Identification of a new antinociceptive alkaloid isopropyl N-methylanthranilate from the essential oil of Choisya ternata Kunth

Article abstract of DOI:10.1016/j.jep.2011.03.035

Ethnopharmacological relevance: Mexican people employed infusion of leaves of Choisya ternata Kunth for their antispasmodic and "simulative properties". Aim of the study: In the present study the detailed GC and GC-MS analyses of the essential oil of Choisya ternata Kunth (Rutaceae) were performed. The presence of a minor constituent isopropyl N-methylanthranilate (1) was revealed among other identified volatiles. A synthesis of 1 was undertaken in order to corroborate this find and obtain gram quantities that would allow the testing of its biological activity (peripheral and central antinociceptive activity). Materials and methods: The oils were investigated by GC and GC-MS. Synthesized compounds were spectrally characterized (UV-Vis, IR, 1D and 2D NMR, MS). The obtained synthetic samples of compounds were assayed for peripheral and central antinociceptive activity in two models (effects on acetic acid induced writhing in mice and the hot plate test for nociception). Results: Detailed GC and GC-MS analyses of the essential oil of Choisya ternata Kunth (Rutaceae) among 157 other identified volatiles revealed the presence of a minor constituent isopropyl N-methylanthranilate (1). Compound 1, named ternanthranin, is therefore detected as a natural product for the first time with a very restricted occurrence (samples of several citrus oils were screened for the presence of 1). The antinociceptive activities were assayed for ternanthranin, the two other synthetic analogs, methyl and propyl N-methylanthranilate, as well as the essential oil and the crude ethanol extract of the leaves. The results clearly demonstrate a very high (even significant at 0.3 mg/kg) dose dependent activity for the anthranilates (and the extracts). Isopropyl N-methylanthranilate showed the highest, while methyl N-methylanthranilate showed the lowest activity (with the methyl ester at 3 mg/kg still better than acetylsalicylic acid, at 200 mg/kg, in the first, or comparable with morphine, at 5 mg/kg, in the second test). Conclusion: This study once again revealed that detailed investigations of plant species with ethnopharmacologically documented activity may yield new natural compounds - a new alkaloid (ternanthranin), a volatile simple anthranilate that can be considered responsible for the antinociceptive activity of the crude plant extracts.

Full text of DOI:10.1016/j.jep.2011.03.035

Journal of Ethnopharmacology 135 (2011) 610–619
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
Identification of a new antinociceptive alkaloid isopropyl N-methylanthranilate
from the essential oil of Choisya ternata Kunth
a,∗
a
b
c
c
Niko S. Radulovi c´ , Ana B. Miltojevi c´ , Michael McDermott , Steve Waldren , John Adrian Parnell ,
d
d
b
Mariana Martins Gomes Pinheiro , Patricia Dias Fernandes , Fabio de Sousa Menezes
Department of Chemistry, Faculty of Science and Mathematics, University of Ni sˇ , Vi sˇ egradska 33, 18000 Ni sˇ , Serbia
Panoz Institute, School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin 23, Westland Row, Dublin 2, Ireland
Department of Botany, School of Natural Sciences, Trinity College, Dublin 2, Ireland
a
b
c
d
Laboratório de Farmacologia da Inflama c¸ ão e do Óxido Nítrico, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Ethnopharmacological relevance: Mexican people employed infusion of leaves of Choisya ternata Kunth
for their antispasmodic and “simulative properties”.
Received 23 December 2010
Received in revised form 26 February 2011
Accepted 13 March 2011
Aim of the study: In the present study the detailed GC and GC–MS analyses of the essential oil of
Choisya ternata Kunth (Rutaceae) were performed. The presence of a minor constituent isopropyl N-
methylanthranilate (1) was revealed among other identified volatiles. A synthesis of 1 was undertaken
in order to corroborate this find and obtain gram quantities that would allow the testing of its biological
activity (peripheral and central antinociceptive activity).
Materials and methods: The oils were investigated by GC and GC–MS. Synthesized compounds were spec-
trally characterized (UV–Vis, IR, 1D and 2D NMR, MS). The obtained synthetic samples of compounds
were assayed for peripheral and central antinociceptive activity in two models (effects on acetic acid
induced writhing in mice and the hot plate test for nociception).
Results: Detailed GC and GC–MS analyses of the essential oil of Choisya ternata Kunth (Rutaceae)
among 157 other identified volatiles revealed the presence of a minor constituent isopropyl N-
methylanthranilate (1). Compound 1, named ternanthranin, is therefore detected as a natural product for
the first time with a very restricted occurrence (samples of several citrus oils were screened for the pres-
ence of 1). The antinociceptive activities were assayed for ternanthranin, the two other synthetic analogs,
methyl and propyl N-methylanthranilate, as well as the essential oil and the crude ethanol extract of the
leaves. The results clearly demonstrate a very high (even significant at 0.3 mg/kg) dose dependent activ-
ity for the anthranilates (and the extracts). Isopropyl N-methylanthranilate showed the highest, while
methyl N-methylanthranilate showed the lowest activity (with the methyl ester at 3 mg/kg still better
than acetylsalicylic acid, at 200 mg/kg, in the first, or comparable with morphine, at 5 mg/kg, in the second
test).
Available online 21 March 2011
Keywords:
Choisya ternata
Essential oil
Synthesis
Esters of N-methylanthranilic acid
Isopropyl N-methylanthranilate
Peripheral and central antinociceptive
activities
Conclusion: This study once again revealed that detailed investigations of plant species with ethnophar-
macologically documented activity may yield new natural compounds—a new alkaloid (ternanthranin),
a volatile simple anthranilate that can be considered responsible for the antinociceptive activity of the
crude plant extracts.
©
2011 Elsevier Ireland Ltd. All rights reserved.
1
. Introduction
Despite its popularity, the volatile oils of this species have only
been investigated in detail on one previous occasion (Respaud
et al., 1997). Other previous phytochemical studies indicated
the importance of its non-volatile anthranilate-derived alkaloid
constituents. In particular, it has previously been shown to con-
tain seven quinoline alkaloids (some of which widespread in the
family Rutaceae): skimmianine, kokusaginine, 7-isopentenyloxy-
c-fagarine, evoxine, choisyine, platydesminium methosalt and
balfourodinium methosalt (Johns et al., 1967; Grundon et al., 1974;
Boyd et al., 2002). Recently, Boyd et al. (2007) have isolated a
range of seventeen quinoline alkaloids, involving several types
Mexican orange (Choisya ternata Kunth, Rutaceae, subfamily
Rutoideae, Bayer et al., 2009) is a small evergreen ornamental
shrub with leaves which when bruised give off a strong and pun-
gent smell. The abundantly produced and highly fragrant flowers
make Mexican orange an important and popular horticultural plant.
∗ Corresponding author. Tel.: +381 18533015; fax: +381 18533014.
E-mail address: vangelis0703@yahoo.com (N.S. Radulovi c´ ).
0
378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2011.03.035

N.S. Radulovi ´c et al. / Journal of Ethnopharmacology 135 (2011) 610–619
611
of oxidations during their biosynthetic pathways, from leaves of
Choisya ternata.
controller C-610 Work-21 pump (Büchi, Switzerland) and was
carried out on pre-packed column cartridges (40 mm × 75 mm),
Silica-gel 60, particle size distribution 40–63 ␮m, Büchi. Silica gel
60 on Al plates, layer thickness 0.2 mm (Kieselgel 60 F254, Merck)
was used for thin layer chromatography (TLC). The spots on TLC
were visualized by UV light (254 nm) and by spraying with 50%
In 1895, Boudoresques studied this species and undertook the
first pharmacological test by experimenting the effects of aqueous
effects of leaves on himself: he found them tonic and appetizing.
On the other hand, the oil is strongly repulsive. In 1923, Stand-
ley reported that Mexican people employed infusion of leaves for
their antispasmodic and “simulative properties”; Choisya ternata
was registered in the 4th and 5th edition of the Mexican Pharma-
copoeia. As far as we know, the present use of Choisya ternata is
not widespread. However, it may be recalled that some Rutaceae
species, such as Ruta graveolens and Ptelea trifoliata, which have
same chemical constitution, are still used pharmaceutically (Creche
et al., 1993).
