Glycocerebroside bearing a novel long-chain base from Sagina japonica (Caryophyllaceae)

Article abstract of DOI:10.1016/j.fitote.2010.01.015

A new glycocerebroside (1), along with one reported one (2), was isolated from the ethanol extract of Sagina japonica (Caryophyllaceae) and was fully characterized. The structures of two compounds were identified as (2S, 3S, 4R, 8E)-1-(β-D-glucopyranosyl-3, 4-dihydroxy-2-[(R)-2′- hydroxypalmitoyl]amino-8-heptadecaene (1) and (2S, 3R, 8E)-1-(β-D- glucopyranosyl-3-hydroxy-2-[(R)-2′-hydroxypalmitoyl]amino-8-octadecaene (2) by using spectroscopic methods (¹H, ¹³C, and 2D NMR, MS) and chemical degradation.

Full text of DOI:10.1016/j.fitote.2010.01.015

Fitoterapia 81 (2010) 540–545
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Glycocerebroside bearing a novel long-chain base from
Sagina japonica (Caryophyllaceae)
a
a
b,
Ai-Qun Jia a, , Xu Yang , Wei-Xin Wang , Yong-Hong Jia
⁎
⁎
a
School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210014, Jiangsu, China
Biological and Environmental College, Zhejiang Wanli University, Ningbo 315100, Zhejiang, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A new glycocerebroside (1), along with one reported one (2), was isolated from the ethanol
extract of Sagina japonica (Caryophyllaceae) and was fully characterized. The structures of two
compounds were identified as (2S, 3S, 4R, 8E)-1-(β-D-glucopyranosyl-3, 4-dihydroxy-2-[(R)-
2′- hydroxypalmitoyl]amino-8-heptadecaene (1) and (2S, 3R, 8E)-1-(β-D-glucopyranosyl-3-
hydroxy-2-[(R)-2′-hydroxypalmitoyl]amino-8-octadecaene (2) by using spectroscopic
methods (1H, 13C, and 2D NMR, MS) and chemical degradation.
Received 3 December 2009
Accepted in revised form 5 January 2010
Available online 18 January 2010
Keywords:
Sagina japonica
Caryophyllaceae
Cerebrosides
© 2010 Elsevier B.V. All rights reserved.
Glycosphingolipid
1. Introduction
Sphingolipids are constituents of the cytoplasmic membrane
of eukaryotic cells, and they are known to function as receptors
Sphingolipids are compounds with a sphingoid base
backbone, an amide linked nonpolar aliphatic “tail”, and a
polar head group. There are more than 70 different sphingoid
base backbones that vary in alkyl chain lengths (14–22 carbon
atoms), degree of unsaturation and position of the double
bonds, the presence or absence of a hydroxyl group at position
4, and branching of the alkyl chain. The amino group of the
sphingoid base is usually substituted with a long-chain fatty
acid. The fatty acids vary in chain length (14–30 carbon atoms),
degree of unsaturation, and the presence or absence of a
hydroxyl group on the carbon atom. Subsequent addition of a
double bond at the 4, 5 carbon–carbon bond of the sphingonine
backbone results in the formation of ceramide (N-acyl-
sphingosine). Structurally versatile sphingolipids are formed
when polar head groups are added at position 1 of ceramide
[1,2].
for certain bioactive compounds and are thus thought to play an
important role in the transportation of cations [3,4]. It has also
been reported that sphingolipids have other physiological
activities such as, activation of certain enzymes [5–7], antimi-
crobial and antitumor activity [8,9], anti-ulcerogenic activity
[10], and nerve growth factor-like activity [11]. A distinct and
novel chemical ecologicalactivity was found by KawaiandIkeda
[12–14] and Kawai et al. [15], who have demonstrated that the
sphingolipids from Schizophyllum commune and other fungi
stimulate fruiting body formation of S. commune. Moreover, the
8E-double bond of the sphingoid has been proposed as one key
element determining the activity. Kawai et al. found deeply that
some plant cerebrosides also show a hormone-like activity
toward fruiting body formation in a mushroom S. commune. A
survey of sphingolipids from various sources, certain cerebro-
sides in wheat grain were found to be active, and Whe II was
determined to be a major active compound [16].
As part of our current interest in bioactive substances from
Caryophyllaceae [17,18], we have chemically analyzed a fraction
previously isolated from the whole plant Sagina japonica. We
now describe the isolation and structural determination of two
glycocerebrosides (1) and (2) from S. japonica (Fig. 3).
⁎
Corresponding authors. Tel./fax: +86 25 84315512.
E-mail addresses: jiaaiqun@gmail.com (A.-Q. Jia), redon@163.com
(Y.-H. Jia).
0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2010.01.015

