Journal of Stored Products Research 43 (2007) 9296

Antifeedant activities of terpenoids isolated from tropical Rutales

S. Omar
a
, M. Marcotte
a
, P. Fields
b
, P.E. Sanchez
c
, L. Poveda
c
, R. Mata
d
, A. Jimenez
d
,
T. Durst
a
, J. Zhang
a
, S. MacKinnon
a
, D. Leaman
a
, J.T. Arnason
a
, B.J.R. Philoge` ne
a,
a
Departments of Biology and Chemistry, University of Ottawa, Ottawa, Ont., Canada ON K1N 6N5
b
Agriculture and Agri-Food Canada, Winnipeg, Canada MB R3T 2M9
c
Herbario Juvenal, Universidad Nacional, Heredia 3000, Costa Rica
d
Departamento de Farmacia, Facultad de Quimica, UNAM, Coyoacan, Mexico, D.F., Mexico
Accepted 30 November 2005
Abstract
Terpenes isolated from tropical species of the Rutales were tested for insect antifeedant activity against rice weevil, Sitophilus oryzae
(L.) using a our disk bioassay that requires only small amounts of compounds (0, 0.05, 0.25 and 0.50% w/w). At 0.50% (w/w) ve
compounds isolated from Lansium domesticum (iso-onoceratriene, 3-keto-22-hydroxyonoceradiene, onoceradienedione, lansiolic acid
and lansiolic acid A) were shown to exhibit signicant antifeedant activity. Humilinolide C and D isolated from Swietenia humilis, and
gedunin from Cedrela odorata, were also active at 0.50% (w/w). The most interesting results were obtained from the spirocaracolitones
from Ruptiliocarpon caracolito which produced total feeding inhibition at 0.50% and potent antifeedant activity at concentrations as low
as 0.05%. In conclusion, the antifeedant bioassay provides a rapid and inexpensive method for screening novel compounds available in
small quantities to assess their activity as insect antifeedants.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Insect control agents; Antifeedants; Sitophilus oryzae
1. Introduction
In the New World tropics as well as in Southeast Asia,
native trees of the order Rutales are well known for the
production of bioactive terpenoids (Isman et al., 1997;
Arnason et al., 1993, 1987). In particular, the plant family
Meliaceae is noted for the production of useful bitter
principles which are insect antifeedant and growth-redu-
cing substances with low mammalian toxicity (Butterworth
and Morgan, 1968; Arnason et al., 1985; Schmutterer,
1990; Isman et al., 1997). In recent years, bioactive
terpenoids have been isolated from different neotropical
species including Cedrela odorata L., Ruptilocarpon car-
acolito L. (MacKinnon, 1995), and Swietenia humilis Zucc.
(Jimenez et al., 1997).
Lansium domesticum Corr. Serr. (Meliaceae) is a tree
native to Southeast Asia producing a sweet and aromatic
fruit, which is a popular dessert (Wong et al., 1994). The
leaves have been used by indigenous people in the
Philippines for the control of mosquitoes (Monzon et al.,
1994). The peel of this fruit is traditionally known to be
toxic to domestic animals. Phytochemical investigations of
the peel revealed the presence of triterpene glycosides, and
seco-onoceranoids such as lansic acid (Nishizawa et al.,
1983). The volatile constituents of the fruit are sesquiter-
pene hydrocarbons including germacrene-D (Wong et al.,
1994). The seed and leaf contain tetranortriterpenoids
named dukunolides (Nishizawa et al., 1985, 1989). The
major triterpenoid compound of the leaf is lansiolic acid
and the minor triterpene is characterized as cycloartanoid
type carboxylic acid.
The bark of L. domesticum is used traditionally as an
antimalarial remedy by the native people of Borneo
(Leaman et al., 1995). Despite the importance of such
use, there was no published phytochemical investigation on
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0022-474X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jspr.2005.11.005

Corresponding author. Tel.: +1 613 562 5800, 4166;

fax: +1 613 562 5765.
E-mail address: bphilog@science.uottawa.ca (B.J.R. Philoge` ne).
the bark until our group successfully isolated six novel
triterpenes namely iso-onoceratriene, 3-keto-22-hydroxyo-
noceradiene, onoceradienedione, lansiolic acid, lansiolic
acid A and 3-keto lansiolic acid (unpublished). In this
paper, we report the bioactivity of these compounds and
related terpenes isolated from neotropical species using a
Sitophilus oryzae L. antifeedant bioassay well suited to
study of small amounts of pure compounds.
2. Material and methods
2.1. Insect rearing
Rice weevils, S. oryzae (Coleoptera: Curculionidae),
were obtained from Agriculture and Agri-Food Canada,
Winnipeg, MB. Insects were reared in a growth chamber
(30 1C, 85% r.h. and 18 h light:6 h dark photoperiod) on
Western Hard Red Spring wheat at 14% moisture content.
