Antifeedant activity of an anthraquinone aldehyde in Galium aparine L. against Spodoptera litura F.

Article abstract of DOI:10.1016/S0031-9422(02)00095-X

The insect antifeedant anthraquinone aldehyde nordamnacanthal (1,3-dihydroxy-anthraquinone-2-al) was identified in Galium aparine L., and isolated from the root powder of akane (Rubia akane), a member of the Rubiaceae. Structure-activity relationship (SAR) studies using a series of anthraquinone analogues suggested that the aldehyde group on the anthraquinone was more important than the quinone moiety for antifeedant activity against the common cutworm (Spodoptera litura). High levels of nordamnacanthal were found in the seed leaf stage and in callus tissue induced from seedlings of G. aparine, but its concentration decreased with plant development. Since these compounds are natural pigments for dying textiles, we also evaluated the antifeedant activity against the carpet beetle (Attagenus japonicus), a textile pest was also evaluated. While nordamnacanthal had strong antifeedant activity against the common cutworm, it did not show any antifeedant activity against the carpet beetle. The most effective antifeedant against the carpet beetle was the major constituent in the extract of R. trictorum, lucidin- 3 - O-primeveroside, a food pigment. 2002 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0031-9422(02)00095-X

Phytochemistry 60 (2002) 163–166
www.elsevier.com/locate/phytochem
Antifeedant activity of an anthraquinone aldehyde in
Galium aparine L. against Spodoptera litura F.
Masanori Morimoto*, Kumiko Tanimoto, Akiko Sakatani, Koichiro Komai
Department of Agricultural Chemistry, Kinki University, Nakamachi 3327-204 Nara, Japan
Received 5June 2001; received in revised form 1 March 2002
Abstract
The insect antifeedant anthraquinone aldehyde nordamnacanthal (1,3-dihydroxy-anthraquinone-2-al) was identified in Galium
aparine L., and isolated from the root powder of akane (Rubia akane), a member of the Rubiaceae. Structure–activity relationship
(SAR) studies using a series of anthraquinone analogues suggested that the aldehyde group on the anthraquinone was more
important than the quinone moiety for antifeedant activity against the common cutworm (Spodoptera litura). High levels of nor-
damnacanthal were found in the seed leaf stage and in callus tissue induced from seedlings of G. aparine, but its concentration
decreased with plant development. Since these compounds are natural pigments for dying textiles, we also evaluated the antifeedant
activity against the carpet beetle (Attagenus japonicus ), a textile pest was also evaluated. While nordamnacanthal had strong anti-
feedant activity against the common cutworm, it did not show any antifeedant activity against the carpet beetle. The most effective
antifeedant against the carpet beetle was the major constituent in the extract of R. trictorum, lucidin-3-O-primeveroside, a food
pigment. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Galium aparine L; Rubia akane; Rubiaceae; Anthraquinones; Nordamnacanthal; Spodoptera litura; Attagenus japonicus
1. Introduction
2. Results and discussion
Galium aparine L. (bedstraw), a member of the
Rubiaceae, is an infamous annual weed in Japan. It is
not harmed by phytophagous insects in the field and
occasionally damages farm crops. On the other hand,
some members of the Rubiaceae have historically been
used as a source of natural dyes for textiles and anthra-
quinone dyes have recently been used commercially.
Anthraquinones found in the Rubiaceae (Halim et al.,
1992; El-Gamal et al., 1995) and iridoids in Galium spp.
(Iavarone et al., 1983) showed weak cytotoxic activity
(Halim et al., 1992; Kawasaki et al., 1992; El-Gamal et
al., 1997). In this study, the structure–activity relation-
ships of a series of anthraquinones as insect antifeedants
against a herbivorous pest, the common cutworm, and a
textile pest, the carpet beetle, are reported.
In the screening of Japanese wild plants against the
common cutworm Spodoptera litura, the hexane extract
of G. aparine was found to have potent insect anti-
feedant activity, but the responsible compound (nor-
damnacanthal 1, see below) was not stable and occurred
only in small quantities in the mature plant, based on TLC
analysis. A large amount of presumably the same insect
antifeedant was present in the hexane extract of the Rubia
akane root, hence this plant material was used for bulk
collection and analysis of the responsible compound 1.
Compound 1 was purified by silica gel column chromato-
graphy, recrystallized from hexane, and its structure con-
firmed to be nordamnacanthal 1, by comparison of NMR
spectral data with literature values (Zhou et al., 1994). The
MS fragmentation pattern and physicochemical proper-
ties of this compound were similar to those of the active
compound in the hexane extract of G. aparine and its calli,
thus identifying the insect antifeedant in G. aparine as the
known compound 1 (Figs. 1 and 2).
Calli produced from seedlings of G. aparine were germi-
nated in the dark at 10 ꢀC, and these also accumulated 1.
* Corresponding author. Fax: +81-742-43-1445.
E-mail address: morimoto@nara.kindai.ac.jp (M. Morimoto).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00095-X

