全 文 ：Received 6 Apr. 2004 Accepted 21 Jun. 2004
* Author for correspondence. E-mail: .
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Acta Botanica Sinica
植 物 学 报 2004, 46 (8): 997-1001
A Novel Polysaccharide from Chrysanthemum morifolium
ZHENG Yun, LIU Liu, FANG Ji-Nian*
(Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Graduate School of The
Chinese Academy of Sciences, Shanghai 201203, China)
Abstract: A novel polysaccharide was isolateded from Chrysanthemum morifolium Ramat. flowers by
successive hot water extraction, anion-exchange chromatography and gel permeation chromatography.
Sugar and methylation analysis, periodate oxidation, partial acid hydrolysis and NMR spectral analysis
revealed that polysaccharide is composed of repeating units with the following structure:
Key words: Chrysanthemum morifolium ; Asteraceae; polysaccharide; galactan
Chrysanthemum morifolium belongs to the family of
Asteraceae and is widely used as food, tea or drug in China
and Japan. Many kinds of low molecular weight constitu-
ents including flavones (Asen and Stewart, 1975),
triterpenes (Ukiya et al., 2002) and volatiles (Storer et al.,
1993), etc. have been isolated from C. morifolium. Their
effects on HIV virus (Olivero-Verbel and Pacheco-Londono,
2002), ocular, metabolism of cholesterol and oxidant (Duh,
1999) have also been studied. However, up to date, no study
on the structure of polysaccharide from this plant has been
reported. This paper reports the structural analysis of a
neutral polysaccharide (F3) from the flowers of C.
morifolium.
1 Results and Discussion
F3 was eluted as a single symmetrical peak correspond-
ing to an average molecular weight of 5.0 × 103 as deter-
mined by the HPSEC method (Fig.1). It had a [a]20D = -16.9°
(c 1.0, water). A negative response to the Lowry method
(Bensadoun and Weinstein, 1976) indicated that F3 con-
tained no protein.
F3 was hydrolyzed with 0.1 and 0.2 mol/L trifluroacetic
acid (TFA) successively to obtain two sub polysaccha-
rides F3L1 and F1L2 (see experimental 2.6 section for details).
Compositional analysis indicated that F3 had arabinose,
galactose and glucose in a molar ratio of 1.0:2.7:1.0, F3L1
Fig.1. HPSEC elute pattern of F3.
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004998
had galactose and glucose in a molecular ratio of 2.5:1.0
and F3L2 only contained galactose. The results of methy-
lation analysis on F3 and F3L2 are shown in Table 1.
According to the methylation results, the molar ratio of
4-linked, 6-linked and 4,6-linked galactose of F3 was about
3:1:1 and F3L2 was composed of 4-linked galactose and 6-
linked galactose in a molar ratio of 4:1, so one of the 4-
linked galactose in F3L2 should originate from 4,6-linked
galactose and the side chain link to the main chain through
the position 6 of 4, 6-linked galactose. Terminal galactose
residues were also found in both GLC-MS spectra of F3
and F3L2 because of their low molecular weights and the
terminal units occupied considerable portions.
F3 was partially hydrolyzed with 0.1 mol/L TFA and
dialyzed. The dialyzate F3S was fractionated by Sephadex
G-10 column chromatography and obtained monosaccha-
ride fraction F3SA and disaccharide fraction F3SB. F3SA
was analyzed by GLC and indicated it only contained free
arabinose originating principally from terminal arabinosyl
residues. The ESI-MS of F3SB indicated an ion peak at m/z
304 corresponding to [M + Na]+ of an oligosaccharide com-
posed of two arabinoses. The linkage was Ara1→ 5Ara
based on methylation analysis (Table 1). GLC analysis
showed that the nondialyzate F3L1 contained no arabinose.
It indicated that all of the arabinofuranosyl residues were
released and that they were distributed in the outer
branches. The nondialyzate F3L1 was further hydrolyzed
by 0.2 mol/L TFA, then dialyzed and the nondialyzate F3L2
was obtained. F3L2 was only contained galactose by GLC
analysis. Thus the glucose residues existed as side chain
and attached directly to the galactan main chain. The pos-
sible structures of F3L2 and F3 are shown in Fig.2.
