Cerebrosides from the roots of Serratula chinensis

Article abstract of DOI:10.3390/11090677

A new cerebroside, 1-O-β-D-glucopyranosyl-(2S,3R,8E)-2-[(2′R)-2- hydroxypalmitoylamino]-8-octadecene-1,3-diol, along with aralia cerebroside and 1-O-β-Dglucopyranosyl-(2S,3S,4R,8E)-2-[(2′R)-2-hydroxybehenoylamino] -8-octadecene-1,3,4-triol were isolated from the roots of Serratula chinensis S. Moore. The structure of the new cerebroside was established by spectroscopic and chemical means. Occurrence of cerebrosides in Serratula is reported here for the first time.

Full text of DOI:10.3390/11090677

Molecules 2006, 11, 677-683
molecules
ISSN 1420-3049
http://www.mdpi.org
Full Paper
Cerebrosides from the Roots of Serratula chinensis
Tiejun Ling 1,*, Tao Xia 1, Xiaochun Wan 1, Daxiang Li 1 and Xiaoyi Wei 2
1
Key Laboratory of Tea Biochemistry and Biotechnology of Ministry of Education and Ministry of
Agriculture, Anhui Agricultural University, Hefei 230036, P.R. China; E-mails: xiatao62@126.com
(Tao Xia); xcwan@ahau.edu.cn (Xiaochun Wan); dxli@ahau.edu.cn (Daxiang Li)
2
South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, P.R. China;
E-mail: wxy@scbg.ac.cn
* Author to whom correspondence should be addressed; e-mail: lingtj@ahau.edu.cn
Received: 12 August 2006; in revised form: 31 August 2006 / Accepted: 31 August 2006 / Published: 7
September 2006
Abstract: A new cerebroside, 1-O-β-D-glucopyranosyl-(2S,3R,8E)-2-[(2’R)-2-hydroxy-
palmitoylamino]-8-octadecene-1,3-diol, along with aralia cerebroside and 1-O-β-D-
glucopyranosyl-(2S,3S,4R,8E)-2-[(2’R)-2-hydroxybehenoylamino]-8-octadecene-1,3,4-triol
were isolated from the roots of Serratula chinensis S. Moore. The structure of the new
cerebroside was established by spectroscopic and chemical means. Occurrence of
cerebrosides in Serratula is reported here for the first time.
Keywords: Serratula chinensis, Compositae, cerebrosides.
Introduction
Serratula chinensis S. Moore (Compositae) is a perennial herbaceous plant growing mainly in
South China [1]. Its roots have long been used as a folk medicine in China for treatment of pharyngitis
and morbilli [2]. During the course of our continuing investigation of bioactive natural products found
in folk medicinal plants from the southern part of mainland China, we have investigated the chemical
constituents of S. chinensis roots and have reported in previous papers [3,4] the isolation of seven
ecdysteroids and a mixture of five ceramides from that source. This paper deals with the isolation and
structure elucidation of some cerebrosides, a novel class of constituents for this plant.

