全 文 ：Received 24 Nov. 2003 Accepted 25 Jun. 2004
Supported by the Knowledge Innovation Program of The Chinese Academy of Sciences (310003).
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Acta Botanica Sinica
植 物 学 报 2004, 46 (9): 1128-1134
Structure of a Bioactive Fructan from the Root of Cyathula officinalis
LIU Ying-Hua, HE Kai-Ze* , YANG Min, MENG Yi-Wen, PU Qiang
(Chengdu Institute of Biology, The Chinese Academy of Sciences, Chengdu 610041, China)
Abstract: The structure of a bioactive fructan RCP isolated from the traditional Chinese medicinal herb
Cyathula officinalis Kuan was determined by NMR, methylation, reductive-cleavage and GC-MS analyses. It
was a highly branched fructan possessing a (2→1)-linked b-D-fructofuranosyl (Fruf ) backbone with (2→6)-
linked b-D-Fruf side chains, belonging to neokestose family. Of the 93.17% b-D-Fruf residues, 24.15% was
terminal, 26.24% was 1-linked, 20.46% was 6-linked, and 22.32% was 1,6-linked. Of the 6.83% a-D-glucopyranosyl
(Glcp) residues, 2.14% was terminal and 4.69% was 6-linked. A structural model was postulated for a degree
of polymerization (DP) of 15.
Key words: Cyathula officinalis ; reductive cleavage; NMR; fructan; structure
The root of Cyathula officinalis is a commonly-used
traditional Chinese herbal medicine with a wide range of
pharmacological activities (Pharmacopoeia Commission of
People’s Republic of China, 2000). The polysaccharide from
C. officinalis could increase mouse red cell immunity func-
tion by strengthening red cell immune adherence and
cleanup of circulating immune complex (Li et al., 1999) and
it could also suppress the growth of mouse S180 tumor and
H22 liver cancer cells, inhibit peripheral white blood cell
decrease caused by Cyclophosphomidum (Cy) in vivo
(Chen and Liu, 2001). Recent report showed that it could
also inhibit growth of Lewis pulmonary carcinoma in vivo
(Chen and Tian, 2003).
We have isolated a low-molecular-weight fructan (RCP)
from the roots of C. officinalis, and demonstrated that it
could improve the proliferation of mouse spleen cell and
the production of IL-2 stimulated by ConA in vitro. Its
sulfated derivative is active against herpes simplex virus
(HSV) in Vero cell culture system (Liu et al., 2004). In this
paper, we report our study on the structure of RCP.
1 Results and Discussion
The crude extract of C. officinalis was applied to the 685
alkalescent anion exchange resin chromatography followed
by Bio-Gel P2 chromatography to give polysaccharide RCP.
Its homogeneity was determined by HPLC and only fruc-
tose and a small amount of glucose were detected in its
complete acid hydrolysates, indicating that RCP is a kind
of fructan (Liu et al., 2003).
In order to convert different residues into different
methylated alditol acetates (Fig.1), RCP was subjected to
complete methylation (Ciucanu and Kerek, 1984) followed
by reductive cleavage (Rolf et al., 1985). The methylated
alditol acetates were analyzed by GC-MS (Table 1) and the
integrated areas were corrected using effective carbon re-
sponse theory (Addison and Ackman, 1968; David et al.,
1975) to obtain quantitative information. Identification of
all significant peaks was carried out according to previ-
ously obtained GC-MS data (Rolf and Gray, 1984; Thomas
et al., 1992).
The reductive cleavage of RCP gave peaks 1-3, 5-9
(Fig.2), indicating that RCP contain a high proportion of
branched residues. The formation of peak 6 suggested the
presence of (2→ 6)-linked residues and peak 7 indicated
the presence of (2→ 1)-linked residues. The peak 4 was
identified as 6-O-acetyl-1,5-anhydro-2,3,4-tri-O-methyl-D-
glucitol according to GC-MS, suggesting that the a-D-Glcp
residue linked with a b-D-Fruf residue also attached at po-
sition 6 as in neokestose (Thomas et al., 1992; Sabine et al.,
2000; Sims et al., 2001). The results of MS are shown in
Table 2 and the linkage information of RCP is shown in
Table 3.
From the methylation it could be concluded that RCP
possesses the average degree of polymerization (DP) of
about 15 on the assumption of a single D-glucosyl group
per molecule, it is consistent with the average molecular
weight of 1.6-2.7 kD by mass spectrum.
