Isolation and structural characterization of a polysaccharide LRP4-A from Lycium ruthenicum Murr.

Article abstract of DOI:10.1016/j.carres.2012.10.013

A complex polysaccharide, termed LRP4-A, was isolated from the fruit of Lycium ruthenicum Murr. and its structure was characterized. The crude polysaccharide LRP was obtained from the fruit of L. ruthenicum Murr. using hot water extraction followed by ethanol precipitation. The water-soluble polysaccharide LRP4-A was purified from LRP by anion-exchange chromatography and gel filtration chromatography. Its molecular weight was 1.05 × 10 ⁵ Da. Monosaccharide composition analysis revealed that LRP4-A mainly consisted of rhamnose, arabinose, glucose, and galactose in the molar ratio of 1:7.6:0.5:8.6, with a trace of xylose. Structure of the polysaccharide LRP4-A was characterized using a series of analytical techniques, including methylation analysis, partial acid hydrolysis, IR, NMR, and ESI-MS. LRP4-A was identified to be a highly branching polysaccharide with a backbone of β-(1→6)- linked galactose partially substituted at O-3 position. The branches were composed of (1→3)-linked-Gal, (1→3)-linked-Ara, (1→5)-linked-Ara, and (1→2,4)-linked-Rha. Arabinose, galactose, and glucose were located at the termini of the branches.

Full text of DOI:10.1016/j.carres.2012.10.013

Carbohydrate Research 365 (2013) 20–25
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Isolation and structural characterization of a polysaccharide LRP4-A
from Lycium ruthenicum Murr.
⇑
⇑
Xiaopeng Lv, Chengjian Wang, Yang Cheng, Linjuan Huang , Zhongfu Wang
Educational Ministry Key Laboratory of Resource Biology and Biotechnology in Western China, Life Science College, Northwest University, Xi’an 710069, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A complex polysaccharide, termed LRP4-A, was isolated from the fruit of Lycium ruthenicum Murr. and its
structure was characterized. The crude polysaccharide LRP was obtained from the fruit of L. ruthenicum
Murr. using hot water extraction followed by ethanol precipitation. The water-soluble polysaccharide
LRP4-A was purified from LRP by anion-exchange chromatography and gel filtration chromatography.
Its molecular weight was 1.05 Â 105 Da. Monosaccharide composition analysis revealed that LRP4-A
mainly consisted of rhamnose, arabinose, glucose, and galactose in the molar ratio of 1:7.6:0.5:8.6, with
a trace of xylose. Structure of the polysaccharide LRP4-A was characterized using a series of analytical
techniques, including methylation analysis, partial acid hydrolysis, IR, NMR, and ESI-MS. LRP4-A was
identified to be a highly branching polysaccharide with a backbone of b-(1?6)-linked galactose partially
substituted at O-3 position. The branches were composed of (1?3)-linked-Gal, (1?3)-linked-Ara, (1?5)-
linked-Ara, and (1?2,4)-linked-Rha. Arabinose, galactose, and glucose were located at the termini of the
branches.
Received 26 July 2012
Received in revised form 22 September
2012
Accepted 16 October 2012
Available online 24 October 2012
Keywords:
Lycium ruthenicum Murr.
Polysaccharide
Structure
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
on Lycium ruthenicum Murr. polysaccharide was merely prelimin-
ary analysis of its extraction method and bioactivity. Li et al.13 ex-
Lycium ruthenicum Murr., which belongs to the genus Lycium of
the family Solanaceae, is a kind of wild resource plant in the north-
west of China. It has high photosynthetic efficiency and strong
resistance to environmental pressure and lives mainly in Qinghai
and Xinjiang Provinces of China.1 Because of the characteristics
of sweetness and juiciness, the fructification of Lycium ruthenicum
Murr. has been eaten as a fruit or been used as a raw material to
produce beverage for a long term. As all know, another species of
the genus Lycium, Lycium barbarum L., has been extensively utilized
as a tonic for thousands of years, which was believed to have a vari-
ety of biological activities, including immunoregulation, anti-aging,
lowering blood-sugar and blood-fat levels, and anti-fatigue.2,3 Com-
pared with Lycium barbarum L., Lycium ruthenicum Murr. has same
types but much higher content of nutritional ingredients.4 More-
over, quite a few biologically active molecules from Lycium ruthen-
icum Murr. have also been proved to have a series of biological
effects, such as cell-mediated immunity enhancement, anti-oxidation,
anti-aging, anti-fatigue, and hypoglycemic activity.5–8
tracted the crude polysaccharide from Lycium ruthenicum Murr.,
and analyzed its physical and chemical properties. Wang et al.14
optimized the extraction conditions for the crude polysaccharide
of Lycium ruthenicum Murr. Many other studies15–17 disclosed the
good biological activities of the crude polysaccharide of Lycium
ruthenicum Murr. in immunoregulation, anti-fatigue, and blood-su-
gar reduction.
