全 文 ：Ｒeceived:July 25，2012 Accepted:January 21，2014
Foundation item:This work was supported by the Industrial Key Project
of Science and Technology Department of Guizhou Province (2009-
3052);and the Ｒesearch Project of Introducing Talent of Guizhou Uni-
versity (2008-017)
* Corresponding author Tel:86-851-3627690;E-mail:wwp931002 @
163． com
天然产物研究与开发 Nat Prod Ｒes Dev 2014，26:745-749，769
文章编号:1001-6880(2014)5-0745-06
野木瓜水溶性多糖的分离纯化及抗氧化活性研究
王文平1* ，田 亮1，吴国卿1，唐维媛1，王明力1，郭祀远2
1贵州大学贵州省发酵工程与生物制药重点实验室，贵阳 550003;2 华南理工大学轻工与食品学院，广州 510640
摘 要:采用水提醇沉法获得了水溶性野木瓜粗多糖(CCCPs)，经过脱蛋白、脱色及透析，得到了野木瓜精多糖
(FCCPs)，再经 DEAE-纤维素和 Sepharose CL-6B 柱层析得到均多糖组分(CCP1)。高效液相色谱法测定 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。体外抗
氧化实验结果表明:不同纯度的野木瓜多糖都具有一定的抗氧化活性，并且随着多糖浓度增加，抗氧化活性增
强，野木瓜粗多糖(CCCPs)对·OH和 O-·2 的清除能力比精多糖(FCCPs)和均多糖(CCP1)强。
关键词:野木瓜;多糖;分离;纯化;抗氧化
中图分类号:Ｒ285. 5 文献标识码:A
Study on the Separation，Purification and Antioxidant Activity of
Water-soluble Polysaccharides from Chaenomeles cathayensis
WANG Wen-ping1* ，TIAN Liang1，WU Guo-qing1，TANG Wei-yuan1，WANG Ming-li1，GUO Si-yuan2
1Guizhou Province Key Laboratory of Fermentation Engineering and Biological Pharmacy，Guizhou University，Guiyang 550003，China;
2College of Light Industry and Food Science，South China University of Technology，Guangzhou 510640，China
Abstract:Crude Chaenomeles cathayensis polysaccharides (CCCPs)were isolated with hot water extraction followed by
ethanol precipitation． The fine C． cathayensis polysaccharides (FCCPs)were obtained by deproteinization，decolorization
and dialysis． FCCPs were further separated to afford a main fraction CCP1 by anion exchange column chromatography on
DEAE-cellulose and Sepharose CL-6B gel filtration column chromatography． The composition of CCP1was determined to
be rhamnose，arabinose，fructose，mannitose，glucose in a molar ratio of 0． 034∶ 0． 228∶ 0． 045∶ 0． 055∶ 0． 638 using high
performance liquid chromatography (HPLC)． The antioxidant activity assay revealed that C． cathayensis polysaccharides
with different purity showed different antioxidant activities． The antioxidant activities of C． cathayensis polysaccharides in-
creased with the increasing of the concentrations of polysaccharides． The results showed that CCCPs had higher scaven-
ging efficiency on hydroxyl radical (·OH)and superoxide anion free radical (O-·2 )than FCCPs and CCP1 ．
Key words:Chaenomeles cathayensis;polysaccharide;separation;purification;antioxidant activity
Introduction
Chaenomeles cathayensis is known as Chinese quince in
China． As one of the most important economic plants，
the fruit of C． cathayensis is a specialized natural re-
source of Zhengan county (Guizhou，China)． It can be
used to produce various types of foods，beverages，
drinks，preserved fruit，wine and fruit vinegar，etc． Ow-
ing to being abundant in carbohydrates，organic acids，
amino acids，proteins，minerals and vitamins as well as
some bioactive components such as trierpenoid sapo-
nins，flavonoids，polysaccharides，pectin and superoxide
dismutase (SOD)［1］，C． cathayensis is a valuable plant
used for both food and medicine．
The chemical constituents of C． cathayensis have been
also investigated within the last few years，but only
some constituents of low molecular weight had been i-