A.-Q. Jia et al. / Fitoterapia 81 (2010) 540–545
541
2. Results and discussion
Seven hydroxyl groups in the original structure of 1 was
determined by acetylation with acetic anhydride/pyridine at
room temperature to give its peracetate derivative 1a, which
showed a molecular ion peak at m/z 1011 [M]+ in its EI-MS,
consistent with the composition C53H89NO17 for 1a. The
existence of a fragment ion peak at m/z 681 [M+H−331
(tetraacetyl hexose)]+ confirmed hexose as the sugar
residue. Meanwhile, the EI-MS data of 1a also displayed the
diagnostic fragments of the sugar moiety at m/z 331 (base
peak), 271, 229, 211, 169, and 109, due to an acetylated
glucopyranoside [20]. Thereby indicating the presence of
seven hydroxyl groups in the original structure of 1.
The EtOH extract of the whole plant of S. japonica was
subjected to silica gel column chromatography (CC), using
CHCl3 and MeOH as eluents. The chloroform-soluble eluent
was separated by normal-phase followed by reversed-phase
CC to give compounds 1 and 2. The following is the structural
elucidation of new compound 1.
Compound 1 wasobtained as white amorphous powder and
was negative to ninhydrin but positive after hydrolysis with
concentrated hydrochloric acid [19]. [α]2D4+10.1° (c 0.26,
MeOH). The molecular formula of C39H75NO10 for 1 was
determined by positive high resolution FAB-MS at m/z
718.5473 [M+H]+ (calcd. 718.5480). In the positive FAB-MS,
compound 1 exhibited significant fragment peaks at m/z 718
[M+H]+, 556 [M−162(glucosyl)]+, 538 [M−162−H2O]+.
The IR spectrum of 1 showed absorption bands ascribable
to hydroxyl at 3400 cm−1, glycosidic (C–O) at 1080 and
1030 cm−1, a secondary amide at 1536 and 1650 cm−1, and
long aliphatic chains at 2965, 2928, 2860 (C–H), 1475, 1309,
On methanolysis [21,22], compound 1 yielded a fatty acid
methyl ester, a mixture of α-and β-anomers of methyl glucoside,
and an LCB (Fig. 1). The methyl ester 1b was identified as methyl
2′-hydroxypalmitate by the help of GC–MS analysis, with a
molecular ion peak at m/z 286, corresponding to the composition
C
17H34O3. Comparison of the optical rotation data ([α]2D4 −1.3°)
with those reported in the literature [23,24] led us to propose
that the relative stereochemistry at C-2′ of the fatty acid methyl
ester was R. That the optical rotation of the methyl glucoside,
[α]2D4 +76.8° (determined on the methanolysis product from 1),
was close to that of the authentic sample, [α]2D5 +77.3° [25],
indicated that glucose was present as its D-isomer.
1120, 965 (trans C C), and 720 [(CH2)n] cm−1. The 1H and 13
NMR spectral data of 1 indicated the presence of a sugar, an
amide, and long-chain aliphatic moieties, strongly suggesting
the glycolipid nature of the molecule (Table 1).
C
Table 1
1H and 13C nuclear magnetic resonance (NMR) data for compound 1 and 2 in pyridine-d5. For abbreviation see Table 1.
Atom no.
1 δ 1H (J in Hz)
δ
13C (ppm)
2 δ 1H (J in Hz)
δ
13C (ppm)
Long-chain base
1
2
3
4
5
6
7
4.76 (m), 4.16 (m)
5.08 (br. s)
4.11 (m)
4.09 (m)
2.20 (m)
70.07 t
4.68 (dd, 3.77, 9.47), 4.17 (m)
4.64 (m)
4.59 (m)
70.28 t
54.58 d
72.55 d
35.67 d
25.83 d
29.54–30.26 t
32.15 t
54.81 d
72.95 d
72.00 d
35.87 t
26.67 t
32.38 t
1.85 (m)
1.13–1.61 (m)
1.13–1.61 (m)
1.85 (m)
1.90 (m)
2.08 (m)
8
9
5.55 (br. s)
5.55 (br. s)
2.08 (m)
1.14–1.62(m)
1.14–1.62(m)
1.14–1.62(m)
0.93 (t, 6.71)
132.76 d
131.42 d
32.38 t
26.08 t
29.82–30.47 t
23.15 t
5.50 (br. s)
5.50 (br. s)
2.06 (m)
1.13–1.61 (m)
1.13–1.61 (m)
1.13–1.61 (m)
1.13–1.61 (m)
0.90 (t, 6.48)
8.21 (d, 8.71)
130.72 d
130.72 d
32.15 t
29.54–30.26 t
29.54–30.26 t
29.54–30.26 t
22.94 t
10
11
12–15
16
17
18
NH
14.48 q
14.26 q
8.34 (d, 8.76)
N-acyl moiety
1′
2′
3′
4′
176.27 s
72.69 d
33.14 t
23.15 t
175.57 s
71.77 d
34.82 t
29.54–30.26 t
4.68 (m)
2.12 (m)
1.14–1.62(m)
4.58 (m)
1,85 (m)
1.76 (m), 1,85 (m)
4′–13′
5′–13′
14′
15′
16′-CH3
1.14–1.62(m)
1.77 (m)
1.14–1.62(m)
0.93 (t, 6.71)
29.82–30.47 t
25.95 t
23.15 t
1.13–1.61 (m)
1.13–1.61 (m)
1.13–1.61 (m)
0.90 (t, 6.48)
29.54–30.26 t
32.98 t
22.94 t
14.48 q
14.26 q
Sugar moiety
1″
2″
3″
4″
5″
6″
4.89 (d, 7.67)
4.01 (dd, 7.81, 8.30)
4.18 (m)
4.10 (m)
3.90 (m)
105.29 d
75.24 d
78.52 d
71.80 d
78.52 d
63.01 t
4.85(d, 8.35)
4.13 (dd, 7.97, 8.51)
4.16 (m)
4.15 (m)
3.98 (m)
105.57 d
75.12 d
78.52 d
71.48 d
78.52 d
62.86 t
4.45 (d, 11.43), 4.29 (d, 11.41)
4.50 (br. d, 11.74), 4.33 (br. d, 11.03)
Assignments were made by distortionless enhancement by polarization transfer (DEPT) and heteronuclear multiple quantum coherence (HMQC) analysis.

