Cerebrosides and a monoacylmonogalactosylglycerol from Clinacanthus nutans

Article abstract of DOI:10.1248/cpb.52.27

A mixture of nine cerebrosides and a monoacylmonogalactosylglycerol were seperated from the leaves of Clinacanthus nutans. The structures of the cerebrosides were characterized as 1-O-β-D-glucosides of phytosphingosines, which comprised a common long-chain base, (2S,3S,4R,8Z)-2-amino-8(Z)- octadecene-1,3,4-triol with nine 2-hydroxy fatty acids of varying chain lengths (C₁₆, C₁₈, C_20-26) linked to the amino group. The glycosylglyceride was characterized as (2S)-1-O-linolenoyl-3-O-β-D- galactopyranosylglycerol. The structures were established on the basis of the spectroscopic data and chemical reactions.

Full text of DOI:10.1248/cpb.52.27

January 2004
Chem. Pharm. Bull. 52(1) 27—32 (2004)
27
Cerebrosides and a Monoacylmonogalactosylglycerol from
Clinacanthus nutans
b
Pittaya TUNTIWACHWUTTIKUL, ,a Yupa POOTAENG-ON,a Photchana PHANSA,a and Walter Charles TAYLOR
*
a Department of Chemistry, Faculty of Science, Silpakorn University; Nakorn Pathom 73000, Thailand: and b School of
Organic Chemistry, University of Sydney; NSW 2006, Australia. Received June 16, 2003; accepted September 19, 2003
A mixture of nine cerebrosides and a monoacylmonogalactosylglycerol were seperated from the leaves of
Clinacanthus nutans. The structures of the cerebrosides were characterized as 1-O-b-D-glucosides of phytosphin-
gosines, which comprised a common long-chain base, (2S,3S,4R,8Z)-2-amino-8(Z)-octadecene-1,3,4-triol with
nine 2-hydroxy fatty acids of varying chain lengths (C16, C18, C20—26) linked to the amino group. The glycosylglyc-
eride was characterized as (2S)-1-O-linolenoyl-3-O-b-D-galactopyranosylglycerol. The structures were estab-
lished on the basis of the spectroscopic data and chemical reactions.
Key words Clinacanthus nutans; Acanthaceae; leave; cerebroside; glycosphingolipid; monoacylmonogalactosylglycerol
The genus Clinacanthus consists of two species, C. nutans pale yellow wax.
LINDAU and C. siamensis BREM. and belongs to the family
The IR spectrum of 1 showed bands at 3315, 1635, 1075
Acanthaceae.1) Both species are small shrubs occurring and 1036 cmϪ1 indicating the presence of hydroxyl, amide
throughout South East Asia. C. nutans (Thai name: phaya yo and C–O functional groups. The 1H- and 13C-NMR spectra of
or phaya plongtong) is often confused with C. siamensis 1 (Table 1) indicated the presence of a sugar moiety (dH 4.30,
(Thai name: lin nguu hao). C. nutans has long been used in 1H, d, Jϭ8.4 Hz, anomeric proton; dC 102.7), an amide func-
Thailand as a traditional medicine for the treatment of insect- tion (dH 7.62, 1H, d, Jϭ9.0 Hz, NH; dC 174.0), and long
and snake-bite and skin rashes, including herpes simplex chain aliphatic and olefinic functions (dH 0.87, t, Jϭ6.6 Hz,
virus (HSV) and varicella-zoster virus (VZV) lesions. The CH3; dH 1.25, br s, CH2, dH 5.33 and 5.34, 1H each, both dt,
anti-inflammatory activity of a n-BuOH-soluble fraction Jϭ9.6, 4.8 Hz; dC 128.9, 129.2). The data were suggestive of
from the leaves has been reported.2) In a series of in vitro a glycosphingolipid structure.
models a crude extract of the leaves showed significant in-
The 13C-NMR spectrum of 1 (Table 1) was assigned by a
hibitory activity on VZV.3) Likewise, ethanol extracts of C. combination of distortionless enhancement by polarization
nutans were found to be virucidal against HSV-2 in vitro.4) transfer (DEPT), heteronuclear multiple quantum coherence
However, negative results have also been reported.5) (HMQC) and heteronuclear multiple bond correlation
Nonetheless, clinical trials have reported the successful use
of a C. nutans preparation (cream) for treatment of genital
herpes and varicella-zoster lesions in patients.6—9) Previous
chemical studies on C. nutans have revealed the presence of
lupeol, b-sitosterol, stigmasterol and myricyl alcohol.10,11)
Six known C-glycosyl flavones, vitexin, isovitexin, shafto-
side, isomollupentin 7-O-b-glucopyranoside, orientin and
isoorientin have been isolated from the n-BuOH- and water-
soluble portion of the methanolic extract of the stems and
leaves of C. nutans collected in Thailand.12) Five sulfur-con-
taining glucosides were isolated from the n-BuOH-soluble
portion of a methanolic extract of the stems and leaves of
plant material said to be C. nutans.13)
As part of our work on bioactive substances from natural
sources, a mixture of cerebrosides (1) and a monoacylmono-
galactosylglycerol (4) were isolated from the EtOAc-soluble
fraction of the ethanolic extract of the fresh leaves of C. nu-
tans. We are now reporting the isolation and structure eluci-
dation of 1 and 4 by spectroscopic methods and chemical re-
actions.
