全 文 ： 2008, Vol. 29, No. 05 食品科学 ※基础研究120
Isolation, Purification and Anti-complement Activity of Water-
soluble Polysaccharides from Chaenom les cathayensis
WANG Wen-ping1，GUO Si-yuan1，LI Lin1，SUN Qian-yun2，TANG Wei-yuan3
(1.College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China；
2. Key Laboratory of Chemistry for Nature Products of Guizhou Province, Chinese Academy of Sciences, Guiyang 550002,
China；3.Guizhou Provincial Key Laboratory of Fermentation Engineering and Biological Pharmacy, Guiyang 550003, China)
Abstract ：The crude water-soluble polysaccharides from Chaen meles c thayensis fruits were isolated with hot water extraction
followed by ethanol precipitation. The polysaccharides were deproteinized using Sevag method, decolorized by macroporous resin
and then dialyzed. The fine water-soluble C aenom les cathayensis polysaccharides (CCP) were obtained and further fraction-
ated to elute for the component CCP1 by anion exchange column chromatography on DEAE-cellulose. The purity of CCP1 was
examined by gel filtration chromatography of Sepharose Cl-6B. The purified polysaccharide CCP1 was proved to be homoge-
neous by HPGPC and its molecular weight (MW) was 2.879×106 D. Analysis of monosaccharide composition by HPLC
indicated that CCP1 consists of rhamnose, arabinose, fructose, mannitose, gluc and their molar ratio is 0.034:0.228:0.045:
0.055:0.638. The main fraction CCP1 was subjected to the complement-fixation experiment. CCP1 expressed s me anti-
complementary activity in vitro and dosage-effect relationship between them existing.
Key words：Chaenomeles cathayensis；polysaccharide；i olation；purification；anti-complement activity
野木瓜水溶性多糖的分离纯化及抗补体活性研究
王文平1，郭祀远1，李 琳1，孙黔云2，唐维媛3
(1. 华南理工大学轻工与食品学院，广东 广州 510640；2.中国科学院 贵州省天然产物化学重点实验室，贵州 贵阳
550002；3.贵州省发酵工程与生物制药重点实验室，贵州 贵阳 550003)
摘 要：采用水提醇沉工艺获得了水溶性野木瓜粗多糖(CCP)，经过Sevag法脱蛋白，大孔树脂脱色及透析，得
到野木瓜精多糖，再经DEAE-纤维素和Sepharose Cl-6B柱层析，从野木瓜中分离得到多糖组分CCP1，经过高效
凝胶渗透色谱法(HPGPC)鉴定为均一组分，测定其相对分子量(MW)为2.879 ×106D。高效液相法测定CCP1的单
糖组成为：鼠李糖、阿拉伯糖、果糖、甘露糖、葡萄糖，其摩尔比为0.034:0.228:0.045:0.055:0.638。补体结合
实验表明，多糖CCP1在体外具有一定抗补体活性，并呈一定的剂量效应关系。
关键词：野木瓜；多糖；分离；纯化；抗补体活性
中图分类号：Q946.3；S852.51 文献标识码：A 文章编号：1002-6630(2008)05-0120-05
收稿日期：2008-01-15
基金项目：贵州省科技基金项目(黔科合J字[2005]2017号)
作者简介：王文平(1966-)，女，教授，在职博士研究生，主要从事天然糖质分离纯化新方法新技术研究。
E-mail：wwp931002@163.com
Chaenomeles cathayensis(Hemsl.) Schneid is a spe-
cialized natural resource of Zheng’an County in Guizhou
Province. Chinese quince is the common name of Chaenomeles
cathayensis fru ts. Owing to being abundant in carbohydrates,
organic acids, amino acids, proteins, minerals and vitamins
and some bioactive components such as trierpenoid
saponins, flavonoids, polysaccharides, pectins and superox-
ide dism tase (SOD)[1], Chaenomeles cathayensisis a valu-
able pla t suitable for both medicinal and food use. This
plant is used for the treatment of a wide range of diseases
such a rheumatic ffection, cholera, dysentery, enteritis and
beriberi[2]etc. Some biological activities of this plant have
121※基础研究 食品科学 2008, Vol. 29, No. 05
been reported: protecting liver, enhancing immune function
of the organism, anticancer, antibacterial and anti-inflamma-
tory properties etc[3].
