Insect antifeedant compounds from Nothofagus dombeyi and N. pumilio

Article abstract of DOI:10.1016/j.phytochem.2004.04.002

A bioassay-guided purification of the extracts of Nothofagus dombeyi and N. pumilio leaves yielded several triterpenes and flavonoids including 2-O-acetylmaslinic acid, 3-O-acetyl 20,24,25-trihydroxydammarane, and 3,20,24,25-tetrahydroxydammarane as new natural products. All the isolated compounds were assessed for antifeeding activity against the 5th instar larvae of Ctenopsteustis obliquana. 12-Hydroxyoleanolic lactone and pectolinarigenin from N. dombeyi and dihydrooroxylin A from N. pumilio, showed significant antifeeding activity.

Full text of DOI:10.1016/j.phytochem.2004.04.002

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 2173–2176
www.elsevier.com/locate/phytochem
Insect antifeedant compounds from Nothofagus dombeyi
and N. pumilio
a
a,
b
c
*
Odile Thoison , Thierry S ꢀe venet , Hermann M. Niemeyer , Graeme B. Russell
a
Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, Bat. 27, F-91198, Gif-Sur-Yvette Cedex, France
b
Departmento de Ciencias Ecol ꢀo gicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
Horticultural Research Institute, Palmerston North, New Zealand
c
Received 17 February 2003; received in revised form 22 March 2004
Available online 8 May 2004
Abstract
A bioassay-guided purification of the extracts of Nothofagus dombeyi and N. pumilio leaves yielded several triterpenes and
flavonoids including 2-O-acetylmaslinic acid, 3-O-acetyl 20,24,25-trihydroxydammarane, and 3,20,24,25-tetrahydroxydammarane
as new natural products. All the isolated compounds were assessed for antifeeding activity against the 5th instar larvae of Cten-
opsteustis obliquana. 12-Hydroxyoleanolic lactone and pectolinarigenin from N. dombeyi and dihydrooroxylin A from N. pumilio,
showed significant antifeeding activity.
Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: Nothofagus dombeyi; Nothofagus pumilio; Fagaceae; Insect antifeeding activity; Flavonoids; Triterpenes
1
. Introduction
Nothofagus dombeyi (Mirb.) Oerst and N. pumilio (Pepp
Endl.) Oerst.
&
The genus Nothofagus Bl. (Fagaceae) constitutes the
most important forest cover in both Chile and New
Zealand (Tanai, 1986) and its wide distribution, lon-
gevity and success in forest formation, make it eminently
suitable for studying the phytochemical interactions that
may have arisen as the genus co-evolved with inverte-
brates. In a recent investigation (Russell et al., 2000) of
bioactivity and patterns of herbivory within the genus
Nothofagus, we identified several species which showed
significant antifeeding activity against the larvae of the
leafrollers, Ctenopsteustis obliquana and Eiphyas post-
vittana. The antifeeding activity of leaf extracts of N.
alessandri Espinosa, from Chile and of N. fusca (Hook
f.) Oerst., from New Zealand was shown to be due to the
combined presence of both the stilbene pinosylvin and
the flavonoid galangin. We have now investigated two
further Chilean species for antifeeding compounds,
2
. Results and discussion
The ethyl acetate extracts of dried leaves of N. dom-
beyi and N. pumilio were chromatographed on silica gel,
eluting with a gradient mixture of n-heptane, EtOAc and
MeOH. Three fractions in each plant extract were found
to have antifeeding activity in the two-way choice bio-
assay using 5th instar larvae of C. obliquana and were
subjected to further chemical analysis.
From the active fractions of N. dombeyi, six com-
pounds were isolated. They were identified from their
MS and NMR spectra and by comparison with litera-
ture data and included, lupeol, cabraleone (1), 2- and
3
1
-O-acetylmaslinic acids (2,3), pectolinarigenin (4), and
2-hydroxyoleanolic lactone (5). The active fractions of
N. pumilio gave, cabraleadiol monoacetate (6), b-sitos-
terol, 5-hydroxytetramethoxyflavone (7), betulonic acid,
dihydrooroxylin A (8), oleanolic acid, ursolic acid, ma-
slinic acid (9), 3-O-acetyl-20,24,25-trihydroxydamma-
rane (10), and its 3-O-deacetyl derivative (11). Of these
*
Corresponding author. Tel.: +33-169-82-31-03; fax: +33-169-07-72-
4
0
7.
