Accepted Manuscript

Title: Preliminary characterizations, antioxidant and

hepatoprotective activity of polysaccharide from Cistanche
deserticola
Author: Yuanheng Guo Lili Cao Qingsheng Zhao Lijun
Zhang Jinjin Chen Boyan Liu Bing Zhao
PII:
DOI:
Reference:

S0141-8130(16)30899-6
http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.09.039
BIOMAC 6506

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17-7-2016
1-9-2016
10-9-2016

Please cite this article as: Yuanheng Guo, Lili Cao, Qingsheng Zhao, Lijun
Zhang, Jinjin Chen, Boyan Liu, Bing Zhao, Preliminary characterizations,
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and
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Cistanche deserticola, International Journal of Biological Macromolecules
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Preliminary characterizations, antioxidant and hepatoprotective

activity of polysaccharide from Cistanche deserticola
Yuanheng Guoa,b, Lili Caoa, Qingsheng Zhaoa , Lijun Zhanga,b, Jinjin Chena, Boyan Liua,b, Bing Zhaoa*

Division of Biorefinery Engineering, State Key Laboratory of Biochemical Engineering,

Institute of Process Engineering, Chinese Academy of Sciences, Beijing , 100190, P. R.
China

University of Chinese Academy of Sciences, Beijing,100049, P. R. China

* Corresponding author: Bing Zhao.

Add: No.1 Bei er tiao, Zhongguancun, Haidian District,
Beijing 100190, P. R. China.
Tel.: +86-010-82627059;
Fax: +86-010-62574372.
E-mail: bzhao@home.ipe.ac.cn;

bingzhaomaca@163.com.

Abstract
To research the preliminary characterizations, antioxidant and hepatoprotective activity of
polysaccharides from Cistanche deserticola (CDPs), three polysaccharide fractions, CDP-A,
CDP-B and CDP-C, were obtained by successively membrane filtration (microfiltration,
ultrafiltration and nanofiltration). Molecular weights, monosaccharide compositions, purities and
IR spectra of the three fractions were analyzed. Results showed that CDP-C contained higher
proportion of galacturonic acid (GalUA) than CDP-B and CDP-A. Antioxidant activities were
also analyzed and the results revealed that CDP-C possessed the highest activity. Thus,
hepatoprotective activity of CDP-C was studied further. In vitro research, CDP-C promoted
viability of HepG2 cells. In vivo research, CDP-C ameliorated the alterations induced by alcohol,
including serological indexes (alanine transaminase, acid phosphatase, -glutamyl transpeptidase
and triglyceride) and hepatic indicators (superoxide dismutase, malondialdehyde, glutathione
S-transferase and triglyceride) in model animals. The prominent microvesicular steatosis and mild
necrosis in hepatic histopathology of model animals were also attenuated by CDP-C
administration. These findings indicated that CDP-C possessed hepatoprotective activity against
chronic hepatic injury induced by alcohol. The underlying mechanism might be that CDP-C can
reduce the contents of MDA and TG, and modulate the activities of the relative enzyme. This
property might associate with GalUA in CDP-C.

Keyword: Cistanche deserticola; Polysaccharides; Preliminary characterizations; Antioxidant;

Hepatoprotective activity.

