Cinnamyl alcohols and methyl esters of fatty acids from Wedelia prostrata callus cultures

Article abstract of DOI:10.1080/14786419.2010.482937

Two methyl esters of fatty acids, namely octadecanoic acid methyl ester (methyl stearate) (1) and hexadecanoic acid methyl ester (methyl palmitate) (2), in addition to four cinnamyl alcohol derivatives, sinapyl alcohol (3), coniferyl alcohol (4), p-coumaryl alcohol (5) and coniferyl alcohol 4-O-glucoside (coniferin) (6), were isolated from callus cultures of Wedelia prostrata. The structure of coniferin was established by spectroscopic and chemical methods, while the other compounds were identified by gas chromatography-mass spectrometry and thin layer chromatography in comparison with standards.

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Cinnamyl alcohols and methyl esters
of fatty acids from Wedelia prostrata
callus cultures
a
a
b
Ahmed M.A. Abd El-Mawla , Salwa F. Farag & Till Beuerle
a
Department of Pharmacognosy, Faculty of Pharmacy, Assiut
University, Assiut 71526, Egypt
b
Institute for Pharmaceutical Biology, Braunschweig University
of Technology, Mendelssohn Street 1, D-38106 Braunschweig,
Germany
Published online: 13 Jan 2011.
To cite this article: Ahmed M.A. Abd El-Mawla , Salwa F. Farag & Till Beuerle (2011): Cinnamyl
alcohols and methyl esters of fatty acids from Wedelia prostrata callus cultures, Natural Product
Research: Formerly Natural Product Letters, 25:1, 45-52
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Natural Product Research
Vol. 25, No. 1, January 2011, 45–52
Cinnamyl alcohols and methyl esters of fatty acids from
Wedelia prostrata callus cultures
a
a
b
Ahmed M.A. Abd El-Mawla *, Salwa F. Farag and Till Beuerle
a
Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526,
Egypt; Institute for Pharmaceutical Biology, Braunschweig University of Technology,
Mendelssohn Street 1, D-38106 Braunschweig, Germany
b
(
Received 9 March 2010; final version received 31 March 2010)
Two methyl esters of fatty acids, namely octadecanoic acid methyl ester
(
(
methyl stearate) (1) and hexadecanoic acid methyl ester (methyl palmitate)
2), in addition to four cinnamyl alcohol derivatives, sinapyl alcohol (3),
coniferyl alcohol (4), p-coumaryl alcohol (5) and coniferyl alcohol
-O-glucoside (coniferin) (6), were isolated from callus cultures of
4
Wedelia prostrata. The structure of coniferin was established by spectro-
scopic and chemical methods, while the other compounds were identified
by gas chromatography–mass spectrometry and thin layer chromatography
in comparison with standards.
Keywords: Wedelia prostrata; MS medium; callus cultures; GC-MS;
coniferin
1
. Introduction
Wedelia prostrata Hook. et Arn. (Hemsl.) (Asteraceae, tribe Heliantheae)
Benson, 1976; Lawrence, 1968) is cultivated in Egypt as an ornamental plant. The
genus Wedelia comprises about 70 species, native to the warmer regions of the world
Bailey, 1963). Economically, the plants of family Asteraceae are of considerable
(
(
importance. They comprise numerous food and drug plants, of which many are
ornamentals and several are noxious weeds (Core, 1962). Many species of Wedelia
have valuable biological activities (Gopalakrishnan, Sadique, & Chandra, 1989;
Perry & Metzger, 1980; Watt & Breyer-Brandwijk, 1962). The isolated kaurene
diterpenes have shown antibiotic activity and thus justify the utilisation of Wedelia
species as antitussive agents (Roque, Giannella, Giesbrecht, & Barbosa, 1987).
Previous phytochemical investigations of W. prostrata have led to the isolation
and
3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid), a sesquiterpene, kaure-
noids, monoterpenes, stigmasterol, stigmasterol glucoside and flavonoids (Farag,
001; Farag, El-Emary, Makboul, Hassanean, & Niwa, 1997; Farag, El-Emary,
Makboul, Hassanean, & Niwa, 1998; Farag, El-Emary, & Niwa, 1996).
identification
of
eudesmanolides,
chalcones,
phenylpropanoids
(
2
*Corresponding author. Email: ahmedpha2000@yahoo.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2011 Taylor & Francis
DOI: 10.1080/14786419.2010.482937
http://www.informaworld.com