E-mail address: sevenet@icsn.cnrs-gif.fr (T. S ꢀe venet).
031-9422/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.04.002

2174
O. Thoison et al. / Phytochemistry 65 (2004) 2173–2176
compounds, 2-O-acetylmaslinic acid (2) and the two
dammarane derivatives (10,11) have not been described
previously from natural sources.
in the 13C NMR spectrum are attributed to two qua-
ternary carbons (C–OH) and two methine carbons (CH–
O). These data, together with HMBC correlations,
indicate 10 is a dammarane-type triterpene similar to
cabraleadiol acetate (6), but with a C-17 side chain like
that of neoalsoside K1 from Neoalsomitra integrifoliola
(Fujita et al., 1995). Attempts to compare this com-
pound with cabraleadiol monoacetate by acid cycliza-
tion failed, and so stereochemistry of C-20 and C-24
cannot be confirmed. The stereochemistry at C-3 is de-
duced from the appearance at d 4.83 ppm of a broad
singlet due to a low coupling constant, consistent with
H-3 being equatorial. An HMBC correlation study
confirmed that the acetyl group was on C-3. Compound
OH
OH
OH
O
OH
R
RO
1
. R = O
1
1
0. R = OAc
1. R = H
6. R = OAc, H
1
0 is therefore 3a-O-acetyl-20,24,25-trihydroxydamma-
þ
rane. Compound 11 exhibits in the CIMS a MH at m=z
478, which is consistent with the molecular formula,
C30H54O4. The H NMR spectrum is similar to that of
OH
O
CO
1
COOH
R1
R2
compound 10 but without a signal for an O-acetyl
group. Compound 11 is 3a,20,24,25-tetrahydroxy-
dammarane.
HO
Ten compounds were tested at 1% concentration, in
the two-way choice bioassay for antifeeding activity
against 5th instar larvae of C. obliquana. The results are
shown in Table 1. There was only enough material of
3-O-acetylmaslinic acid for testing at 0.5% concentra-
tion and its feeding index is not strictly comparable with
those of the other ten compounds.
2
. R1 = OAc, R2 = OH
. R1 = OH, R2 = OAc
. R1 = OH, R2 = OH
5
3
9
R3
R1
MeO
MeO
O
HO
O
O
R2
The fractions of N. dombeyi and N. pumilio that
showed antifeeding activity, contained several common
OMe
O
OH
OH
4
. R1 = R2 = H, R3 = OMe
. R1 = R2 = OMe, R3 = H
8
7
Table 1
Antifeeding activity of Nothofagus compounds against 5th instar lar-
vae of Ctenopsteustis obliquana
The 2- and 3-O-acetylmaslinic acids (2,3) both exhibit
þ
Feeding
a
index
Yield
(%)
a MH peak at m=z 515 in the CIMS matching the
molecular formula, C32H50O5. The IR spectra of both
ꢀ
1
1
From N. dombeyi
2-O-Acetylmaslinic acid (2)
compounds showed strong bands at 1724 and 1695 cm
+22
+12
0.021
0.001
0.002
0.167
0.005
0.003
for acetyl and carboxylic groups, respectively. The H
NMR spectrum of 2 exhibits seven methyl signals, an
olefinic proton at d 5.25 ppm (br s) and two methine
signals at d 4.93 ppm (1H, ddd, J ¼ 4:3, 10.2, 10.2 Hz)
and d 3.18 (1H, d, J ¼ 10:2 Hz), assigned to H-2 and H-
b
3
-O-Acetylmaslinic acid (3)
2-Hydroxyoleanolic lactone (5)
Lupeol
ꢁ
1
+26
+14
Cabraleone (1)
Pectolinarigenin (4)
)1
+35
ꢁ
3
2
, respectively. The O-acetyl moiety was confirmed at C-
From N. pumilio
3-O-Acetylcabraleadiol (6)
Sitosterol
from HMBC correlations that showed that the acetyl
+26
0
0.240
0.012
0.014
0.013
methyl signal at d 2.06 ppm was connected to H-2.