solated and identified［2］． Polysaccharides are one group
of important macromolecular compounds in the fruit of
C． cathayensis，however，few studies have been reported
on them． In recent years，plant polysaccharides have e-
merged as an important class of bioactive natural
DOI:10.16333/j.1001-6880.2014.05.024
products． A wide range of polysaccharides were repor-
ted to exhibit a variety of antioxidant ability ［3，4］． In
addition，further investigations were also shown that
polysaccharides were associated with anticancer，anti-
virus，reducing blood-glucose and blood-fat，anti-in-
flammatory，antibacterial and immunological activities，
etc［5，6］．
The isolation，structure and molecular weight，as well as
the anticomplement activity of the water-soluble poly-
saccharides from C． cathayensis were discussed in our
previous research［7，8］． The aims of the present study
were to isolate and purify polysaccharide from C．
cathayensis and to evaluate the antioxidant activities of
the isolated component as well as its concentration-ac-
tivity relationship． It is expected that C． cathayensis can
be used as a natural antioxidant agent or functional
food．
Materials and Methods
Plant materials and chemicals
Fresh fruits of C． cathayensis were purchased from
Guizhou Tianlou Food Co．，Ltd．(Zunyi，China)． Seph-
arose CL-6B was purchased from Fluka (Neu-Ulm，
Switzerland)． DEAE-Cellulose was obtained from
Shanghai Ｒeagents Company (Shanghai，China)． Other
reagents were of analytical grade and purchased from
local markets． All authentic standards were obtained
from Sigma (St． Louis，USA)．
Extraction and preparation of crude polysaccha-
rides
The dried sample was grinded and subsequently treated
with hot distilled water to extract water-soluble polysac-
charides． The extracts were concentrated by a rotary e-
vaporator at 50 ℃ ． The water-soluble polysaccharides
were precipitated out from the concentrated extracts
with 95% ethanol． The isolated crude water-soluble
polysaccharides were dried in vacuum after being se-
quentially washed by ethanol，acetone and ethyl ether．
Crude C． cathayensis polysaccharides (CCCPs)were
prepared and kept at 5 ℃ for further purification．
Purification of CCCPs
Sevage reagent［9］was added to the crude polysaccha-
rides solution to denature and precipitate protein and
D101 macroporous resin was experimentally selected to
remove pigment，and then dialyzed against distilled wa-
ter in a dialysis bag (molecular weight cut off =
10000 μ)under magnetic stirring for 72 h． The dialy-
sate was vacuum-concentrated，precipitated by 95%
ethanol，and then vacuum-dried． The fine CCPs (FC-
CPs)was obtained finally． FCCPs was fractionated by
anion-exchange chromatography on a pretreated DEAE-
cellulose column． The sample was dissolved in distilled
water and applied onto the column． 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)．
Fractions of 10 mL was collected and monitored for the
presence of polysaccharides using the phenol-sulfuric
acid method ［10］． The elution profile was drawn accord-
ing with the number of testing-tube (X-axis)as well as
the absorbance (Y-axis)． FCCPs were thus separated
into a main fraction CCP1 ． CCP1 was further purified on
a Sepharose CL-6B column，eluted with 0． 1 mol /L of
