Insect antifeedants, pterocarpans and pterocarpol, in heartwood of Pterocarpus macrocarpus Kruz

Article abstract of DOI:10.1271/bbb.60017

The insect antifeedant activities of pterocarpans and a sesquiterpene alcohol from the dichloromethane extract of Pterocarpus macrocarpus Kruz. (Leguminosae) were evaluated against the common cutworm, Spodoptera litura F. (Noctuidae), and the subterranean

Full text of DOI:10.1271/bbb.60017

Biosci. Biotechnol. Biochem., 70 (8), 1864–1868, 2006
Insect Antifeedants, Pterocarpans and Pterocarpol,
in Heartwood of Pterocarpus macrocarpus Kruz.
1
;y
1
1
Masanori MORIMOTO, Hiromi FUKUMOTO, Masaru HIRATANI,
2
1
Warinthorn CHAVASIRI, and Koichiro KOMAI
1
Department of Agricultural Chemistry, Faculty of Agriculture, Kinki University,
Nara City, Nakamachi 3327-204, Japan
2
Department of Chemistry, Faculty of Science, Chulalongkorn University,
Bangkok, 10330, Thailand
Received January 11, 2006; Accepted April 11, 2006; Online Publication, August 23, 2006
[doi:10.1271/bbb.60017]
The insect antifeedant activities of pterocarpans and
logical activities and contribute to the chemical defense
system in plants. Some legumes produce biologically
active pterocarpans, i.e. pisatin and phaseolin, as
phytoalexins to resist infection by phytopathogens and
a sesquiterpene alcohol from the dichloromethane
extract of Pterocarpus macrocarpus Kruz. (Legumino-
sae) were evaluated against the common cutworm,
Spodoptera litura F. (Noctuidae), and the subterranean
termite, Reticulitermes speratus (Kolbe)(Rhinotermiti-
dae). Three pterocarpans, (À)-homopterocarpin (1),
1
)
phytophagous insects (Fig. 1). The insecticidal natural
products, rotenoids, are isoflavones produced by a
Leguminosae (Fig. 1). These phytophenolics act as a
chemical defense system against phytophagous insects
(
(
À)-pterocarpin (2), and (À)-hydroxyhomopterocarpin
2
,3)
3) and the sesquiterpene alcohol, (+)-pterocarpol (5),
and phytopathogens. Flavonoids are widely distribut-
ed in many plants and a large amount is accumulated in
their tissue. While they show insect antifeedant activity
against various phytophagous organisms, this property is
not lethal. With regard to their chemical structure, the
benzopyranone moiety and loss of a hydroxyl group in
the flavonoid appear to be important for their insect
were isolated from the dichloromethane extract of
the heartwood of P. macrocarpus under guidance by
a biological assay. Among these natural products, the
most active insect antifeedant against both S. litura and
R. speratus was 1. On the other hand, sesquiterpene
alcohol 5 showed less insect antifeedant activity than
the other pterocarpans against both insect species.
While its methylated derivative, (À)-methoxyhomo-
pterocarpin (4), showed high biological activity, 3
showed less insect antifeedant activity in this study.
Interestingly, racemic 1 did not show insect antifeedant
activity against S. litura. However, all of the test
pterocarpans and isoflavones showed antifeedant activ-
ity against the test termites. Additionally, since these
compounds were major constituents of P. macrocarpus,
these antifeedant phenolics may act as chemical
defense factors in this tree. In Thailand, lumber made
from this tree is used to make furniture and in
building construction due to its resistance to termite
attack.
4
)
antifeedant activity against S. litura.
The common cutworm Spodoptera litura F. (Noctui-
dae) is the most notorious polyphagous insect and the
most in famous crop pest in the world. This pest insect
seriously damages many crops, and adequate pest
control is required for crop production. The subterranean
termite, Reticulitermes speratus (Kolbe), (Rhinotermiti-
dae) commonly lives in Japanese forests and feeds on
rotten wood. Some flavonoids have been shown to have
antifeedant activity against the termite, Coptotermes
formosaunus, the most active compounds being the
0
0
3 ,4, 5,7-substituted flavonoids, quercetin and taxifolin
5
)
(Fig. 2).
In Thailand, the large deciduous tree, Pterocarpus
macrocarpus Kruz. (Leguminosae) is used to make
furniture and in building construction due to its
resistance to termite attack. The brownish red color of
the heartwood of Pterocarpus spp. may be attributed to
Key words: Pterocarpus macrocarpus Kruz.; insect an-
tifeedant; Spodoptera litura F.; Reticuli-
termes speratus (Kolbe); pterocarpan
6
)
the presence of aurones. In this study, the constituents
of P. macrocarpus were evaluated with regard to their
insect antifeedant activity against S. litura and R. sper-
atus.
Pterocarpans are a member of the isoflavone family
and have two cyclic ether linkage moieties in their
chemical structure (Fig. 1). They show various bio-
y
To whom correspondence should be addressed. Fax: +81-742-43-1445; E-mail: masanori@nara.kindai.ac.jp