1. Introduction
Cistanche is a perennial holoparasite and mainly distribute in the desert region of
northwestern China [1, 2]. The stem of Cistanche species (Orobanchaceae) was first recorded in
Shen Nongs Chinese Materia Medica. Cistanche has long been used as a traditional herbal
medicine for the treatments of kidney deficiency, impotence, senile constipation, soreness and
weakness of waist and knees, and blood deficiency [3, 4]. Pharmacological researches revealed
that Cistanche has anti-inflammatory activity [5, 6], anti-osteoporosis activity [7, 8], sedative
effect [9], antifatigue activity [10], neuroprotective effect [11]. Besides, polysaccharides from
Cistanche deserticola (CDPs) has anti-hyperglycemic and hypolipidemic effects [12],
immunological activity [13] and proliferation effect on lymphocytes [14].
Alcohol, a worldwide consumed beverage and food additive, induced nearly 2.5 million
deaths each year in the world [15, 16]. These deaths mainly related to liver disease induced by
alcohol abuse [17, 18]. Alcohol induced hepatic disease is a complex multistep chronic
progression, normally develops from alcoholic steatosis to alcoholic hepatitis and finally to
alcoholic cirrhosis [19]. Thus, identifying active natural products for those alcohol consumers to
prevent or slow down the progression of alcoholic liver injury in early stage is a beneficial
management strategy.
Cistanche deserticola Y. C. Ma and Cistanche tubulosa (Schrenk) Wight are two of
medicinal species recorded in Chinese Pharmacopoeia [3]. Many papers have elucidated the
hepatoprotective activities of C. tubulosa [20-24] and C. deserticola [25, 26]. But these
hepatoprotective researches always aimed at acute liver injury induced by carbon tetrachloride
(CCl4) or D-galactosamine and lipopolysaccharide, but not concerned about the chronic liver
injury related with alcohol abuse. In addition, these reports were mainly focused on the
mechanism of phenylethanoid glycosides (PhGs) rather than the CDPs. Therefore, in the present
study, the preliminary characterizations of CDPs and their antioxidant activities (in vitro) were
investigated. Moreover, liver injury model of ICR mice induced by white spirit wine was
established to examine the hepatoprotective activity of CDPs against chronic liver injury induced
by alcohol.

2. Materials and methods

2.1. Materials
The stems of C. deserticola were collected from Alashan League, Inner Mongolia of China,
and identified by Prof. Xiaodong Wang (Division of Biorefinery Engineering, Institute of Process
Engineering, Chinese Academy of Sciences, Beijing, P. R. China).
Dimethyl sulfoxide (DMSO), 2, 2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)

(ABTS), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and 1,

1-diphenyl-2-picryl-hydrazyl (DPPH) were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were
purchased from Invitrogen, Inc. Er Guo-tou white spirit was purchased from Beijing Red Star Co.,
LTD. Bicyclol was purchased from Beijing Union Pharmaceutical Factory. Aqueous solutions
were prepared with ultra-pure water from a Milli-Q water purification system (Millipore, Bedford,
MA, USA). All other reagents were of analytical grade.
2.2. CDPs extraction and fractional filtration
The crude CDPs were prepared according to the previous reported method of our group [27]
with slight modification. Briefly, the powder (40 mesh) of C. deserticola (50 kg) was
ultrasonically (40 kHz and 6 kW) extracted with 1000 L of aqueous ethanol solution (50%, v/v) at
60 C for 120 min. Polysaccharides were separated from the extract with macroporous resin
(HPD 300). Polysaccharide solution was concentrated under reduced pressure, deproteinized
using Sevags method [28], and freeze dried the product was called crude CDPs.
Then 50 g of the crude CDPs was dissolved in 2.5 L ultrapure water and successively
separated with the microfiltration, ultrafiltration and nanofiltration (nominal molecular weight
cut-offs were 300 kDa, 10 kDa and 200 Da. Effective membrane area was 0.625 m2). Retained
solution of microfiltration was lyophilized and called CDP-A. Permeated solution of
microfiltration was filtrated with ultra-filtration, while retained solution was lyophilized and
named as CDP-B. Permeated solution of ultra-filtration was filtrated with nanofiltration, retained
solution was lyophilized and named as CDP-C.
2.3. Preliminary characterizations of CDPs
The molecular sizes of CDPs were evaluated according to the previous reported method of

our group [29]. Monosaccharide compositions were analyzed using the method described by P.
zhang et al [30]. Purities were determined according to the phenol-sulfuric acid method [31]. The
protein contents were determined using the method mentioned by Bradford and Maja Kozarski
[32, 33]. FT-IR spectra of CDPs were captured with a Fourier Transform-Infrared Spectrometer
(FT/IR-660 Plus, JASCO) in the range of 400~4000 cm-1.
2.4. Antioxidant activities
The scavenging effects of the CDPs on hydroxyl, superoxide anion, DPPH and ABTS radical
were evaluated according to the methods described by Sun [34],Wang [35], Yap [36] and

