全 文 ： 天然产物研究与开发
2005 Vol.17 No.6 NATURAL PRODUCT RESEARCH AND DEVELOPMENT
Received March 3 , 2005;Accepted April 6 ,2005
Fundation Item:This project was supported by Agricultural Research
Foundat ion of Guangdong Province(2003A203507)
＊Corresponding author E-mail:yangbao@china.com.cn
Characterization of Litchi Pericarp Polysaccharide
YANG Bao＊ ,ZHAO Mou-ming ,LIU Yang ,LI Bao-zhen
(Light Industry and Food College , South China University of Technology , Guangzhou 510640 , China)
Abstract:Poly saccharide was an important functional composition in litchi pericarp.Anion exchange column and gel filtration
column were used to isolate and purify the major polysaccharide extracted from litchi pericarp.The profile of gel permeation
chromatography(GPC)showed its molecular weight as 14000 Dal.Monose composition of the major polysaccharide was deter-
mined by using gas chromatography(GC).It mainly comprised mannose and galactose , and arabinose.Its structure was further
identified by using infrared(IR)spectra.
Key words:litchi pericarp;polysaccharide;GC;infrared specta;composition
荔枝壳多糖特性研究
杨 宝＊ ,赵谋明 ,刘 洋 ,李宝珍
(华南理工大学轻工与食品学院 , 广州 510640)
摘 要:多糖是荔枝壳的重要活性成分 , 本研究采用阴离子交换柱和凝胶过滤柱对荔枝壳多糖进行分离纯化。
凝胶渗透色谱测定其分子量为 14000 Dal。通过气相色谱测定其单糖组成为甘露糖 、半乳糖和少量的阿拉伯糖 ,
分子链由 1 , 2 键 、1 , 3键和 1 , 6键组成 , 不含 1 , 4 键。红外光谱分析表明甘露糖以 β-D-甘露糖形式存在 , 不含羧
基基团。
关键词:荔枝壳;多糖;气相色谱;红外光谱;组成
中图分类号:R931.6;Q946.91
The litchi (Litchi chinensis Sonn.)is an exotic fruit in
southeast Asia , especially in China.In the last 30 years ,
other semitropical regions began to plant this kind of
fruit[ 1] .It has a delicious taste and lovely shape , being
desired by many people
[ 2] .In China , annual litchi produc-
tion is about 1.5 million tons.Litchi contains significant
amounts of polysaccharides.For their important bio-activi-
ties such as blood coagulant-promoting and antitumor ac-
tivities ,polysaccharides development is one of the hot sci-
entific research topics at present.
At present , most papers of litchi pericarp reported the
browning phenomena of litchi pericarp and how to inhibit
it[ 3-5] .Very limited reports dealt with identification of the
polysaccharide in litchi pericarp.This research isolated
and purified the major polysaccharide by using DEAE
sepharose fast flow anion exchange column and sephadex
G50 column , and determined its molecular weight and
monose composition by using GPC and GC , respectively.
Results and Discussion
After isolated by DEAE sepharose fast flow anion ex-
change column , litchi pericarp polysaccharide was classi-
fied into two parts (F1 and F2), amongst non-negative
polysaccharide (F1)was the major part (Fig.1).F1 was
collected and submitted to gel filtration chromatography
(Fig.2).The retention time of major part(F1 )was about
20 min.F1 was collected for further identification.
Monose composition of F1 fraction was determined by
TFA hydrolysis method.Gas chromagram of sample (Fig.
3)revealed the presence of mannose , galactose and small
amounts of arabinose.Their molar percentage were
65.6%,33.0% and 1.4%, respectively.By titrating the
production F1 of sodium metaperiodate oxidation , 1 mol
hexose could develop 0.08 mol formic acid.This meant
the molar percentage of(1 ,6)glycosidic linkage was 8%.
The results of Smith degradation showed the molar per-
centages of glycerol ,mannose and galactose were 16.7%,
53.5% and 29.8%, respcetively.The undetectable ery-
thritol indicated no 1 , 4 glycosidic linkage existed in
685
DOI :10.16333/j.1001-6880.2005.06.003
polysaccharide of F1 .The molar percentages of (1 , 2)
glycosidic linkage , (1 , 3)glycosidic linkage and (1 , 6)
glycosidic linkage were calculated as 8.7%,83.3% and
8.0%, respectively.
Fig.1 Anion exchange chromatogram of crude polysaccha-
ride extracted from litchi pericarp
Fig.2 Superdex G50 gel chromatogram of F1 fraction
Fig.3 Gas chromatogram of monose composition of litchi
pericarp polysaccharide and standard monose
1 arabinose;2 xylose;3 f ructose;4 galactose;5 mannose
The molecular weight of polysaccharide was determined by
gel permeation chromatography.The equation of standard
curve was drawn as LogWt =121.9-9.17t +0.242t 2-
0.00217t 3(Wt (Dal)represents molecular weight , t
(min)represents elution time)by using Millennium 32
software.The elution time of F1 was 36.66 min , so its
molecular weight was calculated as 14000 Dal.
The IR spectrum of the purified polysaccharide F1 (Fig.