Compound 2 was acetylated to give a 2,3-di-O-acetyl
derivative with identical ring A NMR data published for
Betulonic acid
5
)25
)1
-Hydroxy-3,6,7,8-tetramethoxy-
flavone (7)
ꢁ
2
a,3b-di-O-acetylursenes (Kojima and Ogura, 1989).
Dihydrooroxylin A (8)
Maslinic acid (9)
+36
0.003
0.012
0.006
0.006
Thus compound 2 is 2a-O-acetylmaslinic acid and
compound 3 is 3b-O-acetylmaslinic acid.
Compound 10 exhibits in FABMS a [M + Na] at
Not tested
Not tested
Not tested
Acetyltrihydroxydammarane (10)
Tetrahydroxydammarane (11)
þ
a
ðC ꢀ TÞ=ðC þ TÞ ꢂ 100, where C and T are the amounts eaten of
m=z 543 which is consistent with the molecular formula,
control and test squares, respectively.
b
Tested at 0.5% concentration.
1
C H O . The H NMR spectrum shows eight methyl
groups (d 1.6–0.8 ppm) and one acetyl methyl at d 2.06
ppm. The four signals occurring from d 73.3 to 75.0 ppm
3
2
56
5
*
Significant difference between treatment and control (P < 0:05).
Wilcoxon matched pairs test.

O. Thoison et al. / Phytochemistry 65 (2004) 2173–2176
2175
0
and known triterpenes and flavonoids and in this respect
the chemistry of these two species is unspectacular.
However, it is likely that antifeeding activity toward
some Lepidoptera may be due to the matrix of these
compounds, consistent with a generalised resistance
toward insect feeding of a type expected of long lived,
highly apparent plants such as Nothofagus
71°03 W) near Talca, Chile. Herbarium specimens are
kept at the Laboratorio de Qu ꢀı mica Ecol oꢀ gica (NGS-6
and NGS-5, respectively).
3.2. Extraction and isolation from N. dombeyi
Dried and powdered leaves of N. dombeyi (0.9 kg)
were extracted with CH Cl . After removal of the sol-
(
Feeny, 1976; Fox, 1981). Most of the isolated com-
2
2
pounds did not in themselves, show antifeeding activity
at 1%, although fractions and plant extracts were highly
active. This indicates it is the mixtures that are ecolog-
ically important as we discovered with N. alessandrii
when a mixture of both pinosylvin and galangin dem-
onstrated antifeeding activity at 1% whereas the indi-
vidual compounds did not (Russell et al., 2000).
Otherwise, it can be noted that in the active fractions,
the minor compounds were the most active.
The flavanone (8) from N. pumilio and the flavone (4)
from N. dombeyi, showed significant antifeeding activity
but tetramethoxyflavone (7) was inactive. In previous
work (Russell et al., 2000) we found the flavones, gal-
angin, 8-methoxygalangin and 3-O-methyl-8-meth-
oxygalangin from N. alessandri also did not have
significant antifeeding activity at 1%. The three galangin
flavones and flavone 7 all have a 3-oxy substituent and
an unsubstituted Ring C and it seems likely when their
activities are compared to flavone 4, that the absence of
a C-3 substituent and the presence an O-substituted
Ring C may be important for antifeeding activity of
flavones. Of the triterpenes tested, some were inactive (1,
sitosterol, betulonic acid), others bordered on activity
vent by evaporation, the CH2Cl2 extract (150 g) was
chromatographed on a column of silica gel by using
gradient elution with heptane–EtOAc–MeOH (9.5/0.5/0
to 0/0/10). Only the bioactive fractions were subse-
quently worked up. Fraction 3 (2.5 g, heptane–EtOAc,
9:1) gave lupeol (1.5 g). Fraction 8 (1.1 g, heptane–
EtOAc 85:15), was further purified by CC on silica gel
with CH Cl –MeOH 95:5, and afforded cabraleone (1)
(48 mg). Fraction 16 (1.4 g, EtOAc) was purified by CC
on silica gel with CH2Cl2–MeOH (95:5), and afforded 2-
O-acetylmaslinic acid (2) (200 mg), 3-O-acetylmaslinic
acid (3) (8 mg), pectolinarigenin (4) (25 mg) and 12-
hydroxyoleanolic lactone (5) (18 mg).