Molecules 2006, 11,
678
Results and Discussion
The EtOH extract of the powdered dry roots of S. chinensis was successively fractionated with
petroleum ether, CHCl3 and n-BuOH. The CHCl3 fraction was separated by a combination of silica gel,
Sephadex LH-20, and RP-18 silica gel column chromatography (CC) to yield compounds 1, 2, and 3
(Figure 1).
Figure 1. Structures of compounds 1-3.
OH
1'
O
(CH2)n-8CH3
(CH2)8CH3
2'
OH
NH R1
8
O
O
1
3
2
4
HO
HO
OH
OH
1
2
3
R1 = H, n = 16
R1 = OH, n = 16
R1 = OH, n = 22
25
Compound 1 was isolated as a white amorphous powder, [α] D + 8.8 (c 0.17, MeOH). Its positive
ESI-MS showed a [M + K]+ peak at m/z 754, a [M + Na]+ peak at m/z 738 and a [M + H]+ peak at m/z
716, all in accordance with the molecular formula C40H77NO9. The IR spectrum showed strong
absorption bands for hydroxyl (3430 cm-1), amide (1646 and 1540 cm-1) and (CH2)n (721 cm-1) groups.
1
13
The H- and C-NMR spectra of 1 (Table 1) indicated the presence of a β-D-glucopyranosyl moiety
(δH 4.86, 1H, d, J = 7.6 Hz, anomeric proton; δC 105.6, 75.1, 78.6, 71.7, 78.5, and 62.8), an amide
linkage (δH 8.40, 1H, d, J = 9.2 Hz; δC 175.7), an amidomethine (δH 4.69, δC 54.6), an oxygenated
methylene (δH 4.70 and 4.18; δC 70.4), two oxygenated methines (δH 4.16 and 4.59; δC 71.3 and 72.5),
and two long chain aliphatic moieties. The above structural features indicated a dihydrosphingosine
type cerebroside [5]. Methanolysis of 1 afforded a fatty acid methyl ester (FAME) and a long chain
base (LCB) [6]. The FAME was identified as methyl 2-hydroxypalmitate by GC-MS analysis. The
dihydrosphingosine moiety of 1 was derived as 2-amino-octadecene-1,3-diol by analysis of the 1H-1H
COSY of 1 and the positive ESI-MS of the LCB. The absolute configuration of C-2′ was determined to
be R form from the specific rotation of the FAME [7]. The 2S,3R stereochemistry was determined by
comparison of the 13C-NMR chemical shifts of C-2 and C-3 with those of plakosides C and D [5,6]. In
order to determine the position of the double bond in the dihydrosphingosine moiety, the KMnO4
oxidation was performed on the LCB [4]. The oxidation afforded n-decanoic acid which was
determined by GC-MS analysis. This allowed the location of the double bond at C-8. The trans (E)
configuration of the double bond in 1 was indicated by the olefinic proton signals which appeared as
two double triplets (J = 14.4, 5.6 Hz) at δ 5.36 and 5.33 in CD3OD. This was supported by two carbon
signals at δ 33.2/33.1 for the carbons next to the double bond in the 13C-NMR spectrum [8-10], which
1
were assigned by the aid of H-1H COSY and HMQC. In conclusion, 1 was established to be
1-O-β-D-glucopyranosyl-(2S,3R,8E)-2-[(2’R)-2-hydroxylpalmitoylamino]-8-octadecene-1,3-diol.

Molecules 2006, 11,
679
The positive ESI-MS of 3 showed a [M + Na]+ peak at m/z 838, consistent with the composition
C46H89NO10. The IR spectrum of 3 was similar to that of 1. The 1H- and 13C-NMR data of 3 (Table 1)
were essentially identical with those of poke-weed cerebrosides [6], indicating a cerebroside
comprised of monounsaturated (2S,3S,4R)-phytosphingosine, (2R)-2-hydroxy fatty acid and
β-D-glucopyranose moieties. GC-MS analysis of the FAME obtained from the methanolysis of 3
indicated that the chain length of the fatty acid moiety was C22, and the positive ESI-MS data of the
LCB from the methanolysis showed that the chain length of the phytosphingosine moiety was C18. The
double bond was located at C-8, as seen in 1, by the KMnO4 oxidation method. The presence of the
1
two double triplets (J = 14.8, 4.8 Hz) at δ 5.35 and 5.32 in the H-NMR spectrum of 3 (CD3OD) and
the chemical shifts of the carbons next to the double bond (δ 33.4, 33.1) in the 13C-NMR spectrum also
indicated a trans double bond in the phytosphingosine moiety. Thus, compound 3 was identified as
1-O-β-D-glucopyranosyl-(2S,3S,4R,8E)-2-[(2’R)-2-hydroxybehenoylamino]-8-octadecene-1,3,4-triol
[11]. Cerebroside 2 was identified as aralia cerebroside {1-O-β-D-glucopyranosyl-(2S,3S,4R,8E)-2-
[(2’R)-2-hydroxypalmitoylamino]-8-octadecene-1,3,4-triol} by the methods described for 1 and 3, and
by direct comparison of its spectral data with the reported literature values [12].
Conclusions
The present study provides the first report on the presence of cerebrosides in Serratula spp., in
addition to the identification of the new cerebroside 1. Cerebrosides have a wide range of biological
functions, all potentially related to the amphipathic nature of the molecule [13]. The broad bioactivity
spectrum of cerebrosides suggests the further potential utilization of S. chinensis roots as a valuable
crude drug.
Experimental
General
Optical rotations were measured with a Perkin Elmer 343 spectropolarimeter. The IR spectra were
1
13
taken in KBr on a WQF-410 FT-IR spectrophotometer. The H-NMR (400 MHz), C-NMR (100
MHz), and 2D-NMR spectra were recorded on a Bruker DRX-400 instrument. Chemical shifts were
expressed in ppm (δ) with TMS as an internal standard. The positive ESI-MS data were obtained with
a MDS SCIEX API 2000 LC/MS/MS system by direct inlet using MeOH as solvent. The GC-MS
analyses were performed with a Shimadzu QP-5000 instrument [GC conditions: DB-1 capillary
column (30 m × 0.25 mm); column temperature, 60→260°C for 1-2, and 40→260 °C for 3; rate of
temperature increase, 10°C/min; injector temperature, 270°C; He at 15 mL/min]. Silica gel 60
(200-300 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, People’s Republic of China), Develosil
ODS (5 µm, Nomura Chemical Co. Ltd., Japan) and Sephadex LH-20 (Amersham Biosciences,
Sweden) were used for column chromatography (CC). TLC was performed on precoated plates
(Kieselgel 60GF254, Merck) with detection effected by exposure to iodine vapor.

