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