The 13C-NMR spectrum of F3 showed signal at d 108.8,
indicating that Ara adopted an a configuration and fura-
nose conformation. The signals at d 103.9-105.7 indicated
that Gal adopted a b configuration. The anomeric signals
d 4.60, d 4.44, d 5.05 and d 5.12 were assigned to b-Galp, b-
Glcp, terminal a- Araf and 5-linked a-Araf, respectively.
The complete assignment of 1H- and 13C-NMR of F3 and
F3L2 is shown in Table 2 based on 13C-1H HSQC spectrum
and the references (Pinto et al., 2000; Schröder et al., 2001).
F3 was oxidized with sodium metaperiodate and a total
of 1.09 mol NaIO4 was consumed per mol sugar residues
and produced 0.20 mol formic acid based on the averaged
molar mass (156) of a glycosyl residue. It was in approxi-
mate agreement with the expected result based on methyla-
tion analysis (NaIO4 consumption: 1.05 and formic acid
production: 0.15).
2 Experimental
2.1 Materials and general methods
The plant material was purchased from Shanghai Me-
dicinal Materials Co. and identified by Prof. HUANG Xiu-
Lan. The optical rotation was measured with a Shanghai
Spoif W22-1S automatic polarimeter. GLC analysis was
performed on a Shimadzu GC-14B instrument, equipped with
an FID detector and a 5% OV-225 column (2.1 m ×3.2 mm
I.D.) and the column temperature was 210 °C. GLC-MS was
conducted with a Finnigan MD-800 combined with GLC-
MS spectrometry equipped with a HP-1 capillary column.
Table 1 Methylation analysis data for F3 and F3L2
Methylated sugar
Retention time Molar ratio
Mass fragments Linkage
(min) F3 F3L
2,3,5-Me3Ara 10.39 1.0 43,71,87,101,117,129,161 T-Araf
2,3-Me2Ara 13.09 1.1 43,87,101,117,129,189 1,5-Araf①
2,3,4,6-Me2Gal 15.11 0.4 0.4 43,101,117,129,145,161,205 T-Galp
2,3,6-Me3Gal 17.48 3.0 3.8 43,71,87,101,117,129,161,189,233 1,4-Galp
2,3,4-Me3Gal 19.20 1.1 1.0 43,101,117,129,161,189,233 1,6-Galp
2,3-Me3Gal 20.98 1.2 43,85,101,117,127,261 1,4,6-Galp
2,3,6-Me3Glc 17.71 2.0 43,71,87,101,117,129,161,189,233 1,4-Glcp
① The possibility of 1,4-Arap was excluded by 13C-NMR spectrum.
Fig.2. Possible structures of F3L2 and F3. The positions of 4-linked and 4,6-linked galactose in F3 are interchangeable.
ZHENG Yun et al.: A Novel Polysaccharide from Chrysanthemum morifolium 999
The ESI-MS was recorded with VG Quattro MS/MS
spectrometer. Solutions of the sample (1 mg/mL) in aq. 30%
MeOH containing 1% HCl were introduced into the ES
source at 1 mL/min using a Harvard 22 syringe infusing
pump. 13C- and 1H-NMR spectra were recorded at room
temperature with a Bruker AM 400 instrument. All the chemi-
cal shifts are reported relative to Me4 Si as external standard.
2.2 Separation and purification
The flowers (4.5 kg) were defatted with 95% EtOH and
extracted with 100 L hot water (3× 5 h) to give a crude
polysaccharide (378 g). The crude polysaccharide (10 g)
was deproteined five times by Sevag method (Whistler,
1965) then applied to a DEAE-cellulose column and eluted
with water. The eluate was dialyzed (COMW, cut off mo-
lecular weight = 1.2 ×104). The dialyzate was purified by
gel permeation chromatography on a Sephadex G-50
column (90×2.6 cm), giving F3 (12 mg).