Molecules 2006, 11,
680
Plant material
Roots of S. chinensis were collected in Lechang County, Guangdong Province, China, in the
autumn of 2001, and identified by Prof. Zexian Li, South China Botanical Garden, Chinese Academy
of Sciences. A voucher sample (No. 621633) was deposited at the Herbarium of South China Botanical
Garden, Chinese Academy of Sciences.
Extraction and isolation
Ground dry roots of S. chinensis (8 kg) were extracted with 95% EtOH by percolation at room
temperature. The EtOH percolate was concentrated in vacuo to a syrup (500 g). This syrup was
suspended in H2O and the aqueous suspension was successively extracted three times each with
petroleum ether, CHCl3, and n-BuOH. The CHCl3 extract, on concentration, yielded a brown syrup (15
g). This syrup was subjected to CC on silica gel, eluting with CHCl3-MeOH mixtures of increasing
polarities (from 98:2 to 9:1), to obtain three fractions A-C. Fraction B, obtained on elution with 96:4
CHCl3-MeOH was further subjected to silica gel CC, using 92:8 CHCl3-MeOH as eluant, to afford two
subfractions B1 and B2. Subfraction B2 was separated by CC on Sephadex LH-20 with MeOH as
eluant, followed by CC over RP-18 silica gel eluting with 9:1 MeOH-H2O, to yield 1 (20 mg), 2 (18
mg), and 3 (8 mg).
Cerebroside 1
White amorphous powder. [α] D + 8.8 (c 0.17, MeOH). IR (KBr) cm−1: 3430, 2921, 2852, 1646,
25
1540, 1467, 1278, 1164, 1078, 721. 1H- and 13C-NMR data, see Table 1. Positive ESI-MS m/z: 754 [M
+ K]+, 738 [M + Na]+, 716 [M + H]+, 554 [M - Hexose + H]+.
Methanolysis of 1
Compound 1 (2.0 mg) was refluxed with 0.9 N HCl in 82 % aqueous MeOH (15 mL) for 18 h. The
resulting solution was extracted three times with n-hexane. The n-hexane solution was dried over
anhydrous Na2SO4 and then concentrated to yield the FAME 2R-hydroxypalmitic acid methyl ester
25
(0.6 mg), as a white amorphous powder, [α] D -4.2 (c 0.01, CHCl3), GC-MS: GC tR 13.13 min, EI-MS
m/z: 286 [M]+ (4), 268 [M - H2O]+ (0.2), 227 [M - CH3OCO]+ (9), 182 [M - CH3OCO - CH2OHCH2]+
(0.4), 159 (3), 145 (2), 127 [C9H19]+ (5), 125 (3), 111 (7), 97 (20), 90 [CH3OC(OH)=CHOH]+ (19), 83
(26), 69 (30), 57 (85). The H2O layer, after evaporation of MeOH, was adjusted to pH 9 with aqueous
ammonia and extracted with Et2O. The Et2O layer was dried over anhydrous Na2SO4 and evaporated to
yield the LCB 2-aminooctadec-8-ene-1,3-diol (0.7 mg); positive ESI-MS m/z: 300 [M + H]+, 282 [M -
H2O + H]+.
Oxidation of the LCB from 1
The LCB resulting from methanolysis of 1 (0.3 mg) was dissolved in a mixture of 10 % H2SO4 and
acetone (5 mL each). KMnO4 (50 mg) was added and the reaction mixture was stirred overnight at
room temperature. The reaction was then quenched with aqueous Na2S2O3 (5%). The reaction mixture,
after removal of acetone, was extracted with Et2O. The Et2O layer was dried over anhydrous Na2SO4,