4
6
A.M.A. Abd El-Mawla et al.
O
CH3
H C
3
O
O
(
(
1)
2)
CH3
H C
3
O
3
′
2
1′
R1
3
4
1
CH OH
2
2′
6
R2
5
R3
Compounds
R
1
R
2
R
3
3
4
5
6
OCH
OCH
H
3
3
OH
OH
OH
OCH
H
H
3
OCH
3
O-glucose
H
Figure 1. The chemical structures of the isolated compounds.
In this article, we describe the isolation and structural characterisations of six
compounds (1–6) that have been isolated for the first time from the titled plant.
2
. Results and discussion
The acetone extract of W. prostrata callus cultures afforded two methyl esters of
fatty acids (1 and 2) and four cinnamyl alcohol derivatives (3–6) (Figure 1). The
retention time (R ), MS fragmentation pattern and rate of flow (R ) for compounds
t
f
1
–5 were identical to those of the relevant standards. They were identified as
octadecanoic acid methyl ester (methyl stearate) and hexadecanoic acid methyl ester
methyl palmitate), sinapyl alcohol, coniferyl alcohol and p-coumaryl alcohol,
respectively.
The ESI mass spectrum of compound 6 showed a molecular ion peak at m/z
(
–
3
showed absorptions at ꢀ
41 [M–1] , consistent with the molecular formula C H O . Its UV spectrum
16 22 8
228 and 280 nm, indicating its phenolic nature. The
max
1
H-NMR spectrum exhibited one methoxyl group at ꢁ 3.78 (3H, s), methylene
protons at ꢁ 4.09 (2H, br t, J ¼ 5.0 Hz), and trans-olefinic protons at ꢁ 6.25 (1H, dt,
J ¼ 15.7, 5.0 Hz) and 6.50 (1H, d, J ¼ 15.7 Hz). In addition, it displayed three
aromatic protons at ꢁ 6.89 (1H, dd, J ¼ 2.2, 8.0 Hz), 7.03 (1H, d, J ¼ 8.0 Hz) and 7.05
(
(
1H, d, J ¼ 2.2 Hz). A characteristic doublet of an anomeric proton at ꢁ 5.43
J ¼ 7.3 Hz) indicated the presence of a ꢂ-D-glucopyranosyl moiety (Agrawal, 1992;
Falshaw, Ormand, Mongkolsuk, & Podimuang, 1969). The spectral data of
compound 6 were in good agreement with those reported for coniferin (Sugiyama,
Nagayama, & Kikuchi, 1993). Furthermore, acid hydrolysis of compound 6 gave
glucose and coniferyl alcohol, which was detected by TLC comparison with an
authentic sample.