(v/v) aqueous H SO4 followed by heating. Microanalysis of carbon,
2
hydrogen and nitrogen were carried out with a Carlo Erba 1106
microanalyzer; their results agreed favorably with the calculated
values. All the reagents and solvents were obtained from commer-
cial sources (Aldrich, USA; Merck, Germany; Fluka, Germany) and
used as received, except the solvents were purified by distillation.
2.2. Plant material
The antispasmodic properties of the leaves are probably
due to evoxine, which is known as sedative and spasmolytic.
Kokusaganine has gangioplegic effects. The main tertiary alkaloid,
skimmianine, is antidiuretic and hypothermic. Balfourodinium has
been found to produce a slight spasmolytic effect on isolated rat
duodenum (Creche et al., 1993).
Volatiles play a significant role in plant–plant, plant–insect and
other relationships conveying important messages that usually
require minute quantities of volatile secondary metabolites that are
part of an inhomogeneous group of compounds (commonly jointly
termed as essential oils). Many plants emit volatiles to attract pol-
linators or repel herbivores, especially useful at night when visual
clues become insufficient. Essential oil constituents can be biosyn-
thesized by several pathways, wherein benzenoids, isoprenoids
and fatty acid derivatives are their most typical chemical classes
Leaves of Choisya ternata were collected on the 20th October
2008from the TrinityCollege Botanical Gardens, Dartry, Dublin(liv-
ing plant accession number 19850023). One batch of plant material
was dried at room temperature for one week. Another portion
of the material was immediately submitted to hydrodistillation.
Voucher specimens were deposited in the Herbarium of Trinity
College Dublin (TCD) under the collection number SW 10-52.
2.3. Test animals
All experiments were performed with male Swiss mice
(18–25 g) obtained from our own animal facilities (Laboratório de
Farmacologia da Inflama c¸ ão e do Óxido Nítrico). Animals were
◦
maintained in a room with controlled temperature 22 ± 2 C for 12 h
(
Knudsen et al., 1993).
light/dark cycle with free access to food and water. Twelve hours
before each experiment animals received only water, in order to
avoid food interference with substances absorption. Animal care
and research protocols were in accordance with the principles and
guidelines adopted by the Brazilian College of Animal Experimen-
tation (COBEA), approved by the Biomedical Science Institute/UFRJ,
Ethical Committee for Animal Research, and received the number
DFBCICB-015.
Natural substances derived from plants have played an
extremely important role in the development of analgesic drugs
and in the understanding of the complex mechanisms involved in
pain transmission and pain relief (Yunes et al., 2005). For exam-
ple, salicin, a glycoside obtained from the bark of Salix species, was
the lead compound for the synthesis of aspirin based on its activity
and structural properties. Recently discovered antinociceptive sub-
stances include alkaloids, terpenoids and flavonoids (Calixto et al.,
Tested compounds (at 0.3, 1, and 3 mg/kg) were re-suspended
in sterile corn oil and administered by oral gavages (p.o.) to mice.
Essential oil and ethanol leaf extract were tested at 10, 30, and
100 mg/kg doses.
2
000). Such findings have opened new possibilities for research
into new potent analgesic drugs based on structure–activity rela-
tionships.
Due to the obvious lack of detailed studies of the volatile chem-
istry of this Choisya species we set our goal to determine the
chemical composition of the leaf essential oil of Choisya ternata.
The second aim of this study was to assess the activity of the essen-
tial oil, crude ethanol extract, and selected constituents of the oil
in chemical (acetic acid-induced visceral pain) and thermal (hot-
plate test) models of nociception in mice and to possibly collate
these results to the ethnomedical uses of this plant.
2.4. Isolation of essential oils and preparation of the ethanol
extract
Fresh or air-dried, to constant weight, plant material (two
batches of about 250 g) was subjected to hydrodistillation with ca.
2.5 L of distilled water for 2.5 h using the original Clevenger-type
apparatus to produce the yellowish essential oils (about 0.25 ml
per batch) in the yield (%, v/w, typical values): 0.08% essential oil
1
(from fresh leaf tissue), and 0.10% essential oil 2 (from dried
2
. Materials and methods
leaves). The obtained oils were separated by extraction with diethyl
ether (Merck, Germany), dried over anhydrous magnesium sul-
◦
2.1. General
phate (Aldrich, USA) and stored at −18 C until analysis. Ground
air-dried leaves of Choisya ternata were macerated for three days
with ethanol (100 mL of ethanol per 1 g of plant material) in the
dark. The obtained extract was evaporated to dryness in vacuo at
ambient temperature. The resulting oily residue was used in the
antinociception assays.
UV spectra (in acetonitrile) were measured using a UV-1650
PC Shimadzu spectrophotometer (Tokyo, Japan). The IR measure-
ments (ATR-attenuated total reflectance) were carried out using a
Thermo Nicolet model 6700 FTIR instrument (Waltham, USA). The
1
NMR spectra were recorded on a Varian Gemini 200 ( H at 200 MHz,
13
C at 50 MHz) spectrometer, using CDCl3 as the solvent. Chemical
shifts are expressed in ı (ppm) using TMS (Me Si) as an inter-
2.5. Gas chromatography–mass spectrometry (GC–MS) analyses
4
1
1
nal standard. 2D experiments ( H- H COSY, NOESY and HETCOR)
and DEPT were run on the same instrument with the usual pulse
sequences. Preparative medium-pressure liquid chromatography
Analyses of the oils were carried out by GC and GC/MS. The
GC/MS analyses (three repetitions for each sample) were per-
formed on a Hewlett-Packard 6890N gas chromatograph equipped
with fused silica capillary columns HP-5MS and DB-5MS (5%
(
MPLC) was performed with a pump module C-601 and a pump

6
12
N.S. Radulovi ´c et al. / Journal of Ethnopharmacology 135 (2011) 610–619
Table 1
Percentage composition of the essential oil of Choisya ternata Kunth (samples 1 and 2).
Rt/mina
RI
765
%1
%2
IdenMeth
Class
Component
2.256
2.343
2.424
2.439
2.882
2.920
2.931
3.026
3.038
3.317
3.751
3.826
3.954
4.083
4.196
4.370
4.448
4.628
4.677
4.724
4.808
4.861
4.912
5.026
5.040
5.083
0.1
tr
tr
tr
tr
tr
tr
tr
tr
0.1
tr
tr
tr
tr
tr
tr
tr
tr
a,b
a,b
a,b,c
a,b,c
a,b,c
a,b
a,b
a,b
a,b,c
a,b,c
a,b
HT
HT
O
GL
HT/E
GL
GL
GL
GL
HT/E
M
3-Methyl-2-buten-1-ol (syn. prenol)
3-Methyl-2-butenal (syn. prenal)
Octane
Hexanal
Ethyl isovalerate
(E)-2-Hexenal
(E)-3-Hexen-1-ol
(E)-2-Hexen-1-ol
1-Hexanol
Isopropyl isovalerate
Tricyclene
␣-Thujene
␣-Pinene
Propyl isovalerate
Camphene
Benzaldehyde
Isoamyl propionate
Sabinene
␤-Pinene
Methyl 5-methylhexanoate
Myrcene
Dehydro-1,8-cineole
Mesitylene
778
800
802
847
851
852
862
863
892
923
928
936
945
953
964
969
980
984
987
993
996
999
1005
1006
1008
tr
tr
tr
tr
0.5
1.0
tr
0.1
tr
0.5
1.0
tr
0.1
tr
a,b
MT
MP
HT/E
M
a,b,c
a,b,c
a,b,c
a,b,c
a,b,c
a,b,c
a,b,c
a,b
a,b,c
a,b
a,b,c
a,b,c
a,b
O
tr
tr
HT/E
MT
MP
O
MA
MM
O
HT/E
GL
MM
36.5
2.7
tr
7.8
tr
tr
tr
tr
tr
35.1
2.8
tr
8.3
tr
tr
tr
tr
tr
Isobutyl isovalerate
(E)-3-Hexenyl acetate
p-Mentha-1(7),8-diene (syn.