The 1H- and 13C-NMR data of RCP are shown in Figs. 3,
4. Combined with 2D spectrum, the resolution revealed clear
relation in the type OH-3—H-3—H-4—OH-4, H-4—H-
5—H-6—OH-6 and H-1—C-1, H-3—C-3, H-4—C-4,
H-5—C-5, H-6—C-6 (Calub and Waterhouse, 1990; Laine,
LIU Ying-Hua et al.: Structure of a Bioactive Fructan from the Root of Cyathula officinalis 1129
Fig.1. Resulting compounds from the reductive cleavage of fructans.
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041130
1990; Rruyn and Loo, 1991; Liu et al., 1993; Timmermans et
al., 1993; Yosgihiro et al., 1994; James and Engin, 1995;
Wawer et al., 1995; Domenico et al., 1998; Sabine et al.,
2000; Leeflang et al., 2000; Mulloy et al., 2000). Because of
the existence of structurally different glycosyl units, the
1H-signals of RCP are stark overlapped. But the above-
mentioned relationships are still valid when compared with
inulin and levan (Sabine et al., 2000).
There are many reports dealing with the characteriza-
tion of 13C-signals for fructan (Jarrell et al., 1979; Seymour
et al., 1979; Hammer and Morgenile, 1990; Bancal et al.,
1991; Carpita et al., 1991; Heyer et al., 1998), the 13C-sig-
nals assignment of RCP are listed in Table 4. The signals for
a-D-Glcp residues were not clearly detected due to the small
Table 1 GC-MS analysis of permethylated RCP after reductive cleavage
Peak No. Methylated sugar derivatives Retention time (min) Linkage
1 1,5-anhydro-2,3,4,6-tetra-O-methyl-D-glucitol 6.19 Terminal a-D-Glcp
2 2,5-anhydro-1,3,4,6-tetra-O-methyl-D-mannitol 6.41
3 2,5-anhydro-1,3,4,6-tetra-O-methyl-D-glucitol 6.54 Terminal b-D-Fruf
4 6-O-acetyl-1,5-anhydro-2,3,4-tri-O-methyl-D-glucitol 8.37 6-Linked a-D-Glcp
5 1-O-acetyl-2,5-anhydro-3,4,6-tri-O-methyl-D-mannitol 9.07
6 6-O-acetyl-2,5-anhydro-1,3,4-tri-O-methyl-D-glucitol 9.28 6-Linked b-D-Fruf
7 1-O-acetyl-2,5-anhydro-3,4,6-tri-O-methyl-D-glucitol 9.36 1-Linked b-D-Fruf
8 1,6-di-O-acetyl-2,5-anhydro-3,4-di-O-methyl-D-mannitol 11.60
9 1,6-di-O-acetyl-2,5-anhydro-3,4-di-O-methyl-D-glucitol 12.01 1,6-Linked b-D-Fruf
Table 2 Mass spectrum (relative abundance in parentheses)
1,5-anhydro-2,3,4,6-tetra-O-methyl-D-glucitol (Peak 1), 70 eV, m/z: 45 (36%), 58 (15), 71 (61), 88 (32), 101 (100), 115 (12), 125 (4), 133
(1), 143 (20), 158 (4), 175 (23), 188 (4)
2,5-anhydro-1,3,4,6-tetra-O-methyl-D-mannitol (Peak 2), 70 eV, m/z: 45 (80%), 52 (5), 59 (16), 71 (64), 83 (15), 89 (47), 101 (100), 115
(47), 125 (16), 143 (86), 156 (41), 175 (49), 188 (2).
