Isolation and structural characterization of a novel galactomannan from Eremurus anisopterus (Ker. et Kir) Regel roots

Article abstract of DOI:10.1016/j.carbpol.2010.11.054

A novel galactomannan was obtained from the roots of Eremurus anisopterus (Ker. et Kir) Regel, an ephemeroid plant in the Gurbantunggut Desert in Xinjiang. The crude polysaccharide was extracted following the usual separation procedure, including water dissolution, centrifugation and precipitation with ethanol. The crude polysaccharide was then applied to anion-exchange chromatography and gel filtration to obtain a homogeneous polysaccharide E ₁. The average molecular weight of E₁ was estimated to be 6 kDa according to the calibration curve. On the basis of composition analysis, methylation analysis, periodate oxidation analysis, Smith degradation, infrared spectra (IR) and nuclear magnetic resonance (NMR) experiments, E₁ is a novel linear galactomannan with a main chain composed of (1 → 6)-linked α-d-Galp and (1 → 2)-α-d-Manp at a relative molar ratio of 1:3.

Full text of DOI:10.1016/j.carbpol.2010.11.054

Carbohydrate Polymers 84 (2011) 402–406
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Isolation and structural characterization of a novel galactomannan from
Eremurus anisopterus (Ker. et Kir) Regel roots
Changfeng Hu, Qingjun Kong, Deyu Yang, Yuanjiang Pan∗
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel galactomannan was obtained from the roots of Eremurus anisopterus (Ker. et Kir) Regel, an
ephemeroid plant in the Gurbantunggut Desert in Xinjiang. The crude polysaccharide was extracted
following the usual separation procedure, including water dissolution, centrifugation and precipitation
with ethanol. The crude polysaccharide was then applied to anion-exchange chromatography and gel
filtration to obtain a homogeneous polysaccharide E1. The average molecular weight of E1 was estimated
to be 6 kDa according to the calibration curve. On the basis of composition analysis, methylation analysis,
periodate oxidation analysis, Smith degradation, infrared spectra (IR) and nuclear magnetic resonance
(NMR) experiments, E1 is a novel linear galactomannan with a main chain composed of (1 → 6)-linked
␣-d-Galp and (1 → 2)-␣-d-Manp at a relative molar ratio of 1:3.
Received 26 August 2010
Received in revised form
17 November 2010
Accepted 28 November 2010
Available online 3 December 2010
Keywords:
Eremurus anisopterus
Polysaccharide
Galactomannan
Gel filtration
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
isolated from the genus Eremurus (Li, Shi, Zhang, & Zhang, 2000;
Zhang, Zhang, Tao, & Li, 2000). In addition, many polysaccharides
Roots of medical plants are important resources of interesting
bioactive polysaccharide, many of which have been reported to
possess various biological functions. For instance, a glucan isolated
from the roots of Rubus crataegifolius Bge. exhibits strong immuno-
logical activity (Ni et al., 2009). An arabinogalactan extracted from
the roots of Angelica acutiloba Kitagawa was found to possess
anti-complementary activity (Kiyohara & Yamada, 1989); a het-
eropolysaccharide consisting of Ara, Glc, Gal obtained from the
root of Ophiopogon japonicus displays hypoglycemic activity (Chen
et al., 2011); and an arabinoglucogalactan isolated from Panax
notoginseng root has antioxidant activity (Wu & Wang, 2008). More-
over, some polysaccharides isolated from plant roots show special
structure (Dong, Ding, Yang, & Fang, 1999; Matsumoto, Honsono-
Nishiyama, Guo, Ikejima, & Yamada, 2005; Sun & Liu, 2008).
The genus Eremurus (Liliaceae), which includes about 50 species,
mainly distributes in the mountains of central and western Asia.
Four species are known to occur in China (Hou, 1982). Eremu-
rus anisopterus (Ker. et Kir) Regel, one of the four species, is an
ephemeroid plant in the Gurbantunggut Desert in Xinjiang, China.
Its roots can be used as Chinese traditional medicine for the treat-
ment of rheumatism and physical weakness (Liu, Jiang, Dan, Wang,
& Zhao, 2002; Yunnan Materia Medica Co., 1993). Several kinds
of anthraquinones, naphthalenes and other compounds have been
are present in the genus Eremurus as chemical constituents of the
species. However, there is no report on purification and struc-
tural elucidation of the polysaccharides from the roots of the genus
Eremurus. Therefore, further studies on this subject of the genus
Eremurus are desirable.
In this work, we present a detailed study of the purification
and structural characterization of a water-soluble polysaccharide
E1 from E. anisopterus roots based on ion-exchange chromatogra-
phy, gel filtration, acid hydrolysis, methylation analysis, periodate
oxidation, Smith degradation, NMR experiments, and IR.
2. Experimental
2.1. General procedures
The IR spectra (KBr pellet, 4000–400 cm−1) were determined
using a Beckman Acculab 10 instrument. Ultraviolet (UV) absorp-
tions were recorded on a JASCO V-530 UV-Via spectrophotometer.
NMR spectra were obtained at 25 ◦C, using a Brucker 500 MHz
instrument. For NMR spectroscopy, E1 (90 mg) was dissolved in
1 mL of 99.99% D2O and chemical shifts of samples are expressed
in ppm (ı) relative to the signal for Me4Si. GC–MS was carried out
on a Trace DSQ GC-MS instrument (Thermo Fisher Scientific) using
an HP-5 column (30 m × 0.25 mm i.d., Agilent) and a temperature
program (150–300 ◦C, 2 min initial hold, 15 ◦C/min ramp rate, and
hold until the end of run). The ionization potential was 70 eV, and
the temperature of the ion source was 200 ◦C.
∗
Corresponding author. Tel.: +86 571 87951264; fax: +86 571 87951264.
E-mail address: cheyjpan@zju.edu.cn (Y. Pan).
0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.11.054