pseudolimonene)
␣-Phellandrene
a,b
5.086
5.199
5.309
5.457
5.552
5.555
5.615
5.790
5.855
6.132
6.315
6.570
6.762
6.885
6.961
6.984
7.053
7.121
7.291
7.313
7.316
1008
1014
1019
1027
1031
1034
1035
1043
1047
1060
1069
1082
1091
1098
1101
1102
1105
1108
1115
1116
1116
0.2
tr
0.2
tr
a,b
a,b
MM
M
3
ꢀ -Carene
3.2
0.3
2.3
5.4
3.0
tr
0.1
4.7
1.2
tr
1.3
tr
1.2
0.8
tr
2.8
0.2
2.0
6.6
2.2
tr
0.1
4.2
1.3
tr
1.1
tr
1.3
0.8
tr
a,b,c
a,b,c
a,b,c
a,b
a,b,c
a,b,c
a,b
a,b,c
a,b
a,b
MM
MM
MM
MM
MM
HT/E
MA
MM
MT
O
MM
GL
MA
MT
HT/E
HT/E
MM
M
␣-Terpinene
p-Cymene
Limonene
␤-Phellandrene
1,8-Cineole
Butyl isovalerate
(E)-␤-Ocimene
␥-Terpinene
cis-Sabinene hydrate
4-Methylhexyl acetate
Terpinolene
(Z)-3-Hexenyl propionate
Linalool
trans-Sabinene hydrate
Isopentyl isovalerate
2-Methylbutyl isovalerate
1,3,8-p-Menthatriene
(E)-4,8-Dimethyl-1,3,7-nonatriene
3-Methyl-3-butenyl
a,b
a,b
a,b,c
a,b
a,b,c
a,b,c
a,b
tr
tr
tr
tr
tr
tr
–
tr
a,b
a,b
HT/E
3-methylbutanoate
7.513
7.922
7.941
8.052
8.065
8.078
8.280
1124
1142
1142
1147
1148
1148
1157
0.8
0.5
tr
tr
tr
0.7
0.4
tr
tr
tr
a,b
a,b
a,b
a,b
a,b
a,b
a,b
MM
MM
GL
MA
MA
HT/E
MA
cis-p-Menth-2-en-1-ol
trans-p-Menth-2-en-1-ol
(Z)-3-Hexenyl isobutanoate
Ipsdienol
(E)-Myroxide
tr
tr
tr
0.1
3-Methyl-3-butenyl valerate
2,6-Dimethyl-1,5,7-octatrien-3-olb
(
syn. Ocimenol)
8
8
8
8
9
9
9
9
9
9
9
9
9
.409
.595
.714
.896
.022
.048
.103
.167
.281
.420
.560
.649
.988
1162
1170
1175
1183
1188
1189
1192
1194
1199
1205
1210
1214
1228
1229
1232
1235
1243
1255
1255
tr
0.1
tr
10.5
tr
tr
tr
0.1
–
9.9
tr
tr
a,b
a,b
GL
MM
GL
MM
MM
O
MM
MM
MM
HT/E
MM
HT/E
MA
MA
GL
(Z)-3-Heptenyl propionate
␦-Terpineol
1-Nonanol
Terpinen-4-ol
p-Cymen-8-ol
Naphthalene
Cryptone
␣-Terpineol
cis-Piperitol
Isohexyl 2-methylbutyrate
trans-Piperitol
Isoamyl isohexanoate
Citronellol
a,b,c
a,b,c
a,b
a,b,c
a,b
a,b,c
a,b
a,b
a,b
a,b
a,b,c
a,b,c
a,b
tr
tr
1.7
0.2
tr
0.3
tr
tr
tr
tr
tr
1.6
0.2
0.1
0.2
tr
tr
tr
-
tr
1
1
1
1
1
1
0.027
0.087
0.172
0.371
0.661
0.661
Nerol
(Z)-3-Hexenyl 2-methylbutanoate
(Z)-3-Hexenyl 3-methylbutanoate
Ascaridole
Chavicol
Geraniol
a,b
a,b,c
a,b
GL
MM
O
tr
tr
tr
tr
tr
tr
a,b,c
MA

N.S. Radulovi ´c et al. / Journal of Ethnopharmacology 135 (2011) 610–619
613
Table 1 (Continued)
Rt/mina
RI
%1
%2
IdenMeth
Class
Component
10.717
10.844
11.089
11.277
11.506
11.628
11.643
12.148
12.207
12.238
12.319
12.388
12.576
12.900
13.106
13.238
13.286
13.390
13.762
13.793
13.869
13.939
13.974
14.024
14.169
14.340
14.421
14.566
14.885
15.107
15.475
15.620
15.714
15.897
15.935
16.026
16.169
16.235
16.378
16.645
16.685
16.742
16.797
16.810
16.914
16.956
17.044
17.143
17.178
17.347
17.380
17.406
17.562
17.689
17.953
18.025
18.148
18.222
18.402
18.599
18.660
18.788
18.898
18.973
19.030
19.251
19.365
19.491
19.523
19.783
20.073
20.097
20.181
20.247
20.396
20.962
1257
1262
1272
1279
1289
1293
1294
1314
1317
1318
1321
1324
1331
1344
1355
1358
1360
1364
1379
1380
1383
1386
1387
1389
1395
1402
1405
1412
1424
1434
1449
1455
1458
1466
1468
1472
1477
1480
1486
1497
1499
1501
1503
1504
1508
1510
1514
1518
1520
1527
1528
1530
1536
1542
1553
1556
1561
1565
1572
1581
1583
1589
1594
1597
1599
1609
1614
1620
1621
1633
1646
1647
1651
1654
1661
1686
tr
tr
tr
tr
tr
0.1
–
tr
tr
tr
tr
tr
tr
tr
tr
tr
0.6
tr
tr
tr
tr
tr
tr
0.1
tr
–
tr
tr
-
tr
tr
tr
tr
tr
0.8
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
0.2
tr
–
tr
0.2
tr
tr
tr
tr
tr
1.4
–
0.1
tr
0.2
tr
tr
tr
tr
0.1
tr
1.2
tr
tr
tr
tr
tr
tr
tr
a,b
O
GL
MA
GL
M
O
MA
O
O
GL
O
GL
MM
MM
MA
O
O
MA
M
S
MA
O
O
S
S
O
O
O
SCAR
S
S
SCAD
SCAR
SCAR
SCAD
SCAD
SCAD
SCAD
SGER
SCAD
S
SGER
SCAD
SCAD
S
SGER
O
SCAD
S
SCAD
O
SCAD
SCAD
SCAD
SGER
S
SCAD
S
S
SGER
S
SCAR
S
S
S
S
S
9-Methyl-2-decanone
(E)-2-Decenal
Geranial
(Z)-2-Hexenyl valerate
Isobornyl acetate
2-Undecanone
trans-Thio rose oxide
1-Methylnaphthalene
p-Vinylguaiacol
(2E,4E)-2,4-Decadienal
Isoamyl benzyl ether
(Z)-3-Hexenyl tiglate
p-Mentha-1,4-dien-7-ol
exo-2-Hydroxycineole acetate
Citronellyl acetate
10-Methyl-2-undecanone
Eugenol
a,b,c
a,b,c
a,b
a,b,c
a,b,c
a,b
a,b,c
a,b,c
a,b
a,b,c
a,b
a,b
a,b
a,b,c
a,b
a,b,c
a,b,c
a,b,c
a,b
a,b,c
a,b,c
a,b
a,b,c
a,b
a,b,c
a,b,c
a,b,c
a,b,c
a,b
a,b,c
a,b
a,b,c
a,b
a,b
a,b
a,b
a,b
a,b,c
a,b
a,b
a,b
a,b
a,b
a,b
a,b
a,b,c
a,b
a,b
a,b
a,b
a,b
a,b
a,b
a,b
a,b
a,b
a,b,c
a,b
Neryl acetate
Isobornyl propanoate
␣-Copaene
tr
tr
tr
tr
Geranyl acetate
Methyl (E)-cinnamate
(E)-␤-Damascenone
␤-Bourbonene
␤-Elemene
(Z)-Jasmone
tr
0.1
0.1
–
tr
tr
0.3
tr
tr
tr
0.2
tr
tr
tr
tr
tr
1.8
tr
tr
0.1
0.1
–
tr
tr
tr
0.1
tr
1.0
tr
tr
tr
tr
tr
tr
tr
tr
tr
1.1
tr
0.1
tr
tr
Methyl eugenol
Methyl N-methylanthranilate
␤-Caryophyllene
␤-Copaene
Aromadendrene
cis-Muurola-3,5-diene
␣-Humulene
9-epi-(E)-Caryophyllene
cis-Cadina-1(6),4-diene
cis-Muurola-4(14),5-diene
trans-Cadina-1(6),4-diene
␥-Muurolene
Germacrene D
trans-Muurola-4(14),5-diene
epi-Cubebol
Bicyclogermacrene
␣-Muurolene
Epizonarene
