12-h light-and-dark cycles with free access to food and
5
water. A total of 50 mice were evenly and randomly divided into five groups, including a D-galactose
model control group (negative control), a vitamin C (100 mg/kg) group (positive control), and dose-
dependent DDP groups (200, 100, and 50 mg/kg body weight). Each group was induced by a single
intraperitoneal injection of D-galactose (100 mg/kg/day body weight, dissolved in a 0.9% saline
solution) [41]. The mice in the D-galactose model control group were given a 0.2-mL physiological
saline solution (0.9% w/v) once daily for 20 consecutive days by intraperitoneal injection. Twenty-four
hours after the last drug administration, blood samples were obtained from the eyepit of the mice and
processed for serum. The superoxide dismutase (SOD) activity and the malondialdehyde (MDA) level
were also measured.
3
.9. Statistical analysis
The data were presented as mean ± standard deviation. Statistical analysis was conducted with the
SPSS 16.0 software package.
4
. Conclusions
Polysaccharides in many plants are not only energy resources but they play key biological roles in
many life processes as well. The structure and mechanisms of pharmaceutical effects of bioactive
polysaccharides on diseases have been extensively studied, and more natural polysaccharides with
different curative effects have been tested and even applied in therapies [42]. Recent research has
shown that some polysaccharides can play an important role as free radical scavengers for the
prevention of oxidative damage in living organisms [43]. In the present study, the water-extracted
polysaccharide from Dendrobium denneanum was purified with AB-8 and ADS-7 column
chromatography, which yielded the polysaccharide DDP. In the subsequent antioxidant experiments in
vitro, DDP exhibited strong scavenging ability on DPPH radical. The antioxidant effect of DDP is
similar to that of vitamin C. The damaging action of OH radical, which can react with all biological
molecules such as proteins, lipids, and carbohydrates, is very strong [44] and the resulting oxidative
stress can mediate a wide variety of degenerative processes and diseases [45,46]. In the assays above,
the DDP polysaccharide exhibited very strong scavenging ability on hydroxyl radical. At the high dose

Molecules 2011, 16
1589
of 2,000 μg/mL, the antioxidant activity of DDP is higher than that of positive control (vitamin C). For
in vivo assays, the DDP was found to increase the levels of antioxidant enzymes (SOD) and to
decrease the MDA content in blood serum. It was confirmed that DDP could protect tissues against
oxidative damages. Enhanced SOD activity in mice blood serum also can be related to the in vivo
antioxidant activity of DDP. With such strong antioxidant ability, DDP was identified as a potential
antioxidant.
Acknowledgements
This study was supported by the Education Department Foundation of Sichuan Province of China
(
08ZA074).
References and Notes
1
.
Yves, S.Y.H.; Cheng, C.; Sylvian, K.S.L.; Shih, F.L.; Wei, T.H.; Wen, B.Y.; Chih, C.L.; Ting,
J.R.C.; Chia, C.C.; Jim, M.F.; Chi, H.W. Structure and bioactivity of the polysaccharides in
medicinal plant Dendrobium huoshanense. Bioorg. Med. Chem. 2008, 16, 6054-6068.
Majumder, P.L.; Guha, S.; Sen, S. Bibenzyl derivatives from the orchid Dendrobium amoenum.
Phytochemistry 1999, 52, 1365-1369.
2
3
.
.
Yang, L.; Qin, L.H.; Annie Bligh, S.W.; Bashall, A.; Zhang, C.F.; Zhang, M.; Wang, Z.T.; Xu,
L.S. A new phenanthrene with a spirolactone from Dendrobium chrysanthum and its anti-
inflammatory activities. Bioorg. Med. Chem. 2006, 14, 3496-3501.
4
5
6
.
.
.
Wang, Q.; Gong, Q.; Wu, Q.; Shi, J. Neuroprotective effects of Dendrobium alkaloids on rat
cortical neurons injured by oxygen-glucose deprivation and reperfusion. Phytomedicine 2010, 17,
1
08-115.
Tong, H.B.; Xia, F.G.; Feng, K.; Sun, G.R.; Gao, X.X.; Sun, L.W.; Jiang, R.; Tian, D.; Sun, X.
Structural characterization and in vitro antitumor activity of a novel polysaccharide isolated from
the fruiting bodies of Pleurotus ostreatus. Bioresource Technol. 2009, 100, 1682-1686.
Cao, W.; Li, X.Q.; Wang, X.; Fan, H.T.; Zhang, X.N.; Hou, Y.; Liu, S.B.; Mei, Q.B. A novel
polysaccharide, isolated from Angelica sinensis (Oliv.) Diels induces the apoptosis of cervical
cancer HeLa cells through an intrinsic apoptotic pathway. Phytomedicine 2010, 17, 598-600.