The chemical constituents from this plant have also
been investigated within the last few years, but only the
low-molecular weight constituents have been isolated and
identified[4]. Polysaccharides are one group of important
macromolecular compounds in this plant, however, few stud-
ies have been reported on them up to now. In recent years,
plant polysaccharides have emerged as an important class
of bioactive natural products. Various polysaccharides from
different plants were reported to be responsible for the ef-
fects associated with the healing of wounds. Some of these
have an influence on the immune system and are often called
immunomodulators when the complement system is involved
[5]. Due to the important physiological role of the complement
immune system, complement modulation, either inhibition or
stimulation, it is related to various diseases and considered
as an interesting target for drug development. Several plant
polysaccharides are known to possess complement-modu-
lating activity[6]. Such polysaccharides might exit in this plant,
so it would be of some interest to investigate it.
However, the isolation, purification and characterisation
of polysaccharides from Chaenomeles cathayensis fruits and
subsequent evaluation of anticomplementary activity have
not yet been performed. This study will report the extraction
and purification of polysaccharides isolated from
Chaenomeles cathayensis fruit and their effect on the comple-
ment system. The main goal of this experiment is to provide
scientific evidence for development of polysaccharides from
Chaenomeles cathayensis fruits as a potential natural anti-
inflammatory and wound-healing drugs.
1 Materials and Methods
1.1Materials
Fresh C aenomeles cathayensisfruits were purchased
in Zunyi Tianlou Limited Company, Guizhou Province. DEAE-
Cellulose was purchased from Shanghai Reagents Company.
Sepharose Cl-6B was purchased from Waterman Biotech Ltd.
The sheep red blood cell and guinea pigs (200～300 g) were
obtained from Laboratory Animal Center, Guiyang Medical
College, Guiyang, China. Haemolysin is a product of Sigma.
Other analytically pure agents were purchased from local
markets. All authentic standards were obtained from Sigma
or Fluka Company.
1.2Methods
1.2.1Extra tion of polysaccharides from Chaenomeles
cath yens s fruit
Fresh fruits of Chaenomeles cathayensis, previou ly
cut into small piec s, were dried in hot-air oven at 50 ℃，
which gave 14.5% (W/W) of dried material. The dried sample
was ground and sifted by 60 mesh sieve. The ground sample
was efluxed twice to remove lipids with petroleum ether for
2 h. After filtering, the residue was air-dried and socked with
80% e hanol for 24 h in o der to remove alcohol-soluble
solids. The residue was dried and subsequently treated with
hot distilled w ter to i olate cell wall polysaccharides. The
extraction wa performed with distilled water (90 ℃) for 2 h
w th gentle stirring by 1:35 ratio of solid to liquid[7], and this
procedure was peated again. The supernatant was col-
lected and concentrated by a rotary vacuum evaporator (N-
1000V-WB, Japan) at 50 ℃. The water-polysaccharides were
obta ned by prec pitation of the concentrated filtrate with
th ee times volumes 95% ethanol. After being washed with
ethanol, acetone and ethylether, respectively, the isolated
crude water- olysaccharides were vacuum-dried, and crude
Chaenomeles cathayensispolysaccharides named as crude
CCP.
Crude CCP we e purified by deproteinization,
decolorization[8]and dialysis. The dialysate was precipitated
with 95% e hanol, and then vacuum-dried (DZF-0, Shanghai),
an fine Cha nomeles cathayensispolysaccharides named
as fine CCP.
1.2.2Fractionation of polysaccharides
The fine CCP was fractionated by anion-exchange
chromatography on a column (4.8 cm×60 cm) of DEAE-
cellulose which was pretreated and equilibrated with dis-
tilled water for 24 h. The sample was dissolved in distilled
water and filtered through a 0.8μm membrane filter then
applied on to the column coupled to a peristaltic pump. The
column was first eluted with distilled water at a flow rate of
1.0 ml/min followed by NaCl solution (0→1 mol/L). Frac-
tions of 10 ml was collected using an automatic fraction col-
lector and monitored for the presence of polysaccharides
using the phenol-sulfuric acid assay[9]. The elution profile
was drawn according with the number of test-tube (X-axis)
as well as the absorbance (Y-axis). Fractions containing
polysaccharides from the elution step were collected, vacuum-
condensed, dialyzed and lyophilized (ALPHA 1-4, Germany).
The fin CCP was thus separ ted out a main fraction CCP1.
