The investigation of roots of Anthemis cotula (Asteraceae) from cast Serbia revealed, in addition to polyacetylenes previously isolated from the same species, three prenylated 4-hydroxyacetophenones, detected for the first time in any Anthemis species. It is possible that they act as phytoalexins in the plant. From the aerial parts, six linear sesquiterpene lactones (four known and two new ones), as well as two known flavones, apigenin and hispidulin, were isolated.Novo ispitivanje korena biljke A. cotula (Asteraceae) pokazalo je, pored četiri već izolovana poliacetilena, i prisustvo tri prenilovana 4-hidroksiacetofenona koji do sada nisu bili izolovani iz ovog roda. Iz nadzemnog dela biljke izolovano je šest linearnih seskviterpenskih laktona, od koji su dva nova, dok su preostali ranije nađeni u istoj biljci. Takođe su u nadzemnom delu identifikovana dva poznata flavona, apigenin i hispidulin

Janaćković, Peđa T.

Milosavljevic, S

Tešević, Vele

Vajs, Vlatka

Vujisić, Ljubodrag V.

Vučković, Ivan

Journal of the Serbian Chemical Society

English

The investigation of roots of Anthemis cotula (Asteraceae) from east Serbia revealed, in addition to polyacetylenes previously isolated from the same species, three prenylated 4-hydroxyacetophenones, detected for the first time in any Anthemis species. It is possible that they act as phytoalexins in the plant. From the aerial parts, six linear sesquiterpene lactones (four known and two new ones), as well as two known flavones, apigenin and hispidulin, were isolated.Novo ispitivawe korena biqke A. cotula (Asteraceae) pokazalo je, pored ~etiri ve} izolovana poliacetilena, i prisustvo tri prenilovana 4-hidroksiacetofenona koji do sada nisu bili izolovani iz ovog roda. Iz nadzemnog dela biqke izolovano je {est linearnih seskviterpenskih laktona, od koji su dva nova, dok su preostali ranije na|eni u istoj biqci. Tako|e su u nadzemnom delu identifikovana dva poznata flavona, apigenin i hispidulin

Vucković, I

Vujisić, L

Janaćković, P

Milosavljević, Slobodan

 CER - Central Repository of the Institute of Chemistry;  Technology and Metallurgy

