Sphingolipids from Conyza canadensis.

Article abstract of DOI:10.1016/S0031-9422(02)00385-0

Sphingolipid 1 and its corresponding beta-D-glucopyranoside derivative 2 have been isolated from the ethylacetate fraction of Conyza canadensis along with beta-sitosterol 3, stigmasterol 4, beta-sitosterol 3-O-beta-D-glucoside 5 and harmine 6, reported fo

Full text of DOI:10.1016/S0031-9422(02)00385-0

Phytochemistry 61 (2002) 1005–1008
www.elsevier.com/locate/phytochem
Sphingolipids from Conyza canadensis
Naveen Mukhtar, Kiran Iqbal, Itrat Anis, Abdul Malik*
International Centre for Chemical Sciences, HEJ Research Institute of Chemistry University of Karachi, Karachi-75270, Pakistan
Received 14March 2002; received in revised form 19 August 2002
Abstract
Sphingolipid 1 and its corresponding b-d-glucopyranoside derivative 2 have been isolated from the ethylacetate fraction of
Conyza canadensis along with b-sitosterol 3, stigmasterol 4, b-sitosterol 3-O-b-d-glucoside 5 and harmine 6, reported for the first
time from this species. The structures of 1 and 2 were elucidated through spectroscopy including two-dimensional NMR.
# 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Conyza canadensis; Compositae; Sphingolipids
1. Introduction
Conyza canadensis Linn. belongs to the family Com-
positae (commonly called Canada fleabane) is an
annual herb, distributed in Western Himalayas, Pun-
jab, Kashmir and Pakistan (Chopra, 1982). It is
astringent, stimulant, haemostatic and diuretic, also
used in diarrhoea, dysentry, uterine haemorrhages,
dropsy, gravel, cystitis, calculus, bronchial catarrh and
haemoptysis (Sastri, 1952). Literature survey revealed
that only two aromatic carboxylic acids have so far been
reported from this species (Bohlmann et al., 1979).
Phytochemical investigations on the ethylacetate soluble
fraction of C. canadensis have now resulted in the iso-
lation of new sphingolipids 1 and 2 along with b-sitosterol
3, stigmasterol 4, b-sitosterol 3-O-b-d-glucoside 5 and
harmine 6, reported for the first time from this species.
Sphingolipids are a biologically important class of
compounds (Kolter and Sandhoff, 1999) some of which
have been reported to exhibit antihepatotoxic (Kim et
al., 1997), antitumor and immunostimulatory activities
(Natori et al., 1994). These are mainly reported from
different fungi (Lourenco et al., 1996) and sponges
(Hattori et al., 1998; Hirsch and Kashman, 1989; Yun-
ker and Scheuer 1978; Li et al., 1995; Lourenco et al.,
1996; Gao et al., 2001a).
2. Results and discussion
Compounds 1–6 were isolated by column chromato-
graphy of the ethylacetate soluble fraction over silica
gel, as described in Experimental. Compound 1 was
obtained as colorless gummy solid and showed the
molecular ion peak in HRMS at m/z 665.6362 corre-
sponding to molecular formula C42H83NO4 (calcd for
C42H83NO4 665.6321). The IR spectrum showed the
hydroxyl bands at 3340 and 3220 cmꢀ1, bands at 1620
and 1540 cmꢀ1 due to the amide group and a band at
1660 cmꢀ1 for the olefinic bond. The 1H NMR spectrum
of 1 (Table 1) showed the presence of two terminal
methyls at ꢀ 0.86, methylenes at ꢀ 1.25 (br s, 18H) and ꢀ
1.30 (br s, 18H), an amide proton signal at ꢀ 8.55 (d,
J=8.8 Hz), signals of trans-olefinic bond at ꢀ 5.48 (br dt,
J=16.8, 6.5 Hz) and 5.54( br dt, J=16.8, 6.5 Hz). The
13C NMR spectrum of 1 showed a carbon signal at ꢀ
177.0 (CONH), methine carbons at ꢀ 130.8 and 130.6
* Corresponding author. Tel.: +92-21-924-3198; fax: +92-21-924-
3190 or 3191.
E-mail address: hej@biruni.erum.com.pk or hej@digicom.net.pk
(A. Malik).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00385-0