C. Hu et al. / Carbohydrate Polymers 84 (2011) 402–406
403
2.2. Plant material
lyzed by GC–MS and identified by their typical retention times and
electron impact profiles.
Roots of E. anisopterus were collected in August 2008 in Shihezi,
Xinjiang Province, China and identified by Dr. Yongxiang Cheng
(Shihezi University, Shihezi, China).
2.6. Methylation
Per-O-methylation of E1 was performed in dried DMSO–CH3I
using NaOH as a catalyst (Ciucanu & Kerek, 1984). The entire process
was repeated three times. The product showed no –OH absorption
bands in the IR spectrum. After complete acid hydrolysis was car-
ried out with 2 M TFA in a boiling water bath for 8 h, the methylated
E1 was reduced with NaBH4, converted into partially methylated
alditol acetates, and analyzed by GC–MS.
2.3. Extraction and isolation
After defatted with petroleum ether and EtOAc, the powdered
roots (2 kg) were extracted with deionized water at the room tem-
perature. The aqueous was concentrated to 200 mL under vacuum.
To precipitate the polysaccharide, four volumes of 95% ethanol
were gradually added to the solution. After 12 h, the deposit was
collected by centrifugation, and washed with acetone and 95%
ethanol three times. Protein was removed using Sevage reagent
(n-butanol:chloroform at the ratio of 1:5, v/v) according to the
Sevage procedure (Sevag, Lackman, & Smolens, 1938). The solu-
tion was concentrated and subjected to a column of weakly basic
anion-exchange styrene macroreticular resin (D 315) as decol-
orizer, yielding 6.35 g decolorized fraction.
The decolorized fraction (400 mg) was dissolved in deion-
ized water (2.0 mL) and centrifuged. The supernatant was then
applied to a column of fast-flow DEAE-Sepharose (Pharmacia)
(2.6 cm × 40 cm) eluted successively with deionized water, 0.1 M,
0.3 M, and 0.5 M NaCl (300 mL each). The content of polysaccha-
ride was detected by phenol–H2SO4 spectrophotometric method
(Dubois, Gilles, Hamilton, & Rebers, 1956). According to the elution
profile, main part obtained from the deionized water elute was dia-
lyzed against deionized water with a tubular membrane (MWCO:
3500 Da, Huamei Bioengineering Company, Shanghai, China) for
2 d to remove small molecules. The residue within membrane was
then concentrated, lyophilized, and further chromatographed on a
Sephadex G-200 (Pharmacia) column (2.6 cm × 40 cm) using 0.05 M
NaCl as eluent. Appropriate fractions were combined, dialyzed and
lyophilized to yield E1 (92 mg).
2.7. Periodate oxidation and smith degradation
E1 (19.6 mg) was oxidized with 0.04 M NaIO4 (25 mL) in the
dark at 4 ◦C until stabilization of periodate consumed (measured
at 233 nm) (Goldstein, Hay, Lewis, & Smith, 1965; Hay, Lewis, &
Smith, 1965). The excess periodate was destroyed by adding ethy-
lene glycol, and the solution was dialyzed against deionized water
for 2 d. After it was reduced with NaBH4 (50 mg) for 12 h at 25 ◦C,
the residue was subjected to complete hydrolysis with 2 M TFA. The
acid was removed by co-distillation with methanol under vacuum.
Finally, the products were acetylated and analyzed by GC–MS.
3. Results and discussion
The powdered roots were extracted with deionized water at the
room temperature. After it was concentrated, the aqueous solu-
tion was defatted with petroleum ether and EtOAc, respectively.
The resulting fraction was decolorized using a weakly basic anion-
exchange styrene macroreticular resin. Then the decolorized part
was sequentially separated through fast-flow DEAE-Sepharose and
Sephadex G-200 chromatography to yield a purified polysaccharide
E1. The HPGPC profile (Fig. 1) showed a single and symmetrically
sharp peak, indicating that E1 was a homogeneous polysaccharide.
According to the calibration curve, the average molecular weight
of E1 was about 6 kDa. E1 appeared as a white powder and had no
absorption at 280 or 260 nm in the UV spectrum, suggesting the
absence of protein and nucleic acid.
Component analysis was conducted with 2 M TFA for 12 h at
100 ◦C. PC showed that galactose and mannose were present in E1.
In addition, the result of GC–MS indicated only galactose and man-
nose were produced as the neutral sugars at a molar ratio of 1:3.
According to the literature, the absolute configuration of the sugars
was determined by GLC of their acetylated glycosides, using (+)-2-
butanol (Gerwig, Kamarling, & Vliegenthart, 1978). Both of them
had d-configuration.
The IR spectrum of E1 showed strong bands at 3405, 2930, 1635,
1030, 932, 869, 815, and 595 cm−1. The bands at 869 and 815 cm−1
were characteristics of d-mannose present in the polysaccharide.
The broad band at 1635 cm−1 was due to bound water. The band at
2930 cm−1 was ascribed to C–H stretching vibration, whereas the
strong band at 3405 cm−1 was contributed to the hydroxyl stretch-
ing vibration of the polysaccharide (Zhang, 1997).
The oxidation of E1 was completed in 4 d with 0.04 M sodium
periodate at 4 ◦C. The periodate consumption of polysaccharide
was 1.31 mol/mol sugar residue. The oxidized E1 was reduced with
NaBH4 to yield corresponding polyalcohol. After Smith degrada-
tion of the polyalcohol, the glycol and glycerol were found in
GC–MS analysis through conversion to the alditol acetates. The
absence of monoses and erythritol suggested that no (1 → 3)-and
(1 → 4)-linked glycosyl bonds were presented in E1. The amount of
periodate consumed indicated that E1 was a polysaccharide with
(1 → 2)- and (1 → 6)-linked backbone.
2.4. Determination of purity and molecular weight
The purity of E1 was determined by high-performance gel per-
meation chromatography (HPGPC) carried out on a Waters 515
instruments (Milford, MA) fitted with a Waters 2410 refractive
index detector and a TSK G4000-SWXL (Tosoh Biosep, made in
Japan) size exclusion analytical column (7.8 mm × 300 mm). The
mobile phase was deionized water and the sample concentration
was 5 mg/mL. T-series Dextran standards (Sigma), which have def-
inite molecular masses ranging from 10 to 500 kDa, were used to
calibrate the HPGPC system. During the experiment process, the
column was kept at 40 ◦C. All data obtained by the system were
collected and analyzed using the Waters Millenium 32 software
package.
2.5. Component analysis
Polysaccharide E1 was hydrolyzed with 2 M trifluoroacetic acid
(TFA) at 100 ◦C for 6 h, followed by evaporation to dryness. The
product was divided into two portions. One portion was used for
paper partition chromatograph (PC). The PC was performed on
Whatman 3 MM filter paper, and the solvents used were as follows:
A, 3:3:1 EtOAc–HOAc–H2O; B, 6:4:3 n-butanol–pyridine–H2O.
Sugars were detected by aniline–diphenylamine and p-anisidine
(Bailey & Bourne, 1960). Authentic standards including d-glucose,
d-mannose, d-galactose, d-arbinose, d-xylose, l-rhamnose, and d-
glucuronic acid were used as reference. The other portion was
reduced with NaBH4 at room temperature for 12 h and acetylated
with Ac2O-pyridine (1 mL each) at 100 ◦C for 10 h (Wolfrom &
Thrompson, 1963). Finally, the resulting alditol acetates were ana-