J. Serb. Chem. Soc. 71 (2) 127–133 (2006) UDC 582.475:543.9
JSCS – 3406 Original scientific paper
Phytochemical investigation of Anthemis cotula
IVAN VU^KOVI]1#, LJUBODRAG VUJISI]1#, VLATKA VAJS1#, VELE TE[EVI]2#, PEDJA
JANA]KOVI]3 and SLOBODAN MILOSAVLJEVI]2*#
1Institute for Chemistry, Technology and Metallurgy, Njego{eva 12, 11000 Belgrade, 2Faculty of
Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade and 3Faculty of Biology,
University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia and Montenegro (e-mail:
smilo@chem.bg.ac.yu)
(Received 24 May 2005)
Abstract: The investigation of roots of Anthemis cotula (Asteraceae) from east Ser-
bia revealed, in addition to polyacetylenes previously isolated from the same spe-
cies, three prenylated 4-hydroxyacetophenones, detected for the first time in any
Anthemis species. It is possible that they act as phytoalexins in the plant. From the
aerial parts, six linear sesquiterpene lactones (four known and two new ones), as
well as two known flavones, apigenin and hispidulin, were isolated.
Keywords: Asteraceae, Anthemis cotula, polyacetylenes, isopentenylacetopheno-
nes, linear sequiterpene lactones, flavones.
INTRODUCTION
The large genus Anthemis with more than 130 species is widely distributed
over the Mediterranean region. According to the literature, this is one of the best
phytochemically investigated genera of the family Asteraceae (syn. Compositae).
Polyacetylenes, flavonoids and sesquiterpene lactones are the three main classes of
secondary metabolites of the genus.1–4 A. cotula L. is one of nine Anthemis species
that is to be found in Serbia.5 It grows on sandy terrains in the eastern part of the
country.
EXPERIMENTAL
General
The NMR spectra wre recorded using a Varian Gemini 2000 (1H 200 MHz, 13C 50 MHz) in-
strument. The mass spectra were obtained on a Finnigan MAT 8230 (EI, 70 eV and DCI, 150 eV, iso-
butane) and on a VG 70SE (FAB, Cs-ion beam at 20 kV) instruments. The IR spectra were measured
on a Perkin Elmer FTIR Spectrometer 1725X.
127
doi: 10.2298/JSC0602127V
# Serbian Chemical Society active member.
* Corresponding author.
Silica gel (0.063–0.200 mm) was used for column chromatography (CC) and silica gel (<0.063
mm) was used for dry-column flash chromatography. Silica gel G and silica gel F-254 were used for
analytical (0.25 mm) and preparative (0.75 mm) thin layer chromatography (TLC).
Plant material
The plant material was collected during the flowering time near Kladovo in July 2002. A
voucher specimen (AC26052002) was deposited in the Herbarium of the Botanic Garden “Jevre-
movac”, Faculty of Biology, University of Belgrade.
Extraction and isolation
The powdered roots (134 g) were extracted twice with petroleum ether–Et2O (2:1) at room
temperature. The crude extract (600 mg) was subjected to CC on silica gel starting elution with pe-
troleum ether and increasing polarity by adding Et2O up to 50 % to yield 50 fractions (frs
R
1–50).
FrsR5–8 contained 120 mg of 2, frs
R
14–16 gave 20 mg of 1, whereas frs
R
21–23 yielded 20 mg of a mix-
ture of 3 + 4. Prep. TLC (toluene–EtOAc 8:2) of frsR28–29 afforded 3.4 mg of 5. Prep. TLC of
frsR36–40 (toluene–EtOAc 8:2) followed by additional prep. TLC (petroleum ether–Et2O 4:6) af-
forded 6 (2 mg) and 7 (3 mg).
The powdered air-dried aerial parts (1000 g) were extracted twice with petroleum ether–Et2O–MeOH
(1:1:1) at room temperature and then reextracted with MeOH.6 The crude extract ( 30 g) was frac-
tionated by dry-column flash chromatography using petroleum ether–Et2O–MeOH with increasing
polarity to yield 20 fractions (frsA1–20). Frs
A
6–7 (3.18 g) were purified by CC (toluene–EtOAc grad.)
to yield 30 subfractions (sfrs1–30). Srfs8–10 (1.06 g) were subjected to CC (petroleum ether–Et2O
1:2) to afford 120 mg of 8. CC (CH2Cl2–MeOH 95:5) of sfrs11–13 (0.72 g) yielded 47 mg of 11.
FrsA8–9 (1.23 g) were subjected to CC (toluene–EtOAc grad.) and 50 subfractions (sfrs1–50) were ob-
tained. A combination of CC (petroleum ether–Et2O 1:3) and prep. TLC (CH2Cl2–MeOH 95:5) of
sfrs35–39 resulted in the isolation of 10 (14 mg) and 12 (9 mg). Frs
A
11–12 were partly soluble in
CH2Cl2. The soluble part, after CC (petroleum ether–Et2O grad.) and prep. TLC (CH2Cl2–MeOH
95:5, two developments) yielded 9 (26 mg) and 13 (3.7 mg). Prep. TLC of the precipitate
(CH2Cl2–MeOH 95:5, two developments) furnished 14 (4 mg) and 15 (3 mg).
(5R,6R+5S,6S)-Anthecotuloide-5,6-oxide (12): colourless oil; IR film: max: 1764, 1684,
1619, 1270, 817 cm-1; FAB MS m/z (rel. int.): 265 MH+ (100); 1H-NMR in Table I; 13C-NMR in
Table II.
(6R+6S)-6-Hydroxy-4,5-dehydro-5,6-dihydroanthecotuloide (13): colourless oil; IR film: max:
3532, 1765, 1674, 1615, 1115 cm-1; EIMS m/z (rel. int.): 264 M+ (5), 249 M-15+ (5), 98 (20), 83
(100); 1H-NMR in Table I.
RESULTS AND DISCUSSION
The four acetylenes (1 – 4) (Fig. 1) isolated from the roots of A. cotula were al-
ready known constituents of the roots of the species.7,8 Generally, dehydromatrica-
ria ester (1) and its thioether derivatives (e.g. 2 – 4) are characteristic for Anthemis
species.1 Dehydromatricaria ester exhibits significant antimycobacterial9 and alle-
lopathic activities.10
In addition to polyacetylenes, three prenylated acetophenones 5 – 7 were iso-
lated from the roots. Their structures were established by comparison of their spec-
tral data with those reported.11–13 These compounds, common in Asteraceae, have
not hitherto been isolated from any Anthemis species. A previous investigation
showed the presence of a compound with a similar structure, 7-methoxy-6-ace-
tyl-2,2-dimethylchromene, in the aerial parts of A. cotula.14 Isopentenylaceto-
128 VU^KOVI] et al.
PHYTOCHEMICAL INVESTIGATION OF Anthemis cotula 129
Fig. 1. 1. (2Z)-8-dehydromatricaria ester (methyl dec-2Z-en-4,6,8-triynoate); 2. methyl
5-(methylthio)deca-2Z,4E,6Z-trien-8-ynoate; 3. methyl 7-(methylthio)deca-2Z,6E-dien-4,8-diynoate;
4. methyl 7-(methylthio)deca-2Z,6Z-dien-4,8-diynoate; 5. 4-hydroxy-3-isovalerylacetophenone; 6.
4-methoxy-3-isovalerylacetophenone;
7. 2,2-dimethyl-6-acetylchromanone; 8. anthecotuloide; 9. 8-O-dihydroanthecotuloide;
10. 5-oxo-6,13-dehydro-5,6-dihydroanthecotuloide;
11. 5-hydroperoxy-6,13-dehydro-5,6-dihydroanthecotuloide; 12. anthecotuloide-5,6-oxide (new com-
pound); 13. 6-hydroxy-4,5-dehydro-5,6-dihydroanthecotuloide (new compound);
14. apigenin; 15. hispidulin.
phenones, especially benzopyrans (chromenes) and benzofurans, have consider-
able chemotaxonomic and biological significance.15 Several phenylated acetophe-
nones, among them 5 and 7, showed anti-inflammatory activities16 and growth-in-
hibitory activities toward the larvae of the yellow mealworm Tenebrio molitor.17