Insect Antifeedants from Pterocarpus macrocarpus Kruz.
1865
H
H
O
O
O
O
O
O
O
O
O
OH
H
H
H
O
O
O
O
O
O
Pisatin
Phaseollin
Rotenone
Fig. 1. Leguminosae Pterocarpans, Pisatin and Phaseollin, as Phytoalexins and the Insecticidal Isoflavone, Rotenone.
OH
OH
OH
OH
OH
OH
HO
O
HO
O
HO
O
OH
OH
OH
OH O
OH O
OH
Quercetin
Taxifolin
(+)-Catechin
Fig. 2. Antitermite-Active Flavonoids and (+)-Catechin in This Text.
O
O
O
O
O
O
H
H
H
H
H
O
R
O
H
O
O
O
O
O
(-)-Homopterocarpin (1)
(-)-Pterocarpin (2)
R: OH
(-)-Hydroxyhomopterocarpin (3)
R: OMe (-)-Methoxyhomopterocarpin (4)
O
O
O
O
O
HO
O
H
OH
R
R
OH
(+)-Pterocarpol (5)
(±)-Isomedicarpin (6)
R: OH
2',4'-Dihydroxy-7-methoxyisoflavone (7)
R: OMe 2',4',7-Trimethoxyisoflavone (8)
Fig. 3. Pterocarpans and Pterocarpol Used for the Evaluation of Insect Antifeedant Activity.
Material and Methods
Aldrich Co., Ltd. The plant material (P. macrocarpus)
was purchased from a folk-medicine pharmacy in
Bangkok as air-dried heartwood. The plant material
was extracted with dichloromethane at Chulalongkorn
University (Thailand). (ꢁ)-Homopterocarpin (1), (ꢁ)-
pterocarpin (2), (ꢁ)-hydroxyhomopterocarpin (3), and
(+)-pterocarpol (5) were isolated from the dichloro-
methane extract of P. macrocarpus by silica gel column
chromatography (Fig. 3). The extract was separated by
silica gel column chromatography with elution by
hexane:ethyl acetate (10:3) to afford 1 (15.8% yield
from the dichloromethane extract), 3 (6.12% yield from
the dichloromethane extract), and 5 (4.84% yield from
the dichloromethane extract). Compound 2 was isolated
from the fraction that contained 1 by preparative
General. Optical rotation values were determined at
ꢀ
1
13
2
5 C with a Horiba SEPA-300 instrument. H- and C-
NMR data were measured with a Jeol 270EX (270 MHz)
spectrometer, using TMS as an internal standard. Mass
spectra were taken at 70 eV (probe) by a Shimadzu
9
100-MK GCMS instrument. These compounds were
separated by column chromatography on BW-127ZH
and BW-300 silica gel (Fuji Silysia Chemical Ltd.,
Japan). HPLC was performed with a Shimadzu VP-10
system, and TLC used silica gel plates with a fluorescent
indicator (Merck 60 F254 silica gel 0.25 mm thick).
Chemicals. (+)-Catechin was purchased from Sigma-