The fresh leaves of C. nutans were extracted with EtOH at
room temperature. After evaporation of the extract, the
residue obtained was dissolved in H2O and partitioned with
EtOAc and then n-BuOH. The EtOAc-soluble fraction was
subjected to flash column chromatography to give 13 frac-
tions, among which fraction 9 possessed anti-HSV-1 activity
with IC50 value of 7.86 mg/ml. Further chromatographic pu-
rification of this fraction gave 1 as a colorless solid and 4 as a
To whom correspondence should be addressed. e-mail: pittayat@su.ac.th
© 2004 Pharmaceutical Society of Japan

28
Vol. 52, No. 1
Table 1. 1H- and 13C-NMR Spectral Data of 1 (CDCl3–DMSO-d6, 3 : 2) and 1a (CDCl3)
Position
1 (dH)
1 (dC)
68.1
—
49.6
73.7
1a (dH)
1a (dC)
1a
1b
2
3
4
3.58 (dd, 11.0, 3.3)
4.02 (dd, 11.0, 5.9)
4.26 (m)
3.52 (m)
3.53 (m)
3.68 (dd, 11.0, 3.3)
3.86 (dd, 11.0, 3.3)
4.27 (tt, 9.0, 3.3)
5.13 (dd, 9.0, 3.3)
4.88 (dt, 9.0, 3.3)
1.60 (m)
66.7
—
48.2
74.0
73.1
27.9
70.8
33.8
5
1.55 (m)
7 and 10
8 or 9
9 or 8
NH
1Ј
2.05 (m)
26.3, 26.4
128.9
129.2
—
174.0
70.9
1.97 (m)
26.3, 27.2
128.7
130.6
—
169.9
5.33 (dt, 9.6, 4.8)
5.34 (dt, 9.6, 4.8)
7.62 (d, 9.0)
—
5.31 (dt, 9.6, 6.4)
5.36 (dt, 9.6, 6.4)
7.73 (d, 9.0)
—
2Ј
3.95 (m)
5.15 (t, 5.0)
71.6
3Ј
1Љ
2Љ
3Љ
4Љ
5Љ
6aЉ
6bЉ
CH3
(CH2)n
1.55 (m)
33.8
102.7
72.6
75.9
69.5
75.6
61.0
—
13.2, 13.3
21.7, 24.4, 25.2
28.2, 28.6, 30.9
30.9, 31.6
—
1.83 (m)
4.48 (d, 8.0)
4.89 (dd, 9.5, 8.0)
5.18 (t, 9.5)
5.06 (t, 9.5)
3.69 (ddd, 9.5, 4.0, 2.5)
4.13 (dd, 12.5, 2.5)
4.25 (dd, 12.5, 4.0)
0.88 (t, 7.2) (2ϫ)
1.25 (br s)
31.7, 31.9
100.4
71.2
72.7
68.1
71.9
61.7
—
14.1
22.7, 24.9, 25.9
29.2, 29.3, 29.5,
29.7
20.5 (3ϫ), 20.6,
20.7, 20.9, 21.0
169.27, 169.34,
169.7, 170.16,
170.20, 170.6, 171.0
4.30 (d, 8.4)
3.22 (t, 8.4)
3.41 (t, 8.4)
3.35 (t, 8.4)
3.28 (m)
3.68 (dd, 12.1, 4.8)
3.82 (dd, 12.1, 2.2)
0.87 (t, 6.6) (2ϫ)
1.25 (br s)
COCH3
COCH3
—
—
1.99, 2.02, 2.05 (2ϫ), 2.07,
2.09, 2.24, all s
—
—
(HMBC) experiments. Important long-range correlations
were observed between C-1Љ and Hab-1; C-1 and H-1Љ, H-2,
H-3; C-2 and NH and C-1Ј and NH and H-2Ј (Fig. 1). These
results again supported the glycosphingolipid structure.
1
The H-NMR spectrum of the heptaacetate derivative (1a)
of 1 (Table 1) was much clearer, with well-resolved signals.
The signal of the anomeric proton of a b-D-glucopyranose
appeared at d 4.48 as a doublet (J1,2ϭ8.0 Hz, diaxial) and
1
other glucose protons were assigned (Table 1) from H–1H-
COSY spectrum. Signals from two olefinic protons at d 5.31
and 5.36 (each dt, Jϭ9.6, 6.4 Hz), two methyl groups at d
0.88 (t, Jϭ7.2 Hz) and the long-chain methylene protons at d
1.25 (br s) suggested the presence of two long aliphatic
chains, one of which possessed a cis double bond. A doublet
Fig. 1. Selected 1H–1H COSY (Bold Lines) Correlations of 1a and Se-
lected HMBC (Full-Line Arrows) Correlations of 1 and 1a
Methanolysis14) of 1 yielded methyl glucoside, a mixture
at d 7.73 (Jϭ9.0 Hz) was assigned to the NH of the amide of fatty acid methyl esters and a trihydroxy long-chain base
moiety. The spectrum of 1a also showed signals for two oxy- (2). Therefore, 1 must be a mixture of cerebrosides. The fatty
genated methylene protons as two doublets of doublets at d acid methyl esters were identified by GC/MS as methyl 2-hy-
3.68 (Jϭ11.0, 3.3 Hz, Ha-1) and 3.86 (Jϭ11.0, 3.3 Hz, Hb- droxypalmitate (4.6%), methyl 2-hydroxystearate (6.9%),
1), and four methine protons as a triplet of triplets at d 4.27 methyl 2-hydroxyeicosanoate (2.7%), methyl 2-hydroxyhene-
(Jϭ9.0, 3.3 Hz, H-2), a doublet of doublets at d 5.13 (Jϭ9.0, icosanoate (1.1%), methyl 2-hydroxydocosanoate (29.3%),
3.3 Hz, H-3), a doublet of triplets at d 4.88 (Jϭ9.0, 3.3 Hz, methyl 2-hydroxytricosanoate (6.8%), methyl 2-hydroxy-
H-4) and a triplet at d 5.15 (Jϭ5.0 Hz, H-2Ј). These data, to- tetracosanoate (29.0%), methyl 2-hydroxypentacosanoate
1
gether with the other H–1H COSY correlations of 1a (Fig. (4.5%) and methyl 2-hydroxyhexacosanoate (12.4%). The
1), supported the structure as the 1-b-D-glucopyranoside of a MS spectrum of 1 showed a series of molecular ion [MϩH]ϩ
3,4-dihydroxysphingosine-type ceramide possessing a 2-hy- peaks at m/z 872, 858, 844, 830, 816, 802, 788, 760 and 732
droxy fatty acid acyl group.
