Sugar compositional determination of polysaccharides from Dunaliella salina by modified RP-HPLC method of precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone

Article abstract of DOI:10.1016/j.carbpol.2010.05.029

A modified high-performance liquid chromatography method of pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) has been established for high resolution separation and high sensitivity determination of ten monosaccharides simultaneously, w

Full text of DOI:10.1016/j.carbpol.2010.05.029

Carbohydrate Polymers 82 (2010) 629–635
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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Sugar compositional determination of polysaccharides from Dunaliella salina by
modified RP-HPLC method of precolumn derivatization with
1-phenyl-3-methyl-5-pyrazolone
Jun Daia,∗, Yan Wub, Shang-wei Chena, Song Zhua, Hong-ping Yinc, Min Wangc, Jian Tanga
a Sate Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Jiangsu Province, Wuxi 214122, China
b School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China
c College of Life Science, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A modified high-performance liquid chromatography method of pre-column derivatization with 1-
phenyl-3-methyl-5-pyrazolone (PMP) has been established for high resolution separation and high
sensitivity determination of ten monosaccharides simultaneously, which frequently occur in algae
polysaccharides. The effects of volume proportion of acetonitrile and pH value of mobile phase (0.1 M
phosphate buffer–acetonitrile) on retention and separation of the monosaccharide derivatives were
investigated with Eclipse XPB-C18 column screened out. The hydrolyzation condition of polysaccha-
rides and derivatization procedure of hydrolysates were also optimized. The modified analysis method
was used for the determination of monosaccharide compositions in five polysaccharide fractions isolated
from Duanaliella salina. The results showed that PD1 and PD4a were acidic heteropolysaccharide mainly
containing glucose and galactose respectively, and PD4a contained sulfated groups; PD2 and PD3 all were
a glucan; while PD4b was a complex of polysaccharide linked with nucleic acids by covalent bonds.
© 2010 Elsevier Ltd. All rights reserved.
Received 17 April 2010
Accepted 14 May 2010
Available online 11 June 2010
Keywords:
Duanaliella salina
Polysaccharides
Monosaccharide composition
High-performance liquid chromatography
Pre-column derivatization
1-Phenyl-3-methyl-5-pyrazolone
1. Introduction
extraction and purification with DEAE-32 ion-exchange column
and Sephadex G-100 gel filtration column. The three fractions were
Dunaliella salina (D. salina) is a unique unicellular species of
chlorophyta with no cell wall found in saline environments, which
is known for its carotenoids accumulation (mainly beta-carotene),
having various applications in the health and nutritional prod-
ucts (Borowitzka, Borowitzka, & Moulton, 1984; Murthy et al.,
2005). Meanwhile, rich polysaccharides from the residues after ␤-
carotene extraction from D. salina, their content being 12–40% of D.
salina (Brown, 1991), also have been studied for biological activi-
ties (Fabregas, Garcia, & Fernandez-Alonso, 1999; Sun, Wang, Xue,
& Wang, 1997; Zheng, Wang, Shi, & Chu, 2004) such as anti-tumor,
antivirus and immunomodulating properties. However, the study
on monosaccharide compositions of the polysaccharides from D.
salina was rarely reported. Zheng, Wang, Zhang, and Shen (1997)
first reported one polysaccharide isolated from D. salina residue
after extraction of ␤-carotene and its monosaccharide composi-
tion was identified as glucose, galactose, xylose, mannose, and
rhamnose by paper chromatography. Later, Xue, Yin, Wang, and
Xu (2003) and Xie, Yin, Chen, Xie, and Wang (2005) obtained
three polysaccharide fractions from D. salina by hot basic water
determinated as a glucan, a sulfated proteoglycan and a sulfated
heteropolysaccharide mainly containing glucose by GC, IR and bar-
ium sulfate turbidimetry.