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N. Mukhtar et al. / Phytochemistry 61 (2002) 1005–1008
Table 1
1H and 13C NMR data of compounds 1 and 2
Compound 1
Compound 2
Position
1
13Ca (DEPT)
62.0 (CH2)
1Hb multiplicity J (Hz)
13Ca (DEPT)
70.4 (CH2)
1Hc multiplicity J (Hz)
4.41 dd (10.7, 4.8)
4.94 dd (10.5, 4.4)
5.11 m
4.32 dd (10.3, 3.3)
4.60 dd (10.3, 6.2)
5.03 m
2
53.0 (CH)
76.8 (CH)
51.7 (CH)
75.9 (CH)
35.6 (CH2)
72.4(CH)
3
435.7 (CH
4.33 m
2.40 m
4.35 m
2.36 m
)
2
5
73.0 (CH)
33.2 (CH2)
33.0 (CH2)
34.1 (CH2)
33.8 (CH2)
130.8 (CH)
130.6 (CH)
33.0 (CH2)
32.9 (CH2)
4.52 m
1.99 m
4.50 m
1.83 m
6
33.0 (CH2)
32.9 (CH2)
33.8 (CH2)
33.3 (CH2)
130.9 (CH)
130.7 (CH)
32.1 (CH2)
30.5 (CH2)
29.0 (CH2)
28.3 (CH2)
30.0 (CH2)
29.5 (CH2)
7
8,11
1.45 m
1.95–2.01 m (4H)
1.38 m
2.02–2.21 m (4H)
9
10
5.54( br dt 16.8, 6.5)
5.48 (br dt 16.8, 6.5)
1.60 m
5.34( br dt 16.5, 6.2)
5.40 (br dt 16.5, 6.2)
1.58 m
12
13
1.37 m
1.42 m
1430.5 (CH
15
)
2
29.6 (CH2)
)
16–2429.9 (CH
25
1.30 (br s 18H)
1.22 (br s 18H)
2
29.5 (CH2)
17.6 (CH2)
14.2 (CH3)
26
27
1.48 m
0.86 t (7.0)
8.55 d (8.8)
19.4(CH
14.3 (CH3)
)
1.50 m
0.77 t (6.8)
8.45 d ( 8.6)
2
NH
10
20
30
(40–120)
130
140
150
177.0 (C)
175.8 (C)
32.1 (CH2)
30.2 (CH2)
29.9 (CH2)
22.9 (CH2)
19.6 (CH2)
13.8 (CH3)
2.20 t (7.0)
1.34 m
30.1 (CH2)
29.9 (CH2)
30.0 (CH2)
23.6 (CH2)
21.0 (CH2)
13.9 (CH3)
1.89 t (6.3)
1.30 m
1.25 br s (18H)
1.20 m
1.65 m
1.13 br s (18H)
1.35 m
1.72 m
0.86 t (7.0)
0.77 t (6.8)
1. . .
2. . .
3. . .
4. . .
5. . .
6. . .
105.5 (CH)
75.1 (CH)
78.1 (CH)
71.5 (CH)
78.5 (CH)
62.6 (CH2)
4.78 d ( 7.7)
3.85 m
3.91 m
3.95 m
3.78 m
4.27 dd (11.5, 4.5)
4.07 dd (10.0, 3.5)
a
13C NMR carried out at 100 MHz.
1H NMR carried out at 400 MHz.
1H NMR carried out at 500 MHz.
b
c
(C¼C), three methines at ꢀ 53.0 (CHNH), 76.8
(CHOH), 73.0 (CHOH) and one methylene at ꢀ 62.0
be established through characteristic fragment ions in
1
EIMS (Fig. 1) and further confirmed through H–1H
(CH2OH). This suggested that
sphingosine-type sphingolipid (Natori et al., 1994; Gao
et al., 2001b).
1
was
a
phyto-
COSY, HMQC and HMBC correlations. Based on
these cummulative evidence, the compound 1 was
assigned the structure 1,3,5-trihydroxy-2-hexadecanoyl-
amino-9-(E)-heptacosene.
The presence of a two protons triplet in 1H NMR at ꢀ
2.2 due to methylene protons connected to the amide
carbonyl indicated that N-acyl moeity in 1 was a non-
hydroxy fatty acid. Methanolysis (Gao et al., 2001b) of
1 yielded methyl pentadecanoate (1a) detected by
GC–MS. The existence of the pentadecanoyl moeity
was also confirmed by the characteristic ions at m/z 225
[CH3(CH2)13CO]+, 242 [CH3(CH2)13CONH2+H]+,
and 422 [MꢀH2OꢀCH3(CH2)13CO]+ in the EIMS.
Therefore, the base is C27-phytosphingosine containing
three hydroxyls and an amino group. Its structure could
Compound 2 was also obtained as a gummy solid
showing [M+H]+ peak in positive mode HRFABMS
at m/z 828.6934corresponding to the molecular formula
C48H94NO9 (calcd for C48H94NO9 828.6928). The IR
1
spectrum was similar to 1 while the H NMR spectrum
showed additional peaks due to the sugar moeity
[anomeric proton at ꢀ 4.78 (d, J=7.7 Hz), four protons
geminal to the hydroxyl group between ꢀ 3.85–3.95 and
hydroxymethylene protons at ꢀ 4.07 and 4.27]. The 13C
NMR spectrum also revealed the presence of sugar