Natural Product Research
47
Coniferyl alcohol, which is the aglycone of coniferin, is known to be a
biosynthetic precursor of important biologically active compounds (Dewick, 2002).
Two coniferyl alcohol moieties dimerise in early steps in lignan (Broomhead &
Dewick, 1990; Molog et al., 2001; Seidel et al., 2002; Umezawa, Davin, & Lewis,
1
991), e.g. podophyllotoxin. Flavonolignans, e.g. silymarin, arise from a combina-
tion of a flavonoid and a phenylpropanoid, usually coniferyl alcohol (Dewick, 2002;
Wenzig et al., 2005). Coumarinolignans, e.g. silybin, are a combination of a
coumarin with a cinnamyl alcohol, usually coniferyl alcohol (Dewick, 2002). Also,
coniferyl alcohol is a precursor to a number of flavour components of spices, e.g.
eugenol and isoeugenol (Wang, Dudareva, Bhakta, Raguso, & Pichersky, 1997).
The isolated phenylpropanoids (3–6) are pharmacologically active compounds.
(
&
E )-p-coumaryl alcohol has shown antioxidative activity (Ly, Shimoyamada, Kato,
Yamauchi, 2003). Sinapyl alcohol exhibited in vitro anti-inflammatory and
antinociceptive effects; in addition, some sinapyl alcohol derivatives were found to be
cytotoxic agents (Choi et al., 2004; Zhao et al., 2002). Coniferin was shown to have
in vitro anti-inflammatory activity (Diaz Lanza et al., 2001).
The previous phytochemical studies of W. prostrata resulted in the identification
of two phenylpropanoids, 3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid,
from the aerial parts and roots (Farag et al., 1997, 1998; Farag, 2001).
In this article, we report the occurrence of four cinnamyl alcohol derivatives:
sinapyl alcohol, coniferyl alcohol, p-coumaryl alcohol and coniferin.
It has been suggested that phenolic compounds have been formed in the callus
cultures as a response to vitamins and hormones present in the MS medium and as a
response to external stress in the entire plant (Hart, 1981).
L-Phenylalanine and L-tyrosine, as C C building blocks, are precursors for a
3
6
wide range of natural products. In plants, a frequent first step is the elimination of
ammonia from the side-chain by specific lyases to generate the appropriate trans (E )
cinnamic acid. In the case of phenylalanine, this would give cinnamic acid, whilst
tyrosine could yield 4-coumaric (p-coumaric) acid; other cinnamic acids are obtained
by further hydroxylation and methylation reactions, sequentially building up
substitution patterns typical of shikimate pathway metabolites, i.e. an ortho
oxygenation pattern. Some of the more common natural cinnamic acids are
4-coumaric, caffeic, ferulic and sinapic acids. These can be found in plants in free
form and in a range of esterified forms, e.g. with quinic acid, as in chlorogenic acid
(
(
5-O-caffeoylquinic acid, 3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid)
Dewick, 2002).
3
3
. Experimental
.1. General procedures
Gas chromatographic–mass spectrometric (GC-MS) analyses were carried out with
a Finnigan TSQ 700 triple-stage quadrupole mass spectrometer (Thermo Quest,
Bremen, Germany) coupled to a Hewlett Packard 5890A gas chromatograph
(Hewlett-Packard, Waldbronn, Germany). The GC was equipped with a 3 m fused
silica guard column (deactivated, Phenomenex, 0.32 mm ID) and a 30 m analytical
column (ZB 1, Phenomenex, 0.32 mm ID, 0.25 mm film thickness).

4
8
A.M.A. Abd El-Mawla et al.
1
The H-NMR spectrum was measured on a Bruker AM-400 MHz instrument
Bruker, Germany) with tetramethylsilane (TMS) as an internal standard. UV
(
spectra were recorded on an Ultrospec 1000, UV-vis spectrophotometer (Pharmacia,
Biotech., England). The negative-ion electrospray ionisation (ESI) mass spectrum
was performed on a MAT 95 XL Trap hybrid tandem mass spectrometer
(ThermoFinnigan MAT, Bremen, Germany). Column chromatographic separation
was performed with silica gel (60–200 mesh, Merck).
Preparative thin layer chromatography (TLC) was conducted on silica gel layers
using silica gel 60 F254 (Merck). TLC analyses were carried out on a pre-coated silica
gel 60 F254 aluminium sheets (Merck).
The following solvent systems were used for TLC:
(1) n-Hexane–ethyl acetate (85 : 15).
(2) Chloroform–methanol (70 : 30).
(3) n-Butanol–acetone–formic acid–water (60 : 17 : 8 : 15).
Compounds were detected by spraying with 10% H SO followed by heating at
4
2
ꢀ
1
10 C for 2 min.
Melting points were determined by Electrothermal 9100 digital melting point
instrument (Electrothermal Engineering Ltd, UK) and are uncorrected.
3
.2. Chemicals
All the media components were purchased from Merck (Darmstadt, Germany). The
authentic compounds methyl stearate, methyl palmitate, sinapyl alcohol, coniferyl
alcohol and p-coumaryl alcohol were provided by Professor Dr L. Beerhues,
Institute for Pharmaceutical Biology, Braunschweig University of Technology,
Germany.
3
.3. Plant material
The fresh young shoots of W. prostrata Hook. et Arn. (Hemsl.) were obtained from
the plants cultivated in the Experimental Station of Medicinal Plants, Faculty of
Pharmacy, Assiut University, Assiut, Egypt. The identification of the plant was
confirmed by Professor Dr Abdel-Aziz Fayed (Professor of Plant Taxonomy,
Faculty of Science, Assiut University, Assiut, Egypt). A voucher specimen has been
deposited in the Herbarium of the Department of Pharmacognosy, Faculty of
Pharmacy, Assiut University, Assiut, Egypt.
3
.4. Callus induction
Callus cultures were initiated from the fresh W. prostrata young shoots cultivated in
Murashige and Skoog (MS) medium (Murashige & Skoog, 1962) supplemented with
4
.5 mM of 1-naphthaleneacetic acid, 4.5 mM of 6-benzylaminopurine and 1 g of
ꢀ
casamino acid. The calli were maintained in the dark at a temperature of 25 ꢁ 2 C
and subcultured to a new MS medium (50 mL) every four weeks.