Fatiha

[37], respectively.
2.5. Hepatoprotective activity of CDP-C in vitro
On the basis of the results of antioxidant assay, CDP-C was selected to study the
hepatoprotective activity. HepG2 cells (purchased from China Center for Type Culture Collection,
Beijing, China) were cultured in DMEM containing heat inactivited FBS (10%), streptomycin
(0.1 g/mL), penicillin (100 IU/mL) and non-essential amino acid. The cells were incubated in
humidified atmosphere of 5% CO2 at 37, harvested at the exponential growth phase, seeded into
96-well plates (4104 cells per well, 100 L) and incubated for 24 h. Cells of negative group were
treated with alcohol (final concentration was 3.5%, v/v) while naive group treated with water.
Cells of positive group were treated with alcohol and bicyclol (final concentration was 200
g/mL). Cell of the four test groups were treated with alcohol and CDP-C (final concentration of
CDP-C was 0.11, 0.3333, 1.00 and 3.00 mg/mL). All cells were cultured for another 48 h.
The viability of HepG2 cells were determined by MTT assay method mentioned by J. Tong
et al [38] with slight modification. Briefly, MTT (5 mg/mL, 20 L per well) was added into the
cell-seeded 96-well plates, and the cells were incubated for 4 h. The solutions were removed and
DMSO (150 L/well) was added. The absorbance of each well was measured at 492 nm using a
96-well plate reader (Thermo Scientific, America). The cell viability was calculated using the
following formula:
Cell viability (%) = (Asample- Ablank) 100/ (Anaive- Ablank)
Where A sample was the absorbance of the experimental group; A naive was the absorbance of
the control group without sample; A blank was the absorbance of culture medium without any

sample and seeded cell.

2.6. Hepatoprotective activity of CDP-C in vivo
2.6.1. Animals
Adult female ICR mice (22-25 g, purchased from the Beijing Vital River Laboratory Animal
Technology Co. Ltd. Animal license number was 11400700128) were utilized. Animals were
housed in an environmentally-controlled room with feed and water ad libitum (relative humidity
was 40-60%, 22-26), air ventilation was 12-18 times/h, and light irradiation condition was a 12
h light/dark cycle of 150-300 lux. The mice were acclimatized to the animal room conditions for
7 days prior to experiments. All procedures involving animals throughout the experiments were
conducted in strict accordance with the rules for Use of Laboratory Animals as adopted and
promulgated by the United States National Institutes of Health.
2.6.2. Experimental design
ICR mice were randomly divided into the six groups (naive group, negative control group,
positive control group and three test group) with 10 animals in each group. The naive group was
orally administered with distilled water. Negative control group was orally administered with
alcohol (Er Guo-tou white spirit, 56%, 6 mL/kg). Positive control group was orally administered
with bicyclol (300 mg/kg) and 5 hours later with alcohol. Three test groups were orally
administered with CDP-C at a dose of 200, 600, 1800 mg/kg and 5 hours later with alcohol. All
mice were administered for 31 consecutive days.
2.6.3. Determination of serological indexes
About 4 h after last alcoholic treatment, blood was collected from eye socket and centrifuged
at 3000 g for 15 min. After the serum was separated, the enzyme activities of alanine
transaminase (ALT), acid phosphatase (ACP), -glutamyl transpeptidase (-GTP) and triglyceride
(TG) were determined using diagnostic kits.
2.6.4. Determination of hepatic indicators
After the collection of blood, all mice were executed. Liver of each mouse was immediately
excised, washed with physiological saline. A piece of hepatic tissue was separated from left lobe.
Then the tissue was minced and homogenized with aqueous potassium chloride solution (1.15%,
w/v) in a glass homogenizer (Potter Elvehjem Teflon) for 60 seconds to make a liver homogenate