N. Mukhtar et al. / Phytochemistry 61 (2002) 1005–1008
1007
from the combined extracts in vacuo gave a dark resi-
due which was suspended in water and successively
extracted with n-hexane, ethylacetate and n-butanol.
The ethylacetate extract (200 g) was subjected to col-
umn chromatography over silica gel (70–230 mesh),
successively eluting with n-hexane, n-hexane–ethyl-
acetate, ethylacetate, ethylacetate–methanol and finally
methanol in increasing order of polarity. The fraction
which eluted with n-hexane–ethylacetate (8:2) was a
mixture of two components. It was subjected to CC
(flash silica 230–400 mesh) using n-hexane–ethylacetate
(7.5:2.5) as eluent to afford 3 and n-hexane–ethylacetate
(6:4) to obtain 4. The fraction which eluted with n-hexane–
ethylacetate (1:1) was rechromatographed over flash
silica eluting with n-hexane–ethylacetate (4:6) to provide
5. The fraction which eluted with n-hexane–ethylacetate
(2:8) showed one major spot on TLC. It was rechroma-
tographed over flash silica eluting with n-hexane–ethyl-
acetate (1:9) to yield the compound 6. The fraction
which eluted with ethylacetate was rechromatographed
over flash silica eluting with ethylacetate and ethylacetate–
methanol (9:1) to obtain 1 (12 mg) and 2 (10 mg),
respectively.
Fig 1. Mass fragmentation pattern and important HMBC correlations
of compound 1.
moeity [anomeric carbon at ꢀ 105.5 and the hydroxyl
containing methine carbons at ꢀ 75.1, 78.1, 71.5, and
78.5 and a further signal at ꢀ 62.6 (CH2OH)]. This sug-
gested that 2 is a glycoside of 1. The 13C NMR signals
of sugar moeity corresponded to b-d-glucopyranoside.
It was also confirmed by EIMS which showed prominent
peak at m/z 667 due to the elimination of glucosyl moeity.
Further fragmentation pattern was similar to 1. The
position of glucose moeity was evident by downfield
chemical shift of hydroxymethylene carbon at ꢀ 70.4in
13C NMR spectrum by 8 ppm and confirmed by
HMBC experiments in which the additional correlation
was observed between the anomeric proton (ꢀ 4.78) with
the hydroxymethylene carbon (ꢀ 70.4). Thus compound 2
was confirmed as the b-d-glucopyranoside derivative of 1.
3.4. Compound 1
Colorless gummy solid, [ꢁ]2D5 +17.85ꢁ (pyridine; c 0.028);
IR (KBr) nmax cmꢀ1; 3340 (OH), 1660 (C¼C), 1620
(C¼O); EIMS data and important HMBC correlations
1
are illustrated in Fig. 1. H and 13C NMR, see Table 1.
3. Experimental
3.5. Methanolysis of 1
3.1. General
Compound 1 (10 mg) was refluxed with 1.5 mL of
0.9N HCl in 82% aq. methanol for 16 h (Gao et al.,
2001b). The reaction mixture was cooled and extracted
with n-hexane. The n-hexane layer was concentrated
and silica gel column chromatography [n-hexane-ethy-
lacetate (9:1–7:3)] gave the fatty acid methyl ester 1a
which could be identified as methyl pentadecanoate
through GC–MS showing the molecular ion peak at m/z
256 and an intense peak at m/z 197 [MꢀCOOMe]+.
Optical rotations were measured on a Jasco DIP-360
polarimeter. IR spectra were recorded in KBr disk on a
Jasco 320-A spectrophotometer. EI and FABMS were
recorded on JMS HX 110 and JMS-DA 500 mass
spectrometers with a data system. The H, 13C NMR,
1
COSY, HMQC and HMBC spectra were recorded at 500
and 400 MHz. The chemical shift values are reported in
ppm (ꢀ) units and the coupling constants (J) are in Hz.
3.6. Compound 2
3.2. Plant material
Colorless gummy solid, [ꢁ]2D5 +62.5ꢁ (pyridine; c 0.024);
IR (KBr) nmax cmꢀ1; 3340 (OH), 1660 (C¼C), 1620
(C¼O); EIMS data and important HMBC correlations
C. canadensis Linn. (Compositae), whole plant was
collected in July 2000, from Karachi (Pakistan) and
identified by Dr. Javed Zaki, Plant Taxonomist,
Department of Botany, University of Karachi, where a
voucher specimen has been deposited.
1
are illustrated in Fig. 1. H and 13C NMR, see Table 1.
References
3.3. Extraction and isolation
Bohlmann, F., Zdero, C., Robinson, H., King, M., 1979. Neue Acet-
ylenverbindungen Aus Chrysothamnus parryi. Phytochemistry 18,
1519–1521.
The dried plant material (30 kg) was extracted thrice
with MeOH at room temperature. Evaporation of solvent