For monosaccharide composition analysis of polysaccharides,
which is of fundamental importance for the research on polysac-
charide structure and its characteristics, GC, HPLC, GC–MS and CE
have been applied respectively according to different separation
model and detection method. HPLC has been accepted as one of the
major techniques for the analysis of sugars, especially in the form
of anion-exchange chromatography coupled with pulsed amper-
ometric detection of the underivatized sugars (Hardy, Townsend,
& Lee, 1988; Quigley & Englyst, 1994). Owing to requiring more
specialized equipment and fall of the electrochemical response
arisen from the presence of peptides and proteins in sample for
the anion-exchange techniques (Rohrer, Thayer, Weitzhandler, &
Avdalovic, 1998), RP-HPLC (reverse phase-high performance liq-
uid chromatography) method of pre-column derivatization was
often choosed for use of sugar analysis with highly sensitive
detection and high separation selectivity, which may be used to
simultaneous determination of neutral, acidic and basic carbohy-
drates whereas GC could not do usually (Huie & Di, 2004). The
reagent 1-phenyl-3-methyl-5-pyrazolone (PMP) with strong UV
absorbance at 245 nm is one of the popular labels for the HPLC
∗
Corresponding author. Tel.: +86 0510 85919609; fax: +86 0510 85919625.
E-mail address: dj998lc@yahoo.com.cn (J. Dai).
0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.05.029

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J. Dai et al. / Carbohydrate Polymers 82 (2010) 629–635
method that can react with reducing carbohydrates under mild
conditions, requiring no acids catalyst and causing no desialy-
lation and isomerization (Fu & OꢀNeill, 1995; Fu & Zopf, 1999;
Honda et al., 1989; Strydom, 1994; Zhang, Xu, Zhang, Zhang, &
Zhang, 2003). Honda et al. (1989) first reported the application of
PMP labeling for analysis of component monosaccharides of gly-
coproteins and optimized the PMP derivatization chemistry using
glucose as a model compound by independently changing reac-
tion parameters, including the concentrations of PMP and sodium
hydroxide, the reaction temperature, and the reaction time. They
optimized mobile phase (a mixture of pH 5 phosphate buffer
and acetonitrile) on Capcell Pak C18 column by isocratic elu-
tion to make seven neutral sugars resolved basically except two
acetyl-amino sugars. Later, Strydom (1994) established the condi-
tion for reversed-phase chromatographic analysis of glycoproteins
hydrolysates containing eleven sugars with NovaPak column and
gradient elution of triethylamine buffer (pH 4.86 with phosphoric
acid)–acetonitrile, using PMP derivatization procedure described
by Honda et al. (1989), and most of sugars were separated aside
from glucuronic and galacturonic acids. In recent years, the HPLC
method of PMP derivatization has been widely used for the analysis
of monosaccharides and oligosaccharides in many biology samples
such as rat plasma (Aghazadeh-Habashi, Carran, Anastassiades, &
Jamali, 2005), exopolysaccharides including hyaluronic acid (Izawa
et al., 2009), and some factors of the derivatization procedure
were improved, for instance, omitting of drying steps (Fu & Zopf,
1999; Zhang et al., 2003) or selection of extraction solvents (Fu &
OꢀNeill, 1995). In the present study, a RP-HPLC method for simul-
taneous determination of twelve sugars labeled with PMP using
UV detection and isocratic elution on ZORBAX Eclipse XDB-C18
column was established, and the procedure of the pre-column
derivatization reaction was optimized by examination of reac-
tion time and neutralization condition of the reaction system for
eight neutral and two acidic sugars. At the same time, the opti-
mized method and procedure were successfully applied in sugar
compositional analysis of polysaccharide fractions isolated from D.
salina.
then was centrifuged (4000 × g, 20 min), and the supernatant was
adjusted pH to 4.0 for precipitating the proteins and centrifuged.