Kiyohara, H.; Matsumoto, T.; Yamada, H. Intestinal immune system modulating polysaccharides
in a Japanese herbal (Kampo) medicine, Juzen-Taiho-To. Phytomedicine 2002, 9, 614-624.
Sun, Y.X.; Liang, H.T.; Zhang, X.T.; Tong, H.H.; Liu, J.C. Structural elucidation and
immunological activity of a polysaccharide from the fruiting body of Armillaria mellea.
Bioresource Technol. 2009, 100, 1860-1863.
7
.
.
8
9
.
Dwek, R.A. Glycobiology: Toward understanding the function of sugars. Chem. Rev. 1996, 96,
6
83-720.
1
0. Rout, S.; Banerjee, R. Free radical scavenging, anti-glycation and tyrosinase inhibition properties
of a polysaccharide fraction isolated from the rind from Punica granatum. Bioresource Technol.
2
007, 98, 3159-3163.

Molecules 2011, 16
1590
1
1. Luo, A.X.; He, X.J.; Zhou, S.D.; Fan,Y.J.; He, T.; Chun, Z. In vitro antioxidant activities of a
water-soluble polysaccharide derived from Dendrobium nobile Lindl. Extracts. Int. J. Biol.
Macromol. 2009, 45, 359-363.
1
1
1
2. Wang, J.H.; Luo, J.P.; Zha, X.Q. Structural features of a pectic polysaccharide from the stems of
Dendrobium nobile Lindl. Carbohyd. Polym. 2010, 81, 1-7.
3. Zha, X.Q.; Luo, J.P.; Luo, S.Z.; Jiang, S.T. Structure identification of a new immunostimulating
polysaccharide from the stems of Dendrobium huoshanense. Carbohyd. Res. 2007, 69, 86-93.
4. Zhao, Y.P.; Son, Y.O.; Kim, S.S.; Jang, Y.S.; Lee, J.C. Antioxidant and Anti-hyperglycemic
Activity of Polysaccharide Isolated from Dendrobium chrysotoxum Lindl. J. Biochem. Mol. Biol.
2
007, 40, 670-677.
1
5. Fan, Y.J.; He, X.J.; Zhou, S.D.; Luo, A.X.; He, T.; Chun, Z. Composition analysis and antioxidant
activity of polysaccharide from Dendrobium denneanum. Int. J. Biol. Macromol. 2009, 45,
1
69-173.
1
1
1
6. Luo, D.H. Identification of structure and antioxidant acti vity of a fraction of polysaccharide
purified from Dioscorea nipponica Makino. Carbohyd. Polym. 2008, 71, 544-549.
7. Liu, Y.H.; Wang, F.S. Structural characterization of an active polysaccharide from Phellinus ribis.
Carbohyd. Polym. 2007, 70, 386-392.
8. Zhao, M.M.; Yang, N.; Yang, B. Structural characterization of water-soluble olysaccharides from
Opuntia monacanthap cladodes in relation to their anti-glycated activities. Food Chem. 2007, 105,
1
480-1486.
1
2
2
9. Coimbra, M.A.; Goncalves, F.; Barros, A.S.; Delgadillo, I. FTIR spectroscopy and chemometric
analysis of white wine polysaccharide extracts. J. Agr. Food Chem. 2002, 50, 3405-3411.
0. Barker, S.A.; Bourne, E.J.; Stacey, M.; Whiffen, D.H. Infrared spectra of carbohydrates. Part I.
Some derivatives of D-glucopyranose. J. Chem. Soc. 1954, 171-176.
1. Maiti, D.; Chandra, K.; Mondal, S.; Ojha, A.K.; Das, D.; Roy, S.K.; Ghosh, K.; Chakraborty, I.;
Islam, S.S. Isolation and characterization of a heteroglycan from the fruits of Astraeus
hygrometricus. Carbohyd. Res. 2008, 343, 817-824.
2
2
2
2
2
2. Roy, S.K.; Maiti, D.; Mondal, S.; Das, D.; Islam, S.S. Structural analysis of a polysaccharide
isolated from the aqueous extract of an edible mushroom, Pleurotus sajor-caju, cultivar Black
Japan. Carbohyd. Res. 2008, 343, 1108-1113.
3. Cao, W.; Li, X.Q.; Liu, L.; Yang, T.H.; Li, C.; Fan, H.T.; Jia, M.; Lv, Z.G.; Mei, Q.B. Structure of
an anti-tumor polysaccharide from Angelica sinensis (Oliv.) Diels. Carbohyd. Polym. 2006, 66,
1
49-159.
4. He, Y.M.; Liu, C.H.; Chen, Y.X.; Ji, A.C.; Shen, Z.L.; Xi, T.; Yao, Q.S. Isolation and Structural
Characterization of a Novel Polysaccharide Prepared from Arca subcrenata Lischke. J. Biosci.
Bioeng. 2007, 104, 111-116.