2.2. Bioassay
The bioassay was performed according to a method
developed by Xie et al. (1996). Hard red spring wheat our
(200 mg) was added to 1 mL of an aqueous solution
containing the test substance at a concentration of 0.5%
w/w and mixed using a magnetic stirrer. Ten aliquots
(100 mL each) of the stirred suspension were placed in a
100 15 mm plastic Petri dish and allowed to dry overnight
in air at room temperature. The next day, ve of the our
disks were weighed and placed in a new Petri dish with 25
insects. The Petri dish was then sealed with paralm paper
and kept at 30 1C and 85% r.h. for 3 days. Finally, the
uneaten parts of the our disks were weighed. The insect
consumption for the different test substances was com-
pared to the control group. As only very small quantities of
isolated compounds were available, experiments were done
in duplicates.
ARTICLE IN PRESS
H
3
C
CH
3
CH
3
CH
2
H
3
C
CH
3
CH
3
H
3
C
H
3
C
CH
3
H
3
C
CH
2
H
3
C
CH
3
H
3
C
O
CH
3
O
H
3
C
CH
3
H
3
C
CH
2
H
3
C
CH
3
H
3
C
O
CH
3
OH
H
3
C
CH
3
CH
3
CH
2
CH
2
H
3
C
CH
3
H
3
C
O
COOH
H
3
C
CH
3
CH
3
CH
2
CH
2
H
3
C
CH
3
H
3
C
HO
COOH
H
3
C
CH
3
CH
3
CH
2
CH
2
H
3
C
CH
3
H
2
C
O
COOH
HO
ONOCERATRIENE ONOCERADIENEDIONE
3-KETO-22-HYDROXYONOCERADIENE 3-KETOLANSIOLIC ACID
LANSIOLIC ACID LANSIOLID ACID A
Fig. 1. Structures of triterpenes isolated from Lansium domesticum from Borneo.
S. Omar et al. / Journal of Stored Products Research 43 (2007) 9296 93
2.3. Compound isolation
The isolation of all compounds used in this study is
described in detail in Jimenez et al. (1997), MacKinnon
(1995), and Omar (2001). Their chemical structures are
given in Figs. 1 and 2.
3. Results and discussion
To date, numerous triterpenoids are known to possess
strong feeding deterrent activity (Arnason et al., 1987,
1985). The results of feeding deterrence studies against S.
oryzae by the crude extracts and six pure compounds
isolated from L. domesticum further support the phagor-
epressive effect of triterpenoids (Table 1). Flour disks
prepared using the crude extracts (ethyl acetate and hexane
fractions) exhibited total inhibition of diet consumption at
0.50% (w/w), but the water extract was found to be
phagostimulatory. Five of the pure compounds namely,
iso-onoceratriene, 3-keto-22-hydroxyonoceradiene, ono-
ceradienedione, lansiolic acid and lansiolic acid A exhibited
signicant antifeedant activities at 0.5% (w/w); however, 3-
keto lansiolic acid was not active at this concentration. The
crude extracts were more active than the isolated com-
pounds. This may be the result of synergistic effects of the
compounds in a mixture (Xie et al., 1996) and/or the
presence of other unidentied active compounds. However,
no clear structureactivity relationships were evident in this
ARTICLE IN PRESS
MeO
OAc
OAc
OAC
O
O
HO
O
OAc
SPIROCARACOLITONE B
MeO
OAc
OCOPh
OAc
O
O
HO
O
OH
OAc
OAc
O
O
H
3
CO
O
H
3
CO
O
AcOH
2
C
AcO
OR
SPIROCARACOLITONE D
SPIROCARACOLITONE E
O
O
CH
3
CH
3
O
CH
3
OAc
CH
3
O
H
3
C
O
O
OCOC(CH
3
)=CHCH
3
Me
Me
H
MeO
2
C
Me
O
OAc
Me
H
O
O
H
H
O
OCOCH(CH
3
)
2
Me
Me
OAc
MeO
2
C
Me
O
OH
Me
H
O
O
O
H
H
O
OCOCH
3
Me
Me
H
MeO
2
C
Me
O
OH
Me
H
O
O
H
H
R=CO C(CH
3
)=CHCH
3
GEDUNIN
HUMILINOLIDE B HUMILINOLIDE C
HUMILINOLIDE D
Fig. 2. Structures of triterpenes isolated from neotropical plants.