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M. Morimoto et al. / Phytochemistry 60 (2002) 163–166
Fig. 3. Change in nordamnacanthal concentration in aerial parts of
G. aparine with development.
Fig. 1. Nordamnacanthal and alizarin anthraquinones tested for
antifeedant activity.
Table 1
Antifeedant activities of test anthraquinones against S. litura and
A. japonicus
Compound
S. litura
A. japonicus
ED50
(mmol/cm2)
(95% CIa)
FIRb
(%)
1 Nordamnacanthal
2 Lucidin-3-O-primerside
3 Alizarin
0.12
(0.111–0.1342) Inactive
Inactive
Inactive
Inactive
Inactive
88.9
Inactive
Inactive
Inactive
85.6
4 Lucidin
5 Lucidin-3-O-glucoside
6 2-Hydroxymethylanthraquinone Inactive
7 Anthraquinone-2-carboxylic acid Inactive
Inactive
8 Anthraquinone-2-aldehyde
9 2-Methylanthraquinone
) 60–.70In5.8a(20c5.t6iv5e
3.02
(2.026–4.556)
58.3
a
CI: confidence interval.
b
FIR: feeding inhibition rate against carpet beetle. Staining dose: 1–4 (1 mg/
cm2 cloth), 5–9 (3 mg/cm2 cloth). Inactive: antifeedant activity was below 50%
at 0.33 mg/cm2 against S. litura and 3 mg/cm2 against A. japonicus.
weight, and so the concentration of anthraquinone per
plant accordingly decreased (Fig. 3). In a similar man-
ner, it has been reported that in the Cyperaceae, the
total amount of JH III (juvenile hormone III), which is
part of the plant’s chemical defense system, decreases
with plant development (Bede et al., 1999).
We next compared other natural pigments in the
Rubiaceae, including lucidin-3-O-primeveroside 2 and
alizarin 3, as well as authentic anthraquinone analogues
(4–9) in a SAR study of anthraquinones. Among the
natural compounds in Rubiaceae, 1 showed the stron-
gest insect antifeedant activity (Table 1), with complete
inhibition at 83 mg/cm2. Since quinones and compounds
with aldehyde groups have been reported to be insect
antifeedants (Blaney et al., 1987; Morimoto et al., 1999),
the importance of an aldehyde group at C-2 of the
anthraquinone for antifeedant activity against the com-
mon cutworm was evaluated. Anthraquinones 2–7,
Fig. 2. Comparison with MS spectrum fragment pattern of nor-
damncanthal and its hexane extract of calli from G. aparine.
Indeed, both 1 and its decomposition products from
hexane extracts of calli were readily detected by TLC.
On the other hand, while the total amount of 1 in the
plant remained essentially the same based on the HPLC
analysis, plant growth caused an increase in fresh