(10% w/v). The enzyme activities of superoxide dismutase (SOD), malondialdehyde (MDA),
glutathione S-transferase (GST) as well as the triglyceride (TG) contents were measured using the
diagnostic kits.
2.6.5. Histopathologic studies
Residual part of the liver was fixed in Bouin's fixative for 24 h, after dehydrated using
aqueous ethanol solution (50-100%, v/v), the liver tissue was cleared in xylene and embedded in
paraffin. Hepatic sections (5 mm) were stained with alum haematoxylin and eosin (H-E). Images
of liver sections and histopathological changes were captured by a light microscope
(Nikon-DS-L1-5M).
2.7. Statistical analysis
Data were expressed as means standard error of the mean (S.E.M) from three (chemical
composition analysis and antioxidant assay), five (cell viability) or ten (in vivo research)
independent experiments. Statistical significances of differences between groups were analyzed
by one way analysis of variance (ANOVA) followed by a Student-Newman-Keulss multiple
range test using SPSS 19.0 software. In all analyses, p<0.05, p<0.01 or p<0.001 indicated
statistical significance.

3. Results and discussion

3.1. Characterizations of CDPs
As shown in Fig. 1A, the CDP-A contains two groups, the molecular weights are 4000 kDa
and 3946 kDa. The molecular weights of CDP-B and CDP-C are 2400 kDa and 1300 kDa,
The monosaccharide compositions are shown in Fig. 1B. CDP-A, CDP-B and CDP-C
contain six essential monosaccharides with different proportion, including mannose (Man),
rhamnose (Rha), galacturonic acid (GalUA), glucose (Glc), galactose (Gal) and arabinose (Ara).
Besides of above mentioned six monosaccharides, xylose (Xyl) also appears in CDP-C. In
addition, CDP-C contains larger proportion of GalUA than other two fractions.
The purities and protein contents of CDPs are exhibited in Table 1. Table 1 shows that
CDP-A has the highest purity, which is 75.57%, while the purities of CDP-B and CDP-C are
74.72% and 68.92%, respectively. The protein contents of CDP-A, CDP-B and CDP-C are 1.27%,
1.24% and 1.05%, respectively.
The FT-IR spectra of carbohydrates are used to determine their structural features, and
typically used for the qualitative analysis of organic functional groups, especially for O-H, C-O
and C=O [39]. The FT-IR spectra of CDPs are shown in Fig. 1C. Each spectrum shows a strong
and wide stretching peak around 3293 cm1 for O-H stretching vibration as well as a weak
absorption peak at 2939 cm1 for C-H stretching vibration. The absorption band at 1650 cm1 is
caused by C=O asymmetric stretching vibration. The broad absorption band at 1418cm1
attributes to deforming vibration of C-H bond. Each particular polysaccharide has specific band
in the 1000-1200 cm1region, which is dominated by ring vibration overlapped with stretching
vibration of (C-OH) side groups and the (C-O-C) glycosidic band vibration. The absorption at
1036 cm1 indicates a pyranose form of sugar. An absorbance at nearly 829 cm1 suggests the
linkage of -glycosides in the molecular structure of CDPs. As shown in Fig. 1 C, the absorption
band of CDP-C at 1650 cm1 is stronger than CDP-A and CDP-B, that means the content of C=O
in CDP-C is more than those in CDP-A and CDP-B. This result is consistent with the results of
Fig. 1 A, which reveals the proportion of GalUA in CDP-C is larger than those in CDP-A and
CDP-B.
3.2. Antioxidant activities of CDPs