The current study is focused on the structural analysis of the
polysaccharide components of Lycium ruthenicum Murr. The crude
polysaccharide was first extracted from Lycium ruthenicum Murr.,
and then the ion-exchange and gel-permeation chromatography
was used to isolate and purify the crude polysaccharides and a
homogeneous polysaccharide LRP4-A was obtained. Structure of
the polysaccharide LRP4-A was characterized by a series of analyt-
ical techniques including methylation analysis, partial acid hydro-
lysis, GC, GC–MS, ESI-MS, and NMR. These analysis results could
provide a foundation for both the further investigation on the
structure–activity relationship of Lycium polysaccharide and the
deep exploitation of Lycium ruthenicum Murr.
Previously, a lot of studies have been focused on polysaccharide
of Lycium barbarum L., which was revealed to be one of the main
active components.9–12 Polysaccharide from Lycium ruthenicum
Murr., however, has been seldom investigated. Previous research
2. Experimental
2.1. Plant material, chemicals, and instruments
⇑
Corresponding authors. Tel.: +86 29 88305853; fax: +86 29 88303534.
E-mail addresses: huanglj@nwu.edu.cn (L. Huang), wangzhf@nwu.edu.cn (Z.
The fruit of Lycium ruthenicum Murr. was purchased from
Wang).
Jinpenyuan Wolfberry Com. Ltd, Qinghai province. Monosaccharide
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.10.013

X. Lv et al. / Carbohydrate Research 365 (2013) 20–25
21
standards (
D
-galactose,
L
-rhamnose,
L
-arabinose,
D
-glucose,
D
-xy-
weights (5000, 12,000, 25,000, 50,000, 80,000, and 150,000 Da).
Vt and V0 were calibrated with glucose and dextran blue
(2000,000), respectively. The molecular weight of LRP4-A was esti-
mated by reference to the calibration curve made above.
lose, and -galacturonic acid) and dextrans (5000, 12,000, 25,000,
D
50,000, 80,000, and 150,000 Da) were purchased from Sigma (St.
Louis, MO, USA). Sephadex G-100 was purchased from Pharmacia
Co. (Uppsala, Sweden). DEAE-cellulose 52 was purchased from
HengXin Chemical Reagent Co. (Shanghai, China). All other chemi-
cals used were of analytical grade.
2.5. Monosaccharide composition analysis
HPLC analysis was carried out on a waters 2695 HPLC system
(2695 HPLC pump, 2414 refractive index detector). GC analysis
was carried out using a Shimadzu GC-2010 instrument. GC–MS
analysis was carried out using a Shimadzu GCMS-QP 2010 system.
ESI-MS data were acquired on a Thermo Scientific LTQ XL ion trap
mass spectrometer. NMR spectra were recorded using a Varian Ino-
va 400 spectrometer.
The polysaccharide sample was hydrolyzed in 2 mol/L TFA at
121 °C for 2 h. The acid was then completely removed by co-distil-
lation with water. Neutral sugars and uronic acids were simulta-
neously detected by GC utilizing the method described
previously.20 GC was performed on a Shimadzu GC-2010 system
equipped with a capillary column of rtx-50 (30.0 m  0.25 mm Â
0.25 lm). The temperature program was: 180 °C for 2 min, then
to 210 °C at 6 °C/min, then to 215 °C at 0.3 °C/min, then to 240 °C
at 6 °C/min for 30 min. Nitrogen was used as the carrier gas at
0.6 mL/min.