NaCl． Eluent of 5 mL was collected and monitored by
phenol-sulfuric acid method． The absorbance (Y-axis)
was plotted against the number of testing-tube (X-ax-
is)to build the elution profile．
Determination for monosaccharide composition
The monosaccharide components analysis was deter-
mined by high performance liquid chromatography
(HPLC)analysis． Different monosaccharide standards
were dissolved in distilled water at a concentration of
1% (w /v)． 20 mg of CCP1 sample was subjected to
hydrolyze with 2 mol /L of H2SO4 in a dry oven for 8 h
at 110 ℃ ［11］． BaCO3 was used for neutralization of the
hydrolysate followed by centrifugation． The supernatant
was filtered through a 0． 45 μm membrane filter before
sample injection，and the monosaccharide composition
and molar ratio of CCP1 were determined according to
retention time and peak area．
Antioxidant activity of polysaccharides
Assay of hydroxyl radical scavenging activity ［12］
Hydroxyl free radical (·OH)were produced by Fen-
ton reaction under weak basic environment． In the sys-
tem，3． 0 mL of 100 mmol /L phosphate buffer solution
647 Nat Prod Ｒes Dev Vol. 26
(pH 7． 4)，1． 0 mL of 5． 5 mmol /L FeSO4，1． 0 mL of
6． 5 mmol /L EDTA-2Na，1． 0 mL of 0． 2% H2O2，1． 0
mL of 10 mmol /L sodium salicylate and 0． 5 mL of
sample solution with different concentrations were
mixed and topped up to 8． 0 mL using distilled water．
No H2O2 but only solvent was added in the control
group． The mixture was incubated in water bath at 37
℃ for 1 h，and then the absorbance AX of tube-sample
and AC of tube-control were determined at 510 nm． The
clearance rate of ·OH was calculated according to the
following formula:
Hydroxyl free radicals clearance rate = (AC-AX)/AC
× 100%
Assay of superoxide anion free radical scavenging activi-
ty ［13］
The superoxide anion free radical (O-·2 )were genera-
ted by pyrogallol autoxidation method． The amount of
O-·2 was accumulated along with time，so the absor-
bance at 325 nm increased continuously． After added
the sample，a part of O-·2 was scavenged． The variation
rate of the absorbance with time △BC of tube-control
and △BX of tube-sample were determined． The clear-
ance rate of O-·2 was calculated according to the follow-
ing formula:
Superoxide anion free radical clerarance rate = (ΔBC-
ΔBX)/ΔBC × 100%
4． 5 mL of Tris-HCl buffer solution (pH 8． 2)，1． 0 mL
of 1． 0 mmol /L pyrogallol (prepared by 10 mmol /L
HCl)and 0． 5 mL of sample solution with different
concentrations were mixed and topped up to 9． 0 mL u-
sing distilled water． The mixture was incubated in water
bath at 25 ℃ for 20 min． The control group was pre-
pared using solvent instead of sample． The absorbance
variation rate with time △BC of tube-control and △BX
of tube-sample were determined at 325 nm every 30 s
within 5 min．
Data analysis
All experimental results were expressed in terms of
mean ± standard deviation． The UV and IＲ spectra
were obtained using the computer interface with the ori-
gin software．
Ｒesults and Discussion
Isolation of polysaccharide fractions
CCCPs yielded approximately 17． 42% (w /w)accord-
ing to the procedure as described previously． FCCPs
were separated into several fractions by ion exchange
column chromatography on a DEAE-cellulose column．