Molecules 2006, 11,
681
and concentrated, to give n-decanoic acid, GC-MS: GC tR 12.34 min, EI-MS m/z 172 [M]+ (4), 155 [M
- OH]+ (0.5), 143 (3), 129 (27), 73 (70), 60 [CO2H2]+ (98), 55 (100).
Table 1. 1H- and 13C-NMR Data of 1 and 3 (in pyridine-d5).
1
3
Position
δH (J in Hz)
4.70 m
δC
δH (J in Hz)
δC
1a
1b
2
3
4
5
6
70.4
4.71 dd (10.4, 6.8)
4.51 dd (10.4, 4.0)
5.30 m
70.4
4.18 m
4.69 m
4.16 m
2.10 m
1.78 m
1.27-1.60
2.10 m
54.6
71.3
34.9
25.9
51.7
76.0
72.5
34.0
4.29 m
4.20 m
2.10 m
1.70 m
29.7-30.5
33.2
25.9
33.4
33.1
130.9
130.7
7 and 10
2.10 m
33.1
130.8
8 and 9
5.47 m a)
5.48 m b)
11-15
16
17
18
NH
1′
1.27-1.60
1.27-1.60
1.27-1.60
0.88 t (6.8)
8.40 d (9.2)
29.7-30.5
32.3
1.23-1.32
1.23-1.32
1.23-1.32
0.86 t (6.8)
8.58 d (9.2)
29.7-30.1
32.2
23.0
14.4
23.1
14.4
175.7
72.5
35.7
175.8
72.5
35.6
2′
3′
4.59 dd (6.8, 3.6)
2.10 m
4.58 dd (7.6, 3.2)
2.10 m
4′
2.10 m, 1.78 m
1.27-1.60
1.27-1.60
26.3
29.7-30.5
32.3
2.10 m, 1.70 m
1.23-1.32
1.23-1.32
26.9
29.7-30.1
32.2
5′-(n - 3)′
(n - 2)′
(n - 1)′
n′
1″
2″
1.27-1.60
23.1
14.4
105.6
75.1
78.6
1.23-1.32
23.0
14.4
105.5
75.2
78.6
0.88 t (6.8)
4.86 d (7.6)
3.98 t (8.0)
4.18 m
0.86 t (6.8)
4.94 d (7.6)
4.00 t (8.0)
4.20 m
3″
4″
4.18 m
71.7
4.20 m
71.6
5″
3.87 m
78.5
3.87 m
78.5
6″a
6″b
4.48 br d (11.6)
4.30 dd (11.6, 5.6)
62.8
4.48 dd (11.6, 2.0)
4.30 dd (11.6, 6.8)
62.7
a): δ 5.36 and 5.33 (each dt, J = 14.4, 5.6 Hz) when measured in CD3OD.
b): δ 5.35 and 5.32 (each dt, J = 14.8, 4.8 Hz) when measured in CD3OD.
n = 16 for 1; n = 22 for 3.
Cerebroside 3
White amorphous powder; [α] D + 9.4 (c 0.11, MeOH); IR (KBr) cm−1: 3400, 2923, 2854, 1722,
1643, 1537, 1466, 1381, 1261, 1167, 1078, 720; 1H- and 13C-NMR data, see Table 1; positive ESI-MS
m/z: 854 [M + K]+, 838 [M + Na]+, 816 [M + H]+, 654 [M - hexose + H]+ .
25

Molecules 2006, 11,
Methanolysis of 3
682
Methanolysis of 3 (2.0 mg), performed by the same method as described for 1, also yielded a
FAME (0.6 mg) and a LCB (0.5 mg). The FAME, 2-hydroxybehenic acid methyl ester, was a white
amorphous powder; [α] D - 3.7 (c 0.01, CHCl3); GC-MS: GC tR 27.03 min; EI-MS m/z 370 [M]+ (6),
25
352 [M - H2O]+ (0.5), 311 [M - CH3OCO]+ (7), 266 [M - CH3OCO - CH2OHCH2]+ (1), 159 (2), 145
(3), 127 [C9H19]+ (8), 125 (5), 111 (13), 97 (28), 90 [CH3OC(OH)=CHOH]+ (25), 83 (30), 69 (31), 57
(88). The LCB, 2-amino-octadec-8-ene-1,3-diol, had the following ESI-MS: m/z 316 [M + H]+, 298
[M - H2O + H]+, 280 [M - 2H2O + H]+, 262 [M - 3H2O + H]+.
Oxidation of the LCB from 3
The LCB from 3 (0.3 mg) was oxidized with KMnO4 by the same method as described for that
from 1 to give n-decanoic acid, GC-MS: GC tR 14.07 min, EI-MS m/z 172 [M]+ (5), 155 [M - OH]+
(0.1), 143 (5), 129 (34), 115 (10), 101 (5), 73 (72), 60 [CO2H2]+ (100), 55 (55).
Acknowledgements
We thank Prof. Ruiqiang Chen, Guangzhou Institute of Chemistry, Chinese Academy of Sciences,
for NMR measurements. This work was supported by Research Foundation for Young Teachers in
University, Anhui Provincial Department of Human Resources (Grant No. 2006jql118).
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Sample Availability: Samples of compound 1 are available from the authors.
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