2.3 Determination of molecular weight
The homogeneity and molecular weight of polysaccha-
rides were detected on a Waters UltrahydrogelTM 1000 and
UltrahydrogelTM 500 tandem column equipped with a Wa-
ters 515 HPLC pump and a Waters 2410 refractive index
detector, eluted with 3 mmol/L NaAc. The column was
precalibrated by standard T-Dextrans (T-500, T-110, T-80,
T-70, T-40 and T-9.3). All samples were prepared as 1%
(W/V) solutions and 20 mL of solution was injected in each run.
2.4 Sugar analysis
F3 (4 mg) dissolved in 2 mol/L TFA (4 mL) and hydro-
lyzed at 110 °C for 2 h. TFA was removed by repeat co-
evaporation with the addition of MeOH. The hydrolyzate
was reduced with NaBH4 (25 mg) at room temperature for 3
h, excessive NaBH4 was neutralized with HAc and removed
Table 2 Chemical shift (d) data for related glycosyl residues of F3 and F3L2
Glycosyl residues H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6/C-6
→ 4)-b-Galp-(1→ F3 4.60/105.71 3.65/73.17 3.58/75.86 4.14/79.02 3.73/74.66 3.70/62.09
F3L 4.62/105.16 3.63/72.61 3.57/75.31 4.15/78.47 3.73/74.10 3.71/61.22
→ 6)-b-Galp-(1→ F3 4.60/103.85 3.62/73.96 3.69/75.86 3.82/69.63 3.93/74.66 3.91/67.58
F3L 4.62/104.20 3.62/73.39 3.70/75.95 3.84/69.43 3.95/74.53 3.90/70.10
→ 4)-b-Glcp-(1→ F3 4.44/104.76 3.65/73.17 4.24/76.51 4.24/80.57 3.73/74.66 3.77/62.41
a-Araf -(1→ F3 5.05/108.77 4.17/82.62 3.98/77.89 4.01/85.20 3.74/61.80
→ 5)-a-Araf-(1→ F3 5.12/108.77 4.01/85.20 3.65/76.51 4.06/83.59 3.85/68.21
The signals of 4, 6-linked galactose overlapped with those of 4- and 6-linked galactose and were not assigned. The weak signals of terminal
galactose were not assigned.
Fig.3. 13C-NMR spectra of F3 and F3L2.
Acta Botanica Sinica 植物学报 Vol.46 No.8 20041000
by repeat co-evaporation with the addition of HAc/MeOH
(V/V = 5/95) solution, and then acetylated with Ac2O (100
°C, 1 h). The consequent alditol acetates were analyzed by
GLC (Blakeney, 1983).
2.5 Methylation analysis
Samples (8 mg) in Me2SO (1.0 mL) were methylated by
the modified Ciucanu method (Needs and Selvendran,
1993). The permethylated polysaccharides were depolymer-
ized by 90% formic acid (100 °C, 2 h) and then hydrolyzed in
2 mol/L TFA (4 mL, 110 °C, 2 h). The partially methylated
sugars were reduced and acetylated as described in com-
position analysis, then analyzed by GLC-MS (Sweet et al.,
1975).
2.6 Partial acid hydrolysis
F3 (200 mg) was partially hydrolyzed with 0.1 mol/L
trifluroacetic acid (100 mL) at 100 °C for 2 h and dialyzed
(COMW = 3 500) (Wang and Fang, 2001). The mixture was
evaporated to dryness, and the residue was dialyzed against
distilled water (3×1 600 mL). The dialyzate was concen-
trated and separated on a Sephadex G-10 column (90×1.6
cm). The nondialyzate F3L1 (71 mg) was further hydrolyzed
with 0.2 mol/L TFA (100 mL) at 100 °C for 3 h. The mixture
was evaporated to dryness and the residue was dialyzed
against distilled water (3×1 600 mL), obtained nondialyzate
F3L2 (56 mg).
2.7 Periodate oxidation
F3 (20 mg) was dissolved in 0.020 mol/L NaIO4 (15 mL)
and kept in the dark at 4 °C (Abdel-Akher et al., 1952).
Absorption at 223 nm was determined each day. After the
oxidation was complete (72 h), ethylene glycol (0.5 mL) was
added to the solution with stirring for 0.5 h. The solution
was titrated by 0.1 mol/L NaOH.
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(Managing editor: WANG Wei)