2
2
3.2.1. 2-O-Acetylmaslinic acid (2)
1
9
Powder, [a] +1.4° (CHCl3, c 2.3); EIMS m=z (rel.
int.): 514 [M ] (4%), 454 (4%), 248 (100%), 203 (62%);
D
þ
þ
HR-FABMS:
m=z
537.35800
[M + Na]
for
C32H50O5Na (calcd. 537.35559); IR ½mꢃ
cm 1: 2940,
ꢀ
KBr
1
1724, 1695; H NMR (CDCl3): d 5.25 (1H, br s, H-12),
4.93 (1H, ddd, J ¼ 10:2, 10.2, 4.3, H-2), 3.18 (1H, d,
J ¼ 10:2, H-3), 2.82 (1H, dd, J ¼ 13:3, 3.4, H-18), 2.06
(3H, s, OAc), 1.11 (3H, s, Me-27), 1.03 (3H, s, Me-23),
1.01 (3H, s, Me-25), 0.91 (3H, s, Me-30), 0.89 (3H, s,
Me-29), 0.83 (3H, s, Me-24), 0.72 (3H, s, Me-26); 13C
NMR: d 185.0 (C-28), 170.4 (OCOCH3), 144.0 (C-13),
123.0 (C-12), 81.5 (C-3), 73.4 (C-2), 55.5 (C-5), 48.0 (C-
(
3
3, lupeol) and 12-hydroxyoleanolic lactone (5) and
-hydroxycabraleadiol (6) showed significant activity.
3
. Experimental
9), 46.9 (C-17), 46.2 (C-19), 44.8 (C-1), 42.0 (C-14), 41.3
Optical rotations were measured at 20 °C on a Perkin-
(C-18), 40.1 (C-4), 39.7 (C-8), 38.7 (C-10), 34.2 (C-21),
33.5 (C-29), 32.8 (C-7), 31.0 (C-20), 30.0 (C-22), 28.9 (C-
23), 28.0 (C-15), 26.3 (C-27), 24.0 (C-30), 23.3 (C-16),
23.2 (C-11), 21.8 (OCOCH3), 18.6 (C-6), 17.4 (C-26),
17.0 (C-24), 16.7 (C-25).
Elmer 241 polarimeter. IR spectra were recorded on a
Perkin–Elmer Spectrum BX FT-IR spectrometer. HR-
FAB mass spectra were recorded on a MALDI-TOF
spectrometer (Voyager-DE STR; PerSeptive Biosystems)
with a matrix of gentisic acid. CIMS and EIMS were re-
corded on Kratos MS-9 and MS50 mass spectrometers,
respectively. NMR spectra were recorded in CDCl3-pyr-
3.2.2. 2, 3-Di-O-acetylmaslinic acid
idine-d on Bruker AC 250, AC 300 or AMX 400 spec-
Ac O was added to the pyridine solution (pyridine–
Ac O, 1:2) of compound 2 (10 mg) and the mixture
5
2
trometers. Chemical shifts (relative to TMS) are in ppm
and coupling constants (in parentheses) in Hz. Column
chromatography (CC) was performed using silica gel
Merck H60. Preparative plates (PLC) were silica gel 60
F254 (1 cm).