Adjusted again back to pH 7.0, the supernatant was then con-
centrated and mixed with three times volume of ethanol. After
standing for 12 h at 4 ◦C, the sample was centrifuged and the pre-
cipitates were rehydrated. In succession the free proteins were
removed by Sevag method (Wang et al., 2007): the polysaccharide
solution and the Sevag reagent (chloroform:n-butanol = 4:1, v/v)
were mixed (polysaccharide solution:Sevag reagent = 5:1, v/v) and
shaken acutely for 30 min, then centrifuged for removing the denat-
uralized proteins, and the process was repeated 5–6 times. Finally,
the polysaccharides were dialyzed against distilled water (Molec-
ular weight cutoff 3500 Da), then concentrated and lyophilized.
Crude polysaccharide extract (PD) was obtained.
PD dissolved in water to a concentration of about 10 mg/ml,
was fractionated by anion-exchange chromatography on DEAE-
Sepharose Fast Flow column (D 3.5 cm × 30 cm) and eluted by
a linear gradient of NaCl concentration (0.01–1.0 M) in 0.02 M
Tris–HCl buffer at a flow rate of 8.0 ml/min. Four fractions (PD1,
PD2, PD3 and PD4) were collected with a fraction collector and
concentrated, dialyzed for 3 days and lyophilized. PD4 were
then loaded onto a Sepharose CL-6B gel filtration column (D
2.6 cm × 100 cm), and eluted with 0.1 M NaCl at a flow rate of
0.5 ml/min. The eluate obtained (PD4a and PD4b) was pooled, con-
centrated, dialyzed and lyophilized. The eluting fractions were
monitored for the presence of carbohydrate using phenol–sulfuric
acid assay (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956).
Relative molecular mass analysis of polysaccharide fractions
were carried out according to the HPSEC procedure as described
by Sun, Tang, Gu, and Li (2005).
2.3. Hydrolysis of polysaccharides
4 M trifluoroacetic acid (100 ␮l) was added into the polysaccha-
ride solution (100 ␮l, 3–4 mg/ml) in a small ampoule. The ampoule
was sealed under nitrogen atmosphere and kept for 2 h in an oven at
110 ◦C. After the ampoule was cooled to room temperature, it was
opened and 200 ␮l of methanol was added into it for the reaction
mixture to be evaporated to dryness by blowing of nitrogen stream
and heating of water bath. Then the same amount of methanol
was again added and dried by the same method as above, and the
procedure was repeated thrice for TFA to be removed. The dried
hydrolyzed polysaccharide sample was dissolved in 100 ␮l of water
for subsequent derivatization.
2. Experimental
2.1. Chemicals and reagents
Monosaccharides (glucose, galactose, arabinose, xylose, man-
nose, rhamnose, fucose, ribose, glucuronic acid and galacturonic
acid) used for standards were purchased from USA Sigma–Aldrich
Co. 1-Phenyl-3-methyl-5-pyrazolone (PMP), glucosamine and
galactosamine were obtained from USA Acros Organics Co. and
HPLC-grade acetonitrile were purchased from USA Tedia Co. Water
used throughout the experiments was double distilled and puri-
fied on a Milli-Q system (Millipore Inc., Milford, MA, USA). All other
chemicals and solvents used were of analytical grade or HPLC-grade
unless otherwise specified. D. salina powder was provided by Lantai
Biological Engineering Co. Ltd. (Inner Mongolia, China).
2.4. Derivatization of monosaccharides with PMP
100 ␮l of solution of hydrolyzed polysaccharide sample or
monosaccharide standard mixture was mixed with 100 ␮l of 0.6 M
sodium hydroxide. 50 ␮l of the mixture in a small sample tube
with lid was added a 0.5 mol/l methanolic solution (50 ␮l) of PMP
and mixed thoroughly by a vortex mixer. The whole mixture was
heated to 70 ◦C and incubated for 100 min. After the reaction mix-
ture was cooled to room temperature, and was neutralized with
50 ␮l of 0.3 M hydrochloric acid and the resultant solution evapo-
rated to dryness. Water and chloroform (1.0 ml each) were added
to the residue, and the mixture was shaken vigorously. The chloro-
form layer was discarded, and the extraction process was repeated
three times. The aqueous layer was filtered through a 0.45 ␮m pore
membrane filter for HPLC analysis. The derivatization procedure
of hydrolysate samples and standard samples must be carried out
simultaneity under the same condition.