Natural Product Research
49
3
.5. Extraction and isolation of the constituents
About 100 g of fresh W. prostrata cultured cells were ground in 700 mL acetone and
the homogenate was filtered. The residue was extracted twice with 500 mL acetone.
The acetone phases were combined and evaporated under reduced pressure to
dryness (Beerhues & Berger, 1995). The solvent-free residue (10 g) was partitioned
between distilled water and n-hexane (3 ꢂ 100 mL), EtOAc (3 ꢂ 100 mL) and
n-BuOH (3 ꢂ 100 mL) to give the corresponding solubles (2.0 g), (4.0 g), (1.5 g) and
aqueous (2.5 g), respectively. The obtained soluble fractions were separately
concentrated under reduced pressure and screened for different constituents by TLC.
3
.5.1. n-Hexane fraction
About 2.0 g of the n-hexane soluble fraction was chromatographed on silica gel
column (60 g, 30 ꢂ 2 cm) using n-hexane–ethyl acetate step gradients. Fractions of
3
0 mL were collected, concentrated under reduced pressure and monitored by TLC
using solvent system I. Similar fractions were combined. The fractions eluted with
n-hexane–ethyl acetate (90 : 10) were concentrated to give an oily residue (50 mg).
The oily residue contained a crude mixture of compounds 1 and 2. This was
subjected to GC-MS analysis.
3
.5.2. Ethyl acetate fraction
About 4.0 g of the ethyl acetate soluble fraction was subjected to column chroma-
tography on silica gel (120 g, 75 ꢂ 3 cm) with chloroform containing gradually
increasing amounts of methanol. Fractions of 30 mL were collected, concentrated
under reduced pressure and monitored by TLC using solvent system II.
Similar fractions were grouped together to obtain five main groups, A (800 mg,
eluted with chloroform), B (500 mg, eluted with chloroform–methanol 90 : 10), C (1 g,
eluted with chloroform–methanol 85 : 15), D (700 mg, eluted with chloro-
formꢃmethanol 80 : 20) and E (900 mg, eluted with chloroform–methanol 70 : 30).
Both groups B and C were purified separately by preparative TLC on silica gel layers
with solvent system II to obtain pure compounds 3 (15 mg) and 4 (50 mg), respectively.
Group D contained a crude mixture of compounds 5 and 6. It was subjected to
preparative TLC on silica gel layers using solvent system II to afford pure compounds
5
(15 mg) and 6 (20 mg).
3
.6. GC-MS analysis
ꢀ
ꢃ1
ꢀ
The column oven temperature was rapidly raised at the rate of 40 C min to 190 C
and held at this temperature for 2 min. The temperature was then programmed to
ꢀ
ꢃ1
ꢀ
increase linearly at the rate of 5 C min to 320 C, where it was maintained for
ꢀ
5
min. The temperature for the injector was 280 C. The injected volume was 1 mL.
ꢃ1
The carrier gas used was helium with a split ratio of 1 : 20 and 1.6 mL min constant
flow. Mass spectra were obtained in electron impact (EI) mode with an electron
ꢀ
ꢀ
energy of 70 eV. Ion source and transfer-line temperatures were 180 C and 280 C,
respectively. The mass spectrometer was equipped with a digital alpha station
computer data system. The identification of compounds 1–5 was based on sample