Additionally, the prenylated acetophenones could act as phytoalexins, which are
low-molecular-weight antimicrobial compounds which accumulate in plants as a
result of infection or stress.18 They can be chemically diverse: phenylpropanoids,
(iso)flavonoids, sesquiterpenes and polyketides.19 Acetophenone-related phyto-
alexins are rare; only a single species from the family Asteraceae, Polymnia
sonchifolia, exhibits this type of phytoalexins. It has been shown that 4-hydro-
xy-3-isovalerylacetophenone (5) and two similar acetophenone derivatives are
synthesized in the tubers of Polymnia sonchifolia after inoculation with Pseudo-
monas cichorii.20 Since previous investigations of the root of A. cotula from other
localities did not show the presence of acetophenones 5 – 7, it is possible that their
biosynthesis was induced by some external endangering factor, i.e., that they act as
phytoalexins. Further investigation is required to confirm this assumption.
TABLE I. 1H-NMR spectral data of lactones 11 – 13 (200 MHz, CDCl3) (chemical shift in ppm,
multiplicity, coupling constant in Hz)
H 11 12 13
3 3.22 m 3.35 m 3.68 m
4
1.70–1.90 1.60–2.00
5.60/5.56 dd (15.0, 7.2)
4’ –
5 4.46/4.44 dd (10.0, 5.0) 2.84/2.81 dd (6.2, 1.6/4.8, 2.0) 5.74/5.77 d (15.0)
7
3.31 s
2.98/2.93 d (16.4) 2.74 d (17.2)
7’ 2.39/2.35 d (16.4) 2.62 d (17.2)
9 6.18 m 6.06 m 6.01 m
11 1.96 br s 1.91 d (1.4) 1.92 br s
12 2.20 br s 2.16 d (2.8) 2.15 br s
13 5.39 s 1.34 s 1.30/1.31 s
13’ 5.16 s – –
14 4.56/4.54 dd (9.0, 8.0) 4.61 dd (9.2, 8.0) 4.50/4.490 dd (9.0, 9.0)
14’ 4.05/4.02 dd (9.0, 6.0) 4.14 dd (9.2, 6.0) 3.98 dd (9.0, 7.6)
15 6.29/6.23 d (2.8) 6.34/6.22 d (2.8) 6.28/6.29 d (3.0)
15’ 5.65/5.66 d (2.6) 5.14/5.68 d (2.6) 5.53/5.56 d (2.6)
–OH – – 4.71/4.68 s
–OOH 11.02/11.01 s – –
From the aerial parts of A. cotula, six linear sesquiterpene lactones were iso-
lated. The known compounds were identified by comparison of their 1H NMR
spectral data with those reported as: anthecotuloide (8),21 8-O-dihydroanthecotu-
130 VU^KOVI] et al.
} }
}
loide (9),14 5-oxo-6,13-dehydro-5,6-dihydroanthecotuloide (10)14 and 5-hydro-
peroxy-6,13-dehydro-5,6-dihydroanthecotuloide (11).22 The structures of 8 – 11
were also confirmed by 13C NMR spectroscopy (Table II). Anthecotuloide (8) is
considered to be one of the most potent contact allergens.23
The 1H-NMR spectrum of compound 12 was rather similar to that of 8. The most
significant difference between the spectra of these lactones was the absence of a
low-field resonance of a vinyl proton (H-5), occurring in the spectrum of 8 at  5.21. In-
stead of this, a one-proton signal was observed at   2.8 ppm in 1H NMR spectrum of
12 (Table I), indicating the presence of an epoxy function. The FAB mass spectrum ex-
hibiting the MH+ ion at m/z 265, compatible with the molecular formula C15H20O4,
was also in accordance with an additional (epoxy) oxygen in 12. The 13C NMR spec-
trum (Table II) confirmed the latter. The observed doubling of the signals led to the con-
clusion that 12 was a (5R,6R + 5S,6S) diastereomeric mixture in a ratio of ca. 1:1, which
could be formed by epoxidation of the 5 double bond of anthecotuloide. Thus, the
structure of anthecotuloide-5,6-oxide could be assigned to 12.
TABLE II. 13C NMR spectral data of lactones 9 –12 (50 MHz, CDCl3)
C 8 9 10 11 12
1 170.1 170.8 170.4 170.5
170
2 137.4 137.9 137.6 138.0/137.1 137.8
3 37.9 38.6 42.1 35.5/36.8* 37.4/36.8
4 31.4 31.9 34.2 35.4/34.9* 32.4/32.3
5 123.5 122.6 196.7 86.1/87.4 60.7/60.4
6 133.8 135.7 143.1 140.4/140.7 58.1/58.4
7 54.4 47.9 46.6 46.1 53.2/53.3
8 197.9 66.2 198.3 200.4 197.1
9 122.3 127.5 122.5 122.5/122.3* 123.5
10 155.5 135.2 157.2 160.0 157.3
11 26.9 25.7 27.7 28.0 27.8
12 20.0 18.1 20.8 21.3 20.9
13 16.1 16.6 127.5 121.3/121.4* 17.6
14 69.9 70.5 71.4 71.9/70.7 70.6/70.9
15 121.5 122.1 122.9 122.8 122.3/122.7
* The assignments can be interchanged.
The molecular formula of 13, derived from the EIMS, was identical to that of
12 (C15H20O4). The doubling of the signals in the
1H NMR spectrum (Table I)
showed the presence of two diastereomeric forms, this time in the ratio of ca. 1:1.4.
The relative intensity of the low-field signals in the range of  5.45 – 5.82 indicated
the presence of three vinyl protons, one of them assigned as H-15’. A low-field pair
PHYTOCHEMICAL INVESTIGATION OF Anthemis cotula 131
of one-proton doublets (J = 15.0 Hz), at  5.74 and 5.77, in the ratio of 1.4/1, respec-
tively, could be assigned as H-5, coupled vicinally to the E-positioned vinyl proton
(H-4) resonating at a slightly higher field (m,  ca., 5.45 – 5.70). The assignment of
the signal of H-4 was based on its simplification upon irradiating a multiplet ( 3.68)
identified as H-3. In addition, the signals of the allylic methyl (H3-14), observed in 8
at  1.62, were replaced by a pair of three-proton singlets ( 1.30 and 1.31, in the ra-
tio of 1/1.4, respectively) typical for methyls attached to a carbinol carbon (C-6).
This, together with the similarity of the remaining 1H NMR signals (Table I) to those
of 8, led to the structure of 6-hydroxy-4,5-(E)-dehydro-5,6-dihydroanthecotuloide
for this lactone, consisting of a (6R + 6S)-epimeric mixture.
In addition, two flavones, apigenin (14) and hispidulin (15) were isolated from
the aerial parts of A. cotula. This type of flavones is characteristic for the members
of the genus Anthemis.2
Acknowledgements: The authors acknowledge their gratitude to the Ministry for Science and
Environmental Protection of Serbia (Project number 140253).
I Z V O D
FITOHEMIJSKO ISPITIVAWE BIQNE VRSTE Anthemis cotula
IVAN VU^KOVI]
1
, QUBODRAG VUJISI]
1
, VLATKA VAJS
1
, VELE TE[EVI]
2
, PE\A
JANA]KOVI]
3
i SLOBODAN MILOSAVQEVI]
2
1
Institut za hemiju, tehnologiju i metalurgiju, Wego{eva 12, 11000 Beograd,
2
Hemijski fakultet,
Univerzitet u Beogradu, Studentski trg 16, 11000 Beograd i
3
Biolo{ki fakultet, Univerzitet u
Beogradu, Studentski trg 16, 11000 Beograd
Novo ispitivawe korena biqke A. cotula (Asteraceae) pokazalo je, pored ~etiri ve}
izolovana poliacetilena, i prisustvo tri prenilovana 4-hidroksiacetofenona koji
do sada nisu bili izolovani iz ovog roda. Iz nadzemnog dela biqke izolovano je {est
linearnih seskviterpenskih laktona, od koji su dva nova, dok su preostali ranije
na|eni u istoj biqci. Tako|e su u nadzemnom delu identifikovana dva poznata flavo-
na, apigenin i hispidulin.
(Primqeno 24. maja 2005)
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20. M. Takasugi, T. Masuda, Phytochemistry 43 (1996) 1019
21. F. Bohlmann, C. Zdero, M. Lonitz, Tetrahedron Lett. 28 (1969) 2417
22. J. D. Staneva, M. N. Todorova, Lj. N. Evstatieva, Biochem. Syst. Ecol. 33 (2005) 97
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PHYTOCHEMICAL INVESTIGATION OF Anthemis cotula 133