2,5-anhydro-1,3,4,6-tetra-O-methyl-D-glucitol (Peak 3), 70 eV, m/z: 45 (49%), 53 (2), 59 (9), 71 (31), 81 (1), 89 (24), 101 (100), 111 (19),
118 (1), 143 (38), 175 (15)
6-O-acetyl-1,5-anhydro-2,3,4-tri-O-methyl-D-glucitol (Peak 4), 70 eV, m/z: 43 (68%), 59 (16), 71 (100), 87 (43), 101 (82), 111 (51), 129
(28), 147 (30), 156 (4), 171 (15), 185 (3), 203 (12)
1-O-acetyl-2,5-anhydro-3,4,6-tri-O-methyl-D-mannitol (Peak 5), 70 eV, m/z: 43 (86%), 59 (19), 71 (96), 81 (7), 87 (49), 95 (1), 101 (100),
111 (92), 117 (33), 126 (43), 143 (80), 156 (12), 171 (9), 188 (7), 203 (11), 216 (2)
6-O-acetyl-2,5-anhydro-1,3,4-tri-O-methyl-D-glucitol (Peak 6), 70 eV, m/z: 43 (45%), 59 (11), 71 (53), 87 (35), 101 (100), 117 (68), 126
(11), 143 (51), 156 (8), 171 (8), 188 (5), 203 (9)
1-O-acetyl-2,5-anhydro-3,4,6-tri-O-methyl-D-glucitol (Peak 7), 70 eV, m/z: 43 (68%), 53 (4), 59 (14), 71 (72), 81 (5), 87 (45), 95 (1), 101
(100), 111(66), 117 (39), 126 (47), 143 (32), 158 (10), 171 (9), 188 (9), 203 (5)
1,6-di-O-acetyl-2,5-anhydro-3,4-di-O-methyl-D-mannitol (Peak 8), 70 eV, m/z: 43 (100%), 59 (9), 71 (54), 81 (5), 87 (78), 101 (36), 111
(51), 117 (46), 124 (35), 131 (12), 143 (27), 156 (7), 171 (4), 203 (5), 216 (41)
1,6-di-O-acetyl-2,5-anhydro-3,4-di-O-methyl-D-glucitol (Peak 9), 70 eV, m/z: 43 (100%), 59 (9), 71 (54), 87 (86), 101 (59), 109 (3), 117
(96), 129 (32), 143 (22), 153 (12), 161 (22), 171 (5), 203 (5), 216 (90)
Fig.2. GC analysis of the reductive cleavage products of RCP
from Cyathula officinalis. (1-9 is the same as in Table 1) Fig.3. 1H-NMR spectrum of RCP in DMSO.
LIU Ying-Hua et al.: Structure of a Bioactive Fructan from the Root of Cyathula officinalis 1131
proportion of a-D-Glcp in the molecule.
Compared with common methylation analysis, reduc-
tive cleavage could discriminate between (2→ 1)- and (2
→6)-linked b-D-Fruf residues, but the accurate location of
the (2→6) branches is still uncertain.
Based on the evidence mentioned above, the possible
structure of RCP is postulated in Fig.5. RCP consisted of (2
→1)-linked b-D-Fruf residues with highly branching points
at C-6 and an a-D-Glcp residue on the reducing end of the
fructan chain. This result is in accordance with that from
Chen and Tian (2003). Besides this, we also confirmed that
RCP belongs to neokestose family. The structure of an-
other polysaccharide Abs from Achyranthes bidentata
Blume, another traditional Chinese herbal medicine which
has a similar Chinese name and shares some pharmacologi-
cal activities with C. officinalis, has been published (Yu
et al., 1995; Deng and Tian, 2002; Tan and Deng, 2002). Abs
is a mixture of short-chain fructans with an average DP of 8,
containing more (2→ 6) than (2→ 1)-linked b-D-Fru f
residues, branching at O-6 or O-1 of 18% of the
Table 3 Percentages and numbers of b-D-Fruf and a-D-Glcp per molecule of RCP by reductive cleavage
Item Linkage Area sources Percentage (%) Number
b-D-Fruf Terminal Peak 2 + Peak 3 24.15 3.62
1-Linked Peak 7 + 4.1× Peak 7 26.24 3.94
6-Linked Peak 5－ 4.1× Peak 7 + Peak 6 20.46 3.07
1,6-Linked Peak 8 + Peak 9 22.32 3.35
a-D-Glcp Terminal Peak 1 2.14 0.32
6-Linked Peak 4 4.69 0.70
Average DP 15.00
Degree of branching 0.34
Fig.4. 13C-NMR spectrum of RCP in DMSO.
Table 4 Assignments (d) for the 13C-NMR spectrum of RCP
→ 6)- → 1)- Fruf- → 1,6)-
Fruf-(2→ Fruf-(2→ (2→ Fruf-(2→
C－ 1 60.21 61.59 61.30 61.30a
C－ 2 104.86 103.83 104.54 104.76
C－ 3 77.03 77.86 76.60a 75.68a
C－ 4 73.26 72.25 72.25a 72.25a
C－ 5 80.91 81.01a 82.48a 80.91
C－ 6 63.62 62.75a 63.12a 63.62
a, unresolved from other signals.Fig.5. The possible structure of RCP; n = 3 for a DP of 15.