3.2.1. Hydroxyl radical assay

Among the reactive oxygen species, the hydroxyl radical is the most reactive one and
induces severe damage to the adjacent biomolecules [40]. For hydroxyl radical, there are two
types of antioxidation mechanisms: one is scavenging the hydroxyl radical already generated, and
another is suppressing the generation of hydroxyl radical. For the later, hydroxyl radical is
generated by the reaction of Fe (II) complex with hydrogen peroxide. The antioxidant activities of
CDPs may ligate to the metal ions, which do not react with H2O2 and produce hydroxyl radical
but chelate with CDPs and form the metal complex. The metal complex cannot further react with
H2O2 to give hydroxyl radical [41-43]. Therefore, the CDPs show the hydroxyl radical
scavenging effects.
Fig. 2 A shows the scavenging capacities of CDPs on hydroxyl radical in a concentration
dependent manner. The scavenging rate of CDP-A, CDP-B and CDP-C is 29.56%, 33.44% and
38.22%, respectively. CDP-C exhibits higher hydroxyl radical scavenging activity. Considering
that CDP-C contains higher proportion of GalUA than CDP-A and CDP-B, antioxidant of CDPs
may related not only to the metal ions ligating property, but also to the contents of GalUA, Ara
and Gal [44-46].
3.2.2. Superoxide anion radical assay
The superoxide anion radical is a toxic specie which is generated by numerous biological
and photochemical reactions and thereby inducing tissue damage [47]. Although superoxide anion
is a relatively weak oxidant, it plays important role in the formation of other stronger reactive
oxidative species, such as singlet oxygen and hydroxyl radical [48]. Fig. 2 B shows the
relationship between the concentrations and the scavenging capacities of CDPs on superoxide
anion radical. As the concentration increasing, CDP-C exhibits higher scavenging capacity than
CDP-B and CDP-A.
3.2.3. DPPH radical assay
DPPH, one of the stable nitrogen-centered compounds that possesses a proton free radical
with a characteristic absorption at 517nm, decreases significantly on exposure to proton radical
scavengers, therefore, it is widely used to estimate the free-radical scavenging activities of
antioxidants. It is well accepted that the DPPH free radical scavenged by antioxidants is due to

their hydrogen-donating abilities. Antioxidants transfer either electrons or hydrogen atoms to the
DPPH radical and formed DPPH-H, which is the non-radical formation [49].
Total DPPH scavenging effects of all samples were tested and the results are shown in Fig. 2
C. Scavenging abilities of CDPs on DPPH radical display a concentration dependent manner. As
the concentration increasing from 1.0 to 5.0 mg/mL, the scavenging abilities of CDP-C increase
from 52.5% to 58.7%, higher than CDP-B (from 34.2% to 56.8%) and CDP-A (from 12.5% to
52.8%). The DPPH scavenging effect of CDP-C may associated with the carbonyl groups in
GalUA.
3.2.4. ABTS radical assay
ABTS radical assay is often used for measuring the total antioxidant power of a potential
antioxidant of the test samples [50]. ABTS scavenging effects of all samples are shown in Fig. 2
D. The scavenging abilities of CDPs on ABTS radical exhibit a dose dependent manner. The IC50
of CDP-A, CDP-B and CDP-C are about 4.0 mg/mL, 2.5 mg/mL and 1.2 mg/mL, respectively.
The results indicate that CDPs possess ABTS scavenging activity.
In summary, the antioxidant activity of CDP-C was higher than CDP-A and CDP-B. It
reported that some pharmacological activities of polysaccharide were related with its GalUA[51].
In this experiment, the proportion of GalUA in CDP-C was larger than those in CDP-A and
CDP-B, the CDP-C was selected to investigate the hepatoprotective effect against chronic liver
disease induced by alcohol.
3.3. The effects of CDP-C on HepG2 cell viability
Measuring cell viability is a common way to assess the efficacy of natural drugs [52]. Based
on the results that CDP-C possessed the highest antioxidant activity, the effects of CDP-C on
HepG2 cell viability were evaluated. The results are shown in Fig. 3. Alcoholic treatment induced
markedly decrease of HepG2 cell viability. But CDP-C can notably improve the survival rates
when compared with negative group. As shown in Fig. 3, the cell viability does not exhibits an
accurately concentration dependent manner with CDP-C. In contrast, when the concentration of
CDP-C reaches 3.00 mg/mL, the HepG2 cell viability declines dramatically. The reason for the
cell viability decrease might be that low concentration of CDP-C was helpful to the survival of
HepG2 cells. However, when the concentration of polysaccharide in culture medium was high