The main fraction CCP1 was eluted with 0． 2 mol /L
NaCl solution． The eluting profile for CCP was shown in
Fig． 1(a)．
CCP1 was further purified on a Sepharose Cl-6B col-
umn． The purifying identification profile is a single
symmetrical peak as shown in Fig． 1(b)．
6
5
4
3
2
1
0
50% 60% 70% 80% 90 100% 110
Number%of%testing%tube
80% 90 100% 110% 120% 130% 140
Number%of%testing%tube
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0A
bs
or
ba
nc
e%a
t%4
90
nm
Ab
so
rb
an
ce
%at
%4
90
nm （a） （b）
Fig. 1 Fractionation profile of CCP on DEAE-Cellulose column (a)and CCP1 on Sepharose CL-6B column (b)
Monosaccharide composition of CCP1
According to HPLC conditions described previously，5
monosaccharide standards were subjected to HPLC a-
nalysis． The retention time of rhamnose，arabinose，fruc-
tose，mannitose，glucose was 8． 112，11． 971，13． 106，
14． 780 and 17． 908 min，respectively．
The HPLC chromatograms of standards and CCP1 were
shown in Fig． 2(a)and Fig． 2(b)，respectively． The
experimental results indicated that CCP1 consisted of
rhamnose，arabinose，fructose，mannitose，glucose in a
molar ratio of 0． 034∶ 0． 228∶ 0． 045∶ 0． 055∶ 0． 638．
747
Vol. 26 WANG Wen-ping，et al:Study on the Separation，Purification and Antioxidant
Activity of Water-soluble Polysaccharides from Chaenomeles cathayensis
0.0030
0.0025
0.0020
0.0015
0.0010
0.0000
-0.0005
0.0030
0.0025
0.0020
0.0015
0.0010
0.0000
-0.0005
0.0006
0.0004
0.0002
0.0000
-0.0002
-0.0004
0.0006
0.0004
0.0002
0.0000
-0.0002
-0.0004
Vo
lts
Vo
lts Vo
lts
Vo
lts
2% 4% 6% 8% 10% 12% 14% 16% 18% 20% 22
Minutes
2% 4% 6% 8% 10% 12% 14% 16% 18% 20% 22
Minutes
(a) (b)
Rh
am
no
se
%8
.1
12
Ar
ab
in
os
e%
11
.9
71
M
an
ni
ta
se
%1
4.
78
0
Gl
uc
os
e%
17
.9
08Fr
uc
to
se
%1
3.
10
6
Rh
am
no
se
%8
.1
10
Ar
ab
in
os
e%
11
.8
68
Fr
uc
to
se
%1
3.
06
2
M
an
ni
ta
se
14
.7
30
Gl
uc
os
e%
17
.7
86
Fig. 2 HPLC chromatograms of mixed standards of 5 monosaccharides and CCP1
Antioxidant activity of polysaccharides
Scavenging activity of hydroxyl radical
The results showed that C． cathayensis polysaccharides
with different purity had certain effect on scavenging
·OH． The ·OH clearance rate was in positive corre-
lation with concentration of different purity C． cathayen-
sis polysaccharides． In this experiment，the ·OH scav-
enging activity of CCCPs was the highest，followed by
the FCCPs samples and lowest in the CCP1 samples． On
the basis of IC50 value，the ·OH scavenging activity of
C． cathayensis polysaccharides with different purity was
lower than that of ascorbic acid as shown in Table 1．
Table 1 The scavenging effect of water-soluble polysaccharides from C． cathayensis on ·OH
Sample
Concentration of
polysaccharides (μg /mL)
0(control)
A510nm a
0． 639 ± 0． 002
Inhibition
rate (%)
－
ICb50
(g /mL)
－
CCCPs 150 0． 534 ± 0． 003 16． 43 673． 3
300 0． 465 ± 0． 004 27． 23
450 0． 409 ± 0． 002 35． 84
600 0． 332 ± 0． 006 48． 04
750 0． 286 ± 0． 001 55． 09
900 0． 241 ± 0． 002 62． 28
FCCPs 150 0． 554 ± 0． 006 13． 30 942． 9
300 0． 509 ± 0． 004 20． 19
450 0． 461 ± 0． 003 27． 70
600 0． 415 ± 0． 014 35． 05
750 0． 374 ± 0． 002 41． 47
900 0． 337 ± 0． 006 47． 26
CCP1 150 0． 560 ± 0． 001 12． 21 1002
300 0． 525 ± 0． 004 17． 84
450 0． 481 ± 0． 003 24． 57
600 0． 434 ± 0． 007 32． 08
750 0． 386 ± 0． 012 39． 59