2.2. Extraction of crude polysaccharides
The dry fruit of Lycium ruthenicum Murr. was pulverized and
soaked in distilled water at 80 °C for 2 h. After filtration through
a piece of 100-mesh gauze fabric, the obtained solid residue was
reprocessed with distilled water at 80 °C for 2 h. The aqueous ex-
tracts obtained during the two extraction steps were pooled and
concentrated under reduced pressure. A four-fold volume of EtOH
was then added to the concentrated extracts, followed by an incu-
bation step at 4 °C for 24 h. The mixture was subjected to centrifu-
gation, and the resulting precipitate was collected and redissolved
in distilled water. The obtained aqueous solution was deproteinat-
ed according to the Sevage method,18 dialyzed against distilled
water for 48 h, concentrated under reduced pressure and finally
lyophilized. The crude polysaccharides termed LRP was obtained.
LRP was dissolved in distilled water at 40 °C and the solution
was adjusted to pH 9 with aqueous ammonia. H2O2 (30%) was
added dropwise until the color faded. After stirring for several
hours, the color turned yellow. The solution was dialyzed against
distilled water for 48 h, neutralized with 0.5 mol/L HCl and finally
lyophilized.19
2.6. Determination of sugar and protein contents
The carbohydrate content of each sample was determined using
the phenol–sulfuric acid method with glucose as the standard.21
Protein content of each sample was carried out according to the
procedure of Bradford with chicken egg white albumin as the
standard.22
2.7. FT-IR analysis
The IR spectrum of LRP4-A was recorded in the range from 4000
to 400 cmÀ1 using KBr discs on an EQUINOX 55 Fourier transform
infrared spectrophotometer (Bruker, Germany).
2.8. Partial acid hydrolysis
LRP4-A was hydrolyzed in 20 mmol/L H2SO4 at 80 °C for 12 h.
The solution was dialyzed against distilled water for 72 h using a
dialysis bag with a MWCO of 8 kDa. The non-dialyzable (inside
the dialysis bag) sample was concentrated and lyophilized, giving
the partially hydrolyzed polysaccharide LRP4-A-I. The dialyzable
(outside the dialysis bag) fraction was concentrated and neutral-
ized with barium carbonate. After centrifugation, the supernatant
was freeze-dried and designated as LRP4-A-O. Sugar compositions
of LRP4-A-I and LRP4-A-O were analyzed by GC as described above.
2.3. Fractionation of LRP
DEAE-cellulose (DE-52) anion-exchange chromatography was
used to fractionate the decolored LRP. LRP was dissolved in wateÀr
and loaded onto a DEAE-cellulose column (5 Â 25 cm, HCO3
form), followed by elution with water and 0.05, 0.1, 0.25, and
0.5 M NaHCO3 at a flow rate of 1 mL/min, successively. The eluted
fractions were monitored spectrophotometrically at 490 nm using
the phenol–sulfuric acid method. Five fractions designated as LRP1,
LRP2, LRP3, LRP4, and LRP5 were obtained. LRP4 was further frac-
tionated with gel-permeation chromatography on a Sephadex G-
100 column using 0.1 mol/L NaCl solution as an eluent, and two
fractions were obtained. The main fraction, designated as LRP4-A,
was collected, dialyzed, and lyophilized. It was further purified
using the Sephadex G-100 column chromatography. After dialysis
and lyophilization, a white fluffy material termed polysaccharide
LRP4-A was obtained.
2.9. ESI-MS of the dialyzable oligosaccharides
The sample of LRP4-A-O was desalted using a Dowex 50 WX8-
400 cation exchange column according to our previously described
method.23
The desalted sample was detected by a Thermo Scientific LTQ
XL ion trap mass spectrometer (USA) in positive ion mode (sheath
gas N2, flow rate 30.0 arb, auxiliary gas flow rate 5.0 arb, spray volt-
age 5 kV, heated capillary temperature 350 °C, capillary voltage
48 V, tube lens voltage 220 V). Samples were injected into the
ion source by a 2
bile phase was methanol/water (1:1, v/v) solution at a flow rate
of 200 L/min.