10

enough to change the microenvironment of the cells, the HepG2 cell viability was inhibited.
3.4. Pharmacological effects of CDP-C
3.4.1. The effects of CDP-C on serological indexes
Alcohol abuse attributes to nearly 4% of all death rates, which has become a serious social
problem in the world. In human body, alcohol is metabolized in the liver after it is absorbed
through gastric and small intestinal mucosa [53-55]. Alcoholic liver diseases normally occur after
years of alcohol abuse [56]. Commonly pathological conditions such as fatty liver, hepatitis,
fibrosis, and cirrhosis [57], or even cancerous diseases such as hepatocellular carcinoma and
colon cancer [58], are observed in alcohol linked liver disorders. Therefore, alcohol is a typical
hepatoxin and is widely used as an inducer of liver injury in scientific researches [59].
Considering that alcohol induced liver diseases are often caused by alcohol beverages and food
additive rather than industrial ethanol, thus Er Guo-tou white spirit was used to establish liver
injury model in ICR mice in this study. Bicyclol is a common agent used to treat the chronic liver
injury, so it was used to set positive control model.
Hepatic cells contain higher concentrations of ALT in the cytoplasm and mitochondria.
Because of various hepatic cell damages, the leakage of cytosol will cause increment of ALT in
the serum. Thus the promotion of ALT is the indicator of cellular leakage and functional disorders
of the liver [60]. So the serum ALT level is usually set as the indicator to assess the liver health
[61]. For similar reasons, -GTP, ACP and TG are also used as the indicators to assess the healthy
condition of liver [62].
In the present study, the hepatoprotective activity of CDP-C was assessed by determination
of the levels of ALT, ACP, -GTP and TG. The results are shown in Fig. 4 A, B, C and D. Four
serum indicators drastically promoted by alcoholic treatment in negative group. But CDP-C can
attenuate these alteration induced by alcoholic administration. However, not all serological
indexes exhibit dose dependent manner with CDP-C. In Fig. 4 A and B, CDP-C at low
concentration has the significant effect on the restoration of ALT and ACP, but the effect of
CDP-C at the highest concentration was not significant. In fact, many natural products are
beneficial to health at low doses, but harmful for health at high dose [60]. The reason might be
that excessive ingestion of polysaccharide will affect the normal metabolism of organisms, so as

11

to adversely affect the health of body. However, the detailed mechanism needs to be further
researched.
3.4.2. The effects of CDP-C on hepatic indicators
Organisms subject to oxidative stress usually evolved an antioxidant defense mechanism,
and SOD is a typical enzyme based antioxidant systems [63]. SOD catalyzes the dismutation of
superoxide anion into O2 and H2O2 [64]. GST, a soluble protein located in the cytosol, also plays
an important role in the detoxification of liver. For the reasons mentioned above, SOD and GST
become the indicators in the alcohol induced hepatotoxicity. MDA and TG in liver also used as
indicators of hepatotoxicty [65].
In the present study, the effects of CDP-C on liver function are shown in Fig. 5. Picture A, B,
C, and D exhibit the effects of CDP-C on SOD, MDA, GST and TG, respectively. These four
pictures show that the mice treated by alcohol markedly decrease SOD and GST levels, and
increase MDA and TG levels, but CDP-C canevidently attenuated these alteration. Similar with
the phenomena depicted in section 3.4.1, hepatic indicators were also not in dose dependent
manner with the CDP-C. The reason for this may be similar with the mechanism illustrated in
section 3.4.1. Previous studiesreported that the effects of polysaccharides on SOD and catalase might
be associated with the induction of gene expressions of SOD and catalase [66]. However, further
studies are still needed to elucidate the hepatoprotective mechanism of CDP-C and the relationship
between its structure and function.

3.5. Histopathologic effects of CDP-C on alcohol induced mice

On the basis of the histopathologic observations on the liver sections, the naive group
exhibited normal cellular architecture with distinct hepatic cells and no histologic abnormalities
(Fig. 6 A). In comparison, the alcoholic administration caused serious liver damages in mice of
negative group. The liver sections showed hepatocyte fat deposition, hepatocyte necrosis and
swelling, vacuole formation in the cells, and the cellular boundaries also disappeared (Fig. 6 B).
The liver sections of the mice administered with bicyclol (Fig. 6 C), with varying doses of CDP-C
(Fig. 6 D-F) exhibited evidently restoration of those distortions appeared in negative control
group. These results validated that the alcohol induced a certain extent of distortion in
hepatocytes of negative control group. However, CDP-C restored these changes.