1008
N. Mukhtar et al. / Phytochemistry 61 (2002) 1005–1008
Chopra, R.N., 1982. Indian Materia Medica, Vol One. With Auyr-
vedic, Unani and Home Remedies, Appendices and Indexes. New
Delhi, pp. 504–505.
Kolter, T., Sandhoff, K., 1999. Processing of sphingolipid activator
proteins and the topology of lysosomal digestion. Angew. Chem.
Int. Engl. 38, 1532–1563.
Gao, J.M., Dong, Z.J., Liu, J.K., 2001a. A new ceramide from the
basidiomycete Armillaria mellea. Chin. Chem. Lett. 12 (2), 139–
140.
Li, H., Matsunaga, S., Fusetani, N., 1995. Halicyclindrosides, anti-
fungal and cytotoxic cerebrosides from the marine sponge Hali-
chondria cylindrata. Tetrahedron 51, 2273–2280.
Gao, J.M., Yang, X., Wang, C.Y., Liu, J.K., 2001b. Armillaramide, a
new sphingolipid from the fungus Armillaria mellea. Fitoterapia 72,
858–864.
Lourenco, A., Lobo, A.M., Rodriguez, B., Jimeno, M.L., 1996. Cer-
amides from the fungus Phellinus pini. Phytochemistry 43, 617–620.
Natori, T., Morita, M., Akimoto, K., Koezuka, Y., 1994. Agelasphins,
novel antitumor and immunostimulatory cerebrosides from the
marine sponge Agelas mauritianus. Tetrahedron 50, 2771–2784.
Sastri, B.N., 1952. The Wealth of India, Vol III. A Dictionary of
Indian Raw Materials and Industrial Products. CSIR, New Delhi,
pp. 185–186.
Hattori, T., Adachi, K., Shizuri, Y., 1998. New ceramide from marine
sponge Haliclona koremella and related compounds as antifouling
substances against macroalgae. J. Nat. Prod. 61, 823–826.
Hirsch, S., Kashman, Y., 1989. New glycosphingolipids from marine
organisms. Tetrahedron 45, 3897–3906.
Kim, S.Y., Choi, Y.H., Huh, H., Kim, J.W., Kim, Y.C., Lee, H.S.,
1997. New antihepatotoxic cerebroside from Lycium chinese fruits.
J. Nat. Prod. 60, 274–276.
Yunker, M.B., Scheuer, P.J., 1978. Alpha-oxygenated fatty acids
occurring as amides of 2-methylene-b-alanine in a marine sponge.
Tetrahedron Lett. 47, 4651–4652.