900 0． 352 ± 0． 005 44． 91
Vitamin C 50 0． 580 ± 0． 001 9． 10 288． 4
150 0． 448 ± 0． 002 29． 87
250 0． 354 ± 0． 005 44． 54
350 0． 264 ± 0． 001 58． 54
450 0． 149 ± 0． 003 76． 56
aValues were presented as means of three independent experiments (standard deviation．
b IC50，the half maximal inhibitory concentration，represented the concentration of an inhibitor that was required for 50% scavenging effect on ·OH．
847 Nat Prod Ｒes Dev Vol. 26
Scavenging activity of superoxide anion free radical
The A325nm values significantly decreased after treat-
ments with different purities of C． cathayensis polysac-
charides． The O-·2 clearance rate was in positive corre-
lation with concentration of different purity C． cathayen-
sis polysaccharides． Superoxide anion free radical scav-
enging activity of C． cathayensis extracts followed the
order:CCCPs ＞ FCCPs ＞ CCP1 ． On the basis of IC50
value，the O-·2 scavenging activity of C． cathayensis pol-
ysaccharides with different purity was lower than that of
ascorbic acid as shown in Table 2．
Table 2 The scavenging effect of water-soluble polysaccharides from C． cathayensis on O-·2
Sample
Concentration of
polysaccharides
(g /mL)
0(contrd)
△A325nm a
0． 653 ± 0． 004
Inhibition rate (%)
－
ICb50
(g /mL)
－
CCCPs 150 0． 435 ± 0． 006 33． 38
300 0． 354 ± 0． 013 45． 79
450 0． 282 ± 0． 005 56． 81 374． 0
600 0． 221 ± 0． 007 66． 16
750 0． 166 ± 0． 012 74． 58
900 0． 130 ± 0． 008 80． 01
FCCPs 150 0． 471 ± 0． 001 27． 87
300 0． 410 ± 0． 006 37． 21
450 0． 352 ± 0． 013 46． 09 525． 4
600 0． 300 ± 0． 002 53． 91
750 0． 233 ± 0． 001 64． 17
900 0． 191 ± 0． 005 70． 60
CCP1 150 0． 507 ± 0． 003 22． 21
300 0． 446 ± 0． 002 31． 55
450 0． 383 ± 0． 007 41． 34 592． 4
600 0． 311 ± 0． 002 52． 37
750 0． 254 ± 0． 003 60． 95
900 0． 216 ± 0． 006 66． 77
Vitamin C 25 0． 594 ± 0． 002 8． 90
50 0． 565 ± 0． 001 13． 45
100 0． 488 ± 0． 003 25． 12 242． 2
200 0． 347 ± 0． 002 46． 78
400 0． 153 ± 0． 004 76． 52
aValues were presented as the means of three independent experiments ± standard deviation．
b IC50，the half maximal inhibitory concentration，represented the concentration of an inhibitor that was required for 50% scavenging effect on O-·2 ．
Conclusion
The results showed that crude polysaccharides had the
highest scavenging efficiency on superoxide anion free
radical and hydroxyl radical than FCCPs and CCP1 ．
This implied that there were other components such as
vitamin C，polypeptides and oligosaccharides which
may be responsible for the antioxidant activity，and the
purification process resulted in a decrease of the antiox-
idant activities to some extent． Moreover，crude polysac-
charides had higher scavenging efficiency on superox-
ide anion free radical than on hydroxyl radical． The
fruit of C． cathayensis may be a new source of natural
antioxidants．
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(下转第 769 页)
947
Vol. 26 WANG Wen-ping，et al:Study on the Separation，Purification and Antioxidant
Activity of Water-soluble Polysaccharides from Chaenomeles cathayensis
MDA异常增高，这表明 Sch B 对自由基引起的生物
膜脂质过氧化反应有抑制作用;实验亦表明 Sch B
处理组 L02 细胞的抗氧化酶活力有所增强，可能是
H2O2 产生的过多自由基引发。这些结果都可能与
Sch B的抗氧化作用相关，但 Sch B在体内是否有类
似的药理学效应仍需进一步研究。
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967Vol. 26 蔡 晶等:五味子乙素对人肝细胞氧化损伤的保护作用