542
A.-Q. Jia et al. / Fitoterapia 81 (2010) 540–545
Fig. 1. Hydrolysis of compound 1.
The sugar moiety was further indicated as glucose by NMR
homonuclear correlation spectroscopy spectrum, some key
correlations were observed (Fig. 2). These correlations have
thus unambiguously assigned the position of the one double
bond at C-8. The analysis was further supported by HMBC
spectrum of 1 (Fig. 2). The geometry of the C-8/C-9 alkene bond
was determined to be E by the 13C NMR chemical shift of the
methylene carbon C-7 (δ 32.38) and C-10 (32.38) vicinal to the
olefinic carbon [23,27]. It is thus clear that 1 possesses a novel
sphingoid moiety with (8E) geometry, 2-amino-1, 3, 4-
trihydroxy-8-heptadecaene. In addition, treatment of the
methanolysis product of 1 with acetic anhydride/pyridine at
70 °C afforded production of a triacetyl LCB 1c, which we
suggest is 2-acetoamino-1, 3, 4-triacetoxy-8-heptadecaene
on the bases of the molecular ion at m/z 469 and observed
key EI-MS fragments of 1c, 469 [M]+, 349 [M−2×HOAc]+, 152
[CH3(CH2)7CH=CHCH2]+, 144 [AcOCH2CHNHAc+H]+, 102
[144−Ac+l], 84 [144−HOAc]+ [28]. All of the above spectral
evidence further supported that 1 is a cerebroside composed of
a (8E)-2-amino-l, 3, 4-trihydroxy-8-heptadecaene, (2R)-2-
hydroxy fatty acid, and β-D-glucopyranose.
spectrum. In the 1H NMR spectrum of 1 an anomeric signal
indicative of the sugar moiety was observed at δ 4.89, and the
coupling constant (d, J=7.67 Hz) of this signal suggested the
β-configuration of a glycosidic linkage. The six oxygenated
carbon signals at δ 105.29 (CH), 78.52 (CH), 78.52 (CH), 75.24
(CH), 71.80 (CH), and 63.01 (CH2) in the 13C NMR spectrum
also supported the presence of the β-g1ucopyranoside
moiety in 1 by comparison of the observed and reported
chemical shifts [26]. In addition, from the heteronuclear
multiple bond correlation spectrum, the correlation between
H-1″ [δ 4.89 (1H, d)] and C-l [δ 70.07 (CH2)] suggested that
the β-D-glucose was attached to the C-1 position of the LCB.
The 1H NMR data (Table 1) of 1 revealed the presence of two
terminal methyls at δ 0.93 (6H, t, J=6.71 Hz), methylene
protons at δ 1.14–1.62 (m), an amide proton signal at δ 8.34 (d,
J=8.76 Hz), an anomeric proton at δ 4.89 (d, J=7.67 Hz), and
carbinol protons appearing as multiplets between δ 3.90 and
4.68. A signal appearing at δ 5.08 (m, H-2) was assigned as a
methine proton vicinal to the nitrogen atom, clearly suggesting
a cerebroside containing a 2-hydroxy fatty acid [21,23]. The 13
C
The relative stereochemistry at C-2, C-3, and C-4 in 1 was
presumed as 2S, 3S, and 4R which was shown to be same as
that of aralia cerebrosides 1 [28]. On the basis of the above
evidence, the structure of 1 was therefore established as (2S,
3S, 4R, 8E)-1-(β-D-glucopyranosyl)-3, 4-dihydroxyl-2-[(R)-
2′-hydroxyl palmitoyl] amino-8-heptadecaene (Fig. 3).
NMR spectrum of 1 exhibited carbon signals at δ 176.27
(carbonyl carbon), 54.81 (CHNH, C-2), 29.82–30.47 (methylene
carbons), 14.48 (two terminal methyls, C-17 and C-16′). One
olefinic carbon signal observed at δ 132.76 (CH) and 131.42
(CH) suggested that 1 possessed one double bond. In the 1H–1H
Fig. 2. Key HMBC, and H–H COSY correlations of compound 1.