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C. Hu et al. / Carbohydrate Polymers 84 (2011) 402–406
Fig. 1. The HPGPC profile of E1 isolated from E. anisopterus roots.
Table 1
GC–MS results of partially O-methylalditol acetates of methylation analysis of E1 isolated from E. anisopterus roots.
Alditol acetate
Retention timea
Approximate molar ratiob
Fragments (diagnostic ions, m/z)
Proposed structure
2,3,4-Tri-O-methylgalactitol
3,4,6-Tri-O-methylmanitol
2,3,4,6-Tetra-O-methylglucitol
1.02
1.00
0.95
1.00
3.18
Trace
87,99,101,117,129,161,189,233
87,101,129,130,145,161,189, 190
87,88,101,117,129,145,161,205
→ 6)-Galp-(1 →
→ 2)-Manp-(1 →
Glc-(1 →
a
Relative to 3,4,6-tri-O-methylmanitol.
Relative to 2,3,4-tri-O-methylgalactitol.
b
Fig. 2. 500 MHz 1H NMR spectrum of E1 isolated from E. anisopterus roots in D2O at 25 ◦C.
Fig. 3. 125 MHz 13C NMR spectrum of E1 isolated from E. anisopterus roots in D2O at 25 ◦C.

C. Hu et al. / Carbohydrate Polymers 84 (2011) 402–406
405
Fig. 4. The proposed structure of E1.
Permethylated E1 was obtained by repeating the procedure of
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