Фитохемијско испитивање биљне врсте Anthemis cotula

Ivan Vuckovic

Ljubodrag Vujisic

Vlatka Vajs

Vele Tesevic

Pedja Janackovic

Slobodan Milosavljevic

Crossref

Phytochemical investigation of Anthemis cotula

Central Repository of the Institute of Chemistry, Technology and Metallurgy (CER)

J. Serb. Chem. Soc. 71 (2) 127–133 (2006) UDC 582.475:543.9JSCS – 3406 Original scientific paperPhytochemical investigation of Anthemis cotulaIVAN VU^KOVI]1#, LJUBODRAG VUJISI]1#, VLATKA VAJS1#, VELE TE[EVI]2#, PEDJAJANA]KOVI]3 and SLOBODAN MILOSAVLJEVI]2*#1Institute for Chemistry, Technology and Metallurgy, Njego{eva 12, 11000 Belgrade, 2Faculty ofChemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade and 3Faculty of Biology,University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia and Montenegro (e-mail:smilo@chem.bg.ac.yu)(Received 24 May 2005)Abstract: The investigation of roots of Anthemis cotula (Asteraceae) from east Ser-bia revealed, in addition to polyacetylenes previously isolated from the same spe-cies, three prenylated 4-hydroxyacetophenones, detected for the first time in anyAnthemis species. It is possible that they act as phytoalexins in the plant. From theaerial parts, six linear sesquiterpene lactones (four known and two new ones), aswell as two known flavones, apigenin and hispidulin, were isolated.Keywords: Asteraceae, Anthemis cotula, polyacetylenes, isopentenylacetopheno-nes, linear sequiterpene lactones, flavones.INTRODUCTIONThe large genus Anthemis with more than 130 species is widely distributedover the Mediterranean region. According to the literature, this is one of the bestphytochemically investigated genera of the family Asteraceae (syn. Compositae).Polyacetylenes, flavonoids and sesquiterpene lactones are the three main classes ofsecondary metabolites of the genus.1–4 A. cotula L. is one of nine Anthemis speciesthat is to be found in Serbia.5 It grows on sandy terrains in the eastern part of thecountry.EXPERIMENTALGeneralThe NMR spectra wre recorded using a Varian Gemini 2000 (1H 200 MHz, 13C 50 MHz) in-strument. The mass spectra were obtained on a Finnigan MAT 8230 (EI, 70 eV and DCI, 150 eV, iso-butane) and on a VG 70SE (FAB, Cs-ion beam at 20 kV) instruments. The IR spectra were measuredon a Perkin Elmer FTIR Spectrometer 1725X.127doi: 10.2298/JSC0602127V# Serbian Chemical Society active member.* Corresponding author.Silica gel (0.063–0.200 mm) was used for column chromatography (CC) and silica gel (<0.063mm) was used for dry-column flash chromatography. Silica gel G and silica gel F-254 were used foranalytical (0.25 mm) and preparative (0.75 mm) thin layer chromatography (TLC).Plant materialThe plant material was collected during the flowering time near Kladovo in July 2002. Avoucher specimen (AC26052002) was deposited in the Herbarium of the Botanic Garden “Jevre-movac”, Faculty of Biology, University of Belgrade.Extraction and isolationThe powdered roots (134 g) were extracted twice with petroleum ether–Et2O (2:1) at roomtemperature. The crude extract (600 mg) was subjected to CC on silica gel starting elution with pe-troleum ether and increasing polarity by adding Et2O up to 50 % to yield 50 fractions (frsR1–50).FrsR5–8 contained 120 mg of 2, frsR14–16 gave 20 mg of 1, whereas frsR21–23 yielded 20 mg of a mix-ture of 3 + 4. Prep. TLC (toluene–EtOAc 8:2) of frsR28–29 afforded 3.4 mg of 5. Prep. TLC offrsR36–40 (toluene–EtOAc 8:2) followed by additional prep. TLC (petroleum ether–Et2O 4:6) af-forded 6 (2 mg) and 7 (3 mg).The powdered air-dried aerial parts (1000 g) were extracted twice with petroleum ether–Et2O–MeOH(1:1:1) at room temperature and then reextracted with MeOH.6 The crude extract ( 30 g) was frac-tionated by dry-column flash chromatography using petroleum ether–Et2O–MeOH with increasingpolarity to yield 20 fractions (frsA1–20). FrsA6–7 (3.18 g) were purified by CC (toluene–EtOAc grad.)to yield 30 subfractions (sfrs1–30). Srfs8–10 (1.06 g) were subjected to CC (petroleum ether–Et2O1:2) to afford 120 mg of 8. CC (CH2Cl2–MeOH 95:5) of sfrs11–13 (0.72 g) yielded 47 mg of 11.FrsA8–9 (1.23 g) were subjected to CC (toluene–EtOAc grad.) and 50 subfractions (sfrs1–50) were ob-tained. A combination of CC (petroleum ether–Et2O 1:3) and prep. TLC (CH2Cl2–MeOH 95:5) ofsfrs35–39 resulted in the isolation of 10 (14 mg) and 12 (9 mg). FrsA11–12 were partly soluble inCH2Cl2. The soluble part, after CC (petroleum ether–Et2O grad.) and prep. TLC (CH2Cl2–MeOH95:5, two developments) yielded 9 (26 mg) and 13 (3.7 mg). Prep. TLC of the precipitate(CH2Cl2–MeOH 95:5, two developments) furnished 14 (4 mg) and 15 (3 mg).(5R,6R+5S,6S)-Anthecotuloide-5,6-oxide (12): colourless oil; IR film: max: 1764, 1684,1619, 1270, 817 cm-1; FAB MS m/z (rel. int.): 265 MH+ (100); 1H-NMR in Table I; 13C-NMR inTable II.