2
heated at 100 °C for 1 h to give a di-O-acetyl compound
in high yield. The H-2 and H-3 shifts were observed in
1
the H NMR at 5.10 (ddd) and 4.75 ppm (d), corre-
sponding to H-2a and H-3b, respectively, as observed
for data published for 2a,3b-di-O-acetylursenes (Kojima
and Ogura, 1989). The comparison with compound 2
shows clearly that in the di-O-acetyl compound, H-3
shifts from 3.18 to 4.75 ppm due to the O-acetyl group
attached to C-3.
3
.1. Plant collection
Leaves of N. dombeyi and N. pumilio were collected
0
from Altos de Lircay National Reserve, (35°37 S;

2176
O. Thoison et al. / Phytochemistry 65 (2004) 2173–2176
3
.3. Extraction and isolation from N. pumilio
Dried and powdered leaves of N. pumilio (1 kg) were
(C-9), 51.5 (C-5), 51.3 (C-17), 51.2 (C-14), 43.3 (C-13),
1.5 (C-8), 40.0 (C-22), 38.7 (C-4), 38.2 (C-10), 36.3 (C-
7), 35.0 (C-1), 32.2 (C-15), 30.0 (C-29), 28.7 (C-16), 27.3
(C-2), 27.1 (C-23), 27.0 (C-21), 27.0 (C-27), 26.3 (C-26),
26.0 (C-12), 23.2 (C-28), 22.4 (C-11), 19.2 (C-6), 18.2 (C-
30), 17.3 (C-19), 16.4 (C-18).
4
extracted with CH2Cl2. After removal of the solvent by
evaporation, the CH2Cl2 extract (84 g) was chromato-
graphed on a column of silica gel by using gradient
elution with heptane–EtOAc–MeOH (10/0/0 to 0/0/10).
Only the bioactive fractions were subsequently worked
up. Fraction 8 (6 g, heptane–EtOAc, 8:2) gave, after
purification by CC on silica gel with heptane–Et2O (8:2)
and repeated CC with CH2Cl2–EtOAc (1:1), cabralea-
diol monoacetate (6) (2.4 g), b-sitosterol (0.12 g), and
3.4. Antifeeding test
In a two way test design (Blaney et al., 1990) 5th
instar larvae of the brown headed leafroller (C. obliqu-
ana) were presented with diet squares incorporating the
extracts or fractions or pure compounds from the plants
to make a final concentration of 1% w/w on the moist-
ened squares (Russell and Lane, 1993). The compounds
or extracts were applied to the dry diet squares in
MeOH or CH2Cl2 (10 ll) and the solvent evaporated.
Control diet squares were treated with solvent only.
Antifeeding activity was assessed by recording the
weight of the test square after 24 h exposure to the
larvae compared to a control square. A feeding index
[FI ¼ ðC ꢀ T=C þ T Þꢂ100] was calculated from the
amount eaten of control (C) and test (T ) squares, re-
spectively. Antifeedant activity shows as a positive in-
dex. The treatment versus control data of replicates were
analyzed by the two sided, Wilcoxon matched-pairs test.
5
-hydroxy-3,6,7,8-tetramethoxyflavone (7) (0.13 g).
Fraction 11 (6 g, heptane–EtOAc, 1:1), was purified by
CC on silica gel with heptane–acetone (1:1) and afforded
betulonic acid (144 mg), dihydrooroxylin A (8) (28 mg).
Fraction 18 (1 g, EtOAc) was purified by CC on silica
gel with toluene–acetone (3:1) then EtOAc–isopropanol
(
95:5) and afforded a mixture of ursolic and oleanolic
acids (0.4 g), maslinic acid (9) (0.12 g), 3-O-acetyl-
0,24,25-trihydroxydammarane (60 mg) (10) and its 3-
O-acetyl derivative, 3,20,24,25-tetrahydroxydammarane
11) (60 mg).
2
(
3
.3.1. 3-O-Acetyl-20,24,25-trihydroxydammarane (10)
2
5
Powder, [a]D +37° (MeOH, c 0.9); HR-FABMS:
þ
m=z 543.40135 [M + Na] for C32H56O5Na (calcd.