(E,E)-␣-Farnesene
Germacrene A
Isopropyl N-methylanthranilate
␥-Cadinene
Cubebol
␦-Cadinene
Chavibetol acetate
Zonarene
trans-Cadina-1,4-diene
␣-Cadinene
Hedycaryol
Mintoxide
cis-Muurol-5-en-4␤-ol
(E)-Nerolidol
1,5-Epoxysalvial-4(14)-ene
Germacrene D-4-ol
Spathulenol
Caryophyllene oxide
4-epi-Cubebol
Globulol
Salvial-4(14)-en-1-one
Ledol
␤-Oplopenone
1,10-di-epi-Cubenol
(E)-Isoeugenyl acetate
1-epi-Cubenol
epi-␣-Cadinol (syn. ␶-cadinol)
epi-␣-Murrolol (syn. ␶-muurolol)
␣-Muurolol (syn. torreyol)
␤-Himachalol
tr
tr
1.5
tr
0.1
–
a,b
a,b,c
a,b,c
a,b
a,b
a,b
a,b
a,b
a,b
a,b,c
a,b
a,b,c
a,b
a,b
a,b
a,b
–
tr
tr
0.1
0.5
tr
0.1
0.6
0.1
tr
0.1
0.5
1.5
0.4
–
SCAD
O
tr
0.1
0.5
1.1
0.3
tr
SCAD
SCAD
SCAD
SCAD
S
3.2
tr
4.0
tr
SCAD
S
␣-Cadinol
Eudesma-4(15),7-dien-1␤-ol
a,b

6
14
N.S. Radulovi ´c et al. / Journal of Ethnopharmacology 135 (2011) 610–619
Table 1 (Continued)
Rt/mina
RI
%1
%2
IdenMeth
Class
Component
21.087
21.199
22.214
22.726
27.402
27.702
27.832
29.415
38.788
41.566
44.153
1692
1697
1744
1769
2003
2020
2027
2116
2700
2900
3100
0.6
0.3
0.1
tr
0.1
0.1
tr
tr
tr
tr
tr
0.9
0.4
–
a,b
a,b
a,b
a,b,c
SGER
S
S
O
D
D
D
D
O
Germacra-4(15),5,10(14)-trien-1␣-ol
Shyobunol
Mint sulphide
–
Benzyl benzoate
0.1
0.1
tr
tr
tr
Unidentified diterpene 1c
d
Unidentified diterpene 2
Unidentified diterpene 3
e
a,b,c
a,b,c
a,b,c
a,b,c
(E)-Phytol
Heptacosane
Nonacosane
Hentriacontane
tr
tr
O
O
9
0
tr
9.0
.1
98.5
0.2
0.1
Total identified
Hemiterpenoids (HT)
Esters (HT/E)
8
6
2
3
3
9
3
1
3
8
0
6
3
0
6.4
83.8
65.0
18.8
37.7
3.8
Monoterpenoids (M)
Hydrocarbons
Oxygenated
Thujane (MT)
Pinane (MP)
6.1
0.3
9.0
.7
.1
9.8
Acyclic (MA)
4.5
1.8
.8
.0
.6
.4
.6
.2
32.4
13.6
3.3
10.3
0.5
8.1
3.8
0.2
Menthane and related (MM)
Sesquiterpenoids (S)
Hydrocarbons
Oxygenated
Caryophyllane and related (SCAR)
Cadinane and related (SCAD)
Germacrane and related (SGER)
Diterpenoids (D)
tr
.7
tr
0.9
“Green leaf” volatiles (GL)
Others (O)
0
a
Compounds listed in order of elution on HP-5MS column (Rt: retention time (in min) and RI: experimentally determined retention indices on the mentioned column by
co-injection of a homologous series of n-alkanes C7–C31).
b
Correct stereoisomer not determined.
c
◦
MS, 70 eV, 230 C, m/z(rel. int.): 39(16), 40(3), 41(62), 43(19), 53(26), 55(56), 57(7), 65(13), 67(32), 68(11), 69(47), 77(34), 78(8), 79(52), 80(35), 81(49), 82(5), 83(9), 91(50),
9
1
2
2(18), 93(100), 94(26), 95(36), 96(3), 105(40), 106(15), 107(73), 108(26), 109(29), 115(1), 117(4), 119(45), 120(18), 121(39), 122(19), 123(20), 131(5), 133(24), 134(31),
35(30), 136(25), 137(20), 145(9), 147(23), 148(14), 149(9), 150(2), 159(12), 160(2), 161(20), 162(7), 173(5), 175(2), 187(3), 189(10), 201(2), 203(1), 229(14), 244(6), 257(12),
72(14).
d
◦
MS, 70 eV, 230 C, m/z(rel. int.): 39(24), 41(84), 43(25), 44(6), 53(43), 55(66), 56(3), 57(6), 65(20), 66(2), 67(69), 68(19), 69(38), 77(54), 78(11), 79(89), 80(17), 81(86),
8
1
2
2(8), 83(8), 91(84), 92(20), 93(92), 94(20), 95(48), 96(5), 97(3), 105(78), 106(24), 107(73), 108(24), 109(35), 117(14), 118(15), 119(89), 120(34), 121(100), 122(22), 123(25),
31(29), 132(24), 133(83), 134(47), 135(40), 136(12), 145(36), 146(20), 147(36), 148(18), 149(12), 159(37), 160(5), 161(51), 162(9), 173(40), 175(26), 187(23), 189(15),
01(25), 203(2), 229(28), 243(10), 257(61), 258(10), 272(18).
e
◦
MS, 70 eV, 230 C, m/z(rel. int.): 39(21), 41(75), 43(31), 53(43), 55(58), 67(55), 68(18), 69(38), 77(61), 79(76), 80(6), 81(78), 91(89), 92(11), 93(86), 95(47), 105(92),
1
1
06(27), 107(88), 108(38), 109(51), 119(97), 120(41), 121(100), 122(26), 123(15), 133(100), 134(80), 135(68), 136(26), 145(15), 147(34), 148(19), 159(18), 161(50), 162(30),
75(16), 189(28), 229(6), 257(66), 272(6).
tr: trace (<0.05%); syn.: synonym; IdenMeth—identification method: a, constituent identified by mass spectra comparison; b, constituent identified by retention index
matching; c, constituent identity confirmed by co-injection of an authentic sample. Class: HT: hemiterpenoids, HT/E: hemiterpenoid esters, M: monoterpenoids, MT: Thujane
monoterpenoids, MP: Pinane monoterpenoids, MA: acyclic monoterpenoids, MM: menthane and related monoterpenoids, S: sesquiterpenoids, SCAR: caryophyllane and
related sesquiterpenoids, SCAD: cadinane and related sesquiterpenoids, SGER: germacrane and related sesquiterpenoids, D: diterpenoids, GL: “Green leaf” volatiles, O:
otherwise unclassified constituents (others).
phenylmethylsiloxane, 30 m × 0.25 mm, film thickness 0.25 ␮m,
Agilent Technologies, USA) and coupled with a 5975B mass selec-
tive detector from the same company. The injector and interface
Wiley 6, NIST02, MassFinder 2.3, and a homemade MS library with
the spectra corresponding to pure substances and components of
known essential oils, and wherever possible, by co-injection with
an authentic sample (as indicated in Table 1).