(6R+6S)-6-Hydroxy-4,5-dehydro-5,6-dihydroanthecotuloide (13): colourless oil; IR film: max:3532, 1765, 1674, 1615, 1115 cm-1; EIMS m/z (rel. int.): 264 M+ (5), 249 M-15+ (5), 98 (20), 83(100); 1H-NMR in Table I.RESULTS AND DISCUSSIONThe four acetylenes (1 – 4) (Fig. 1) isolated from the roots of A. cotula were al-ready known constituents of the roots of the species.7,8 Generally, dehydromatrica-ria ester (1) and its thioether derivatives (e.g. 2 – 4) are characteristic for Anthemisspecies.1 Dehydromatricaria ester exhibits significant antimycobacterial9 and alle-lopathic activities.10In addition to polyacetylenes, three prenylated acetophenones 5 – 7 were iso-lated from the roots. Their structures were established by comparison of their spec-tral data with those reported.11–13 These compounds, common in Asteraceae, havenot hitherto been isolated from any Anthemis species. A previous investigationshowed the presence of a compound with a similar structure, 7-methoxy-6-ace-tyl-2,2-dimethylchromene, in the aerial parts of A. cotula.14 Isopentenylaceto-128 VU^KOVI] et al.PHYTOCHEMICAL INVESTIGATION OF Anthemis cotula 129Fig. 1. 1. (2Z)-8-dehydromatricaria ester (methyl dec-2Z-en-4,6,8-triynoate); 2. methyl5-(methylthio)deca-2Z,4E,6Z-trien-8-ynoate; 3. methyl 7-(methylthio)deca-2Z,6E-dien-4,8-diynoate;4. methyl 7-(methylthio)deca-2Z,6Z-dien-4,8-diynoate; 5. 4-hydroxy-3-isovalerylacetophenone; 6.4-methoxy-3-isovalerylacetophenone;7. 2,2-dimethyl-6-acetylchromanone; 8. anthecotuloide; 9. 8-O-dihydroanthecotuloide;10. 5-oxo-6,13-dehydro-5,6-dihydroanthecotuloide;11. 5-hydroperoxy-6,13-dehydro-5,6-dihydroanthecotuloide; 12. anthecotuloide-5,6-oxide (new com-pound); 13. 6-hydroxy-4,5-dehydro-5,6-dihydroanthecotuloide (new compound);14. apigenin; 15. hispidulin.phenones, especially benzopyrans (chromenes) and benzofurans, have consider-able chemotaxonomic and biological significance.15 Several phenylated acetophe-nones, among them 5 and 7, showed anti-inflammatory activities16 and growth-in-hibitory activities toward the larvae of the yellow mealworm Tenebrio molitor.17Additionally, the prenylated acetophenones could act as phytoalexins, which arelow-molecular-weight antimicrobial compounds which accumulate in plants as aresult of infection or stress.18 They can be chemically diverse: phenylpropanoids,(iso)flavonoids, sesquiterpenes and polyketides.19 Acetophenone-related phyto-alexins are rare; only a single species from the family Asteraceae, Polymniasonchifolia, exhibits this type of phytoalexins. It has been shown that 4-hydro-xy-3-isovalerylacetophenone (5) and two similar acetophenone derivatives aresynthesized in the tubers of Polymnia sonchifolia after inoculation with Pseudo-monas cichorii.20 Since previous investigations of the root of A. cotula from otherlocalities did not show the presence of acetophenones 5 – 7, it is possible that theirbiosynthesis was induced by some external endangering factor, i.e., that they act asphytoalexins. Further investigation is required to confirm this assumption.TABLE I. 1H-NMR spectral data of lactones 11 – 13 (200 MHz, CDCl3) (chemical shift in ppm,multiplicity, coupling constant in Hz)H 11 12 133 3.22 m 3.35 m 3.68 m41.70–1.90 1.60–2.005.60/5.56 dd (15.0, 7.2)4’ –5 4.46/4.44 dd (10.0, 5.0) 2.84/2.81 dd (6.2, 1.6/4.8, 2.0) 5.74/5.77 d (15.0)73.31 s2.98/2.93 d (16.4) 2.74 d (17.2)7’ 2.39/2.35 d (16.4) 2.62 d (17.2)9 6.18 m 6.06 m 6.01 m11 1.96 br s 1.91 d (1.4) 1.92 br s12 2.20 br s 2.16 d (2.8) 2.15 br s13 5.39 s 1.34 s 1.30/1.31 s13’ 5.16 s – –14 4.56/4.54 dd (9.0, 8.0) 4.61 dd (9.2, 8.0) 4.50/4.490 dd (9.0, 9.0)14’ 4.05/4.02 dd (9.0, 6.0) 4.14 dd (9.2, 6.0) 3.98 dd (9.0, 7.6)15 6.29/6.23 d (2.8) 6.34/6.22 d (2.8) 6.28/6.29 d (3.0)15’ 5.65/5.66 d (2.6) 5.14/5.68 d (2.6) 5.53/5.56 d (2.6)–OH – – 4.71/4.68 s–OOH 11.02/11.01 s – –From the aerial parts of A. cotula, six linear sesquiterpene lactones were iso-lated. The known compounds were identified by comparison of their 1H NMRspectral data with those reported as: anthecotuloide (8),21 8-O-dihydroanthecotu-130 VU^KOVI] et al.} }}loide (9),14 5-oxo-6,13-dehydro-5,6-dihydroanthecotuloide (10)14 and 5-hydro-peroxy-6,13-dehydro-5,6-dihydroanthecotuloide (11).22 The structures of 8 – 11were also confirmed by 13C NMR spectroscopy (Table II). Anthecotuloide (8) isconsidered to be one of the most potent contact allergens.23The 1H-NMR spectrum of compound 12 was rather similar to that of 8. The mostsignificant difference between the spectra of these lactones was the absence of alow-field resonance of a vinyl proton (H-5), occurring in the spectrum of 8 at  5.21. In-stead of this, a one-proton signal was observed at   2.8 ppm in 1H NMR spectrum of12 (Table I), indicating the presence of an epoxy function. The FAB mass spectrum ex-hibiting the MH+ ion at m/z 265, compatible with the molecular formula C15H20O4,was also in accordance with an additional (epoxy) oxygen in 12. The 13C NMR spec-trum (Table II) confirmed the latter. The observed doubling of the signals led to the con-clusion that 12 was a (5R,6R + 5S,6S) diastereomeric mixture in a ratio of ca. 1:1, whichcould be formed by epoxidation of the 5 double bond