and fragment ions at m/z 710 [872Ϫglc]ϩ, 696 [858Ϫglc]ϩ,
The 13C-NMR of 1a (Table 1) was assigned by a combina- 682 [844Ϫglc]ϩ, 668 [830Ϫglc]ϩ, 654 [816Ϫglc]ϩ, 640
tion of DEPT, HMQC and HMBC experiments. In particular, [802Ϫglc]ϩ, 626 [788Ϫglc]ϩ, 598 [760Ϫglc]ϩ and 570
the long-range correlations which were observed in the [732Ϫglc]ϩ. Identification of the nine fatty acids mentioned
HMBC spectrum (Fig. 1) also supported the substitution pat- above indicated that 1 was comprised of a common long-
tern in 1a.
chain base (2) acylated by 2-hydroxy fatty acids of varying

January 2004
29
chain lengths. The absolute configuration at C-2 of the 2-hy-
droxy fatty acid was presumed to be R from the specific rota-
tion of the mixture of fatty acid methyl esters ([a]D
Ϫ11.0°).15—17)
The long-chain base tetraacetate (2a) showed a [M]ϩ peak
1
at m/z 483. The H-NMR spectrum of 2a (Experimental)
(well-resolved signals) contained a doublet at d 5.92
(Jϭ9.0 Hz) of the NH of the amide function, two doublets of
triplets at d 5.30 (Jϭ10.8, 7.2 Hz) and 5.38 (Jϭ10.8, 6.8 Hz)
of the two cis olefinic protons and a triplet of a methyl group
at d 0.88 (Jϭ6.6 Hz) and a broad singlet at d 1.28 of the
Fig. 2. Selected 1H–1H COSY (Bold Lines) and HMBC (Full-Line Ar-
rows) Correlations of 2a
droxydocosanoyl]-2-amino-8(Z)-octadecene-1,3,4-triol,
(2S,3S,4R,8Z)-1-O-(b-D-glucopyranosyl)-2N[(2ЈR)-2Ј-hy-
droxytricosanoyl]-2-amino-8(Z)-octadecene-1,3,4-triol,
(2S,3S,4R,8Z)-1-O-(b-D-glucopyranosyl)-2N[(2ЈR)-2Ј-hydro-
xytetracosanoyl]-2-amino-8(Z)-octadecene-1,3,4-triol,
(2S,3S,4R,8Z)-1-O-(b-D-glucopyranosyl)-2N[(2ЈR)-2Ј-hy-
droxypentacosanoyl]-2-amino-8(Z)-octadecene-1,3,4-triol
and (2S,3S,4R,8Z)-1-O-(b-D-glucopyranosyl)-2N[(2ЈR)-2Ј-
hydroxyhexacosanoyl]-2-amino-8(Z)-octadecene-1,3,4-triol.
The spectral data of 4 indicated the presence of a sugar
and a long-chain unsaturated aliphatic system strongly sug-
gesting a glycolipid. The 1H- and 13C-NMR spectra of 4 (Ex-
perimental), together with 1H–1H COSY, HMQC and HMBC
1
methylene protons. The H-NMR spectrum also had signals
of two doublets of doublets at d 4.00 (Jϭ11.7, 3.2 Hz, Ha-1)
and 4.29 (Jϭ11.7, 4.5 Hz, Hb-1), a multiplet centered at d
4.47 (H-2), a doublet of doublets at d 5.10 (Jϭ8.1, 3.2 Hz,
H-3), a doublet of triplets at d 4.94 (Jϭ9.9, 3.2 Hz, H-4) and
four acetoxy methyl groups apparent as three singlets at d
2.03, 2.05 (2ϫ) and 2.15. The assignments were supported
1
by the H–1H COSY spectrum (Fig. 2). The 13C-NMR spec-
tral data of 2a (Experimental) was assigned by combination
of DEPT, HMQC and HMBC experiments. The 1H- and 13C-
NMR spectra of 2a were shown to be almost identical to
(2S,3S,4R,9Z)-2-acetamino-1,3,4-triacetoxy-9-docosene,15)
and (2S,3S,4R,13Z)-2-acetamino-1,3,4-triacetoxy-13-doco-
1
spectra, led to the assignments of all the H- and 13C-NMR
)
sene.18 The optical rotations of 2a, (2S,3S,4R,9Z)-2-aceta-
signals for the sugar and glycerol moieties. The signals at d
103.4, 71.1, 73.1, 68.6, 74.7 and 61.1 in the 13C-NMR spec-
trum and peaks at d 4.25 (d, J1Ј,2Јϭ7.6 Hz, diaxial, H-1Ј) and
3.51 (dd, J3Ј,4Јϭ3.0 Hz, axial-equatorial, 9.0 Hz, H-3Ј) in the
1H-NMR spectrum suggested that the sugar in 4 was a b-D-
galactopyranose. Signals at d 64.9, 68.2 and 70.9 and a dou-
blet at d 4.14 (Jϭ6.8 Hz), a multiplet at d 4.00 and two dou-
blets of doublets at d 3.71 (Jϭ10.5, 3.5 Hz) and 3.89
mino-1,3,4-triacetoxy-9-docosene,15) and (2S,3S,4R,13Z)-2-
acetamino-1,3,4-triacetoxy-13-docosene18) (ϩ25.4°, ϩ17.6°,
ϩ26.5°, respectively) suggests that 2a also has 2S, 3S, 4R
configuration.