5
0
A.M.A. Abd El-Mawla et al.
retention time data, comparison with the relevant standards and electron
impact–mass spectrometry (EI-MS) data.
3
.7. Acid hydrolysis
Compound 6 (5 mg) was dissolved in 5 mL methanol to which an equal volume of
N/2 methanolic sulphuric acid was added. The mixture was refluxed on a boiling
water bath for 3 h and cooled; the aglycone was extracted with chloroform, purified
and subjected to TLC (using solvent system II). The resultant sugar was identified by
TLC using solvent system III.
3
.8. Spectral data of compounds
Octadecanoic acid methyl ester (methyl stearate) (1): R ¼ 0.75 (solvent system I);
f
þ
R ¼ 18.75 min; EI-MS (70 eV) m/z (relative intensity): 298 [M] for C H O (45),
t
19 38
2
2
(
67 (10), 255 (15), 241 (4), 227 (14), 213 (6), 199 (13), 185 (15), 171 (5), 157 (13), 143
44), 129 (18), 115 (15), 97 (35), 87 (95), 74 (100), 69 (52), 55 (90), 43 (90).
Hexadecanoic acid methyl ester (methyl palmitate) (2): R ¼ 0.60 (solvent system I);
f
þ
R ¼ 18.32 min; EI-MS at (70 eV) m/z (relative intensity): 270 [M] for C H O (40),
t
17 34
2
2
9
39 (10), 227 (14), 213 (2), 199 (4), 185 (5), 171 (5), 157 (2), 143 (17), 129 (8), 115 (2),
7 (5), 87 (73), 83 (6), 74 (100), 69 (12), 55 (15), 43 (15).
Sinapyl alcohol (3): colourless gum; R ¼ 0.60 (solvent system II); R ¼ 18.96 min; UV
f
t
þ
ꢀ (MeOH) nm: 222, 273; EI-MS (70 eV) m/z (relative intensity): 210 [M] for
max
C H O (100), 192 (6), 182 (28), 167 (68), 154 (24), 133 (12), 121 (14), 103 (11),
1
1
14
4
9
1 (9), 77 (14), 65 (6), 55 (8), 39 (7).
Coniferyl alcohol (4): colourless gum; R ¼ 0.58 (solvent system II); R ¼ 16.38 min;
f
t
þ
UV ꢀ (MeOH) nm: 229, 280; EI-MS (70 eV) m/z (relative intensity): 180 [M] for
C H O (82), 162 (4), 147 (9), 137 (100), 131 (13), 124 (47), 119 (22), 103 (13), 91
max
1
0
12
3
(26), 77 (13), 65 (10), 55 (10), 39 (4).
p-Coumaryl alcohol (5): colourless gum; R ¼ 0.41 (solvent system II);
f
R ¼ 18.69 min; UV ꢀ
(MeOH) nm: 226, 273; EI-MS (70 eV) m/z (relative
max
t
þ
intensity): 150 [M] for C H O (100), 132 (10), 121 (15), 103 (9), 91 (6), 77 (10),
9
10
2
6
5 (5), 55 (6), 39 (4).
Coniferyl alcohol 4-O-glucoside (coniferin) (6): colourless needles; R ¼ 0.33 (solvent
f
ꢀ
system II); m.p. 186–188 C; UV ꢀ
[
(MeOH) nm: 228, 280; ESI–MS at m/z: 341
max
–
1
Mꢃ1] for C H O ; H-NMR spectrum (400 MHz, DMSO-d ): ꢁ 3.1–3.3 (4H, m,
16
22
8
6
00
00
00
H-2 -5 ), 3.52 (1H, dd, J ¼ 11.7, 5.5 Hz, H-6 b), 3.72 (1H, dd, J ¼ 11.7, 2.0 Hz,
0
0
0
0
H-6 a), 3.78 (3H, s, OMe), 4.09 (2H, br t, J ¼ 5.0 Hz, H-3 ), 5.43 (1H, d, J ¼ 7.3 Hz,
0
0
0
H-1 ), 6.25 (1H, dt, J ¼ 15.7, 5.0 Hz, H-2 ), 6.50 (1H, d, J ¼ 15.7 Hz, H-1 ), 6.89 (1H,
dd, J ¼ 8.0, 2.2 Hz, H-6), 7.03 (1H, d, J ¼ 8.0 Hz, H-5), 7.05 (1H, d, J ¼ 2.2 Hz, H-2).
Acknowledgements
The authors are grateful to Prof. Dr L. Beerhues (Institute for Pharmaceutical Biology,
Braunschweig University of Technology, Germany) for providing the authentic samples,

Natural Product Research
51
and Prof. Dr L. Ernst and Dr U. Papke (Institute for Organic Chemistry, Braunschweig
University of Technology, Germany) for NMR and ESI-MS measurements, respectively.
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