1866
M. MORIMOTO et al.
crystallization, using a hexane-ethyl acetate solvent
system. Separation was monitored by measuring the
melting point and by an HPLC analysis. The analytical
conditions for HPLC were an Imtakt cadenza column
test compound was evaluated in terms of the ED50 value
for the rate of feeding inhibition calculated from the area
of the leaf disk consumed. A straight line was fitted to
the points obtained by the bioassay, and the ED50 value
was calculated as the dose corresponding to the
midpoint between complete inhibition and no effect.
The leaf disks were replaced by paper disks of 6 mm in
diameter for the termites made from filter paper Toyo
No. 1). Treated paper disks were placed in a petri dish of
5 cm in diameter on moistened vermiculite ca. 5 mm
thick. Four paper disks were placed in alternating
positions in the same Petri dish. Twenty workers from
the same colony were released into the dish and fed on
(
CD-C18, 100 mm ꢂ 4.6 i.d.), eluent of 50% acetoni-
trile in water, flow rate of 0.8 ml/min, detection at UV
80 nm. These natural products were identified by
2
comparing their spectral data with those in the liter-
ature.7 Compound 4 was obtained by methylating 3
,8)
0
0
with methyl iodide and K CO3 (95.7% yield). 2 ,4 -
2
Dibenzyloxy-7-methoxyisoflavone and (ꢃ)-isomedicar-
9
)
pin (6) were prepared by known synthetic methods.
0
0
2
,4 -Dibenzyloxy-7-methoxyisoflavone was hydrolyzed
ꢀ
0
0
ꢀ
with HCl–AcOH (1:2) at 60 C to obtain 2 ,4 -dihy-
droxy-7-methoxyisoflavone (7, 29.6% yield). Com-
pounds 6 and 7 were methylated with methyl iodide in
acetone with K CO to obtain racemic homopterocarpin
the disks for 2 weeks in darkness at 27 C. To keep the
paper from drying, the disks were occasionally sprayed
with water. Partially consumed paper disks were pasted
on black paper and their images digitized. Data were
analyzed on a PC using NIH Image after black and white
inversion. For each experiment, the data for an intact
disk were measured and compared to those of a treated
disk. To measure the activity of a test compound, we
used the antifeedant index AFI = % of treated disks
consumed/(% of treated disks consumed + % of control
disks consumed) ꢂ 100. The AFI value was converted
to the feeding inhibition rate FI (%) = (50-AFI) ꢂ 2.
An FI value under 40% when treated at 1 mg/disk (ca.
2
3
0
0
[
(ꢃ)-1, 54.4% yield] and 2 ,4 ,7-trimethoxyisoflavone
(
8, 48.6% yield). These synthetic analogues have had
their chemical structures confirmed by comparison with
1
literature data on the basis of their EI-MS, and H- and
1
3
10)
C-NMR spectra.
Insects. Common cutworms (Spodoptera litura F.)
were purchased from Sumika Technoservice Co., Ltd.
Takarazuka, Japan). The insects were reared on an
(
2
artificial diet (Insecta LF, Nihon Nosan Kogyo Co.,
ꢀ
1.2 mmol/cm ) is indicated as inactive in this study.
Japan) in a controlled environment at 26.5 C and 60%
humidity. Subterranean termites, Reticulitermes spera-
tus (Kolbe), were collected from a pine forest near the
Results and Discussion
ꢀ
0
00
Enju coast (Wakayama Pref. Japan, N35 53 15 ,
ꢀ
The antifeedant effects of the test compounds greatly
differed between the phytophagous worms and termites.
In this study, the subterranean termites, R. speratus,
were highly sensitive to all of the test compounds,
except for sesquiterpene alcohol 5 and the phytophenol,
(+)-catechin. (+)-Catechin was used as a negative
control in the termite feeding test, and did not
significantly inhibit termite feeding at 100 mg/disk
(Table 1).
0
00
E135 07 51 ) in 2004, brought to our university, and
fed on pieces of pine wood in a controlled environment
ꢀ
at 26.5 C until bing used in this test.
Antifeedant bioassays. The antifeedant test against
common cutworms used leaf disks of 2 cm in diameter
that had been prepared with a cork borer from the leaves
of fresh sweet potato (Ipomoea batata cv. narutokintoki)
cultivated on the farm at Kinki University (Nara Pref.,
Japan). Two disks were treated with test compounds in
an acetone solution and two other disks were treated
only with acetone as a control. The four disks were set in
alternating positions in the same petri dish on moistened
filter paper at the bottom. After the acetone had
completely evaporated, 15 larvae (3rd instar) were
released into the dish. The dishes were then kept in an
The insect antifeedant activities of 1, 2, 3 and 5 from
Table 1. Insect Antifeedant Activities of the Test Flavonoids against
S. litura Larvae and R. speratus
S. litura
R. speratus
Compound
2
ED50 (95% CI) (mmol/cm ) FI (%) (SD) 50 mg/disk
ꢀ
11)
1
2
3
4
5
6
7
8
0.14 (0.098–0.1839)
0.37 (0.322–0.419)
0.91 (0.798–1.053)
0.10 (0.058–0.158)
1.05 (0.892–1.255)
inactive
89.5 (0.80)
86.2 (2.36)
80.9 (3.43)
85.8 (0.32)
16.4 (7.21)
93.1 (0.24)
79.9 (1.36)
94.2 (0.12)
insect-rearing room at 26.5 C in the dark for 5–6 h.
Images of the partially consumed leaf disks were
digitized. Data were analyzed on a PC by using NIH
Image (http://rsb.info.nih.gov/nih-image/). The data for
an intact disk were measured and compared to those of a
treated disk for each experiment. To measure the
activity of a test compound, we used the antifeedant
index AFI = % of treated disks consumed/(% of treated
disks consumed + % of control disks consumed) ꢂ
inactive
inactive
1 (racemic) inactive
a
+)-Catechin NT
86.4 (2.19)
38.4a
(
a
Used as negative control for the termite antifeedant test at 100 mg/disk
1
00. The AFI value was converted to the feeding
2
Inactive, FI < 40% (1 mg/disk, ca. 1.2 mmol/cm )
NT, Not Tested
inhibition rate (%) = (50-AFI) ꢂ 2. The potency of a