In order to establish the position of the double bond, the
tetraacetate (2a) was treated with dimethyl disulfide (DMDS)
and I2 and the product subjected to electron impact (EI)-MS
analysis. The EI-MS spectrum of the DMDS derivative
showed a molecular ion at m/z 577 and significant fragment
ions at m/z 530, 482, 422, 390, 330 and 187 arising from se-
lective fragmentation at the C-8–C-9 position of C18 chain,
thus confirming the position of the double bond at C-8–C-9
in the long-chain base. Similar results have been reported for
another cerebroside containing 2 as the long-chain base.19)
The D8 double bond in 2a was determined to be cis (Z) by
the upfield shifted carbon chemical shifts of C-7 (d 26.8) and
C-10 (d 27.3)17,20) and the relative small coupling constant of
H-8 at d 5.30 (dt, Jϭ10.8, 7.2 Hz) and H-9 at d 5.38 (dt,
Jϭ10.8, 6.8 Hz).
The above evidence led to the assignment of 2a as
(2S,3S,4R,8Z)-2-acetamino-1,3,4-triacetoxy-8(Z)-octadecene
and therefore 2 as the long-chain base present in 1. This base
is also present in a cerebroside isolated from Euphorbia
biglandulosa19) and in cerebrosides from Phytolaccae
Radix.17) The (E)-isomer is present in aralia cerebroside from
Aralia elata.21)
1
(Jϭ10.5, 6.3 Hz) in the 13C- and H-NMR spectra of 4, re-
spectively, were indicative of a glycerol moiety. A carbonyl
carbon signal was observed at d 174.1. A broad triplet of
four protons at d 2.81 (Jϭ5.8 Hz), a quartet of two protons at
d 2.06 (Jϭ8.0 Hz), a quintet of two protons at d 2.09
(Jϭ8.0 Hz) and a triplet of two protons at d 2.35 (Jϭ8.0 Hz)
were due to the methylene hydrogens lying between two dou-
ble bonds, a double bond and a methylene group, a double
bond and a methyl group and next to a carbonyl moiety, re-
spectively. Six olefinic carbons at d 126.8, 127.4, 127.9,
128.0, 129.9 and 131.6 were observed in the 13C-NMR spec-
trum of 4, indicating the presence of three double bonds in
the structure. This was in good agreement with the presence
of a multiplet (triplet-like) signal of six protons of the three
1
double bonds at d 5.36 in the H-NMR spectrum of 4. The
1
narrow width of the olefinic protons in the H-NMR spec-
trum at 5.36 (triplet-like) and the absence of IR absorption at
965—975 cmϪ1 in 4 indicates that the three double bonds are
cis.23,24) These spectral data suggested the long chain fatty
acid in 4 was linolenic acid. Treatment of 4 with NaOMe in
MeOH gave methyl ester of linolenic acid which was identi-
fied by GC/MS and (2R)-1-O-b-D-galactopyranosylglycerol
([a]D Ϫ7.1°)22,24,25) (Experimental).
Important long-range correlations were observed between
C-1Ј and Hab-3, C-1Љ and H-1 and H-2Љ and C-3 and H-1Ј in
the HMBC spectrum of 4 (Fig. 3). These results suggested
that linolenic acid and b-D-galactopyranose were connected
to C-1 and C-3 of glycerol, respectively. Compound 4 was
thus characterized as (2S)-1-O-linolenoyl-3-O-b-D-galac-
Therefore
1
was characterized as
a
mixture of
(2S,3S,4R,8Z)-1-O-(b-D-glucopyranosyl)-2N[(2ЈR)-2Ј-hy-
droxypalmitoyl]-2-amino-8(Z)-octadecene-1,3,4-triol,
(2S,3S,4R,8Z)-1-O-(b-D-glucopyranosyl)-2N[(2ЈR)-2Ј-hy-
droxysteroyl]-2-amino-8(Z)-octadecene-1,3,4-triol,
(2S,3S,4R,8Z)-1-O-(b-D-glucopyranosyl)-2N[(2ЈR)-2Ј-hy-
droxyeicosanoyl]-2-amino-8(Z)-octadecene-1,3,4-triol,
(2S,3S,4R,8Z)-1-O-(b-D-glucopyranosyl)-2N[(2ЈR)-2Ј-hy-
droxyheneicosanoyl]-2-amino-8(Z)-octadecene-1,3,4-triol,
(2S,3S,4R,8Z)-1-O-(b-D-glucopyranosyl)-2N[(2ЈR)-2Ј-hy-

30
Vol. 52, No. 1
Fig. 3. Selected 1H–1H COSY (Bold Lines) and HMBC (Full-Line Arrows) Correlations of 4
[MϩH]ϩ, C42H82NO10 requires 760.5934. Other molecular ion peaks were
too weak to allow the measurement of high resolution data. 1H- and 13C-
NMR data see Table 1.