CCP1 was further purified on a column (1.5 cm × 90 cm)
of Sepharose Cl-6B, eluted with 0.1 mol/L NaCl. Eluent of 5 ml
2008, Vol. 29, No. 05 食品科学 ※基础研究122
was collected using an automatic fraction collector and
monitored for the presence of polysaccharides using the
phenol-sulfuric acid assay. The absorbance (Y-axis) was plot-
ted against the number of test-tube (X-axis) to make the elu-
tion profile. Polysaccharides solutions with the same eluting
peak were collected, vacuum-concentrated, dialyzed and then
lyophilized.
1.2.3Monosaccharide composition analysis of CCP1
The monosaccharide components analysis was deter-
mined by a HPLC apparatus (Shimadzu, Japan). The differ-
ent standard monosaccharides were dissolved in distilled
water at a concentration of 1% (W/W), respectively. 20 mg
of CCP1 sample was subjected to hydrolyze with 2 mol/L
H2SO4 in a dry oven for 8 h at 110 ℃[10]. BaCO3 was used for
neutralization of the hydrolyzing solution followed by
centrifugation. The supernatant fluid was vacuum-concen-
trated and filtered by a 0.45 μm millipore and degassed before
use for HPLC analysis. The chromatographic conditions
were: Shodex Asahipak NH2P-50 4E column(250 mm×4.6
mm，5μm); acetonitrile-water(75:25) as mobile phase; the
flow rate 0.8 ml/min; the system operated at 30 ℃ with refrac-
tive index detector. The monosaccharide composition and
proportion of CCP1 were determined according to the reten-
tion time and the area of peaks of the monosaccharide.
1.2.4Assay of CCP1 molecular weight
The molecular weight of CCP1 as determined by gel
permeation chromatography (GPC) with a HPLC apparatus
(Agilent, USA), equipped with a Ultrahydrogel column (300
mm×7.5 mm, 8μ ), a model 410 refractive index detector
and a Millennium 32 chromatography Workstation. The chro-
matographic conditions were: 0.05 mol/L Na2SO4 as mobile
phase; the flow rate 1.0 ml/min; the system operated at 30℃;
column pressure 39 Bar and the injection volume 50μl. T
Dextrans (Pharmacia) with different molecular weights (4400,
21400, 124000, 196000, 277000 D) were used as calibration
standards. The sample CCP1 wa dissolved in 0.05 mol/L
Na2SO4 at a concentration of 1.0 mg/ml. The molecular weight
of CCP1 was calculated by the retention time and calibration
curve.
1.2.5Assay for CCP1 anti-complement activity
1.2.5.1Experimental principle
Anti-complement activity was evaluated by the effect
on the complement system using the widely used haemolytic
complement assay. In cases of sheep red blood cell (SRBC,
antigen) and hemolysin (antibody) complex formation, the
activation f the complement system in the blood serum can
induce hemolysis of red blood cells. As levels of SRBC and
hemolysin are constont, the hemolytic degree of red blood
cells is positively correlated with the complement content
and activity. The hemolysis degree will be decreased in the
reaction system after adding samples with anti-complement
activity. The effect of the polysaccharides on the comple-
ment system is expressed as inhibition rate of haemolysis.
1.2.5.2Experimental procedure
Anti-com lement act vity of CCP1 was assa ed accord-
ing to the complement-fixation assay[11]. CCP1 was dissolved
in phosphate buffer solution (PBS, 1 mol/L NaOH adjusting
pH 7.0) at different concentrations. Guinea pig serum was
diluted (1:100) with glucose-gelatin veronal buffer (GGVB) [12].
0.1 ml f each sample and 0.1 ml guinea pig serum were
blended and preincubated at 37 ℃ water bath for 30 min. 0.1
ml of she p red blood cells (5×108 cells/m ) in GGVB buffer
sensi ized by anti-sheep red blood cell serum were added
and in ubated for a further 15 min at 37 ℃ with occasional
shaking. Aft r that, 1 ml of cold phosphate-buffered saline
were added to each tube and centrifuged for 10 min at 2000 r/
min. The control group w s prepared as above, but contain-
ing PBS instead of the sample. And then the supernatant
was assayed the absorbance Ax of tube-sample and Ac of
tube-standard at 412 nm. The inhibition rate of haemolysis
according to the following formula:
AC－AX
The inhibition rate of haemolysis=————×100%
AC
1.2.5.3Data analysis
Data is expressed by means (X±SD). Student’s t-
tes is u ed t assess s atistical significance between con-
trol and experiment group, p＜0.05 as c nsidered to be stati-
cally signific nt.