2.2. Extraction, purification and relative molecular mass analysis
of polysaccharides from D. salina
After carotenoids were extracted from dried D. salina powder
using acetone, other polar organic compounds with low molecu-
lar weight in the extraction residue were removed by reflux with
ethanol. Then the organic solvent was vaporized and the residue
was dried in a vacuum state. The pretreated dry powder was
extracted twice with alkaline water (pH adjusted by 2.0 M NaOH
to 9.0) under constant stirring for 3.5 h in an 80 ◦C water bath.
After cooling, the mixture was adjusted pH to 7.0 by 2.0 M HCl,

J. Dai et al. / Carbohydrate Polymers 82 (2010) 629–635
631
3. Analysis of monosaccharide composition of
polysaccharide fractions from D. salina
200–1000 m/z; photomultiplier voltage: 700 V; analyser vacuum:
2.6e−5 mbar; gas flow: 4.2 l/h.
3.1. HPLC analysis of monosaccharide-PMP derivatives
3.3. Ion chromatography analysis of hydrolysate of PD4a
The HPLC system (Agilent1100, USA) used consisted of a G1311A
liquid chromatograph pump, G1379A online degas unit, G1313A
automatic injector with a 20 ␮l loop, and G1315B diode array detec-
tor. The data were collected using an Agilent1100 ChemStation
system provided by Agilent Company. A special ZORBAX Eclipse
XDB-C18 HPLC column (250 mm length, 4.6 mm i.d., and 5 ␮m
particle size) (Agilent, USA), optimized for the separation of PMP
derivatives, was used at ambient temperature of 30 ◦C. The PMP
derivatives elution was performed with a mixture of 0.1 M phos-
phate buffer (pH 6.7) and acetonitrile in a ratio of 83:17 (v/v, %)
at a flow rate of 1 ml/min, and UV absorbance of the effluent was
monitored at 245 nm. The PMP derivatives were quantified by com-
paring their integration values of peak area to a calibrated standard
curve.
Except the method of precolumn derivatization with PMP,
monosaccharide composition of polysaccharide fraction PD4a
isolated from D. salina was determined and validated yet by
hydrolyzing samples in trifluoroacetic acid, using a Dionex HPAEC
system (ICS3000) equipped with pulsed amperometric detection
(PAD) and eluting by a gradient of H2O, 0.25 M NaOH and 1 M NaAC
solutons according to the procedure as describled by Quigley and
Englyst (1994).
3.4. Chemical analysis of polysaccharide components
Total sugar content was determined by the reaction with phenol
in the presence of sulfuric acid (Dubois et al., 1956; Sun et al., 2005)
using glucose as a standard. Proteins in the solution were quanti-
fied by the method of binding of Coomassie Brilliant Blue G-250
to protein (Bradford, 1976) using bovine serum albumin as a stan-
dard. The total uronic acid content was colorimetrically determined
by the m-hydroxydiphenyl assay (Blumenkrantz & Asboe-Hansen,
1973) using galacturonic acid as a standard. The sulfated group
content was determined by barium sulfate turbidimetry (Dodgson,
1961).