Acetylation of 1 A mixture of 1 (19 mg), pyridine (0.5 ml) and acetic
anhydride (0.5 ml) was heated at 85 °C for 2 h. After the usual work up, the
acetate (1a) was obtained as a light yellow wax (23 mg) which was purified
by column chromatography using silica gel (3 g) and hexane/EtOAc as the
eluent to give 1a as a colorless gum (17 mg). IR nmthaixn film cmϪ1: 2924, 2853,
1752, 1686, 1522, 1467, 1435, 1370, 1226, 1041. 1H- and 13C-NMR data see
Table 1.
topyranosylglycerol. Unfortunately, the mixture of cerebro-
sides (1) did not show antiviral (HSV-1) and antiinflamma-
tory (COX-1 and COX-2) activities. The monoacylmono-
galactosylglycerol (4) was not tested because the sample had
decomposed.
Experimental
Melting point: uncorrected. IR spectra: Jasco A-302 Spectrophotometer.
1H-NMR: CDCl3, CDCl3/DMSO-d6, CDCl3/CD3OD Bruker Avance 400
(400 MHz), TMS as internal standard. 13C-NMR: CDCl3, CDCl3/DMSO-d6,
CDCl3/CD3OD, 100 MHz, TMS as internal standard. MS: VG 7070 mass
spectrometer operating at 70 eV for EI-MS and a VG Quattro triple quadru-
pole instrument for the electrospray MS (LSI). GC/MS was performed with
a Hewlett Packard 5890 Series II gas chromatograph attached to a Hewlett
Packard 5989B MS employing the EI mode (70 eV). Conditions: HP-5 capil-
lary column (30 mϫ0.25 mmϫ0.25 mm); column temperature: 150—270 °C
(3 °C minϪ1), then held at 270 °C; carrier gas: He; injector and detector tem-
peratures: 250 °C and 280 °C, respectively. Optical rotation: MeOH or
CHCl3. TLC: precoated PLC254 plate (Merck); spots were detected by spray-
ing with 1% CeSO4 in 10% aq. H2SO4 followed by heating. CC: silica gel
70—230 mesh (Merck), silica gel 230—400 mesh (Merck), Lichroprep RP-
18 25—40 mm. A voucher specimen (BRU. 350) was deposited at the Na-
tional Center for Genetic Engineering and Biotechnology (BIOTEC), 113
Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand.
Extraction and Isolation The fresh leaves of Clinacanthus nutans
(2.45 kg) were extracted with 95% EtOH at room temperature. After filtra-
tion, the filtrate were evaporated to give a dark green thick oil which was
partitioned between water (300 ml) and EtOAc (3ϫ300 ml) and the water
layer then extracted with n-BuOH (3ϫ250 ml). Removal of the solvent of
each fraction gave the EtOAc fraction as a dark green thick oil (20.9 g), the
n-BuOH fraction as a brown thick oil (15.8 g) and the water fraction as a
brown thick oil (111.4 g). The EtOAc fraction (20.0 g) was separated by
flash column chromatography using silica gel (230—400 mesh, diameter
13.0 cmϫheight 5.5 cm) and the column was eluted with (500 ml each)
hexane, hexane/EtOAc (4 : 1, 3 : 2, 2 : 3, 1 : 4), EtOAc, EtOAc/%MeOH (5,
10, 20, 40, 60, 80) and MeOH to give 13 fractions. Fraction 9 (2.1 g) which
possessed strong anti-HSV-1 activity (IC50ϭ7.86 mg/ml) was purified by
flash column chromatography using silica gel (230—400 mesh, diameter
7.0 cmϫheight 3.0 cm) and the column was eluted with (3ϫ50 ml each)
EtOAc and EtOAc/%MeOH (2, 4, 6, 8, 10, 20). The fractions were com-
bined on the basis of their behaviour on TLC and evaporated to give 7 frac-
tions. Fraction 4 (771 mg) was further chromatographed on a column of sil-
ica gel (70—230 mesh, 75 g) using CH2Cl2/MeOH/H2O (lower layer)
(40 : 3 : 1, 30 : 3 : 1, 20 : 3 : 1, 15 : 3 : 1) as the eluent to give a mixture of cere-
broside 1 and monoacylmonogalactosylglycerol 4 as a colorless solid
(332 mg). The mixture (152 mg) was separated on PLC (silica gel, layer
thickness 1.0 mm) using CH2Cl2/MeOH/H2O (lower layer) (15 : 3 : 1, 4 runs
and 10 : 3 : 1, 2 runs) to give cerebroside 1 as a colorless solid (83 mg). The
mixture (123 mg) was separated by C-18 reverse-phase gravity column chro-
matography eluting with MeOH/H2O (5 : 1), MeOH/H2O (10 : 1) and MeOH
to give 4 as a pale yellow wax (50 mg) and 1 as a colorless solid (41 mg).