Insect Antifeedants from Pterocarpus macrocarpus Kruz.
1867
2
P. macrocarpus were between 0.1 and 1.04 mmol/cm
based on the ED50 values against S. litura. However,
isoflavones. Pterocarpans 1–4 showed a slight correla-
tion between their biological activity against the
common cutworm and that against the termite.
0
0
natural pterocarpan 1 corresponding to 2 ,4 ,7-trisubsti-
tuted isoflavones 7 and 8 did not show insect antifeedant
Insect antifeedant 1 moderately inhibited the activities
of both ꢀ-amylase and 5ꢀ-reductase (data not shown).
0
0
activity. 2 ,4 ,7-Trisubstituted isoflavones 7 and 8 have
been predicted to be biosynthetic precursors of their
1
3)
Some insect antifeedants inhibit ꢀ-amylase and other
enzymes that participate in metamorphosis and physio-
logical functions. However, the mode of action of these
pterocarpans for insect antifeedant activity was not
demonstrated in this study.
Finally, the fact that lumber made from P. macro-
carpus is used to make furniture and in construction in
Thailand is reasonable considering its resistanse to
termite attack. Pterocarpans 1–3 found in P. macro-
carpus may be useful as a natural termite repellent and
could be used as lead compounds for the development of
termite repellents and other agents for the protection of
wood products.
1
0)
respective pterocarpans (1–3). The results with the
corresponding isoflavones (7 and 8) did not show
biological activity, suggesting that the presence of a
hydrofuran moiety played an important role. We have
previously reported that various simple dihydrobenzo-
furans showed insect antifeedant activity against S. lit-
ura larvae.2
)
Interestingly, racemic 1 showed no biological activity,
even though 1 showed strong insect antifeedant activity
against S. litura larvae. A similar change in the bio-
logical activity of flavonoids attributed to an optical
isomer or racemic form has been reported, in that (ꢁ)-
medicarpin showed insect antifeedant activity, but
racemic medicarpin did not show such activity against
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