◦
were operated at 250 and 290 C, respectively. Oven tempera-
◦
◦
ture was raised from 70 to 290 C at a heating rate of 5 C/min
and then isothermally held for 10 min. As a carrier gas helium
at 1.0 ml/min was used. The samples, 1 ␮l of the oil solutions in
diethyl ether (1:100), were injected in a pulsed split mode (the
flow was 1.5 ml/min for the first 0.5 min and then set to 1.0 ml/min
throughout the remainder of the analysis; split ratio 40:1). Mass
selective detector was operated at the ionization energy of 70 eV,
in the 35–500 amu range and scanning speed of 0.34 s. GC (FID)
analysis was carried out under the same experimental conditions
using the same columns as described for the GC/MS. The percent-
age composition was computed from the GC peak areas without
the use of correction factors. Qualitative analyses of the essen-
tial oil constituents were based on the comparison of their linear
retention indices relative to retention times of C7–C31 n-alkanes on
the HP-5MS (Van den Dool and Kratz, 1963) with those reported
in the literature (Adams, 2007), and by comparison of their mass
spectra with those of authentic standards, as well as those from
2.6. Reductive methylation of methyl anthranilate
Reductive methylation was accomplished following a literature
method (da Silva et al., 2007) employing it for the first time on an
ortho substituted aniline. A mixture of 1.51 g of methyl anthrani-
late (10 mmol), 2.40 g of glacial acetic acid (40 mmol), 0.92 g of 35%
aqueous formaldehyde (11 mmol), 1.31 g of zinc dust (20 mmol)
◦
and 20 ml of dioxane was stirred and heated at 50–60 C for the
duration of 4 h. After completion of the reaction (monitored by
TLC) aqueous ammonia was added and the reaction mixture was
extracted with chloroform, followed by a usual work-up (drying
with MgSO4 and solvent evaporation) and gave 1.66 g of the crude
product (consisting of ca. 80% of the wanted product, 8% of methyl
N,N-dimethylanthranilate and 12% of the starting ester, as inferred
from a GC–MS analysis). A portion of this product, 0.77 g, was
directly subjected to the transesterification procedure as described

N.S. Radulovi ´c et al. / Journal of Ethnopharmacology 135 (2011) 610–619
615
Table 2
NMR spectroscopic data (200 MHz, CDCl3) for compounds 1, 2 and 3.
Position
Isopropyl N-methylanthranilate (1)
Methyl N-methylanthranilate (2)
Propyl N-methylanthranilate (3)
ıC, mult.
110.5a
ıH (J in Hz)
ıC, mult.
ıH (J in Hz)
ıC, mult.
ıH (J in Hz)
1
2
3
4
5
6
7
C
109.8 C
152.0 C
110.6 CH
134.5 CH
114.2 CH
131.5 CH
169.0 C
51.1 CH3
–
110.2 C
152.1 C
152.0 C
110.5a CH
134.3 CH
114.1 CH
131.4 CH
168.2 C
6.67, dd (1.0, 8.7)
6.66, dd (1.0, 8.6)
110.7 CH
134.5 CH
114.3 CH
131.5 CH
168.8 C
65.7 CH2
22.0 CH2
10.4 CH3
29.4 CH3
6.66, dd (1.2, 8.6)
7.39, ddd (1.7, 7.1, 8.7)
6.62, ddd (1.0, 7.1, 8.0)
7.96, dd (1.7, 8.0)
7.38, ddd (1.6, 7.1, 8.6)
6.60, ddd (1.0, 7.1, 8.0)
7.92, dd (1.6, 8.0)
7.38, ddd (1.6, 7.2, 8.6)
6.59, ddd (1.2, 7.2, 8.0)
7.93, dd (1.6, 8.0)
ꢀ
1
67.2 CH
5.23, septet (6.2)
1.38, d (6.2)
1.38, d (6.2)
2.92, s
3.85, s
-
–
2.90, s
7.68, s
4.21 t (7.1)
1.77, sextet (7.1)
1.02, t (7.1)
2.90, br s
ꢀ
2
21.8 CH3
21.8 CH3
29.3 CH3
ꢀ
3
–
N-CH3
N-H
29.3 CH3
7.75, s
7.68, s
a
Overlapping signals.
+
below. The yield (76%) of the methylation step was determined
gravimetrically after an MPLC fractionation using a gradient of hex-
ane and diethyl ether. This also gave an analytical sample to be
utilized for spectral characterization.
Table 2; EIMS m/z 165 [M]+ (100.0), 105 [M−CH OH–CO] (85.8),
3
+
+
104 [M−CH –H O–CO] (84.4), 132 [M−CH –H O] (54.4), 77
3
2
3
2
+
+
+
[M−C H O N] (47.9), 133 [M−CH OH] (29.7), 91 [M−C H O ]
3
6
2
3
3
6
2
(10.0); Anal. C 65.52, H 6.69, N 8.47%, calcd for C H NO , C 65.44,
9
11
2
H 6.71, N 8.48, O 19.37%; Rt 14.584 min, RI (HP-5MS) 1412.
2.7. Alkaline transesterification of methyl N-methylanthranilate
2.10. Propyl N-methylanthranilate
The same procedure was exploited for the synthesis of both
isopropyl (1) and propyl N-methylanthranilates (3). A solution of
the corresponding alkoxide was prepared by dissolving 1.50 g of
metallic sodium (80 mmol) in refluxing rigorously anhydrous alco-
hol (40 ml, 1- or 2-propanol) and cooled to room temperature. The
crude methyl N-methylanthranilate (6.5 mmol, calculated accord-
ing to the results of a GC–MS run) was added to this solution and
brought to reflux (CaCl2 tube) and quenched with excess ice after
Propyl 2-(methylamino)benzoate; yellowish liquid; UV
(
(
(
CH CN) ꢁmax(log ε) 354 (4.42), 256 (4.02), 219 (3.86) nm; FTIR
3
neat) ꢂmax 3376.1 (N–H), 2965.6 (C–H), 2818.4 (NCH –H), 1677.0
2
C
O), 1606.1, 1578.8, 1518.4 (C C), 1256.7 (C-O, asym.), 1234.3 (C-
−1
1
13
O, sym.), 747.4, 702.0 cm ; H and C NMR data, see Table 2; EIMS
m/z 193 [M]+ (83.3), 105 [M−H C CH–CH –H O–CO]+ (100.0),
2
3
2
+
+
1
51 [M−H C CH–CH ] (20.0), 133 [M−H C CH–CH –H O]
2
3
2
3
2
2
0 min. This was followed by immediate extraction of the reaction
+
(
[
[
39.0),
104
[M−H C CH–CH –H O–CO–H]
(61.6),
77
2
3
2
mixture with Et O, the solvent evaporated under reduced pressure
+
+
2
M−C H7O–CH NH–CO] (39.1), 134 [M−C H7O] (30.6), 132
3
2
3
and the residue chromatographed (MPLC, gradient Et O:hexane,
+
+
2
M−H C CH–CH –H O–H] (43.2), 106 [M−COOC H7] (20.4);
2
3
2
3
from pure hexane to pure diethyl ether with an increment step of
Anal. C 68.39, H 7.81, N 7.21%, calcd for C11
H
15
NO , C 68.37, H 7.82,
2
5
%). In the case of the isopropyl ester (1) 0.70 g of the pure product
N 7.25, O 16.56%; Found: Rt 18.734 min, RI (HP-5MS) 1586.
was obtained (a 56% conversion of the methyl to isopropyl ester
was achieved), while the other one (3) was obtained in the yield of
6
6% (0.82 g).
2.11. Isopropyl N,N-dimethylanthranilate
Isopropyl
2-(dimethylamino)benzoate;
EIMS
(100.0),
m/z
2
.8. Isopropyl N-methylanthranilate
+
+
2
[
(
1
07
[M]
(38.0),
164
[M−(CH ) CH]
148
3
2
+
+
M−(CH ) CHO]
(56.1), 132 [M−H C CH–CH –CH OH–H]
Isopropyl 2-(methylamino)benzoate; yellowish liquid; UV
3 2
2
3
3
+
58.0),
118
[M−H C CH–CH –H O–CO–H]
(42.7),
(
(
(
CH CN) ꢁmax(log ε) 353 (3.62), 256 (3.78), 222 (4.24) nm; FTIR
neat) ꢂmax 3374.7 (N–H), 2978.4 (C–H), 2818.8 (NCH –H), 1672.8
C
2
3
+
2
3
05
[M−H C CH–CH –CH OH–CO]
(18.9),
77
[M−
2
3
3
2
+
+
(
CH ) CHCOO–C H5N] (28.9), 133 [M−H C CH–CH –CH OH]
O), 1605.7, 1579.0, 1517.1 (C C), 1256.3 (C-O, asym.), 1230.9 (C-
3 2
2
2
3
3
+
−1
1
13
(20.9), 104 [M−H C CH–CH –CH OH–CO–H]
(21.4); Rt
O, sym.), 746.1, 701.8 cm ; H and C NMR data, see Table 2; EIMS
2
3
3
m/z 193 [M]+ (53.8), 105 [M−H C CH–CH –H O–CO]+ (100.0),
16.731 min, RI (HP-5MS) 1500.