12

It is well accepted that the hepatoprotective effect of polysaccharides is associated with their
antioxidant activity [67, 68]. The high uronic acid contents had been proved to be beneficial for
antioxidant effects of polysaccharides [44, 45]. Polysaccharides rich in Gal and Ara also had been
verified to possess higher antioxidant effects [46]. However, it is difficult for large molecular
polysaccharide to enter the intestinal epithelial cells directly. For the CDP-C (with the molecular
weight of 1300kDa), it is not easily to enter the body directly and play the hepatoprotective role.
Published reports revealed that the molecular weights of the polysaccharides was decreased after
gastric and intestinal digestion [69]. Thus, we assumed that CDP-C maybe degraded into
polysaccharides with low molecular weights and absorbed by intestinal epithelial cells. Reducing
ends of polysaccharides can be increased due to the breakdown of glycosidic bonds [69, 70].
According to the monosaccharide composition of CDP-C, those degraded polysaccharides with
low molecular weights should contain GalUA, Ara and Gal. Hence these low molecular weight
polysaccharides, which possess reducing ends and contain GalUA, Ara and Gal, may play the role
of liver protection in body. However, the detailed mechanism needs to be further researched.
4. Conclusions
According to the monosaccharide composition analyses and FT-IR spectra, all of three
fractions of CDPs contained Man, Rha, GalUA, Glc, Gal and Ara. CDP-C contained large
proportion of GalUA. CDP-C had the highest antioxidant activity. The results of both in vitro and
vivo researches illustrated that CDP-C possessed hepatoprotective activity against chronic hepatic
injury induced by alcohol. The underlying mechanism was related with modulating relative
enzyme activities, reducing MDA and TG in liver. The physiological activity of CDP-C might
associate with GalUA. These findings provide new insights into the pharmacological targets of C.
deserticola in the prevention of alcoholic liver disease.

5. Acknowledgements
This work was financially supported by the Science & technology supporting project from
the New Technology Research Institute of Alashan (Zhong Ke) (No. KFA2013-189). The authors
are also grateful for the financial support of the National Natural Science Fund of China (No.
21506220). The work also received assistance from Hong Kui Cong Rong Group.

13

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16

Table 1. Purities and protein contents of CDPs. Data are the means S.E.M of three replicates.
Polysaccharide purities (%)

Protein contents (%)

CDP-A

75.57 2.35

1.27 0.04

CDP-B

74.72 2.53

1.24 0.06

CDP-C

68.92 1.28

1.05 0.03

17

Intensity (mV)

6000
5000

250
3946 kDa

150

>4000 kDa

3000

1300 kDa

2000

Glc

Ara

100

1000

Man

50

GalUA Gal
Rha
Xyl

0
0

Transmittance %

CDP-A
CDP-B
CDP-C

200

4000

2400 kDa

CDP-A
CDP-B
CDP-C

Intensity (mV)

7000 A

6
8
Time (min)

10

12

10

20

30
40
Time (min)

50

60

CDP-A
CDP-B
CDP-C

4
3
2

829
2939

14181036
1650
3293
0
4000 3500 3000 2500 2000 1500 1000 500
-1
Wavenumbers (cm )

Fig. 1. The preliminary characterizations of CDPs, including molecular sizes (A),

monosaccharide compositions (B) and FT-IR spectra (C).
35

30
25
20
15
1

2
3
4
Concentration (mg/mL)

60
40
30
20
10
1

2
3
4
Concentration (mg/mL)

25
20
15
10
1

100

50

CDP-A
CDP-B
CDP-C

30

CDP-A
CDP-B
CDP-C

70 C

Scavenging rate (%)

Scavenging rate (%)

CDP-A
CDP-B
CDP-C

35

Scavenging rate (%)

Scavenging rate (%)

40 A

2
3
4
Concentration (mg/mL)
CDP-A
CDP-B
CDP-C

5
D

80
60
40
20
1

2
3
4
Concentration (mg/mL)

Fig. 2. Scavenging effects of CDPs on hydroxyl radical (A), superoxide anion radical (B), DPPH
(C) and ABTS (D). Data are the means S.E.M of three replicates.