lL fixed quantity (Rheodyne) loop, and the mo-
2.4. Determination of homogeneity and molecular weight
l
The homogeneity and molecular weight of LRP4-A were evalu-
ated and determined by high performance gel-permeation chro-
matography (HPGPC). The sample solution was subjected to a
Waters 2695 High Performance Liquid Chromatography (HPLC)
2.10. Methylation analysis
Both the samples of LRP4-A and LRP4-A-I were methylated
three times according to the method of Needs and Selvendran.24
The methylated products were extracted using chloroform and
examined by IR spectroscopy. The absence of the absorption peak
corresponding to hydroxyl indicated the complete methylation.
The per-methylated polysaccharide was hydrolyzed by treatment
system equipped with
a
TSK-GEL G4000 SWXL column
(7.8 Â 300 mm) and a Waters 2414 Refractive Index Detector.
The mobile phase was 0.1 mol/L phosphate buffer (pH 6.0) contain-
ing 0.1 mol/L Na2SO4 at a flow rate of 0.4 mL/min. The linear regres-
sion was calibrated with dextran standards of known molecular

22
X. Lv et al. / Carbohydrate Research 365 (2013) 20–25
with 90% formic acid at 100 °C for 3 h. Excess acid was removed by
co-distillation with methanol for three times after the hydrolysis
was completed. The products were hydrolyzed in 2 mol/L TFA at
121 °C for 2 h, followed by reduction with NaBH4 and acetylation.
The resulting partially methylated alditol acetates were analyzed
by GC and GC–MS. GC–MS was performed using a Shimadzu GC–
MS-QP2010 system equipped with a capillary column of rtx-5 ms
(30.0 m  0.25 mm  0.25
lm), and the temperature program
was: 140 °C for 2 min, then to 250 °C at 2 °C/min for 20 min. He-
lium was used as the carrier gas and maintained at 1.24 mL/min.
2.11. NMR spectroscopy
Figure 2. Chromatographic elution profile of LRP4 on a Sephadex G-100 column
with 0.1 mol/L NaCl as the eluant.
Before NMR analysis, the polysaccharide samples were ex-
changed three times with 99.9% D2O, lyophilized and finally dis-
solved in 99.9% D2O. 1H and 13C NMR spectra were recorded at
30 °C on a Varian Inova 400 spectrometer.
R2 = 0.998, in which Mw denoted the molecular weight of the stan-
dard dextran and tR represented the retention time. Based on the
calibration with standard dextrans, the average molecular weight
(MW) of LRP4-A was estimated to be 1.05 Â 105 Da.
3. Results and discussion
3.1. Isolation and fractionation of LRP
3.3. Monosaccharide composition, character, and partial acid
hydrolysis analysis of LRP4-A
The water-soluble crude polysaccharide, designed as LRP, was
obtained from the fruit of Lycium ruthenicum Murr. by hot water
extraction, ethanol precipitation, deproteinization, dialysis, and
lyophilization. LRP was a black polysaccharide, the pigment of
which could not be removed by column chromatography. To avoid
the influence of the pigment on structure analysis, LRP was decol-
ored with 30% H2O2, followed by dialysis and lyophilization, giving
a pale yellow solid material (yield is 0.96% of the crude herb).
Decolored LRP was fractionated by anion-exchange chromatog-
raphy. As shown in Figure 1, five sub-fractions, designed as LRP1,
LRP2, LRP3, LRP4, and LRP5, respectively, were obtained.
The polysaccharide component LRP4 was further fractionated
by gel-permeation chromatography on a Sephadex G-100 column,
and two sub-fractions, LRP4-A and LRP4-B, were obtained (see
Fig. 2). LRP4-A, the dominant sub-fraction, was further purified
by gel-permeation chromatography on a Sephadex G-100 column,
and a symmetrical sugar peak was obtained (LRP4-A) with a yield
of 0.19% of the crude extracts.