A.-Q. Jia et al. / Fitoterapia 81 (2010) 540–545
543
Fig. 3. The chemical structures of compound 1, 1a, and 2.
Compound 2 obtained as white amorphous powder was
also negative to ninhydrin but positive after hydrolysis with
concentrated hydrochloric acid [19]. The HR-FAB-MS data, 1H
and 13C NMR (Table 1) data, and IR absorptions indicated that
compound 2 was one known gylcosphingolipid as (2S, 3R, 8E)-
1-(β-D-glucopyranosyl-3-hydroxy-2-[(R)-2′-hydroxypalmi-
toyl]amino-8-octadecaene (2) (Fig. 3), which were previously
obtained from the Roots of Serratula chinensis [29,30].
Sphingolipids are ubiquitous membrane constituents of
animals, plants, and also lower forms of life, the principal
components of which are the LCBs or sphingoid bases. In
nature, the most widely occurring LCB is D-erythro-4(E)-
sphingenine containing even carbon atoms, whereas LCBs
containing odd carbon atoms are less common in higher plants
[31,32]. We have demonstrated in S. japonica a previously
unrecognized cerebroside containing a novel LCB, (8E)-hepta-
decaene. The cerebrosides containing odd-numbered LCBs
have been found in marine animals, such as starfish [33], sea
cucumber [34], sponge [35], and coral [36]. From the viewpoint
of comparative biochemistry, these glycocerebrosides will be of
considerable interest to elucidate fully their distribution and
also to investigate the physiological significance of the odd-
numbered LCBs, as well as the biosynthesis pathway.
was uncorrected. Optical rotations were taken on a Horiba
SEPA-300 automatic polarimeter (Horiba, Tokyo, Japan). The
nuclear magnetic resonance (NMR; 1H, 13C, and two-
dimensional NMR) spectra were acquired on Bruker AM
400 spectrometer (Karlsruhe, Germany); tetramethylsilane
was used as an internal standard, and coupling constants
were represented in Hertz. Mass spectra were measured with
a VG Autospec3000 mass spectrometer (VG, Manchester,
England). Infrared (IR) spectra were obtained in KBr pellets
on a Bio-Rad FTS-135 IR spectrophotometer (Bio-Rad,
Richmond, CA). Gas chromatography mass spectrometry
(GC–MS) was performed with a Finnigan 4510 GC–MS
spectrometer (San Jose, CA) employing the electron impact
(EI) mode (ionizing potential 70 eV) and a capillary column
(30 mm×0.25 mm) packed with 5% phenyl/95% methylsili-
cone on 5% phenyl-dimethylsilicone (HP-5) (Hewlett-Pack-
ard, Palo Alto, CA). Helium was used as carrier gas; column
temperature 160–240 °C (rate of temperature increase: 5 °C/
min).
3.2. Materials
Column chromatography (CC) was performed on silica gel
(200–300 mesh; Qingdao Marine Chemical Ltd., Qingdao,
China) and Sephadex LH-20 gel (25–100 μm, Amersham
Pharmacia Biotech AB, Uppsala, Sweden). Reversed-phase
chromatography was carried out on LiChroprep® RP-8 (40–
63 μm) (Merck, Darmstadt, Germany). Thin layer chroma-
tography (TLC) was carried out on plates precoated with
Merck RP-18 and silica gel (Qingdao Marine Chemical Ltd.,
China), and detection was achieved by spraying with 10%
H2SO4 followed by heating, and accompanied with cyclopep-
tidic TLC detection method [19]. All solvents were distilled
before use. The whole plants of S. japonica were collected in
Songming county of Yunnan province, China, in September
2005. It was identified by Prof. M. J. Qin, and a voucher
specimen was preserved in the Herbarium of Kunming
Institute of Botany (CAS).
Both of these two compounds possess the acid amide
linkage and 8E-double bond, which are essential for the
achievement of high hormone-like activity [16]. Moreover, an
acyl moiety with a carbon chain of fewer than 24 units and/or a
2-hydroxy group increases such activity [14]. To confirm such a
relationship between structure and activity and get further
insight, it is necessary to carry out further experiments of
hormone-like activity towards higher fungi shows necessary.
3. Experimental part
3.1. Chromatographic and instrumental methods
Melting points were obtained on an XRC-1 apparatus
(Sichuan University, Sichuan, China) and the thermometer