S. Omar et al. / Journal of Stored Products Research 43 (2007) 9296 94
study. The concentration used to observe antifeedant
effects was also much higher than for the commercially
available products such as Margosan-O, active at a
3.75 ppm azadirachtin level (Xie et al., 1996) or toosenda-
nin at 20 ppm (Champagne et al., 1992). Thus, the
commercial application of L. domesticum compounds as a
stored-product antifeedant may not be practical. Never-
theless, the bioassay employed in this study clearly allowed
a fast screening and isolation of bitter compounds present
at low concentrations in the active crude extracts. For
example, other secondary compounds were also isolated
from tropical sources (Liche xanthone, Formylorcinol-
carboxyl, methyl orsellinate, gyrophoric acid) but were
found inactive when tested with this bioassay (data not
shown).
The chemical structures of other triterpenes isolated
from the Rutales are shown (Fig. 2). The CD spirocar-
acolitones B, D and E isolated from R. carocolito caused
total feeding inhibition at 0.50% (w/w) and were sig-
nicantly active at 0.25 % (w/w) (Table 2). Spirocaraco-
litone B and D also showed signicantly different activity
at 0.05%. Other studies also revealed that these com-
pounds were very active when tested against the European
corn borer, Ostrinia nubilalis Hu bner (MacKinnon, 1995)
and showed modest antifungal and antimalarial activities
(MacKinnon et al., 1997).
The main active compound in the C. odorata wood,
gedunin, had previously shown moderate antifeedant
activity against many insect species (Arnason et al., 1987;
Kubo and Klocke, 1986; Champagne et al., 1992).
Humilinolides isolated from Swietenia humilis showed
signicant growth-reducing activity including delay in time
of pupation and adult emergence against O. nubilalis
(Jimenez et al., 1997). In this study, gedunin and
humilinolide C and D were active but humilinolide B was
inactive at 0.50% (w/w) (Table 3). The most active
compound was humilinolide C. A similar spectrum of
activity was noted with O. nubilalis, humilinolide C
exhibiting the highest growth reducing activity (Jimenez
et al., 1997). These studies suggested the mode of action of
these compounds to be a combination of antifeedant action
and post-digestive toxicity as seen in other limonoids
(Isman et al., 1997; Xie et al., 1995).
Kubo and Klocke (1986) indicated that most antifeedant
compounds they had isolated exhibited pharmacological
activities as well and suggested that the antifeedant
bioassay has unexpectedly provided them with a unique
system for screening bioactive compounds. Studies con-
ducted in our laboratory have demonstrated that the
extracts from L. domesticum and C. odorata exhibited
potent in vitro antiplasmodial activities (Leaman et al.,
1995; MacKinnon et al., 1997). Furthermore, the anti-
feedant bioassay provides a rapid and inexpensive method
for screening such compounds.
Acknowledgements
Funding was provided by the Natural Science and
Engineering Research Council of Canada and Ontario
Ministry of Science and Technology.
ARTICLE IN PRESS
Table 2
Antifeedant activity of spirocaracolitones against Sitophilus oryzae
Compound Concentration
(% w/w)
Consumption
of diet
(% control7SEM)
P value
Control 0 100.078.5a
Spirocaracolitone B 0.05 67.176.1b o 0.05
0.25 8.873.9c o0.001
0.50 0.0 o0.001
Spirocaracolitone D 0.05 74.676.3b 0.05
0.25 2.971.3c o0.001
0.50 0.0 o0.001
Spirocaracolitone E 0.05 78.3710.1a,b 40.08
0.25 19.079.9d o0.001
0.50 0.0 o0.001
Multiple range test using Tukeys test (Po0.05). The same letters denote
treatments not signicantly different from each other.
Table 1
Antifeedant activity of crude extracts and pure compounds isolated from
Lansium domesticum against Sitophilus oryzae (0.5% w/w)
Treatment Consumption of diet
(% control7SEM)
P value
Control 100.0710.2a
3-ketolansiolic acid 68.9717.4a,b 40.05
Iso-onoceratriene 64.777.5b o0.05
Lansiolic acid 63.273.8b o0.01
Lansiolic acid A 56.174.5b o0.01
3-keto-22-hydroxyonoceradiene 53.873.4b o0.01
Onoceradienedione 40.176.2b o0.001
Ethyl acetate fraction 0.0c o0.001
Hexane fraction 0.0c o0.001
Water fraction 184.6729d o0.001
Multiple range test using Tukeys test (Po0.05). The same letters denote
treatments not signicantly different from each other.
Table 3
Antifeedant activity of humilinolides and gedunin against Sitophilus
oryzae
Compound Concentration
(% w/w)
Consumption
of diet
(% control7SEM)
P value
Control 10077.4a
Humilinolide B 0.50 79.7716.7a 40.05
Humilinolide C 0.50 24.871.0b o0.05
Humilinolide D 0.50 65.2711.1c o0.05
Gedunin 0.50 31.276.3b o0.05
Multiple range test using Tukeys test (Po0.05). The same letters denote
treatments not signicantly different from each other.
S. Omar et al. / Journal of Stored Products Research 43 (2007) 9296 95
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