of anthecotuloide. Thus, thestructure of anthecotuloide-5,6-oxide could be assigned to 12.TABLE II. 13C NMR spectral data of lactones 9 –12 (50 MHz, CDCl3)C 8 9 10 11 121 170.1 170.8 170.4 170.51702 137.4 137.9 137.6 138.0/137.1 137.83 37.9 38.6 42.1 35.5/36.8* 37.4/36.84 31.4 31.9 34.2 35.4/34.9* 32.4/32.35 123.5 122.6 196.7 86.1/87.4 60.7/60.46 133.8 135.7 143.1 140.4/140.7 58.1/58.47 54.4 47.9 46.6 46.1 53.2/53.38 197.9 66.2 198.3 200.4 197.19 122.3 127.5 122.5 122.5/122.3* 123.510 155.5 135.2 157.2 160.0 157.311 26.9 25.7 27.7 28.0 27.812 20.0 18.1 20.8 21.3 20.913 16.1 16.6 127.5 121.3/121.4* 17.614 69.9 70.5 71.4 71.9/70.7 70.6/70.915 121.5 122.1 122.9 122.8 122.3/122.7* The assignments can be interchanged.The molecular formula of 13, derived from the EIMS, was identical to that of12 (C15H20O4). The doubling of the signals in the1H NMR spectrum (Table I)showed the presence of two diastereomeric forms, this time in the ratio of ca. 1:1.4.The relative intensity of the low-field signals in the range of  5.45 – 5.82 indicatedthe presence of three vinyl protons, one of them assigned as H-15’. A low-field pairPHYTOCHEMICAL INVESTIGATION OF Anthemis cotula 131of one-proton doublets (J = 15.0 Hz), at  5.74 and 5.77, in the ratio of 1.4/1, respec-tively, could be assigned as H-5, coupled vicinally to the E-positioned vinyl proton(H-4) resonating at a slightly higher field (m,  ca., 5.45 – 5.70). The assignment ofthe signal of H-4 was based on its simplification upon irradiating a multiplet ( 3.68)identified as H-3. In addition, the signals of the allylic methyl (H3-14), observed in 8at  1.62, were replaced by a pair of three-proton singlets ( 1.30 and 1.31, in the ra-tio of 1/1.4, respectively) typical for methyls attached to a carbinol carbon (C-6).This, together with the similarity of the remaining 1H NMR signals (Table I) to thoseof 8, led to the structure of 6-hydroxy-4,5-(E)-dehydro-5,6-dihydroanthecotuloidefor this lactone, consisting of a (6R + 6S)-epimeric mixture.In addition, two flavones, apigenin (14) and hispidulin (15) were isolated fromthe aerial parts of A. cotula. This type of flavones is characteristic for the membersof the genus Anthemis.2Acknowledgements: The authors acknowledge their gratitude to the Ministry for Science andEnvironmental Protection of Serbia (Project number 140253).I Z V O DFITOHEMIJSKO ISPITIVAWE BIQNE VRSTE Anthemis cotulaIVAN VU^KOVI]1, QUBODRAG VUJISI]1, VLATKA VAJS1, VELE TE[EVI]2, PE\AJANA]KOVI]3i SLOBODAN MILOSAVQEVI]21Institut za hemiju, tehnologiju i metalurgiju, Wego{eva 12, 11000 Beograd,2Hemijski fakultet,Univerzitet u Beogradu, Studentski trg 16, 11000 Beograd i3Biolo{ki fakultet, Univerzitet uBeogradu, Studentski trg 16, 11000 BeogradNovo ispitivawe korena biqke A. cotula (Asteraceae) pokazalo je, pored ~etiri ve}izolovana poliacetilena, i prisustvo tri prenilovana 4-hidroksiacetofenona kojido sada nisu bili izolovani iz ovog roda. Iz nadzemnog dela biqke izolovano je {estlinearnih seskviterpenskih laktona, od koji su dva nova, dok su preostali ranijena|eni u istoj biqci. Tako|e su u nadzemnom delu identifikovana dva poznata flavo-na, apigenin i hispidulin.(Primqeno 24. maja 2005)REFERENCES1. L. P. Christensen, Phytochemistry 31 (1992) 72. C. A. Williams, J. Greenham, J. B. Harborne, Biochem. Syst. Ecol. 29 (2001) 9293. V. Vajs, V. Bulatovic, K. Fodulovic-Savikin, N. Menkovic, S. Macura, N. Juranic, S. Milosav-ljevic, Phytochemistry 2 (1999) 2874. M. Bruno, S Rosselli, M. L. Bondian, T. E. Gedris, W. Herz, Biochem. Syst. Ecol. 9 (2002) 8915. M. Josifovi} Ed., in Flora of Serbia VII, SANU, Belgrade, 1975, p. 876. F. Bohlmann, C. Zdero, R. M. King, H. Robinson, Phytochemistry 23 (1984) 19797. F. Bohlmann, K.-M. Kleine, C. Arndt, S. Köhn, Chem. Ber. 98 (1965) 16168. F. Bohlmann, T. Burkhardt, C. Zdero, in Naturally occurring acetylenes, Academic Press, Lon-don, 1973, p. 3839. T. Lu, C. L. Cantrell, S. L. Robbs, S. G. Franzblau, N. H. Fischer, Planta Med. 64 (1998) 66510. T. Rong, E. Morifusa, Chemosphere 32 (1996) 130711. F. Bohlmann, N. Rao, Chem. Ber. 106 (1973) 3035132 VU^KOVI] et al.12. F. Bohlmann, M. Grenz, Chem. Ber. 110 (1977) 29513. F. Bohlmann, C. Zdero, M. Lonitz, Phytochemistry 16 (1977) 57514. R. N. Baruah, F. Bohlmann, R. M. King, Planta Med. 6 (1985) 53115. P. Proksch, E. Rodriguez, Phytochemistry 22 (1983) 233516. L. Favier, C. Tonn, E. Guerreiro, A. Rotelli, L. Pelzer, Planta Med. 64 (1998) 65717. R. F. Carrizo, M. E. Sosa, L. S. Favier, F. Penna, E. Guerreiro, O. S. Giordano, C. E. Tonn, J. Nat.Prod. 61 (1998) 120918. J. Ku}, Annual Review of Phytopathology 33 (1995) 27519. J. B. Harborne, Biochem.Syst. Ecol. 27 (1999) 33520. M. Takasugi, T. Masuda, Phytochemistry 43 (1996) 101921. F. Bohlmann, C. Zdero, M. Lonitz, Tetrahedron Lett. 28 (1969) 241722. J. D. Staneva, M. N. Todorova, Lj. N. Evstatieva, Biochem. Syst. Ecol. 33 (2005) 9723. B. M. Hausen, E. Busker, R. Carle, Planta Med. 50 (1984) 229.PHYTOCHEMICAL INVESTIGATION OF Anthemis cotula 133