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A.-Q. Jia et al. / Fitoterapia 81 (2010) 540–545
The dried whole plants of S. japonica (20 kg) were
analytical group in the State Key Laboratory of Phytochemistry
and Plant Resources in West China for spectral measurements,
Professor Min-Jian Qin for the identification of plants.
extracted 3 times with 95% EtOH under reflux (3×100 L)
for 3, 2 and 1 h, respectively. After evaporation of the
combined extracts, the residue was suspended in H2O and
then extracted with petroleum ether (60–90 °C), AcOEt, and
BuOH. The AcOEt extract (620.0 g) was decolored on Diaion
HP 20 eluting with a gradient H2O/MeOH 0:1→1:0, The 90%
MeOH elute (200.0 g) was subsequently subjected to CC
(silica gel, CHCl3/MeOH 50:1→5:1), and resubmitted to CC
(silica gel, CHCl3/MeOH 15:1→9:1) to give glycocerebroside
1 (21.0 mg) and glycocerebroside 2 (28.0 mg).
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[(R)-2′-hydroxylpalmitoyl]amino-8-heptadecaene (1). White
amorphous powder (methanol); [α]2D4 +10.1° (c 0.26,
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(C O), 1536 (NH), 1475, 1320, 1075 (C–O), 965 (trans C C), 720
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3.3. Acetylation of 1
The method of acetylation was the same as Refs. [21,22]. The
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[M−HOAc]+ (7.5), 891 [M−2×HOAc]+ (3.3), 681 [M+H−
Glc(OAc)4, glucose=Glc]+ (0.9), 390 [H2N+ =CHCH2OGlc
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white solid. [α]2D4−1.3 (c 0.04, n-hexane) [23]; EI-MS (70 eV)
m/z (relative intensities, %) 286 [M]+ (4.5), 254 [M−CH3OH]+
(5.3), 227 [M−CH3COO]+ (3.0). 2-Acetoamino-1, 3-diacetoxy-
4-methoxyl-8-heptadecaene (1c) was white solid. EI-MS
(70 eV) m/z (relative intensities, %) 469 [M]+ (5.2), 410 [M−
Ac]+ (4.5), 392 [M−Ac−H2O]+ (5.8), 349 [M−2×HOAc]+
(6.9). 1-O-Methyl-D-glucopyranoside was obtained as de-
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We wish to acknowledge the financial support from Nanjing
University of Science and Technology. We are grateful to the

A.-Q. Jia et al. / Fitoterapia 81 (2010) 540–545
545
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