Cerebroside 1 A colorless solid, IR nmNauxjol cmϪ1: 3315 (broad), 2921,
2851, 1635, 1588, 1075, 1036. Liquid secondary ion (LSI)-MS m/z (rel. int.
%): 872 [MϩH]ϩ (13%), 858 [MϩH]ϩ (6), 844 [MϩH]ϩ (37), 830
[MϩH]ϩ (15), 816 [MϩH]ϩ (71), 802 [MϩH]ϩ (5), 788 [MϩH]ϩ (11), 760
[MϩH]ϩ (25), 732 [MϩH]ϩ (5), 710 [872Ϫglc]ϩ (20), 696 [858Ϫglc]ϩ (5),
682 [844Ϫglc]ϩ (54), 668 [830Ϫglc]ϩ (24), 654 [816Ϫglc]ϩ (100), 640
[802Ϫglc]ϩ (5), 626 [788Ϫglc]ϩ (12), 598 [760Ϫglc]ϩ (27), 570
[732Ϫglc]ϩ (4). High resolution (HR)-LSI-MS m/z: 872.7165 [MϩH]ϩ,
C50H98NO10 requires 872.7185; 844.6854 [MϩH]ϩ, C48H94NO10 requires
844.6873; 816.6567 [MϩH]ϩ, C46H90NO10 requires 816.6560; 760.5914
Methanolysis of 1 Compound 1 (43 mg) was refluxed with 0.9 M HCl in
82% aq. MeOH (10 ml) for 18 h. The mixture was extracted with hexane and
the combined organic phase was washed with water and dried over Na2SO4.
Removal of the solvent gave a colorless wax (22 mg) which was chro-
matographed on silica gel [hexane/EtOAc (20 : 1, 5 : 1)] to yield a mixture of
fatty acid methyl esters as a colorless wax (15 mg). [a]D25 Ϫ11.0° (cϭ0.80,
CHCl3). The mixture of the esters was analyzed by GC/MS. Peak 1 (tR
8.50 min, 2-hydroxypalmitic acid methyl ester), EI-MS m/z: 286 [M]ϩ, 254
[MϪCH3OH]ϩ, 227 [MϪCH3COO]ϩ, 208, 182, 159, 145, 127 [C9H19]ϩ,
111 [C8H15]ϩ, 97 [C7H13]ϩ, 90 [CH3OC(OH)ϭCHOH]ϩ, 83, 69 [C5H9]ϩ, 57.
Peak 2 (tR 9.822 min, 2-hydroxystearic acid methyl ester), EI-MS m/z: 314
[M]ϩ, 282 [MϪCH3OH]ϩ, 255 [MϪCH3COO]ϩ, 236, 210, 159, 146, 127
[C9H19]ϩ, 111 [C8H15]ϩ, 97 [C7H13]ϩ, 90 [CH3OC(OH)ϭCHOH]ϩ, 83, 69
[C5H9]ϩ, 57. Peak 3 (tR 11.01 min, 2-hydroxyeicosanoic acid methyl ester),
EI-MS m/z: 342 [M]ϩ, 310 [MϪCH3OH]ϩ, 283 [MϪCH3COO]ϩ, 238, 207,
159, 146, 127 [C9H19]ϩ, 111 [C8H15]ϩ, 97 [C7H13]ϩ, 90 [CH3OC(OH)ϭ
CHOH]ϩ, 83, 69 [C5H9]ϩ, 57. Peak 4 (tR 11.57 min, 2-hydroxyheneicosanoic
acid methyl ester), EI-MS m/z: 356 [M]ϩ, 297 [MϪCH3COO]ϩ, 278, 236,
159, 127 [C9H19]ϩ, 111 [C8H15]ϩ, 97 [C7H13]ϩ, 90 [CH3OC(OH)ϭCHOH]ϩ,
83, 69 [C5H9]ϩ, 57. Peak 5 (tR 12.13 min, 2-hydroxydocosanoic acid
methyl ester), EI-MS m/z: 370 [M]ϩ, 338 [MϪCH3OH]ϩ, 311 [MϪ
CH3COO]ϩ, 266, 160, 127 [C9H19]ϩ, 111 [C8H15]ϩ, 97 [C7H13]ϩ, 90
[CH3OC(OH)ϭCHOH]ϩ, 83, 69 [C5H9]ϩ, 57. Peak 6 (tR 12.63 min, 2-hy-
droxytricosanoic acid methyl ester), EI-MS m/z: 384 [M]ϩ, 352
[MϪCH3OH]ϩ, 325 [MϪCH3COO]ϩ, 280, 207, 127 [C9H19]ϩ, 111
[C8H15]ϩ, 97 [C7H13]ϩ, 90 [CH3OC(OH)ϭCHOH]ϩ, 83, 69 [C5H9]ϩ, 57.