5. Yoon, S.; Kim, M.K.; Lee, I.Y.; Yun, M.; Nam Shin, J.E. Production and structural features of a
water-soluble polysaccharide from a mutant strain of Agrobacterium sp. J. Ind. Eng. Chem. 2008,
1
4, 759-764.
6. Kath, F.; Kulicke, W.M. Mild enzymatic isolation of mannan and glucan from yeast
Saccharomyces cerevisiae. Angew. Makromol. Chem. 1999, 268, 59-68.

Molecules 2011, 16
1591
2
2
2
3
3
3
3
3
3
7. Wu, L.C.; Hsu, H.W.; Chen, Y.C.; Chiu, C.C.; Lin, Y.I.; Ho, J.A. Antioxidant and
antiproliferative activities of red pitaya. Food Chem. 2006, 95, 319-327.
8. Oyanagui, Y. Reevaluation of assay methods and establishment of kit for superoxide dismutase
activity. Anal. Biochem. 1984, 142, 290-301.
9. Asakawa, T.; Matsuhita, S. Colouring conditions of Thiobarbituric acid test for detecting lipid
hydroperoxides. Lipids 1980, 15, 137-140.
0. Navarini, L.; Gilli, R.; Gombac, V. Polysaccharides from hot water extracts of roasted Coffea
arabica beans: isolation and characterization. Carbohyd. Polym. 1999, 40, 71-81.
1. Xia, W.; Lv, Q.; Zhang, W.Q. Study on the Decoloration Of Polvsaccharides from M ulberry
Leaves by M acro-resin Adsorption. Food Ferment. Ind. 2007, 33, 141-144.
2. Li, R.J.; Li, D.Y.; Zhang, X.F. Deproteinization from Epimedium Polysaccharide by Macroporous
Adsorption Resin. Chem. J. Chin. Univ. 2006, 27, 67-70.
3. Dubois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F. Colorimetric method for
determination of sugars and related substances. Anal. Chem. 1956, 28, 350-356.
4. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254.
5. Yamamoto, Y.; Nunome, T.; Yamauchi, R.; Kato, K.; Sone, Y. Structure of an exocellular
polysaccharide of Lactobacillus helveticus TN-4, a spontaneous mutant strain of Lactobacillus
helveticus TY1-2. Carbohyd. Res. 1995, 275, 319-332.
3
6. Pang, X.B.; Yao, W.B.; Yang, X.B.; Xie, C.; Liu, D.; Zhang, J.; Gao, X.D. Purification,
characterization and biological activity on hepatocytes of a polysaccharide from Flammulina
velutipes mycelium. Carbohyd. Res. 2007, 70, 291-297.
3
7. Luo, A.X.; He, X.J.; Zhou, S.D.; Fan, Y.J.; Luo, A.X.; Chun, Z. Purification, composition analysis
and antioxidant activity of the polysaccharides from Dendrobium nobile Lindl. Carbohyd. Polym.
2
010, 79, 1014-1019.
3
3
4
8. Shimada, K.; Fujikawa, K.; Yahara, K.; Nakamura, T. Antioxidative properties of xanthan on the
autoxidation of soybean oil in cyclodextrin emulsion. J. Agr. Food Chem. 1992, 40, 945-948.
9. Smirnoff, N.; Cumbes, Q. Hyroxyl radical scavenging activity of compatible solutes.
Phytochemistry 1989, 28, 1057-1060.
0. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant
activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med.
1
999, 26, 1231-1237.
4
4
4
1. Lv, L.S.; Gu, X.H.; Tang, J.; Ho, C.T. Antioxidant activity of stilbene glycoside from Polygonum
multiflorum Thunb in vivo. Food Chem. 2007, 104,1678-1681.
2. Wang, D.F.; Xie, X.F.; Cai, C.Y.; Yang, M. Analysis of pharmacological components of coarse
tea curing diabetes. Chin. Trad. Herbal Drugs 1995, 26, 255-257.
3. Tsiapali, E.; Whaley, S.; Kalbfleisch, J.; Ensley, H.E.; Browder, I.W.; Williams, D.L. Glucans
exhibit weak antioxidant activity, but stimulate macrophage free radical activity. Free Radic. Biol.
Med. 2001, 30, 393-402.
4
4. Santanam, N.; Ramachandram, S.; Parthasarathy, S. Oxygen radicals, antioxidants, and lipid
peroxidation. Semin. Reprod. Endocrinol. 1998, 16, 275-280.

Molecules 2011, 16
1592
4
4
5. Dhalla, N.S.; Temsah, R.M.; Netticadam, T. Oxidative stress and cardiovascular diseases. J.
Hypertens. 2000, 18, 655-663.
6. Sayre, L.M.; Smith, M.A.; Perry, G. Chemistry and biochemistry of oxidative stress in
neurodegenerative disease. Curr. Med. Chem. 2001, 8, 721-738.
Sample Availability: Samples are available from the authors.
©
2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(