Sugar composition of LRP4-A was determined by GC and the re-
sults were shown in Figure 3. LRP4-A mainly consisted of rham-
nose, arabinose, glucose, and galactose in the molar ratio of
1:7.6:0.5:8.6, with a trace of xylose. No uronic acid was detected
in LRP4-A, and arabinose and galactose accounted for 42.9% and
48.6% of the total sugar content, respectively. As determined by a
colorimetric method, the total carbohydrate content was 95.7%,
and the protein content was 1.4%. A UV scan in the region of
200–400 nm showed strong absorbance at about 200 nm but no
distinct absorbance at 280 nm, indicating further that the LRP4-A
sample contained a trace of protein. In the FT-IR spectrum, a typi-
cal major broad stretching peak in the region of 3600–3200 cmÀ1
was attributed to the O–H stretching and bending vibrations; the
small band at around 2926 cmÀ1 was attributed to the C–H stretch-
ing and bending vibrations; the relatively strong absorption peak
at 1642 cmÀ1 reflected the absorption of the C–O group that is part
of glycosides;25 the weak absorbance at 895 cmÀ1 suggested that
pyranoses existed in the b-configuration. The IR spectrum showed
no specific signature of uronic acid in consistence with the data of
GC analysis. These results showed that LRP4-A was a neutral poly-
saccharide which was rich in arabinose and galactose.
Partial acid hydrolysis was carried out for the existence of abun-
dant arabinose in LRP4-A, because furanose has a hydrolysis rate
nearly two orders of magnitude higher than that of pyranose in di-
lute sulfuric acid.26 LRP4-A was partially hydrolyzed with
20 mmol/L H2SO4 at 80 °C for 12 h and then dialyzed against dis-
tilled water. The sugar compositions of the non-dialyzable fraction
LRP4-A-I and the dialyzable fraction LRP4-A-O were separately
analyzed. The results were shown in Figure 4. The dialyzable frac-
tion LRP4-A-O was composed of rhamnose, arabinose, glucose, and
galactose in a molar ratio of 1:12.5:0.4:1.8 and contained a trace of
xylose, indicating that they were present on the outer chains. The
non-dialyzable fraction LRP4-A-I contained Gal and a trace of Glc,
suggesting that Gal and a trace of Glc were present as the main
chain or brink of the main chain. LRP4-A-I had no rhamnose, arab-
inose or xylose, suggesting that all rhamnose, arabinose, and xylose
were present on the branches of the polysaccharide.
3.2. Homogeneity and molecular weight
The homogeneity and molecular weight of LRP4-A were deter-
mined by high performance gel-permeation chromatography
(HPGPC). LRP4-A was eluted with 0.1 mol/L phosphate buffer (pH
6.0) containing 0.1 mol/L Na2SO4, and a single symmetrical peak
was obtained, indicating that the polymer was homogeneous.
The calibration curve equation was: lgMw = 7.925–0.145tR,
3.4. Methylation analysis
Figure 1. Chromatographic elution profile of LRP on DEAE-cellulose column
(5 Â 25 cm). LRP1–LRP5 were obtained by elution with H2O, 0.05, 0.1, 0.25, and
0.5 mol/L NaHCO3 solution, respectively. A490 was determined by sulfate–phenol
method.
Methylation of LRP4-A and LRP4-A-I was performed three times
to obtain per-methylated polysaccharides. After complete acid

X. Lv et al. / Carbohydrate Research 365 (2013) 20–25
23
identified by their retention times and mass spectra. The molar ra-
tio of partially methylated alditol acetates was calculated based on
both peak area and molar response factors, which were evaluated
by the ‘effective carbon response’, originally according to the pre-
dicted ionization potential of organic constituents in an FID.27
The results of methylation analysis of LRP4-A and LRP4-A-I were
listed in Table 1.