3.2. LC–MS analysis of PMP derivatives of hydrolysate of PD4b
LC-ESI/MS measurements were performed using a Waters
Alliance 2690 HPLC coupled with Platform ZMD 4000 sin-
gle quadrupole mass spectrometer. Because the mobile phase
for LC–MS only can contain volatile salt, the mobile phase
used to liquid chromatography was 20 mM ammonium acetate
buffer–acetonitrile (volume proportion is 84:16). The same chro-
matographic column as HPLC-UV was used. The flow rate was
0.8 ml/min, but eluent from the column was split to interface of
liquid chromatography–mass spectrometry with 20 ␮l of injection
volume and a post column split ratio of 1/3. The experiment was
carried out in positive ion mode. The capillary voltage and sample
cone voltage were 4.4 kV and 67 V respectively. The temperature
of ion resource was 100 ◦C and desolvation 250 ◦C, mass range:
4. Results and discussion
4.1. Optimization of separation of monosaccharide-PMP
derivatives
Separation of PMP derivatives of monosaccharides was studied
by using various stationary and mobile phases. The stationary phase
most suitable for the rapid and effective separation of eight neutral,
two acidic and two amino sugars labeled with PMP was found to be
ZORBAX Eclipse XDB-C18 column compared with Waters Atlantis
dC18 (Waters, USA), Waters Symmetry C18 (Waters, USA), ZORBAX
Extend-C18 (Agilent, USA), Hypersil AA-ODS (SMI-LabHut Ltd., UK)
and Venusil XBP-C18 (Agela Technologies Inc., USA). The separation
result obtained with the most suitable column was shown as Fig. 1.
The retention behaviors of the PMP derivatives were examined
by using mixtures of 0.1 M phosphate buffer with various pH values
and acetonitrile at varying proportions as mobile phase. The effects
of acetonitrile proportions on the retention time in the separation
of eight neutral, two acidic sugars labeled with PMP was given in
Fig. 2(a) (buffer pH 6.0). The result showed with the increase of
acetonitrile proportion the elution process of ten PMP derivatives
became faster but the elution order of derivatives keep changeless.
17% (v/v) of acetonitrile in the mobile phase presented better sep-
aration effect. Because affecting charge state of PMP-sugars with
alkaline group, pH also has obvious effect on retetion and separa-
tion of PMP-sugars. At 17% of acetonitrile proportion, the effects
of phosphate buffer pH on the retention time were investigated,
as shown in Fig. 2(b). The result showed an increase of phos-
phate buffer pH resulted in rapid elution and the elution order of
derivatives remained changeless. The phosphate buffer of pH 6.7
brought better separation and relatively rapid elution simultane-
ously. Furthermore, when methanol was used as organic solvent
in the mobile phase, the separation effect of the monosaccharide
derivatives was obviously poorer than acetonitrile. As a result, the
mixture of 0.1 M phosphate buffer (pH 6.7) and acetonitrile to a
concentration of 17 (v/v%) gave optimal separation condition for
PMP derivatives of eight neutral, two acidic sugars. Two aminosug-
ars shown in Fig. 1 was not detected in polysaccharide samples
Fig. 1. Chromatograms of PMP derivatives of monosaccharide standard samples (a)
and hydrolysate of fraction PD1 from D. salina (b).

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J. Dai et al. / Carbohydrate Polymers 82 (2010) 629–635
Fig. 2. Effects of volume proportions of acetonitrile (a) and phosphate buffer pH
values (b) in mobile phase on retention time of PMP derivatives of ten monosaccha-
rides.
Fig. 3. Effects of derivatization reaction time on peak area of PMP monosaccharide
derivatives.
from D. salina, so retention behaviors of the two sugars at different
mobile phase composition were not investigated.
ric acid used to neutralization) most PMP derivatives exhibited
maximal peak areas and highest detective sensitivity except glu-
cose.
4.2. Optimization of PMP derivatization reaction
PMP derivatization reaction time was usually selected to be
30 min (Honda et al., 1989; Strydom, 1994) or 2 h (Fu & OꢀNeill,
1995) according to the literatures. The effects of derivatization
reaction time on peak area of derivative products in HPLC were pre-
sented in Fig. 3 (the peak area is in proportion to yields of derivative
products). The graph data were average of thrice repeated determi-
nation, and the RSD were 2.1–4.8%. The result showed the peak area
of derivative of each sugar was not the same at different derivati-
zation reaction time. While the reaction time was 100 min the peak
area of derivative of each sugar exhibited the maximum, which was
higher by 30% at least than that at reaction time of 30 min. PMP
derivatization reaction time was determined to be 100 min with
highest sensitivity.