2 Results and Discussion
2.1Extraction and fractionation by ion-exchange and gel
filtration chromatography
　Crude water-soluble polysaccharides were further
frac ionated by ion exchange column chromatography on a
DEAE-cellulose column eluted with water and NaCl solution
(0→1 mol/L). CCP was separated out a main fractions CCP1
which as eluted with 0.2 mol/L NaCl solution. The eluting
profile for CCP1 is sh wed as shown in Fig.1.
CCP1 was further pur fied on a Sepharose Cl-6B column.
123※基础研究 食品科学 2008, Vol. 29, No. 05
Fig.1 Fractionation eluting curve of CCP1 on DEAE-cellulose
5
4
3
2
1
0
50 60 70 80 90 100110
A4
9
0
Number of tube
The purifying identification profile is a single symmetrical
peak as shown in Fig.2.
2.2Monosaccharide composition of CCP1
The mixed standard monosaccharide and the sample
CCP1 were subjected to HPLC analysis and the chromatogra-
phy was given in Fig. 3 and Fig. 4, respectively. The experi-
mental results indicated that CCP1 consists of rhamnose,
arabinose, fructose, mannitose, glucose and their molar ratio
is 0.034:0.228:0.045:0.055:0.638.
2.3Molecular weight of CCP1
The GPC calibration curve was drawn according to the
regression of logarithm of standard Dextrans with different
molecular weights to elution volumes (V). Millennium 32
software was utilized for the data acquisition and analysis.
The GPC calibration curve and the GPC chromatogram of the
sample CCP1 were given in Fig. 5 and Fig. 6, respectively. The
elution time of the sample CCP1 was 10.408 min, so its weight-
average molecular weight (MW) was calculated as 2.879 ×
106 D.
Concentration of
pH Value
Inhibition rate of
CCP1 (mg/ml) haemolysis (%)
0.10 7.0 10.65±0.2*
0.20 7.0 21.38±0.5*
0.30 7.0 33.27±0.3*
0.40 7.0 45.51±0.8*
0.50 7.0 52.76±0.6*
0.60 7.0 57.01±0.3*
Table 1 Anti-complement activity of CCP1 (X±SD, n=3)α
Note: αValues are presented as the means of three independent experiments ±
standard deviation; *p＜0.05 vs control.
2.4Determination of CCP1 anti-complement activity
The results showed that the polysaccharide CCP1
possessed anti-complement activity. The inhibition rate of
complement activity has positive correlation with the con-
centration of CCP1. The results from the complement-fixation
assay are shown in Table 1.
Fig.2 Gel column chromatography of CCP1 on Sepharose Cl-6B
80 90 100110120130140
A4
9
0
Number of tube
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Fig.3 Chromatogram of mixed standard monosaccharide
2 4 6 8 10121416182022
V
Time(min)
0.0030
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
-0.0005
G
l
u
c
o
s
e

1
7
.
9
0
8
I
s
o
d
u
l
c
i
t
o
l
8
.
1
1
2
A
r
a
b
i
n
o
s
e
1
1
.
9
7
1
F
r
u
c
t
o
s
e
1
3
.
1
0
6
M
a
n
n
i
t
o
s
e

1
4
.
7
8
0
Fig.4 Chromatogram of CCP1 sample
2 4 6 8 10121416182022
V
Time(min)
0.0006
0.0004
0.0002
0.0000
-0.0002
-0.0004
G
l
u
c
o
s
e

1
7
.
7
8
6
I
s
o
d
u
l
c
i
t
o
l
8
.
1
1
0
A
r
a
b
i
n
o
s
e
1
1
.
8
6
8
F
r
u
c
t
o
s
e
1
3
.
0
6
2
M
a
n
n
i
t
o
s
e

1
4
.
7
3
0
Fig.5 GPC calibration curve
5
4
7.47.67.88.08.28.48.6
l
g
M
W
Elution volume(ml)
Fig.6 GPC chromatogram of sample CCP1
2000
0
－2000
－4000
－6000
－8000
－10000
0 2 4 6 8 10 12
n
R
I
u
Time(min)
10.408
2008, Vol. 29, No. 05 食品科学 ※基础研究124
3 Conclusion
After isolation by anion exchange column and gel
filtration column, the purified polysaccharide CCP1 was
obtained. It comprises rhamnose, arabinose, fructose,
mannitose, glucose with a molar ratio of 0.034:0.228:0.045:
0.055:0.638. GPC showed the molecular weight of CCP1 is 2.879
×106 D. The results of anti-complement activity indicated
that CCP1 possesses anti-complement action in vitro, and
suggested that CCP1 may be used as a potential anti-inflam-
matory and wound-healing agent in the future.
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