M. Morimoto et al. / Phytochemistry 60 (2002) 163–166
165
which lack an aldehydic group at the C-2 position, did
not show insect antifeedant activity, whereas anthra-
quinone-2-aldehyde 8 and 2-methylanthraquinone 9,
still had an antifeedant activity against common cut-
worms (Table 1). These results suggest that aldehydic
and/or methyl groups at the C-2 position, are important
for antifeedant activity against the common cutworms.
The extract of R. tinctorum protect textiles from the
carpet beetle (Nakashima and Doi, 1997). However, our
antifeedant assays for anthraquinones using carpet bee-
tles showed that only 2 (1 mg/cm2 ) significantly inhib-
ited feeding, whereas 1 and 8 had no antifeedant activity
against the carpet beetle (Table 1). While the carpet
beetle was sensitive to 2, this activity disappeared with
its conversion to 5. These results suggest that dyes from
R. akane and R. tinctorum may protect against textile
pests partially due to the presence of 2. These com-
pounds cannot, however, be used commercially since
they need to be applied in very high doses. Nevertheless,
the results may be useful for the development of other
functional textile pigments.
chromatography, eluted with n-hexane–EtOAc (3:1),
and purified by recrystallization from n-hexane.
3.4. Preparation of test compounds
Compound 2 purified in crystalline form, was
obtained from the food pigment made from the extract
ꢀ
of R. tinctorum dissolved in glycerol, and stored at 4 C
for 1 month. Compound 4 was prepared by hydrolysis
ꢀ
of 5 using 6 M HCl aq. at 90 C, whereas compound 5
was obtained by hydrolysis of 2 using 1.2 M HCl aq.,
compound 2 (140 mg, 0.25mmol) dissolved in 1.2 M
ꢀ
HCl aq. (30 ml) at 90 C for 1.5h. After neutralization
using NaOH aq. we obtained the crystalline 5 by filtra-
tion (89.5mg, yield 83.5%).
Compound 5 (5.3 mg, 12.3 nmol) dissolved in 6 M
HCl aq. (15ml) and hydrolysed at 90 ꢀC for 4 h. We
obtained the crystalline 4 by filtration (3.0 mg, yield
90.6%).
Compound 8 was prepared by oxidation of 4 with
PCC. Compound 6 (630 mg, 2.64 mmol) was dissolved
in CH2Cl2 (12.5ml), and PCC (1.97 g, 9.2 mmol) was
added to the solution along with 3A molecular sieves
(2 g). After 1 h, the products were extracted with ethyl
acetate, concentrated by evaporation, passed through
silica gel, and recrystallized from hexane to give pure
crystalline 8 (60 mg, 9.6%).
3. Experimental
3.1. General
ꢀ
1H and 13C NMR: CDCl3 with TMS as an int. stan-
dard. IR spectra were measured using a KBr tablet.
TLC was performed on silica gel F254 (Merck) using
n-hexane–EtoAc (3:1). Spots were visualized by fluor-
escence at 254 and 365 nm or by spraying with 50%
H2SO4. Nordamnacanthal has an orange fluorescence
when irradiated at 365nm. Authentic anthraquinones
3 and 6 were purchased from Tokyo Kasei Co., Ltd., 9
was purchased from Wako Chemical Co., Ltd., and 7
was purchased from Aldrich Chemical Co., Ltd. The
food pigment from R. tinctorum was provided by San-Ei
Gen F.F.I. Co., Ltd.
White crystal. mp 188–191 C EIMS (probe) 70 eV,
m/z (rel. int.): 236 (M+, 100), 207 (24.2), 151 (34.7).
HR-EIMS (probe) m/z: 236.11799 (C15H8O3, requires
236.04732). 1H NMR (270 MHz CDCl3): ꢀ 10.25(1H, s,
CHO), 8.81 (1H, s, Ar-H), 8.49 (1H, d, J=7.9 Hz),
8.39–8.33 (2H m., Ar-H), 8.30 (1H, s, Ar-H), 7.90–7.83
(2H, m, Ar-H). 13C NMR (270 MHz CDCl3): ꢀ 190.9,
182.3, 182.3, 139.9, 134.7, 133.2, 129.7, 128.3, 127.6.
3.5. Insect rearing
Common cutworms (Spodoptera litura Lepidoptera,
Noctuidae) purchased from Sumika Techno Service Co.
Ltd. (Takarazuka, Japan), were reared on an artificial
diet (Insecta LF, Nihon Nosan Kogyo Co. Ltd.) in a
controlled room environment at 26.5 ꢀC and 60%
humidity. Carpet beetles (Attagenus japonicus, Dermes-
tidae) were a gift from Dr. T. Nakashima (Kinki Uni-
versity, Japan) and reared on dried bonito shavings in a
3.2. Plant material
Bedstraw, G. aparine L., was collected near Kinki
University, Nara, Japan, from November to May 1998–
2000.
ꢀ
3.3. Extraction and isolation
controlled room environment at 28 C.
Fresh aerial portions of G. aparine (3 kg) were
extracted with n-hexane for 3 days at 4 ꢀC, and the
resulting extract was concentrated under reduced pres-
sure to give a yellowish green oil (4.19 g). The root
powder of R. akane (2 kg), was also extracted with
n-hexane at 4 ꢀC for 3 days, with the extract con-
centrated under reduced pressure to give a brown oil (5.37
g). The extracts were separated by silica gel column
3.6. Antifeedant bioassay
The experimental conditions have been described
previously (Morimoto et al., 1999). Leaf-disks, 2 cm in
diameter, were prepared from fresh sweet potato (Ipo-
moea batatas) leaves using a cork borer. Two disks were
treated with a specified amount of the plant extract or
test compounds in an acetone solution and two other

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M. Morimoto et al. / Phytochemistry 60 (2002) 163–166
disks that were treated only with acetone were used as a
control. Acetone-insoluble test compounds were applied
using an arabic gum paste. The four disks were placed
in alternating positions in the same petri dish. After
complete removal of the solvent, 15larvae (third instar)
were released into the dish. To evaluate antifeedant
activity against textile pests, the substrate was changed
from sweet potato leaves to swatches of wool fabrics ( l
cm  1 cm). The incubation period was 6 h in the dark
for the common cutworm. The slower-feeding carpet
beetle was allowed to feed for 2 weeks.
AFI (antifeedant index)=% of treated disks con-
sumed/(% of treated disks consumed+% of control
disks consumed ) Â 100. A value of less than 30 was
considered to reflect antifeedant activity. The ED50
values of the test compounds were calculated by the
probit method after changing the AFI value to a feeding
inhibition rate.
Acknowledgements
We thank Dr. Teruo Nakashima (Kinki University)
for providing the carpet beetle (Attagenus japonicus),
Morihiro Kinoshita (Nihon Funmatsu Yakuhin Co.,
Ltd., Japan) for supplying the dried root chips of
R. akane and San-Ei Gen F.F.I. Co., Ltd. for supplying
the food pigment from R. tinctorum.
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