18

Survival rate (%)

100

90
80
70
60

)
)
)
)
e
e
e
NaivNegativPositivmg/mLmg/mLmg/mLmg/mL
1
3
0
0
1
3
0
0
.
.
.
.
0
0
1
3
-C ( -C ( -C ( -C (
CDP CDP CDP CDP

Fig. 3. Effects of CDP-C on survival rates of alcohol treated HepG2 cell, Bar represents mean
S.E.M, n = 5 well of cell. p < 0.05, p < 0.01, compared with alcohol treated group
(one-way ANOVA followed by Student-Newman-Keulss multiple range test ).
B

80

ACP (U/L)

60

60

40

45
30

20

15

0
e
e
e
g)
g)
g)
NaivNegativ Positiv0 mg/k 0 mg/k 0 mg/k
0
0
0
6
2
8
-C ( P-C( C (1
CDP CD CDP30 C

0
g)
g)
g)
ve
ve
ve
Nai Negati Positi 0 mg/k 0 mg/k 0 mg/k
0
0
0
8
2
6
-C ( P-C( -C (1
CDP CD CDP
2000 D

25

1600

20
15

10
5
0
e
e
e
g)
g)
g)
NaivNegativ Positiv0 mg/k 0 mg/k 0 mg/k
0
0
0
6
2
8
-C ( P-C( C (1
CDP CD CDP-

TG (mg/L)

-GTP (U/L)

ALT (U/L)

100 A

1200

800
400
0
g)
g)
g)
ve
ve
ve
Nai Negati Positi 0 mg/k 0 mg/k 0 mg/k
0
0
0
8
2
6
-C ( P-C( -C (1
CDP CD CDP

Fig. 4. Effects of CDP-C on serological indexes of ICR mice treated by alcohol, including ALT
(A), ACP (B), -GTP (C) and TG (D). Bar represents means S.E.M, n = 10 mice per group. p
< 0.05, p < 0.01, p < 0.001 as compared with the negative group (one-way ANOVA
followed by Student-Newman-Keulss multiple range test).

19

400

300
200
100
0

GST (U/mg protein)

70 C
60
50
40
30
20
10
0
e
e
e
g)
g)
g)
NaivNegativ Positiv0 mg/k 0 mg/k 0 mg/k
60
20
80
-C ( P-C( C (1
CDP CD CDP-

3.0

2.5
2.0

1.5
1.0
0.5
0.0

e
e
e
g)
g)
g)
NaivNegativ Positiv0 mg/k 0 mg/k 0 mg/k
60
20
80
-C ( P-C( C (1
CDP CD CDP-

40

TG (mg/g)

SOD (U/mg protein)

MDA (n mol/mg protein)

500 A

g)
g)
g)
ve
ve
ve
Nai Negati Positi 0 mg/k 0 mg/k 0 mg/k
0
0
0
8
2
6
-C ( P-C( -C (1
CDP CD CDP
D

30

20

10
0

g)
g)
g)
ve
ve
ve
Nai Negati Positi 0 mg/k 0 mg/k 0 mg/k
80
20
60
-C ( P-C( -C (1
CDP CD CDP

Fig. 5. Effects of CDP-C on liver biochemical parameters of ICR mice treated by alcohol,
including SOD (A), MDA (B), GST (C) and TG content (D). Bar represents means S.E.M, n =
10 mice per group. p < 0.05, p < 0.01, p < 0.001 as compared with the
negative group (one-way ANOVA followed by Student-Newman-Keulss multiple range test).

Fig. 6. Histological photos of liver tissues of naive group (A), negative group (B), positive group
(C), CDP-C at 200 mg/kg (D), CDP-C at 600 mg/kg (E) and CDP-C at 1800 mg/kg (F).

20