2
3
2
+
+
1
51 [M−H C CH–CH ] (62.3), 133 [M−H C CH–CH –H O]
2
104
3
2
3
2
+
(
37.3),
[M−H C CH–CH –H O–CO–H]
(57.6),
77
2
3
2
2
.12. Isopropyl anthranilate
+
+
[
(
M−(CH ) CHO–CH NH–CO]
(34.7), 134 [M−(CH ) CHO]
3
2
2
3 2
+
25.5),
132
[M−H C CH–CH –H O–H]
(22.2),
106
2
3
2
+
Isopropyl 2-aminobenzoate; EIMS m/z 179 [M] (30.2), 119
+
[
M−COOCH(CH ) ] (17.4); Anal. C 68.30, H 7.85, N 7.19%, calcd
3
2
+
+
[
[
[
M−(CH ) CHOH] (100.0), 137 [M−H C CH–CH ] (32.9), 92
3
2
2
3
for C
H NO , C 68.37, H 7.82, N 7.25, O 16.56%; Rt 17.057 min, RI
15 2
11
+
+
M−COOCH(CH ) ] (29.9), 120 [M−(CH ) CHO] (25.5), 91
3
2
3 2
(
HP-5MS) 1515.
+
M−(CH ) CHOH–CO] (5.2); Rt 15.532 min, RI (HP-5MS) 1451.
3
2
2.9. Methyl N-methylanthranilate
2.13. Methyl N,N-dimethylanthranilate
Methyl 2-(methylamino)benzoate; yellowish liquid; UV
Methyl 2-(dimethylamino)benzoate; EIMS m/z 179 [M]+
(
(
1
1
CH CN) ꢁmax(log ε) 354 (3.73), 256 (3.89), 219 (4.32) nm; FTIR
3
+
+
(
[
64.0), 164 [M−CH ] (100.0), 148 [M−CH O] (88.1), 132
neat) ꢂmax 3379.3 (N–H), 2948.6 (C–H), 2818.1 (NCH –H),
3
3
2
+
+
M−CH OH–CH ]
(97.1), 77 [M−C H O N]
(57.6), 91
678.3 (C O), 1606.0, 1577.2, 1518.1 (C C), 1233.5 (C-O, asym.),
3
3
4
8
2
+
−1
1
13
[M−C H O ] (56.4); Rt 14.445 min, RI (HP-5MS) 1406.
258.5 (C-O, sym.), 746.7, 702.0 cm
;
H and C NMR data, see
4
8
2

6
16
N.S. Radulovi ´c et al. / Journal of Ethnopharmacology 135 (2011) 610–619
2.14. Methyl 2-(methyleneamino)benzoate
Prism Software 5.0 (GraphPad Prism Software). Statistical signif-
icance between groups was determined by the ANOVA analysis
of variance followed by Bonferroni’s test. p values less than 0.05
(p < 0.05) were used as the significance level (* when comparing a
treated group with a vehicle-treated group and # when comparing
a treated group with an ASA/morphine-treated group).
EIMS m/z 163 [M]+ (30.3), 148 [M−CH ] (100.0), 132
+
3
+
+
+
[
(
M−CH O] (33.8), 105 [M−C H O ] (40.9), 77 [M−C H O N]
3
2
2
2
3
4
2
57.1); Rt 13.372 min, RI (HP-5MS) 1363.
.15. Methyl 2-(hydroxymethylamino)benzoate
EIMS m/z 181 [M]+ (0.1), 119 (100.0), 148 (75.6), 92 (62.0), 151
2
3. Results and discussion
(
(
1
60.0), 77 (45.6), 120 (34.3), 105 (33.0), 65 (28.2), 132 (24.8), 163
22.9), 181 (0.1); Rt (broad peak, apex) 15.150 min, RI (HP-5MS)
435.
A portion of the plant material was extracted for essential oils
immediately upon collection. The other portion was allowed to air
dry in a dark unheated room and after one week the sample was
extracted. This provided two oils from this plant, oil 1 (fresh mate-
rial) and 2 (after drying). In general, the two samples of Choisya
ternata gave rather poor yields of their respective leaf oils (0.08%
v/w[1] and 0.10% v/w[2]).
Component identification along with their percent composition,
RI values and methods of identification are summarized in Table 1.
The GC and GC–MS analyses of the hydrodistillate from Choisya ter-
nata leaves revealed a complex mixture of compounds of which 157
were identified (the extent of identification of components being
more than 98% of the total detected GC peak areas). The main dif-
ference between the two samples (1 and 2) was the fact that sample
2
.16. Propyl N,N-dimethylanthranilate
Propyl
2-(dimethylamino)benzoate;
EIMS
(100.0),
m/z
+
+
2
07
[M]
(51.4),
(97.6),
148
132
[M−C H7O]
164
3
+
+
[
(
M−C H7]
[M−H C CH–CH –CH OH–H]
3
2
3
3
+
70.4),
05
118
[M−H C CH–CH –H O–CO–H]
(54.7),
91
(41.5),
2
3
2
+
1
[M−H C CH–CH –CH OH–CO]
(29.1),
2
3
3
+
+
[
1
M−C H O ]
(51.3), 77 [M−C H7COO–C H5N]
6
12
2
3
2
+
33
[M−H C CH–CH –CH OH]
(21.3),
104
[M−H C
2
3
+
3
2
CH–CH –CH OH–CO–H] (31.6); Rt 18.224 min, RI (HP-5MS)
1
3
3
565.
2
showed a slightly high concentration of sesquiterpenoids. Sam-
ple 2 had a total of 157 compounds identifiable including 0.2%
hemiterpenoids, 83.8% monoterpenoids, 13.6% sesquiterpenoids,
2.17. Propyl anthranilate
0
.2% diterpenoids and 0.9% other unclassified molecules (Table 1).
Propyl 2-aminobenzoate; EIMS m/z 179 [M]+ (39.8),
The essential oils from leaves of Choisya ternata show sabinene (ca.
one third of the oils), terpinen-4-ol (around 10%), myrcene (7.8-
8
major oil components. In contrast, the previously investigated leaf
oil of Choisya ternata (Respaud et al., 1997) showed the presence
of ␣-phellandrene (64–71%) and myrcene (11.5%) as the predomi-
nant components. The previously mentioned study (Respaud et al.,
+
+
1
9
[
19 [M−C H7OH] (100.0), 137 [M−H C CH–CH ] (13.4),
3
2
3
+
+
2
[M−COOC H7]
(29.9), 120 [M−OC H7]
(29.4), 91
3
3
.3%), ␤-phellandrene (5.4-6.6%) and ␥-terpinene (4.2-4.7%) as the
+
M−C H7OH–CO] (4.9); Rt 17.140 min, RI (HP-5MS) 1518.
3
2
.18. Acetic acid-induced abdominal writhing test
Mice were treated according to Whittle (1964) and adapted by
1
997) on the oil of this species resulted in the identification of only
Matheus et al. (2005). Briefly, the total number of writhing follow-
ing intraperitoneal administration of 2% (v/v) acetic acid (AA) was
recorded over a period of 20 min, starting 5 min after AA injection.
Mice were pre-treated with the test substances or acetylsalicylic
acid (ASA, 200 mg/kg) or vehicle, 60 min before administration of
AA.
eighteen components (all of which were also detected by us).