Faculty of Chemistry Repository - Cherry

J. Serb. Chem. Soc. 71 (2) 127–133 (2006) UDC 582.475:543.9JSCS – 3406 Original scientific paperPhytochemical investigation of Anthemis cotulaIVAN VU^KOVI]1#, LJUBODRAG VUJISI]1#, VLATKA VAJS1#, VELE TE[EVI]2#, PEDJAJANA]KOVI]3 and SLOBODAN MILOSAVLJEVI]2*#1Institute for Chemistry, Technology and Metallurgy, Njego{eva 12, 11000 Belgrade, 2Faculty ofChemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade and 3Faculty of Biology,University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia and Montenegro (e-mail:smilo@chem.bg.ac.yu)(Received 24 May 2005)Abstract: The investigation of roots of Anthemis cotula (Asteraceae) from east Ser-bia revealed, in addition to polyacetylenes previously isolated from the same spe-cies, three prenylated 4-hydroxyacetophenones, detected for the first time in anyAnthemis species. It is possible that they act as phytoalexins in the plant. From theaerial parts, six linear sesquiterpene lactones (four known and two new ones), aswell as two known flavones, apigenin and hispidulin, were isolated.Keywords: Asteraceae, Anthemis cotula, polyacetylenes, isopentenylacetopheno-nes, linear sequiterpene lactones, flavones.INTRODUCTIONThe large genus Anthemis with more than 130 species is widely distributedover the Mediterranean region. According to the literature, this is one of the bestphytochemically investigated genera of the family Asteraceae (syn. Compositae).Polyacetylenes, flavonoids and sesquiterpene lactones are the three main classes ofsecondary metabolites of the genus.1–4 A. cotula L. is one of nine Anthemis speciesthat is to be found in Serbia.5 It grows on sandy terrains in the eastern part of thecountry.EXPERIMENTALGeneralThe NMR spectra wre recorded using a Varian Gemini 2000 (1H 200 MHz, 13C 50 MHz) in-strument. The mass spectra were obtained on a Finnigan MAT 8230 (EI, 70 eV and DCI, 150 eV, iso-butane) and on a VG 70SE (FAB, Cs-ion beam at 20 kV) instruments. The IR spectra were measuredon a Perkin Elmer FTIR Spectrometer 1725X.127doi: 10.2298/JSC0602127V# Serbian Chemical Society active member.* Corresponding author.Silica gel (0.063–0.200 mm) was used for column chromatography (CC) and silica gel (<0.063mm) was used for dry-column flash chromatography. Silica gel G and silica gel F-254 were used foranalytical (0.25 mm) and preparative (0.75 mm) thin layer chromatography (TLC).Plant materialThe plant material was collected during the flowering time near Kladovo in July 2002. Avoucher specimen (AC26052002) was deposited in the Herbarium of the Botanic Garden “Jevre-movac”, Faculty of Biology, University of Belgrade.Extraction and isolationThe powdered roots (134 g) were extracted twice with petroleum ether–Et2O (2:1) at roomtemperature. The crude extract (600 mg) was subjected to CC on silica gel starting elution with pe-troleum ether and increasing polarity by adding Et2O up to 50 % to yield 50 fractions (frsR1–50).FrsR5–8 contained 120 mg of 2, frsR14–16 gave 20 mg of 1, whereas frsR21–23 yielded 20 mg of a mix-ture of 3 + 4. Prep. TLC (toluene–EtOAc 8:2) of frsR28–29 afforded 3.4 mg of 5. Prep. TLC offrsR36–40 (toluene–EtOAc 8:2) followed by additional prep. TLC (petroleum ether–Et2O 4:6) af-forded 6 (2 mg) and 7 (3 mg).The powdered air-dried aerial parts (1000 g) were extracted twice with petroleum ether–Et2O–MeOH(1:1:1) at room temperature and then reextracted with MeOH.6 The crude extract ( 30 g) was frac-tionated by dry-column flash chromatography using petroleum ether–Et2O–MeOH with increasingpolarity to yield 20 fractions (frsA1–20). FrsA6–7 (3.18 g) were purified by CC (toluene–EtOAc grad.)to yield 30 subfractions (sfrs1–30). Srfs8–10 (1.06 g) were subjected to CC (petroleum ether–Et2O1:2) to afford 120 mg of 8. CC (CH2Cl2–MeOH 95:5) of sfrs11–13 (0.72 g) yielded 47 mg of 11.FrsA8–9 (1.23 g) were subjected to CC (toluene–EtOAc grad.) and 50 subfractions (sfrs1–50) were ob-tained. A combination of CC (petroleum ether–Et2O 1:3) and prep. TLC (CH2Cl2–MeOH 95:5) ofsfrs35–39 resulted in the isolation of 10 (14 mg) and 12 (9 mg). FrsA11–12 were partly soluble inCH2Cl2. The soluble part, after CC (petroleum ether–Et2O grad.) and prep. TLC (CH2Cl2–MeOH95:5, two developments) yielded 9 (26 mg) and 13 (3.7 mg). Prep. TLC of the precipitate(CH2Cl2–MeOH 95:5, two developments) furnished 14 (4 mg) and 15 (3 mg).(5R,6R+5S,6S)-Anthecotuloide-5,6-oxide (12): colourless oil; IR film: max: 1764, 1684,1619, 1270, 817 cm-1; FAB MS m/z (rel. int.): 265 MH+ (100); 1H-NMR in Table I; 13C-NMR inTable II.(6R+6S)-6-Hydroxy-4,5-dehydro-5,6-dihydroanthecotuloide (13): colourless oil; IR film: max:3532, 1765, 1674, 1615, 1115 cm-1; EIMS m/z (rel. int.): 264 M+ (5), 249 M-15+ (5), 98 (20), 83(100); 1H-NMR in Table I.RESULTS AND DISCUSSIONThe four acetylenes (1 – 4) (Fig. 1) isolated from the roots of A. cotula were al-ready known constituents of the roots of the species.7,8 Generally, dehydromatrica-ria ester (1) and its thioether derivatives (e.g. 2 – 4) are characteristic for Anthemisspecies.1 Dehydromatricaria ester exhibits significant antimycobacterial9 and alle-lopathic activities.10In addition to polyacetylenes, three prenylated acetophenones 5 – 7 were iso-lated from the roots. Their structures were established by comparison of their spec-tral data with those reported.11–13 These compounds, common in Asteraceae, havenot hitherto been isolated from any Anthemis species. A previous investigationshowed the presence of a compound with a similar structure, 7-methoxy-6-ace-tyl-2,2-dimethylchromene, in the aerial parts of A. cotula.14 Isopentenylaceto-128 VU^KOVI] et al.PHYTOCHEMICAL INVESTIGATION OF Anthemis cotula 129Fig. 1. 1. (2Z)-8-dehydromatricaria ester (methyl dec-2Z-en-4,6,8-triynoate); 2. methyl5-(methylthio)deca-2Z,4E,6Z-trien-8-ynoate; 3. methyl 7-(methylthio)deca-2Z,6E-dien-4,8-diynoate;4. methyl 7-(methylthio)deca-2Z,6Z-dien-4,8-diynoate; 5. 4-hydroxy-3-isovalerylacetophenone; 6.4-methoxy-3-isovalerylacetophenone;7. 2,2-dimethyl-6-acetylchromanone; 8. anthecotuloide; 9. 8-O-dihydroanthecotuloide;10. 5-oxo-6,13-dehydro-5,6-dihydroanthecotuloide;11. 5-hydroperoxy-6,13-dehydro-5,6-dihydroanthecotuloide; 12. anthecotuloide-5,6-oxide (new com-pound); 13. 