A large excess PMP was used for derivatization reaction, there-
fore removal of residuary reagent by chloroform extraction before
HPLC analysis was necessary. PMP with alkaline group in a strongly
acidic solution, were hardly extracted completely by chloroform,
due to protonation of PMP, whereas at high pH significant amounts
of the PMP-sugars were also extracted (Strydom, 1994). Because
quantitative recovery of monosaccharides was essential for accu-
rate monosaccharide composition analysis, pH of PMP derivative
solution before extraction was a important parameter. Honda
et al. (1989) neutralized the reaction solution using equal mol
hydrochloric acid with sodium hydroxide, and Strydom (1994)
and Fu et al. (1995) selected excess hydrochloric acid for neu-
tralization of solution (pH 3–4 or 4–5). The effects of different
volume of HCl (0.3 M) for neutralization on peak area of PMP deriva-
tives were examined in the present study. The result indicated the
peak areas of PMP derivatives decreased with increasing HCl vol-
ume, perhaps because the derivatives were instable in the acidic
solution. When using HCl volume of 50 ␮l (equal mol hydrochlo-
4.3. Validation of the analysis method
To achieve accurate analysis results of the monosaccharide com-
position, the detection linearity of PMP derivatives was verified by
the analysis of seven data points (n = 3, take the means) of different
concentration of mixture standard sugars, and the linear regres-
sion parameters of the calibration curves were shown in Table 1.
Excellent response linearity was obtained for all ten monosaccha-
rides over tested concentration ranges. The limits of detection at
the signal-noise ratio of 3 were determined to be 0.081–0.271 nmol
(listed as Table 1) by successive dilutions of PMP-labeled monosac-
charide mixture.
The method repeatability was also determined by measuring
relative standard deviations (RSD) of retention time and peak area
for each tested monosaccharide derivatives. Results of repeated
determination (n = 5) of mixture standard sugars (including deriva-
tization and HPLC analysis) showed the relative standard deviations
were less than 1.15% for the retention time and 2.89% for the
peak areas, indicating that the method precision was satisfac-
tory.
In the experiments, known amounts of each monosaccharide
at a close concentration with the sample were added to the
hydrolyzed polysaccharide sample (PD4a), and the resulting spiked
sample in triplicate were subjected to the derivatization reaction
and HPLC determination.The recoveries were calculated based on
the difference between the total amount determined in the spiked
samples and the amount observed in the non-spiked samples. The
results showed that the recoveries of all the ten monosaccharides
ranged between 95.3% and 103.2% and the RSD values fell within
2.7–4.9% (Table 2).

J. Dai et al. / Carbohydrate Polymers 82 (2010) 629–635
633
Table 1
Calibration curves and detection limit of ten monosaccharides.
Sugar
Regression equationa
Determination
Liner range (␮mol/ml)
Detection limit
(nmol)
coefficient R2
Man
Rham
Rib
GlcUA
GalUA
Glc
Gal
Xyl
Ara
Fuc
Y = 373.81X + 2.07
Y = 371.06X − 5.54
Y = 373.40X + 5.05
Y = 342.33X − 21.73
Y = 406.76X − 12.71
Y = 336.34X − 6.54
Y = 406.73X − 7.07
Y = 312.22X − 10.08
Y = 389.78X − 6.50
Y = 375.58X − 3.03
0.9999
0.9998
0.9998
0.9983
0.9996
0.9998
0.9998
0.9995
0.9999
0.9998
0.0104–8.33
0.0117–9.38
0.0114–9.09
0.00964–7.72
0.0101–8.06
0.0102–8.15
0.0103–8.21
0.0124–9.91
0.0122–9.76
0.0114–9.08
0.081
0.12
0.17
0.20
0.20
0.13
0.16
0.27
0.14
0.10
a
Y: Peak area; X: sugar concentration (mmol/l).
Table 2
Recoveries of the ten monosaccharides in process of the derivatization reaction and HPLC determination (n = 3).