Peak 7 (tR 13.15 min, 2-hydroxytetracosanoic acid methyl ester), EI-MS m/z:
398 [M]ϩ, 366 [MϪCH3OH]ϩ, 339 [MϪCH3COO]ϩ, 294, 159, 127
[C9H19]ϩ, 111 [C8H15]ϩ, 97 [C7H13]ϩ, 90 [CH3OC(OH)ϭCHOH]ϩ, 83, 69
[C5H9]ϩ, 57. Peak 8 (tR 13.62 min, 2-hydroxypentacosanoic acid methyl
ester), EI-MS m/z: 412 [M]ϩ, 353 [MϪCH3COO]ϩ, 281, 207, 145, 127
[C9H19]ϩ, 111 [C8H15]ϩ, 97 [C7H13]ϩ, 90 [CH3OC(OH)ϭCHOH]ϩ, 83, 69
[C5H9]ϩ, 57. Peak 9 (tR 14.54 min, 2-hydroxyhexacosanoic acid methyl
ester), EI-MS m/z: 426 [M]ϩ, 394 [MϪCH3OH]ϩ, 367 [MϪCH3COO]ϩ,
322, 281, 207, 159, 127 [C9H19]ϩ, 111 [C8H15]ϩ, 97 [C7H13]ϩ, 90
[CH3OC(OH)ϭCHOH]ϩ, 83, 69 [C5H9]ϩ, 57.
The aq. MeOH layer was neutralized with NH4OH and extracted with
EtOAc. The combined EtOAc extract was washed with H2O, dried over
Na2SO4 and evaporated to give the long-chain base (2) as a slightly yellow
wax (12 mg). The aq. MeOH layer was then evaporated to dryness and chro-
matographed on silica gel [CH2Cl2/MeOH/H2O (lower layer) (20 : 3 : 1,
10 : 3 : 1, 7 : 3 : 1)] to give methyl glucopyranoside (mixture of a- and b-
anomer) as a colorless solid (3 mg). TLC [silica gel, CH2Cl2/MeOH/H2O
(lower layer) (10 : 3 : 1)] of the resulting methyl glucopyranoside (a- and b-
anomer) was identical to that of the standard methyl a-D-glucopyranoside
and methyl b-D-glucopyranoside.
Acetylation of 2 A mixture of 2 (10 mg), pyridine (0.5 ml) and acetic
anhydride (0.5 ml) was stirred at room temperature overnight. After the
usual work up, the tetraacetate 2a was obtained as a light yellow oil (14 mg)
which was chromatographed on silica gel [hexane/EtOAc (10 : 1, 5 : 1, 4 : 1,
3 : 1, 2 : 1, 1 : 1, 2 : 1)] to give 2a as a colorless wax (4 mg). [a]D25 ϩ25.4°
(cϭ0.35, CHCl3). 1H-NMR (CDCl3): d 0.88 (3H, t, Jϭ6.6 Hz, CH3), 1.28

January 2004
31
10% fetal vovine serum, in 96-well flat-bottomed tissue culture plates at
83 ml/well. Cells were incubated at 37 °C for 72 h in a humidified incubator
with 5% CO2. Subsequently, cells were washed with phosphate buffer saline
solution and incubated for 30 min in 83 ml serum-free DMEM medium con-
taining test compounds. DMEM media containing drug vehicle, DMSO
(0.1%), and aspirin were used as a control for 100% COX activities and a
positive control, respectively. The medium was then replaced with serum-
free DMEM containing the same amount of drugs or DMSO and 2 mM of
calcium ionophore A23187, and cells were incubated for 30 min. Culture su-
pernatants were collected at the end of incubation time and assayed for
prostaglandin E2 (PGE2) concentrations by the radioimmunoassay method
previously described by Kirtikara and coworkers.28) The inhibition of COX
activity was determined from the percent reduction of PGE2 produced by
drug treated cells relative to PGE2 produced by cells treated with DMSO
alone. IC50 values of COX-1 and COX-2 were determined using SOFTmax
software (Molecular Devices, Sunnyvale, CA, U.S.A.). Aspirin was used as
a positive control and almost equally effective against COX-1 and COX-2.
IC50 values of aspirin for COX-1 and COX-2 are 2.06 mg/ml and 3.57 mg/ml,
respectively.
(14H, br s, 7ϫCH2), 1.40 (2H, m, H-6), 1.65 (2H, m, H-5), 2.01 (4H, m, H-
7, H-10), 2.03, 2.05 (2ϫ), 2.15 (12H, all s, 4ϫOAc), 4.00 (1H, dd, Jϭ11.7,
3.2 Hz, Ha-1), 4.29 (1H, dd, Jϭ11.7, 4.5 Hz, Hb-1), 4.47 (1H, m, H-2), 4.94
(1H, dt, Jϭ9.0, 3.2 Hz, H-4), 5.10 (1H, dd, Jϭ8.1, 3.2 Hz, H-3), 5.30 (1H,
dt, Jϭ10.8, 7.2 Hz, H-8 or H-9), 5.38 (1H, dt, Jϭ10.8, 6.8 Hz, H-9 or H-8),
5.92 (1H, d, Jϭ9.0 Hz, NH). 13C-NMR (CDCl3): d 14.1 (CH3), 20.7 (2ϫ),
21.0, 23.3 (4ϫOCOCH3) 22.7, 29.3 (2ϫ), 29.6 (2ϫ), 29.7, 31.9 (7ϫCH2),
25.6 (C-6), 26.8, 27.3 (C-7, C-10), 27.9 (C-5), 47.7 (C-2), 62.8 (C-1), 72.0
(C-3), 72.9 (C-4), 128.8 (C-8 or C-9), 130.7 (C-9 or C-8), 169.8, 170.1,
170.8, 171.1 (4ϫOCOCH3). EI-MS m/z (rel. int. %): 483 [M]ϩ (0.5%),
423 [MϪCH3COOH]ϩ (14), 364 [423ϪCH3COO]ϩ (12), 304 [364Ϫ
CH3COOH]ϩ (13), 262 (12), 244 (13), 184 (44), 144 (20), 102 (70), 84
(100), 67 (24).