4)displayed a broad stretching intense peak P1 at around
3407 cm
-1
characteristic for hydroxyl group
[ 6] , and a weak
C-H stretching band P2 at 2931 cm-1.The peak P3 around
2360 cm
-1
also indicated aliphatic C-H bonds.No peak in
the range of 1600 cm
-1
to 1800 cm
-1
meant no carboxyl
group existed.Two stretching peak (P4 , P5)at 1154 cm-1
and 1077 cm-1 suggested the presence of C-O bonds.The
absorption of 932 cm-1(P6)was due to antisymmetrical
ring vibration.The peak P7 at around 893 cm-1 was char-
acteristic forβ-D-mannose.A band of absorption at 771
cm-1(P8)represented symmetrical ring vibration.The peak
P9 at 600 cm
-1was due to O-H out-of -plane vibration[ 7] .
Fig.4 Infra-red spectra of litchi pericarp polysaccharide
Conclusion
After isolation by using anion exchange column and gel
filtration column , the purified polysaccharide F1 was ob-
tained.It comprised mannose , galactose and arabinose with
the ratio of 65.6%:33.0%:1.4%.Smith degradation
indicated there were (1 , 2), (1 , 3)and (1 , 6)glycosidic
linkage existing in F1 molecule with the ratio of 8.7%:
83.3%:8.0%.GPC showed the molecular weight of F1
as 14000 Dal.
Materials and Methods
Plant material
Fresh litchi fruit (Litchi chinensis Sonn.)cv.Huaizhi at
the commercially mature stage were picked from a com-
mercial orchard in Guangzhou ,China.Fruits were selected
for uniformity of shape and colour , then stored at 4 ℃ in
refrigerator.
Extraction of the polysaccharide
Extraction of the polysaccharide was done according to the
method reported by Qin ,Huang and Xu[ 8] with some mod-
ifications.Litchi pericarp (5.0 g)was extracted three
times with distilled water (100 mL)at 30 ℃ for 2 h.The
three extracts were pooled and filtered on buchner funnel
under vacuum before concentrating to 25 mL under vacu-
um at 30 ℃.Then protein was removed by using Sevag
reagent for six times
[ 9] .After removing the Sevag reagent ,
100mL anhydrous ethanol was added to the water phase
and kept at 4 ℃ overnight in a refrigerator to precipitate
686 天然产物研究与开发 2005 Vol.17 No.6
carbohydrate compounds.
Isolation and purification of the polysaccharide
A column (16×100 mm)of DEAE sepharose fast flow
was used to isolate negative polysaccharide and non-nega-
tive polysaccharide.The column was first eluted with H2O
and then 0.5M NaCl at a flow rate of 0.3 mL/min.The
major fraction was collected and concentrated under vacu-
um for further purification with Sephadex G50 gel column
(10×300 mm).H2O was used as eluent at a flow rate of
0.5mL/min.The fractions was determined by phenol-sul-
furic acidmethod[ 10] .The major fraction was collected and
freeze-dried.
Measurement of molecular weight
The molecular weight of the polysaccharide was deter-
mined by GPC[ 11] with a Waters HPLC apparatus (Wa-
ters ,USA), equipped with a Ultrahydrogel column , a mod-
el 410 Refractive index detector and a Millennium 32
Workstation.The column was eluted with H2O at a flow
rate of 0.6 mL/min.The Dextran standards with different
molecular weights (4400 , 9900 , 21400 , 43500 , 124000 ,
196000 ,277000 ,845000)were used for calibration curve.
Infrared spectra analysis of the polysaccharide
The polysaccharide was characterized using a Fourier
transform infrared (FT-IR)spectrophotometer (Bruker ,
German)equipped with OPUS 3.1 software.The dried
polysaccharide was ground with KBr powder and pressed
into pellets for FT-IR spectra measurement in the frequen-
cy range of 4000 ～ 500 cm-1[ 12] .
Analyses of monose composition and glycosidic link-
age
10 mg polysaccharide was hydrolyzed with 2 M TFA at
120 ℃ for 6 h[ 13] prior to being derived by the
trimethylsilylated reagent according to the method reported
by Guentas , et al [ 14] .The trimethylsilylated derivatives
were submitted to gas chromatography (GC)analysis , us-
ing inositol as internal base.The operation was performed
using a HP5 capillary column.The conditions were as fol-
lows:H2:16 mL/min;air:150 mL/min;N2:20 mL/min;
injection temperature:230 ℃;detector temperature:230
℃;column temperature programmed from 130 to 180 ℃
at 5 ℃/min ,holding at 180 ℃ for 2min , then to 220 ℃
at 5 ℃/min and holding at 220 ℃for 3 min.
Smith degradation was used to analyse glycosidic link-
age[ 15] .Polysaccharide(20 mg)was treated with 15 mM
sodium metaperiodate(40 mL)for 120 h at 4 ℃.Excess
sodium metaperiodate was destroyed by addition of ethy-
lene glycol (50 μL), and the product was reduced with
NaBH4(30 mg),dialyzed against distilled water , and then
was hydrolyzed in 2 M TFA for 6 h at 120 ℃.The re-
leased sugars were trimethylsilylated.The resulting
trimethylsilylated ether derivatives were analyzed by GC.
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6872005 Vol.17 No.6 杨 宝等:荔枝壳多糖特性研究