Sugar component
Man
Rib
39.5
Rham
GlcUA
GalUA
Glc
Gal
Xyl
642.7
500
1158.7
103.2
4.5
Ara
Fuc
Content in sample (nmol)
Spiked amount (nmol)
Found amount (nmol)
Recovery (%)
280.2
100
375.4
95.2
4.9
200.2
200
397.4
98.6
2.7
134.0
100
230.7
96.7
4.1
292.5
200
487.3
97.4
3.8
299.1
200
494.7
97.8
3.5
2337.5
1000
3328.5
99.1
1083.1
1000
2066.1
98.3
251.3
200
448.7
98.7
2.9
50
90.7
102.4
4.3
RSD (%)
3.2
3.8
4.4. Chemical components of polysaccharide fractions from D.
salina
Griffiths, & Wilkinson, 1985) was 49.26%. In addition, PD4b was
obtained by anion exchange and gel filtration chromatography,
and the analysis results of HPSEC and agarose gel electrophoresis
(Motlagh, Ravines, Karamallah, & Ma, 2006) of PD4b only presented
a single peak and a remarkable spot respectively, so PD4b was a
complex of polysaccharide linked with nucleic acids by covalent
bonds.
The crude polysaccharides (PD) from D. salina were fraction-
ated to five fractions (PD1, PD2, PD3, PD4a and PD4b) by anion
exchange and gel filtration chromatography as stated above. Total
carbohydrate, protein, uronic acid and sulfated group content of the
polysaccharide fractions and their relative molecular mass were
determined using aforementioned analysis methods. The results
showed that total carbohydrate contents of PD1, PD2, PD3, PD4a
and PD4b are 91.67, 94.21, 85.01, 80.35 and 57.09% (w/w) respec-
tively, PD1, PD4a and PD4b contained uronic acids of 5.28, 8.53 and
3.57% (w/w), and the three fractions and PD3 all contained small
amounts of proteins (respectively 1.92, 5.17, 6.90 and 10.34%, w/w).
PD4a containing sulfated group (8.36%, w/w) belonged to sulfated
polysaccharides. Weight mean molecular mass (Mw) of PD1, PD2,
PD3, PD4a and PD4b were determined to be 1548.3 kDa, 33.5 kDa,
66.8 kDa, 424.2 kDa, 10.3 kDa respectively.
4.5. Monosaccharide composition of polysaccharide fractions
from D. salina
The results of sugar compositions of five polysaccharide frac-
tions from D. salina obtained by the HPLC analysis based on
precolumn PMP derivatization are shown in Table 3, indicating PD2
and PD3 were all glucan but PD4a, PD4b and PD1 were found to
be acidic heteropolysaccharides. The HPLC profile of PMP deriva-
tives of hydrolysate from PD1 are shown in Fig. 1, chromatograms
of other fractions are not shown. The data in Table 3 also shown
monosaccharide composition of PD4a by RP-HPLC was in good
agreement with the result obtained by HPAEC-PAD detection.
Based on Table 3, the ribose content in PD4b was much higher
than any other monosaccharide. In addition, PMP derivatives of
PD4b were detected by selective ion monitoring using LC–MS,
and the result showed there was no deoxyribose in fraction PD4b.
The UV spectra of PD4b and PD4b treated by nuclease (and
dialyzed) have been measured. The results showed PD4b had the
remarkable absorbance at the wavelength of 260 nm, and however,
the absorbance of PD4b after hydrolysis by nuclease at 260 nm dis-
appeared, indicating PD4b contained the nucleic acids. The nucleic
acids content determined by measuring phosphate (Alexander,
Table 3
Sugar composition of three polysaccharide fractions from D. salina (mol%, n = 3).