As shown in Table 1, the non-reducing ends of LRP4-A con-
tained arabinose, glucose, and galactose. Branches were present
on galactosyl residues and on a trace of rhamnosyl residues. The
types of arabinosyl residues included terminal Ara, (1?3)-linked-
Araf, (1?3)-linked-Arap, and (1?5)-linked-Ara. The glucosyl residues
were present at the non-reducing end. In addition, the galactosyl
residues were composed of terminal-, (1?3)-linked, (1?6)-linked,
and (1?3,6)-linked galactose. The branching unit was ?3,6)
Gal(1?, which accounted for about 40.9% of all hexoses. Obviously,
the glycan branches highly. Termini of LRP4-A-I were composed of
galactose. The branching unit of LRP4-A-I was (1?3,6)-linked Gal,
which indicated that the backbone of LRP4-I was composed of
either (1?3)-linked Gal or (1?6)-linked Gal. The amount of total
terminal sugars was approximately equal to that of branched
sugars, suggesting the completeness of methylation. Compared
with LRP4-A, LRP4-A-I had no terminal arabinose, terminal glucose,
(1?3)-linked Araf, (1?3)-linked Arap, (1?5)-linked Ara or
(1?2,4)-linked Rha, indicating that they existed as branches. The
Table 1
Results of methylation analysis of LRP4-A and LRP4-A-I
Peak
Partially
methylated sugar
Deduced
linkage
Relative molar ratio
numbera
LRP4-A
LRP4-A-I
Figure 3. Gas chromatograms of the monosaccharides derived from LRP4-A, LRP4-
A-I, and LRP4-A-O. (1) Rhamnose (Rha); (2) Fucose (Fuc); (3) Arabinose (Ara); (4)
Xylose (Xyl); (5) Mannose (Man); (6) Glucose (Glc); (7) Galactose (Gal); (8)
Glucuronic acid (GlcA); (9) Galacturonic acid (GalA).
1
2
3
4
5
6
7
8
9
2,3,5-Me3-Ara
2,5-Me2-Ara
2,3-Me2-Ara
2,3,4,6-Me4-Glc
2,3,4,6-Me4-Gal
2,4-Me2-Ara
3-Me-Rha
2,4,6-Me3-Gal
2,3,4-Me3-Gal
2,4-Me2-Gal
Ara(1-
8
2
6
1
2
2
2
7
3
9
-3)Araf(1-
-5)Ara(1-
Glc(1-
Gal(1-
5
-3)Arap(1-
-2,4)Rha(1-
-3)Gal(1-
-6)Gal(1-
-3,6)Gal(1-
4
7
5
10
a
Number of the peaks are the same as those designated in Figure 4.
Figure 4. Gas chromatograms of partially methylated alditol acetates derived from
LRP4-A and LRP4-A-I. Column: rtx-5 ms (30 m  0.25 mm  0.25
lm); tempera-
ture: 140 °C (2 min)–2 °C/min, 250 °C (20 min). (1) 2,3,5-Me3-Ara; (2) 2,5-Me2-Ara;
(3) 2,3-Me2-Ara; (4) 2,3,4,6-Me4-Glc; (5) 2,3,4,6-Me4-Gal; (6) 2,4-Me2-Ara; (7) 3-
Me-Rha; (8) 2,4,6-Me3-Gal; (9) 2,3,4-Me3-Gal; (10) 2,4-Me2-Gal.
hydrolysis and alditol acetylation, the obtained partially methyl-
ated alditol acetates were analyzed by GC and GC–MS. The GC
chromatograms of LRP4-A and LRP4-A-I were shown in Figure 4.
The chromatogram of LRP4-A exhibited ten peaks, while that of
LRP4-A-I showed only four peaks. Peaks of methylated sugars were
Figure 5. 1H (A) and 13C (B) NMR spectra of LRP4-A.

24
X. Lv et al. / Carbohydrate Research 365 (2013) 20–25
Figure 6. ESI-MS profile of LRP4-A-O.
Figure 7. Scheme of the structural unit of LRP4-A.
increase of four ?6)Gal(1? in LRP4-A-I was concomitant with the
decrease of four ?3,6)Gal(1? branching unit in LRP4-A, showing
that the main chain was composed of ?6)Gal(1?. In addition,
the three new terminal Gal(1? in LRP4-A-I was accompanied with
the decrease of three ?3)Gal(1?, implying that (1?3)-linked Gal
was located at branches. It can be deduced from the results that the
backbone of LRP4-A was composed of (1?6)-linked Gal.
5.22 and 5.06 ppm was 1.2:1. Therefore, (1?3)-linked Ara and
(1?5)-linked Ara were of b-configuration.