6-hydroxy-4,5-dehydro-5,6-dihydroanthecotuloide (new compound);14. apigenin; 15. hispidulin.phenones, especially benzopyrans (chromenes) and benzofurans, have consider-able chemotaxonomic and biological significance.15 Several phenylated acetophe-nones, among them 5 and 7, showed anti-inflammatory activities16 and growth-in-hibitory activities toward the larvae of the yellow mealworm Tenebrio molitor.17Additionally, the prenylated acetophenones could act as phytoalexins, which arelow-molecular-weight antimicrobial compounds which accumulate in plants as aresult of infection or stress.18 They can be chemically diverse: phenylpropanoids,(iso)flavonoids, sesquiterpenes and polyketides.19 Acetophenone-related phyto-alexins are rare; only a single species from the family Asteraceae, Polymniasonchifolia, exhibits this type of phytoalexins. It has been shown that 4-hydro-xy-3-isovalerylacetophenone (5) and two similar acetophenone derivatives aresynthesized in the tubers of Polymnia sonchifolia after inoculation with Pseudo-monas cichorii.20 Since previous investigations of the root of A. cotula from otherlocalities did not show the presence of acetophenones 5 – 7, it is possible that theirbiosynthesis was induced by some external endangering factor, i.e., that they act asphytoalexins. Further investigation is required to confirm this assumption.TABLE I. 1H-NMR spectral data of lactones 11 – 13 (200 MHz, CDCl3) (chemical shift in ppm,multiplicity, coupling constant in Hz)H 11 12 133 3.22 m 3.35 m 3.68 m41.70–1.90 1.60–2.005.60/5.56 dd (15.0, 7.2)4’ –5 4.46/4.44 dd (10.0, 5.0) 2.84/2.81 dd (6.2, 1.6/4.8, 2.0) 5.74/5.77 d (15.0)73.31 s2.98/2.93 d (16.4) 2.74 d (17.2)7’ 2.39/2.35 d (16.4) 2.62 d (17.2)9 6.18 m 6.06 m 6.01 m11 1.96 br s 1.91 d (1.4) 1.92 br s12 2.20 br s 2.16 d (2.8) 2.15 br s13 5.39 s 1.34 s 1.30/1.31 s13’ 5.16 s – –14 4.56/4.54 dd (9.0, 8.0) 4.61 dd (9.2, 8.0) 4.50/4.490 dd (9.0, 9.0)14’ 4.05/4.02 dd (9.0, 6.0) 4.14 dd (9.2, 6.0) 3.98 dd (9.0, 7.6)15 6.29/6.23 d (2.8) 6.34/6.22 d (2.8) 6.28/6.29 d (3.0)15’ 5.65/5.66 d (2.6) 5.14/5.68 d (2.6) 5.53/5.56 d (2.6)–OH – – 4.71/4.68 s–OOH 11.02/11.01 s – –From the aerial parts of A. cotula, six linear sesquiterpene lactones were iso-lated. The known compounds were identified by comparison of their 1H NMRspectral data with those reported as: anthecotuloide (8),21 8-O-dihydroanthecotu-130 VU^KOVI] et al.} }}loide (9),14 5-oxo-6,13-dehydro-5,6-dihydroanthecotuloide (10)14 and 5-hydro-peroxy-6,13-dehydro-5,6-dihydroanthecotuloide (11).22 The structures of 8 – 11were also confirmed by 13C NMR spectroscopy (Table II). Anthecotuloide (8) isconsidered to be one of the most potent contact allergens.23The 1H-NMR spectrum of compound 12 was rather similar to that of 8. The mostsignificant difference between the spectra of these lactones was the absence of alow-field resonance of a vinyl proton (H-5), occurring in the spectrum of 8 at  5.21. In-stead of this, a one-proton signal was observed at   2.8 ppm in 1H NMR spectrum of12 (Table I), indicating the presence of an epoxy function. The FAB mass spectrum ex-hibiting the MH+ ion at m/z 265, compatible with the molecular formula C15H20O4,was also in accordance with an additional (epoxy) oxygen in 12. The 13C NMR spec-trum (Table II) confirmed the latter. The observed doubling of the signals led to the con-clusion that 12 was a (5R,6R + 5S,6S) diastereomeric mixture in a ratio of ca. 1:1, whichcould be formed by epoxidation of the 5 double bond of anthecotuloide. Thus, thestructure of anthecotuloide-5,6-oxide could be assigned to 12.TABLE II. 13C NMR spectral data of lactones 9 –12 (50 MHz, CDCl3)C 8 9 10 11 121 170.1 170.8 170.4 170.5 1702 137.4 137.9 137.6 138.0/137.1 137.83 37.9 38.6 42.1 35.5/36.8* 37.4/36.84 31.4 31.9 34.2 35.4/34.9* 32.4/32.35 123.5 122.6 196.7 86.1/87.4 60.7/60.46 133.8 135.7 143.1 140.4/140.7 58.1/58.47 54.4 47.9 46.6 46.1 53.2/53.38 197.9 66.2 198.3 200.4 197.19 122.3 127.5 122.5 122.5/122.3* 123.510 155.5 135.2 157.2 160.0 157.311 26.9 25.7 27.7 28.0 27.812 20.0 18.1 20.8 21.3 20.913 16.1 16.6 127.5 121.3/121.4* 17.614 69.9 70.5 71.4 71.9/70.7 70.6/70.915 121.5 122.1 122.9 122.8 122.3/122.7* The assignments can be interchanged.The molecular formula of 13, derived from the EIMS, was identical to that of12 (C15H20O4). The doubling of the signals in the1H NMR spectrum (Table I)showed the presence of two diastereomeric forms, this time in the ratio of ca. 1:1.4.The relative intensity of the low-field signals in the range of  5.45 – 5.82 indicatedthe presence of three vinyl protons, one of them assigned as H-15’. A low-field pairPHYTOCHEMICAL INVESTIGATION OF Anthemis cotula 131of one-proton doublets (J = 15.0 Hz), at  5.74 and 5.77, in the ratio of 1.4/1, respec-tively, could be assigned as H-5, coupled vicinally to the E-positioned vinyl proton(H-4) resonating at a slightly higher field (m,  ca., 5.45 – 5.70). The assignment ofthe signal of H-4 was based on its simplification upon irradiating a multiplet ( 3.68)identified as H-3. In addition, the signals of the allylic methyl (H3-14), observed in 8at  1.62, were replaced by a pair of three-proton singlets ( 1.30 and 1.31, in the ra-tio of 1/1.4, respectively) typical for methyls attached to a carbinol carbon (C-6).This, together with the similarity of the remaining 1H NMR signals (Table I) to thoseof 8, led to the structure of 6-hydroxy-4,5-(E)-dehydro-5,6-dihydroanthecotuloidefor this lactone, consisting of a (6R + 6S)-epimeric mixture.In addition, two flavones, apigenin (14) and hispidulin (15) were isolated fromthe aerial parts of A. cotula. This type of flavones is characteristic for the membersof the genus Anthemis.2Acknowledgements: The authors acknowledge their gratitude to the Ministry for Science andEnvironmental Protection of Serbia (Project number 140253).I Z V O DFITOHEMIJSKO ISPITIVAWE BIQNE VRSTE Anthemis cotulaIVAN VU^KOVI]1, QUBODRAG VUJISI]1, VLATKA VAJS1, VELE TE[EVI]2, PE\AJANA]KOVI]3i SLOBODAN MILOSAVQEVI]21Institut za hemiju, tehnologiju i metalurgiju, Wego{eva 12, 11000 Beograd,2Hemijski fakultet,Univerzitet u Beogradu, Studentski trg 16, 11000 Beograd i3Biolo{ki fakultet, Univerzitet uBeogradu, Studentski trg 16, 11000 BeogradNovo ispitivawe korena biqke A. cotula (Asteraceae) pokazalo je, pored ~etiri ve}izolovana poliacetilena, i prisustvo tri prenilovana 4-hidroksiacetofenona kojido sada nisu bili izolovani iz ovog roda. Iz nadzemnog dela biqke izolovano je {estlinearnih seskviterpenskih laktona, od koji su dva nova, dok su preostali ranijena|eni u istoj biqci. 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