Fractions
PD4a
Methods
Man
Rib
rha
glcUA
galUA
Average
RSD (%)
Average
RSD (%)
Average
RSD (%)
Average
RSD (%)
Average
RSD (%)
PMP
IC
5.04
4.18
3.54
2.72
0.71
0
3.17
–
3.60
3.06
3.22
3.98
2.41
3.33
5.22
4.87
5.26
5.89
4.80
4.32
PD4b
PD1
PMP
PMP
3.48
2.32
4.87
3.87
67.8
0
2.92
–
1.34
3.67
3.04
4.35
2.36
1.95
4.06
3.24
2.70
2.00
3.95
2.83
Fractions
PD4a
Methods
glc
gal
xyl
ara
fuc
Average
RSD (%)
Average
RSD (%)
Average
RSD (%)
Average
RSD (%)
Average
RSD (%)
PMP
IC
5.38
5.95
3.31
2.16
42.04
41.49
3.24
2.57
11.56
12.24
2.93
2.84
19.48
19.35
3.29
3.05
4.52
4.51
2.87
3.19
PD4b
PD1
PMP
PMP
4.69
73.1
2.07
2.38
8.40
7.40
2.22
3.04
4.25
4.01
3.16
3.41
3.61
3.35
3.73
3.60
1.37
2.18
3.45
4.02

634
J. Dai et al. / Carbohydrate Polymers 82 (2010) 629–635
Fig. 4. Peak area change of PMP-monosaccharide derivatives from hydrolysates of PD4a (a) and PD4b (b) by HPLC in different hydrolysis conditions.
Therefore, the nucleic acids contained in PD4b should be ribonu-
cleic acids (RNA).
of polysaccharide linked with nucleic acids by covalent bonds. Com-
position of PD4a (Mw = 424.2 kDa) confirmed the deduction made
by Fabregas et al. (1999) that water extracts with antivirus activity
from D. salina contained sulfated polysaccharides with molecular
weight of hundreds thousand Da. For the polysaccharide linked
with nucleic acids such as PD4b, little result was reported until
now.
4.6. Hydrolysis of polysaccharide fractions from D. salina
PD1, PD2 and PD3 with simple carbohydrate chains (see Table 3)
could be essentially hydrolyzed completely using 2 M TFA in 110 ◦C
for 2 h (Strydom, 1994). However, PD4a and PD4b containing
sulfated group or nucleic acid were difficult to be hydrolyzed
completely in above condition. The peak area changes of monosac-
charide derivatives of their hydrolysates from HPLC with different
hydrolysis conditions (Fu & OꢀNeill, 1995; Strydom, 1994; Sun et al.,
2005) are shown in Fig. 4. The results were presented: (a) for PD4a,
to make the hydrolysis complete basically and the loss least, it was
appropriate to hydrolyze by 2 M TFA in 110 ◦C for 4 h; (b) owing to
alike reason, PD4b may be hydrolyzed using 2 M TFA in 121 ◦C for
2 h.
Acknowledgements
Gratitude is expressed to Dr. Liang Lina of Research Center for
Eco-environmental Sciences, Chinese Acadamy of Science, engi-
neer Liu Yan-fang of Institute of Edible Fungi, Shanghai Academy of
Agriculture Sciences, China for fellowship support to us in ion chro-
matography analysis, and Sci. & Tech. Office, Jiangnan University for
financial support of the project.
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Jun Dai was born in Yancheng city, Jiangsu province, China. He entered the Depart-
ment of Chemistry Engineering, Wuxi University of Light Industry in Sep 1978,
and obtained his bachelor degree in July 1982. In the same year, He started his
research and teaching career in the Analysis and Testing Centre of Wuxi Univer-
sity of Light Industry (renamed Jiangnan University in Jan 2001), and worked in
the State Key Laboratory of Food Science and Technology of Jiangnan University
from May 2007 to present. He completed MS program (March 1996 to Decem-
ber 1999) and PhD program (March 2002 to June 2007) respectively, and awarded
MS and PhD degree in Food Science and Technology School of Wuxi University
of Light Industry and Jiangnan University. He was promoted to engineer in 1989
and then as senior engineer in 1996 in Wuxi University of Light Industry. In 2007,
he was promoted as researcher. He has been mainly teaching “Application and
Progress of Advanced Chromatography Analysis” for the graduate students. At
present, he is active in studying analytical technology of functional constituents
in foods and isolation and identification of bioactive compounds in natural prod-
ucts. More than sixty papers have been published in Chinese and international
journals.
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