G Model
CCLET-3623; No. of Pages 5
2
C.-L. Liu et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
a Tibetan medicine factory (Lhasa of Tibet, China). All samples were
sliced and ground into fine powder in a mill before extraction.
DEAE-Sephadex A-25, Sepharose CL-4B, and MW standards of
T-series Dextran were purchased from Amersham Pharmacia
detector (KNAUER Germany) [8]. The elution phase was 0.1 mol/
L NaNO3 and the flow rate was 0.8 mL/min. The polysaccharide
sample was dissolved in the elution solvent and centrifuged. A
20
mL of the supernatant was injected in each run. The mean
Biotech (China) Ltd. Pure monosaccharide standards of
D
-mannose
-galactose (Gal), -xylose
-fucose (Fuc), -glucose (Glc) and
molecular weight was estimated according to the calibration curve
made with the Dextran T-series standards (molecular weight 5000,
10,000, 20,000, 40,000, 70,000, 110,000 and 500,000). During the
process of experiment, the column was kept at 35 8C. The
molecular weight of HM41 was estimated with reference to the
calibration curve prepared above.
(Man),
(Xyl),
L
-rhamnose (Rha),
D
-ribose (Rib),
D
D
D-arabinose (Ara),
L
D
D-
galacturonic acid (GalA) were obtained from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai). All other reagents used were of
analytical grade.
2.1. General experimental procedures
2.4. Monosaccharide analysis
UV absorption spectra were recorded with a UV-Jasco53
spectrophotometer between 190 and 290 nm. IR spectra (KBr)
were recorded with a Fourier transform infrared spectrometer of
Magna-IR 750 in a range of 400–4000 cmꢁ1. The hydrolysates were
determined by GC (GC-2010) equipped with Rtx-225 capillary
The polysaccharide HM41 (1 mg) was hydrolyzed with 1 mL
4.0 mol/L TFA at 100 8C in a sealed tube for 4 h. After that, the
removal of the excess amount of TFA was accomplished by co-
evaporation at reduced pressure with methanol added after
reaction. The hydrolyzates were concentrated and dried, followed
by successive reduction with NaBH4 and acetylation with Ac2O-
pyridine (1:1, v/v, 1 mL) at 100 8C for 30 min. The resulting alditol
acetates obtained were analyzed by GC as indicated above and
identified by their typical retention time and electron impact
profiles [9].
column (30 m ꢀ 0.32 mm ꢀ 0.25
mm) and a flame-ionization
detector. Identification of acetate sugar was carried out from the
retention time relative to standard sugars. GC–MS analyses were
performed on a GC-MS-QP2010A instrument equipped with a OV-
210 capillary column (30 m ꢀ 0.32 mm ꢀ 0.25
mm). For a solution
of HM41 (2%) in D2O, NMR spectra were recorded at 25 8C with a
Bruker Avance 400 type magnetic resonance instrument. X-ray
diffraction of HM41 was determined by a Japan D/Max-YB type X-
ray diffraction instrument. Uronic acids were determined colori-
metrically using a method described in the literature [4].
2.5. Periodate oxidation and smith degradation
HM41 (50 mg) was mixed with 0.04 mol/L NaIO4 (50 mL), kept
at 4 8C for 8 days in the dark [10], and 0.1 mL aliquots were
withdrawn at 3–6 h intervals, diluted to 25 mL with distilled water
and read in a spectrophotometer at 223 nm. Excess periodate was
decomposed by the addition of ethylene glycol (2 mL). The solution
of periodate product (2 mL) was sampled to calculate the yield of
formic acid by 0.01 mol/L NaOH. The rest was dialyzed against
distilled H2O for 24 h. The solution was concentrated and reduced
with NaBH4 (60 mg), and the mixture was left for 24 h at room
temperature, neutralized to pH 6.0–7.0 with 50% acetic acid,
dialyzed as described above, and was concentrated to a final
volume (10 mL). The solutions were added to the same volume of
1 mol/L sulfuric acid, maintained for 40 h at 25 8C, neutralized to
pH 6.0 with barium carbonate, and filtered. The filtrate was
dialyzed as mentioned previously, and the content out of the sack
was desiccated for GC analysis; the content inside was diluted with
ethanol, and after centrifugation, the supernatant and precipitate
were also dried out for the GC analysis [11].
2.2. Isolation and purification
Dried and ground H. elliptica D. Don (10 g) was previously
defatted with light petroleum and 95% EtOH under reflux
conditions, and successively extracted with hot water. The
supernatant was collected, condensed to 300 mL with a rotary
evaporator, and mixed with 95% (v/v) EtOH (4 volumes). The
mixture was left for 24 h at room temperature. The mixture was
then centrifuged (3000 r/min, 15 min) to separate the supernatant
and the precipitate. The precipitate was dried under vacuum at
37 8C to afford the crude polysaccharide (HMc, yield: 2.3%, based
on the original dried material). The crude polysaccharide was
deproteinated by a combination of proteinase (37 8C, 3 h) and the
Sevage method (1:4, n-butanol: chloroform, v/v) [5]. The depro-
teinated extract was dialyzed using a regenerated cellulose
membrane tube (MW cutoff 1000) against H2O for 48 h and
distilled with H2O for 24 h. The concentrated nondialysate was
precipitated with 5 volumes of dehydrated EtOH. The precipitate
was washed with absolute EtOH, acetone and ether, respectively.
The washed precipitate was collected as the crude polysaccharide
and named as HM. The deproteined polysaccharide HM was
fractionated by acidic EtOH, to obtain the fraction containing
HM4. The polysaccharide HM4 was further purified on DEAE-
Sephadex A-25 column (2.5 cm ꢀ 90 cm) and eluted with 0.1 mol/L
NaCl at a flow-rate of 1.1 mL/min. The eluent was monitored by the
phenol–sulfuric acid method [6,7]. The 0.1 mol/L NaCl eluent was
collected, concentrated, dialyzed in flowing tap-H2O (24 h) and
distilled H2O (24 h) to remove salts. The retentate was lyophilized
to obtain the purified polysaccharide termed HM41 for further
study.
2.6. Methylation analysis of HM41
The vacuum-desiccated HM41 (6 mg) was dissolved in anhy-
drous DMSO (2 mL), and methylated with MeI and anhydrous NaOH
as a catalyst according to a modified Hakomori procedure [12],
repeating the whole process twice. The fully methylated product
showed no OH absorption bands in the regionof 3600–3300 cmꢁ1 in
the IR spectrum. The methylated HM41 was first hydrolyzed with
90% formic acid for 6 h at 100 8C and then with 2 mol/L TFA for 3 h at
the same temperature. The hydrolyzate was then reduced with
NaBH4, and the alditol acetate was prepared as usual [13]. The
alditol acetate of the methylated sugar was analyzed by GC-MS
using a DB-5MS fused silica capillarycolumn (30 m ꢀ 0.25 mm, film
thickness: 0.25
mm). Partially methylated alditol acetates were
identified by their fragment ions in EIMS, and the molar ratios were
estimated from the peak areas and response factors [14].
2.3. Homogeneity and molecular mass determination of HM41
The homogeneity and molecular weight of HM41 were
identified by high performance liquid chromatography equipped
with a OHpak SB-805-HQ series gel column (molecular weight
<4 ꢀ 106, 8 ꢀ 300 mm) and an RI2041 parallactic refraction
2.7. NMR analysis
For NMR measurements HM41 was dried in a vacuum over P2O5
for several days, and then exchanged with deuterium by
Please cite this article in press as: C.-L. Liu, et al., Isolation, purification and structural characterization of a water-soluble polysaccharide
Liu, Chun-Lan