Dimethyl Disulfide Derivative (3) of 2a Tetraacetate 2a (6 mg) was dis-
solved in carbon disulfide (0.5 ml) and dimethyl disulfide (0.5 ml) and iodine
(10 mg) added. The reaction mixture was then kept at 60 °C for 48 h in a
small sealed vial. The reaction was quenched with 5% aq. Na2S2O3 and the
mixture was extracted with EtOAc. The EtOAc layer was dried over Na2SO4,
filtered and concentrated to give the dimethyl disulfide derivative (3) as a
light yellow solid (6 mg). [a]D25 ϩ14.6° (cϭ0.32, CHCl3). EI-MS m/z (rel.
int. %): 577 [M]ϩ (5%), 530 [MϪCH3S]ϩ (5), 482 [MϪCH3SϪCH3S]ϩ (6),
422 [MϪ95Ϫ60]ϩ (8), 390 [MϪ187]ϩ (33), 330 [MϪ187Ϫ60]ϩ (100), 187
[C11H23S]ϩ (25).
Acknowledgements We are indebted to the Biodiversity Research and
Training Program (BRT), BIOTEC/NSTDA for financial support. Thailand-
Tropical Diseases Research Program (T-2)’s support for the anti-HSV-1 and
antiinflammatory activity (COX-1 and COX-2) assay is gratefully acknowl-
edged. We thank Dr. Noel Davies of the Central Science Laboratory, Univer-
sity of Tasmania, Australia for the high-resolution mass spectra. Mrs. Panit
Vedkanchana and Mr. Witoon Ngow, Silpakorn University, Thailand are
gratefully acknowledged for the GC/MS spectra. We are grateful to Mrs.
Jaree Bansiddhi, the Division of Medicinal Plant Research and Develop-
ment, Department of Medical Science, Nonthaburi, Thailand, for providing
the plant material.
Monoacylmonogalactosylglycerol (4): A pale yellow wax, [a]D25 Ϫ3.3°
(cϭ0.70, MeOH). IR nmneaaxt cmϪ1 3369, 3010, 1738, 1069. 1H-NMR
:
(CDCl3–CD3OD): d 0.98 (3H, t, Jϭ8.0 Hz, CH3), 1.32 (6H, br s, H-5Љ, H-6Љ,
H-7Љ), 1.34 (2H, m, H-4Љ), 1.62 (2H, quintet, Jϭ8.0 Hz, H-3Љ), 2.06 (2H,
br q, Jϭ8.0 Hz, H-8Љ), 2.09 (2H, br quintet, Jϭ8.0 Hz, H-17Љ), 2.35 (2H, t,
Jϭ8.0 Hz, H-2Љ), 2.81 (4H, br t, Jϭ5.8 Hz, H-11Љ, H-14Љ), 3.51 (1H, dd,
Jϭ9.0, 3.0 Hz, H-3Ј), 3.52 (1H, m, overlapped signal, H-5Ј), 3.58 (1H, dd,
Jϭ9.0, 7.6 Hz, H-2Ј), 3.71 (1H, dd, Jϭ10.5, 3.5 Hz, Ha-3), 3.76 (1H, dd,
Jϭ11.2, 5.3 Hz, Ha-6Ј), 3.82 (1H, dd, Jϭ11.2, 6.0 Hz, Hb-6Ј), 3.89 (1H, dd,
Jϭ3.0, 1.2 Hz, H-4Ј), 3.89 (1H, dd, Jϭ10.5, 6.3 Hz, Hb-3), 4.00 (1H, m, H-
2), 4.14 (2H, d, Jϭ6.8 Hz, H-1), 4.25 (1H, d, Jϭ7.6 Hz, H-1Ј), 5.36 (6H, m,
triplet-like signal, H-9Љ, H-10Љ, H-12Љ, H-13Љ, H-15Љ, H-16Љ). 13C-NMR
(CDCl3–CD3OD, 6 : 1): d 13.8 (C-18Љ), 20.2 (C-17Љ), 24.5 (C-3Љ), 25.2, 25.3
(C-11Љ, C-14Љ), 26.9 (C-8Љ), 28.8 (2ϫ), 28.9 (C-5Љ, C-6Љ, C-7Љ), 29.3 (C-4Љ),
33.8 (C-2Љ), 61.1 (C-6Ј), 64.9 (C-1), 68.2 (C-2), 68.6 (C-4Ј), 70.9 (C-3), 71.1
(C-2Ј), 73.1 (C-3Ј), 74.7 (C-5Ј), 103.4 (C-1Ј), 126.8, 127.4, 127.9, 128.0,
129.9, 131.6 (C-9Љ, C-10Љ, C-12Љ, C-13Љ, C-15Љ, C-16Љ), 174.1 (C-1Љ). MS
m/z (rel. int. %): 515 [MϩH]ϩ (20%), 353 (100), 309 (37), 291 (50), 270
(75), 235 (50), 219 (70). HR-LSI-MS m/z: 515.3205 [MϩH]ϩ, C27H47O9 re-
quires 515.3217. Found: 515.3205.
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32
Vol. 52, No. 1
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