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041132
D-fructofuranosyl residues. It is obvious that RCP and Abs
have very different structures.
2 Experimental
2.1 General experimental procedures
Et3SiH and Me3SiO3SCF3 were purchased from Sigma
Chemical Co. All other chemicals with analytical purity (P.
A.) were obtained from China. NMR spectrum was recorded
on Bruker Advance 600 spectrometer at 20 ℃ (1H: 600 MHz,
13C: 150 MHz, DMSO-d6, TMS as internal standard). Mass
spectrum (MS) with electron spray ionization (ESI) were
performed on a Finnigan LC-QDECA mass spectrometer.
HPLC analysis was carried out on OHpak KB-803 chroma-
tography column（30 cm×8 mm） with ELSD detection.
The eluent is degassed water and the separation was per-
formed at a flow-rate of 0.8 mL/min at 30 ℃.
2.2 Plant materials
The roots of Cyathula officinalis Kuan were collected
from Jinkouhe Town, Leshan City, Sichuan Province, China,
in springtime and authenticated by Prof. ZHAO Zuo-Cheng
in Chengdu Institute of Biology, The Chinese Academy of
Sciences.
2.3 Extraction of RCP
The roots of C. officinalis were washed and cut into
thin pieces, then dried at 60 ℃ and powered. The detailed
method for crude polysaccharide extract was described else-
where (Meng, 1996). The crude extract was further purified
by two-step chromatography. First, 685 alkalescent anion
exchange resin chromatography was performed on
Pharmacia chromatography column (70 cm×26 mm) with
LKB system at a flow-rate of 1.0 mL/min. Two eluent sys-
tems were used. A: 20 mmol/L Tris-HCl (pH 8.0); B: 0.5
mol/L NaCl. Second, Bio-Gel P2 chromatography was car-
ried out on Pharmacia chromatography column (100 cm×
16 mm) with LKB system at a flow-rate of 0.5 mL/min. Eluent
was distilled water and Cl-1 was detected by AgNO3
solution. The saccharide component was detected at 206
nm combined with a-naphthol/H2SO4 method. The sugar
peaks were collected and freeze-dried.
2.4 Determination of monosaccharide composition
To get the condition for complete hydrolysis of RCP,
TLC-orthogonal experiments concerning concentration of
RCP and H2SO4, time and temperature of hydrolysis were
carried out. According to the results, relatively mild hydro-
lytic conditions were selected to get the complete hydroly-
sate of RCP due to the sensitivity of fructose to acid (Liu
et al., 2003). HPLC was carried out on a carbohydrate analy-
sis column（Waters, 30.0 cm× 3.9 mm）with MeCN/
H2O (75:25) as eluent at a flow rate of 1.0 mL/min at 30 ℃
with ELSD detection.
2.5 Determination of structure
Methylation and reductive cleavage were performed as
described (Ciucanu and Kerek, 1984; Rolf et al., 1985). The
permethylated RCP was subjected to reductive cleavage
into partially methylated alditol acetates and finally the prod-
ucts were adopted directly to GC-MS analysis. The peaks
were corrected by effective carbon response theory
(Addison and Ackman, 1968; David et al., 1975). GC analy-
sis was performed on a SUP-PET-S gas chromatograph with
FID detector. The column was a capillary one with 0.25 mm
×30 m×0.25 mm (film thickness) with N2 as carrier gas
and temperature program was 152→182 ℃ at 3 ℃/min and
to 250 ℃ at 12 ℃/min. GC-MS was performed on a HP-5MS
with temperature program 100→280 ℃ at 12 ℃/min, injec-
tor temperature 260 ℃ and detector temperature 290 ℃.
Acknowledgements: We are grateful to Dr. Toshiaki Iso
of POLA Japanese Company for recording 2D spectrum, to
Dr. ZHANG Guo-Lin of Chengdu Institute of Biology, The
Chinese Academy of Sciences, for his critical assessment
of the manuscript and to Dr. TAN Hong of Chengdu Insti-
tute of Biology, The Chinese Academy of Sciences, for the
enthusiastic discussion.
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