However, our attention was mostly focused on the minor com-
ponents detected in the oils of this species. Two peaks in the GC
chromatograms (detected in both samples 1 and 2) that were ten-
tatively identified (based on mass spectral comparison with the MS
from the Wiley-NIST data base) as methyl (2) and isopropyl (1) N-
methylanthranilates were of great interest since nitrogen volatiles
are known as potent floral volatiles that are difficult to detect and
are frequently missed out during the analyses (Jabalpurwala et al.,
2.19. Hot-plate test
Mice were tested according to the method described by Sahley
2
009). A literature survey on these two compounds gave the follow-
and Berntson (1979) and adapted by Matheus et al. (2005). Ani-
mals were placed on a hot plate (Insight equipments, Brazil) set
ing results: the methyl ester of N-methylanthranilic acid (2) was
previously reported from a number of Rutaceae species (belonging
to one of the two sister subfamilies, Rutoideae and Aurantioideae,
namely the genera Zanthoxylum (Dreyer and Brenner, 1980; Detoni
et al., 2009), Citrus (Lota et al., 2000; Brophy et al., 2001; Tomi
et al., 2008; Jabalpurwala et al., 2009), Fortunella (Umano et al.,
◦
at 55 ± 1 C. Reaction times were recorded when the animals licked
their fore- and hind-paws and jumped at several intervals of 30 min
after oral administration of the test substances, vehicle or morphine
(
5 mg/kg). Baseline was considered as the mean of reaction time
obtained at 60 and 30 min before administration of the substances,
vehicle or morphine and defined as normal reaction of animal to
the temperature. Increase in baseline (%) was calculated by the for-
mula: ((reaction time × 100)/baseline) − 100. Antinociception was
quantified as area under the curve (AUC) of responses from 30 to
1
994; Quijano and Pino, 2009a,b) and Murraya (Imai et al., 1989),
respectively) with some sporadic (most frequently singular) occur-
rence (in low amount) in the unrelated genera Chamaecyparis
(Thomas et al., 1966), Corydalis (Olesen and Knudsen, 1994), Jas-
minum (Calvarano, 1966; Cheng and Chao, 1979; Toda et al., 1983;
Kaiser, 1988; Ito et al., 2002), Mangifera (Pino et al., 2005), Miche-
lia (Kaiser, 1991), Siphonochilus (Viljoen et al., 2002), and Trifolium
1
80 min after drug administration. The following formula, based on
the trapezoidal rule, was used to calculate the AUC: AUC = 30 × IB
(min 30)/2 + (min 60) + · · · + (150 min) + (min 180)/2], where IB is
the increase in baseline (in %).
[
(Honkanen et al., 1969). However, the isopropyl ester (1) has
never been detected in a sample of natural origin. Methyl N-
methylanthranilate (2) is regarded as the marker compound of
Citrus recutita (mandarin) essential oil, and the value of its iso-
topic 14N/ N ratio has been proposed as a means of natural sample
authentification (Faulhaber et al., 1997). This compound was found
to be important in the aroma of mandarin peel oil and the flavor of
2.20. Statistical analysis
15
All experimental groups were composed of 6–10 mice. The
results are presented as mean ± S.D. The AUC was calculated by

N.S. Radulovi ´c et al. / Journal of Ethnopharmacology 135 (2011) 610–619
617
3
'
step into account. After the co-injection of these three synthesized
compounds with the oil of Choisya ternata, the originally proposed
hypothesis was corroborated and compounds 1 and 2 were proved
to be isopropyl- and methyl N-methylanthranilates.
O
O
O
6
3
1
'
(i)
(ii)
5
4
1
O
O
O
2'
N
H
2
2 N
H
The structural assignment of the synthesized compounds was
achieved by spectral means (UV–Vis, IR, 1 and 2D NMR and MS).
NH2
1
1
The H NMR spectra of compounds 1, 2 and 3 all displayed two
doublet of doublets (dd 6.66–6.67, 7.92–7.96 ppm) and two ddd
(6.59–6.62, 7.38–7.39 ppm) signals in the chemical shifts range of
aromatic protons. This pattern was indicative of an aromatic core
ortho substituted with one electron donating group and an electron
withdrawing carbonyl group (due to the anisotropic deshielding
of the ortho H to the C O group). Additional common features
of the three compounds were the signals originating from the
NHMe protons. The N-methyl group unambiguously resonated at
2.90–2.92 ppm as a singlet. The exchangeable protons attached to
the nitrogen atom pointed out to a subtle distinction between the
methyl and propyl esters (appearing at ı 7.68 as a broad singlet
for both 2 and 3) on one side and the isopropyl (ı 7.75) on the
other suggesting a somewhat different in strength intramolecular
hydrogen bonding (again probably as a consequence of the more
sterically demanding isopropyl group that tips the carbonyl of the
ester outside of planarity with the aromatic ring). This is also evi-
dent from the corresponding IR vibrations of the N–H (3375, 3376,
Scheme 1. Synthesis of isopropyl N-methylanthranilate (1). Reagents and condi-
◦
tions: (i) 1.1 eq HCHO(aq), 2 eq Zn, 4 eq AcOH(aq), stirring at 50 C, 4 h, yield 76%;
+
−
(
ii) 10 eq Na OiPr, iPrOH, 20 min of reflux, yield 56%.
mandarin juice (Wilson and Shaw, 1981; Fanciullino et al., 2006),
has been attributed with the attractiveness to Mexican fruit flies
(
Anastrepha ludens, Massa et al., 2008) and thrips (Thrips coloratus,
Imai et al., 2001), as well as the repellency in bird species (e.g. Stur-
nus vulgaris, Clark et al., 1991; other species, Schafer et al., 1983), to
possess the inhibitory effect on the l-alanine-induced initiation of
spore germination in Bacillus subtilis (Prasad, 1974), and induce a
chemotactic response of Escherichia coli (Ohba and Hayashi, 1979).
It is also claimed to be phototoxic (Api, 1997) and to show an
acute and short-term toxicity in rats (Gaunt et al., 1970). The N-
demethylated ester (methyl anthranilate) is also a characteristic
feature of the citrus oils (Jabalpurwala et al., 2009) but was not
identified as the constituent of the currently investigated Choisya
ternata oils. Also noteworthy is the fact that it serves an important
biological role in plant defense as a bird repellent. The chemosen-
sory irritation caused by this compound has been employed to not
only protect crops (Curtis et al., 1994; Avery et al., 2001) but also
prevent bird-aircraft accidents (Engeman et al., 2002).
Having all of this in mind we decided to corroborate our ten-
tative structure assignments by comparing the chromatographic
properties of the two oil components (supposedly methyl and
isopropyl N-methylanthranilates, 2 and 1) to that of authentic
material—synthetic samples of 1 and 2. Isolation from the at hand
oil samples has been dismissed as an option due to the complex
oil matrix and their low relative abundance in the oils. We have
undertaken a synthetic effort to produce gram quantities of the
mentioned two esters, and since the procedure already present
in the literature (Staiger and Miller, 1959; Dembele et al., 1988)
was not convenient (low yields and complex reaction mixtures) a
new strategy was envisaged. The commercially available methyl
anthranilate was the starting material and a two step transfor-
mation (N-methylation and transesterification) of this molecule to
isopropyl N-methylanthranilate (1) has been achieved in 43% over-
all yield. The reductive methylation, followed by transesterification
with the isopropoxide was the preferred order of synthetic events
−
1
and 3379 cm , respectively for 1, 3, and 2). Thus, the spectra are
consistent with the anthranilic acid core as the base structural frag-
ment and the alcohol moieties making up the rest of the spectra.
13
The ester groups appeared at ı 168.2–169.0 in the C NMRs, and
−
1
gave strong IR absorptions at 1672.8–1678.3 cm confirming the
connection modes of the alcohols and the acid, i.e. the presence
of a conjugated and strongly hydrogen bonded ester group. This is
also strengthened by the fragmentation patterns visible in the MS
spectra (with intense m/z corresponding to M+ ions) of 1, 2 and 3
that all possessed an important contribution of an ion correspond-
ing to the loss of the OR groups, and the McLafferty fragmentation
•
+
of the propyl and isopropyl esters giving the [M−C H ] ion of N-
3
6
methylanthranilic acid. The usual multiplicity and chemical shifts
of hydrogens of the methyl, propyl and isopropyl esters of car-
boxylic acids can be readily seen from the H NMRs. All data on
1
1
the H NMR of these three compounds are summarized in Table 2.
13
The C NMRs contained the expected number of signals for com-
pounds 2 (nine signals) and 3 (eleven signals), but in the case of the
isopropyl derivative it appeared to have one carbon atom signal
missing (nine instead of ten). This situation was resolved by simple
comparison of the spectrum of 1 with those of 2 and 3, that sug-
gested that the signal at ı 110.5 was arising from an overlap of two
Cs (these are at 109.8 and 110.6 ppm for 2, and 110.2 and 110.7 ppm
for 3). The assignation of all proton and carbon NMR signals was
made possible only by the use of 2D NMR ( H- H COSY and HET-
COR spectra) as well as comparison with literature data (Yoshikawa
et al., 1994). Correlations observed in these spectra give rise to the
assignations given in Table 2. For example, the overlapping signals
at 110.5 ppm for 1 corresponded to C-1 and C-3.