The signals at 110.07–111.95 ppm, which could be attributed to
non-reducing a
-Araf units, were present in the 13C NMR spectrum
of LRP4-A (see Fig. 5B) but absent in that of LRP4-A-I. Signals at
105.29–105.83 ppm were assigned to b-Galp, corresponding to
the characteristic absorption at 895 cmÀ1 in IR spectrum. The d
103.44 ppm resonance signal was assigned to the anomeric carbon
of b-Araf.
3.5. NMR analysis
Signals in the 1H and 13C NMR spectra of LRP4-A and LRP4-A-I
were assigned as completely as possible, based on the analysis of
component and linkage as well as previously reported values.28–
3.6. ESI-MS analysis of LRP4-A-O
The MS profiling of LRP4-A-O was shown in Figure 6. Molecular
ions at m/z 305.17, 437.17, 569.25, 701.33, 833.33, and 965.33, cor-
responding to [M+Na]+ adducts of pentose polymers with degrees
of polymerization ranging from two to seven, respectively, sug-
gested that the side chains of LRP4-A contain different degrees of
polymerization of arabinose, consisting with the results analyzed
by GC. Ions at m/z 319.08, 451.25, 583.17, 715.17, 847.08, and
979.17, corresponding, respectively, to [M+Na]+ adducts of dHex-
In the 1H NMR spectrum of LRP4-A (see Fig. 5A), signals for
30
two anomeric protons occurred near 5.22 and 5.06 ppm, respec-
tively. The signals at 1.24 ppm was assigned to
a-Rhap residues.
The signals at 1H 5.22 ppm of LRP4-A were not found in the spec-
trum of LRP4-A-I, indicating that Ara (1- was a-furanose. The sig-
nals of LRP4-A at 1H 5.22 and 5.06 ppm disappeared in the
spectrum of LRP4-A-I and the ratio of integral area between 1H

X. Lv et al. / Carbohydrate Research 365 (2013) 20–25
25
Pent(1–6), manifested that the side chains of LRP4-A contain rham-
nose and different degrees of polymerization of arabinose ranging
from one to six. Ions at m/z 335.17, 467.17, 599.25, 731.25, 863.33,
and 995.25, corresponding to [M+Na]+ of HexPent(1–6), respec-
tively, demonstrated the side chains of LRP4-A contain one hexose
(galactose or glucose) and different degrees of polymerization of
arabinose ranging from one to six. Ions at m/z 349.08, 481.25,
613.17, 745.33, 877.25, 1009.33, 365.17, 497.25, 629.25, 761.25,
893.17, and 1025.33, were ascribed to [M+Na]+ adducts of Hexd-
Hex, Pent1HexdHex, Pent2HexdHex, Pent3HexdHex, Pent4Hexd-
Hex, Pent5HexdHex, Hex2, Pent1Hex2, Pent2Hex2, Pent3Hex2,
Pent4Hex2, Pent5Hex2, respectively (Pent: arabinose; dHex: rham-
nose; Hex: galactose or glucose).
Ara, (1?5)-linked Ara, Glc(1?, and (1?2,4)-linked Rha. This kind
of structure is commonly found in plant arabinogalactan, which
comprises generally arabino-3,6-galactan (type II). The research
on the biological activities of LRP4-A is proceeding steadily.
Acknowledgment
This study is carried out with financial support from the Na-
tional Natural Science Foundation of China (No. 31071506,
31170773).
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The polysaccharides isolated from the fruit of Lycium Barbarum
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In this study, a complex polysaccharide LRP4-A was isolated
and purified from the Lycium ruthenicum Murr., and then its struc-
ture was characterized. LRP4-A, a heteropolysaccharide with a
molecular weight of 1.05 Â 105 Da, was mainly composed of rham-
nose, arabinose, xylose, glucose, and galactose, among which arab-
inose and galactose accounted for 42.9% and 48.6% of the total
carbohydrate content, respectively. LRP4-A was identified to be a
highly branching polysaccharide with a backbone of b-(1?6)-
linked-Galactose. The backbone was partially substituted at O-3
of galactose residues for (1?3)-linked Gal and Gal(1?. The
branching side chain was comprised of Ara (1?, (1?3)-linked