ꢀ
cm : 1740; H NMR (CDCl3): d
KBr
1
1
5
43.40254); IR ½mꢃ
References
4
.83 (1H, br s, H-3), 3.80 (1H, d, J ¼ 8, H-24), 2.06 (3H,
s, OAc), 1.52 (3H, s, Me-26), 1.48 (3H, s, Me-27), 1.43
3H, s, Me-21), 0.97 (3H, s, Me-18), 0.92 (3H, s, Me-29),
.85 (3H, s, Me-30), 0.84 (3H, s, Me-28), 0.81 (3H, s,
Blaney, W.M., Simmonds, M.S., Ley, S.V., Anderson, J.C., Toogood,
P.L., 1990. Antifeeding effects of azadirachtin and structurally
related compounds on Lepidopteran larvae. Entomologia Exper-
imentalis et Applicata 55, 149–160.
(
0
1
3
Me-19); C NMR: d 170.4 (OCOCH3), 80.5 (C-24),
Feeny, P., 1976. Plant apparency and chemical defense. Recent
Advances in Phytochemistry 10, 1–40.
7
9
8.6 (C-3), 75.0 (C-20), 73.3 (C-25), 51.8 (C-5), 51.5 (C-
), 51.4 (C-17), 51.2 (C-14), 43.1 (C-13), 41.4 (C-8), 39.8
Fox, L., 1981. Defense and dynamics in plant herbivore systems.
American Zoologist 21, 833–844.
(
3
(
2
C-22), 37.9 (C-10), 37.5 (C-4), 36.0 (C-7), 35.1 (C-1),
2.2 (C-15), 28.6 (C-29), 28.8 (C-16), 27.3 (C-23), 26.7
C-21), 26.7 (C-27), 26.4 (C-26), 26.0 (C-2), 24.5 (C-12),
2.4 (C-28), 22.3 (C-11), 21.7 (COCH ), 19.0 (C-6), 17.4
Fujita, S., Kasai, R., Ohtani, K., Yamasaki, K., Chiu, M.H., Nie,
R.L., Tanaka, O., 1995. Dammarane glycosides from aerial part of
Neoalsomitra integrifoliola. Phytochemistry 39, 591–602.
Kojima, H., Ogura, H., 1989. Configurational studies on hydroxy
groups at C-2,3 and 23 or 24 of oleanene and ursene-type
triterpenes by NMR spectroscopy. Phytochemistry 28, 1703–
3
(
C-30), 17.0 (C-19), 16.3 (C-18).
1
710.
3
.3.2. 3,20,24,25-Tetrahydroxydammarane (11)
2
5
Russell, G.B., Lane, G.A., 1993. Insect antifeedants. A New Zealand
perspective. In: Proceedings of the 46th New Zealand Plant
Protection Conference, pp. 179–186.
Powder, ½aꢃ +150° (MeOH, c0.11); HR-FABMS:
D
þ
m=z 501.39381 [M+Na]
for C30H54O4Na (calcd.
ꢀ
1
1
5
01.39198); IR ½mꢃ
cm : 2931; H NMR (CDCl3): d
Russell, G.B., Bowers, W.S., Keesing, V., Niemeyer, H.M., Sevenet,
T., Visanthaverni, S., Wratten, S.D., 2000. Patterns of bioactivity
and herbivory on Nothofagus species from Chile and New Zealand.
Journal of Chemical Ecology 26, 41–56.
KBr
3.80 (1H, d, J ¼ 8, H-24), 3.60 (1H, br s, H-3), 1.52 (3H,
s, Me-26), 1.49 (3H, s, Me-27), 1.43 (3H, s, Me-21), 1.21
(
0
8
3H, s, Me-29), 1.00 (3H, s, Me-18), 0.92 (3H, s, Me-28),
Tanai, T., 1986. Phytogeographic and phylogenetic history of the
genus Nothofagus Bl. (Fagaceae) in the southern hemisphere.
Journal of the Faculty of Science (Hokkaido) 21, 505–582.
.90 (3H, s, Me-19), 0.83 (3H, s, Me-30); 13C NMR: d
0.5 (C-24), 76.1 (C-3) 75.0 (C-20), 73.4 (C-25), 51.7