(
as depicted in Scheme 1).
1
1
The reversed order gave a poor yield probably due to the steric
hindrance of the isopropyl group rendering the necessarily tran-
sient ortho iminium cation unreactive to the in situ generated
reducing agent (Zn + AcOH, da Silva et al., 2007). This relatively
recent approach to the reductive methylation of primary and sec-
ondary amines was used since the classical ones gave unsatisfactory
The obtained synthetic samples of compounds 1–3, as well as
the essential oil and the ethanol extract of leaves were assayed
for peripheral and central antinociceptive activity in two mod-
els used to evaluate the potential analgesic activity of drugs. The
results for the first time show that the essential oil and crude
ethanol leaf extract of Choisya ternata, and all of the anthranilates,
when given orally, produce dose-related and significant antinoci-
ception according to assessment of the abdominal constrictions
elicited by acetic acid and the hot plate test (Fig. 1). The tested
natural product isopropyl N-methylanthranilate (at 3 mg/kg) was
more potent and efficacious than aspirin (at 200 mg/kg) in the
acetic acid induced writhings assay and all the anthranilates were,
at 3 mg/kg, more potent, and at a dose of 0.3 mg/kg, of a simi-
results (formaldehyde (aq) and NaBH —the main isolated product
4
were the imine—methyl N-methylanthranilate and aminal—N-
(
hydroxymethyl)-derivative, while the direct methylation with MeI
gave an irresolvable mixture of the mono- and dialkylated products
along with a significant amount of the unreacted methyl anthrani-
late). In order to exclude the possibility that compound 1 is a
propyl rather than the isopropyl ester we employed an analogous
reaction sequence and obtained propyl N-methylanthranilate (3)
in 50% total yield. To obtain pure samples of the three prepared
anthranilates final reaction mixtures, after the usual workup, were
subjected to a gradient MPLC (100% hexane to 100% Et O, with
2
the increment step of 5%). All reported yields take this purification

6
18
N.S. Radulovi ´c et al. / Journal of Ethnopharmacology 135 (2011) 610–619
(anthranilates), using antagonists and antibodies, is necessary in
order to understand their mechanism of action.
4. Conclusion
Hence, these arguments confirm 1 as a new and biologi-
cally/ecologically interesting natural product. It is our proposal
that this compound should be named ternanthranin for its very
restricted occurrence in the natural world. We have screened com-
mercially available samples of various oils of Citrus sp. for the
presence of methyl and isopropyl N-methylanthranilates. Neither
of the analyzed oils had the new alkaloid as its constituent, and
only the oil of mandarin possessed a certain quantity (less than
1
%) of the methyl ester. (The volatile oils from peel of grapefruit,
bergamot, lemon and orange contained neither the methyl nor the
isopropyl esters.) This constituent is, thus, a strong marker com-
pound, and might have additional chemotaxonomical implications
as new investigations provide further information on its natural
distribution.
Yet another aspect of the natural occurrence of this compound
deserves comment. It is by no means an accident that the plant
species that represents a rich source of anthranilate-derived quino-
line alkaloids would be the one to produce two volatile alkaloids
that probably originate from the commencement of the same path-
way (the enzyme that is considered to perform the N-methylation
step has been isolated and characterized, Maier et al., 1995) either
as a side product or as, and this is a mere speculation, a regu-
latory molecule of this metabolism. One other possibility is that
1 is merely an artefactual compound formed during hydrodis-
tillation from the mentioned quinoline alkaloids or 2, but this
seems unlikely due to two facts: (1) the methyl ester is an estab-
lished natural product (whose biosynthesis has been investigated,
Maier et al., 1995) and (2) the acid catalyzed (trans)esterification
of N-methylanthranlic acid/esters (mimicking the hydrodistillation
conditions) with isopropanol was entirely unsuccessful (this was
our first attempt in the synthesis of 1) giving not even trace amounts
of the desired ester.
Fig. 1. (A) Effects of the anthranilates 1–3, essential oil and ethanol leaf extract of
Choisya ternata on acetic acid-induced writhings in mice; (B) hot-plate test results.
The vehicle and acetylsalicylic acid (ASA, 200 mg/kg) or morphine (5 mg/kg) were
p.o. administered to the control groups. *p < 0.05 when comparing a treated group
with a vehicle-treated group and p < 0.05 when comparing a treated group with an
ASA/morphine-treated group.
#
lar efficacy in relation to morphine (at 5 mg/kg) in inhibiting the
heat-induced nociceptive response. The results of the first model
(
acetic acid induced writhing) showed that the activity was depen-
dent on the substitution pattern in the following crescent order:
< 3 < 1 (Fig. 1). The responses to the thermal stimuli in the hot
2
plate test (the second model) revealed that the substitution pat-
tern also plays an important role in this case (the same crescent
order: 3 < 1 again with methyl N-methylanthranilate displaying
the least activity (Fig. 1)). The maximum response for isopropyl
N-methylanthranilate was achieved at 60 min after the adminis-
tration of the drug (at 10 mg/kg) and the effect goes down up to
In summary, the results of the present study demonstrate for the
first time that the ethanol extract and essential oil, as well as one
of its minor alkaloid constituents from the leaves of Choisya ternata
produce dose-related antinociceptive action in chemical (acetic
acid-induced visceral pain) and thermal (hot-plate test) models of
nociception in mice. The mechanism by which the isopropyl N-
methylanthranilate produces antinociception still remains unclear,
but pharmacological studies are continuing so as to characterize
the mechanism(s) responsible for the antinociceptive action. Fur-
thermore, the antinociceptive action demonstrated in the present
study supports, at least partly, the ethnomedical uses of this
plant.
1
20 min when no effect can be seen. With morphine the maximum
effect is shown at 90 min after the administration of the drug and
although it goes down with time it is still high and significant after
1
80 min.
Another interesting result of this current study was the fact
that p.o. administration of the essential oil of the leaves of Choisya
ternata exhibited one hundred times less potency and efficacy
when compared with isopropyl N-methylanthranilate in prevent-
ing the acetic acid-induced pain. This fits nicely with idea that the
antinociceptive activity of the oil (that can be roughly regarded
as a 100–1000 fold dilution of the anthranilates) is caused by the
presence of the two anthranilates (the summed content of the
methyl and isopropyl N-methylanthranilates is ca. 0.1%). Possibly
the higher activity of the ethanol extract could be linked to other
non-volatile (alkaloid) constituents of Choisya ternata leaves.
Since the nociceptive neurons are sensitive to non-steroidal
anti-inflammatory drugs (NSAIDs) and to narcotics and other
centrally acting drugs (Calixto et al., 2000; Yunes et al., 2005),
and although the anthranilates posses a high degree of struc-
tural similarity to the known antinociceptive molecule ASA and
have a different time dependent nature of action when compared
with morphine, a deeper evaluation of these simple compounds
Acknowledgements
The authors are grateful to the Botany Department, Faculty of
Natural Sciences, Trinity College Dublin as well as to the Staff of
the Botanic Gardens, TCD. The authors from Brazil thank the Con-
selho Nacional de Desenvolvimento Científico e Tecnológico (CNPq,
grant support and fellowship to PDF), Funda c¸ ão Carlos Chagas Filho
de Apoio à Pesquisa do Estado do Rio de Janeiro (FAPERJ, grant sup-
port to PDF), and Conselho de Administra c¸ ão de Pessoal de Ensino
Superior (CAPES, fellowship to MMGP). FSM is grateful to the Start
up grant No. 51002.516.R01336 from Trinity College Dublin. The
authors from Serbia acknowledge funds received from the Min-
istry of Science and Technological Development of Serbia (Project
1
72061).

N.S. Radulovi ´c et al. / Journal of Ethnopharmacology 135 (2011) 610–619
619
Appendix A. Supplementary data
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Lawrence, B.M., Mookherjee, B.D., Willis, B.J. (Eds.), Flavors and Fragrances: A
World Perspective. Proceedings of the 10th International Congress Essential Oils.
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paca L. Journal of Essential Oil Research 3, 129–146.
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