Insecticidal sesquiterpene from Alpinia oxyphylla against Drosophila melanogaster

Article abstract of DOI:10.1021/jf000325z

In the course of screening for novel naturally occurring insecticides from Chinese crude drugs, an MeOH extract of Alpinia oxyphylla was found to possess insecticidal activity against larvae of Drosophila melanogaster Meigen. From the extract, an insecticidal compound was isolated by bioassay-guided fractionation and identified as nootkatone (1) by GC, GC-MS, and ¹H and ¹³C NMR spectroscopy. In bioassays for insecticidal activity, I showed an LC₅₀ value of 11.5 μmol/mL of diet against larvae of D. melanogaster and an LD₅₀ value of 96 μg/adult against adults. Epinootkatol (1A), however, showed slight insecticidal activity in both assays, indicating that the carbonyl group at the 2-position in I was the important function for enhanced activity of 1.

Full text of DOI:10.1021/jf000325z

J. Agric. Food Chem. 2000, 48, 3639−3641
3639
In secticid a l Sesqu iter p en e fr om Alpin ia oxyph ylla a ga in st
Dr osoph ila m ela n oga ster
,
†
†
‡
Mitsuo Miyazawa,* Yuji Nakamura, and Yukio Ishikawa
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae,
Higashiosaka-shi, Osaka 577-8502, J apan, and Laboratory of Applied Entomology, Graduate School of
Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, J apan
In the course of screening for novel naturally occurring insecticides from Chinese crude drugs, an
MeOH extract of Alpinia oxyphylla was found to possess insecticidal activity against larvae of
Drosophila melanogaster Meigen. From the extract, an insecticidal compound was isolated by
1
13
bioassay-guided fractionation and identified as nootkatone (1) by GC, GC-MS, and H and C NMR
spectroscopy. In bioassays for insecticidal activity, 1 showed an LC50 value of 11.5 µmol/mL of diet
against larvae of D. melanogaster and an LD50 value of 96 µg/adult against adults. Epinootkatol
(1A), however, showed slight insecticidal activity in both assays, indicating that the carbonyl group
at the 2-position in 1 was the important function for enhanced activity of 1.
Keyw or d s: Alpinia oxyphylla; Drosophila melanogaster Meigen.; sesquiterpene; nootkatone;
epinootkatol; insecticidal activity; structure-activity relationship
INTRODUCTION
Biological activity of A. oxyphylla against D. melano-
gaster has not been reported.
The present paper deals with the isolation of the
active principles, their derivatives, and their insecticidal
activity against D. melanogaster.
In our search for new naturally occurring insecticidal
compounds, we have used Chinese crude drugs having
a history of safe use as medicines (Miyazawa et al.,
1
991, 1992, 1993, 1996a,b).
The crude drug “yakuchi” in J apan, prepared from the
fruits of Alpinia oxyphylla, originated in southern
China. This plant has been used for gastrointestinal
disturbances as a folk remedy in J apan and other
Oriental countries. As for its chemical components,
monoterpenes (pinene, camphor, 1,8-cineole), sesquit-
erpenes (zingiberene, zingiberol, nootkatone, nootkatol,
oxyphyllenonic acid A and B, oxyphyllenone A and B,
and oxyphyllenodiol), and vanillyl compounds (yakuchi-
none A and B) have been reported (Namba, 1980;
Itokawa et al., 1981, 1982; Shoji et al., 1984; Muraoka
et al., 1998).
In screening for the development of new drugs from
natural products, an MeOH extract of A. oxyphylla was
found to have insecticidal activity against Drosophila
melanogaster Meigen. Further analysis of the extract
indicated strong activity in the fraction containing
nootkatone, which was confirmed to be the active
constituent.
The sesquiterpene nootkatone, the principal flavoring
constituent in grapefruit and J apanese summer orange,
was reported to be an antiulcer compound (Yamahara
et al., 1990). Insecticidal and antifeeding effects of
sesquiterpenes against Heliothis armigera, Aphis gos-
sypii, Pieris rapae, Plutella xylostella, Brevicoryne bras-
sicae, Spodoptera littoralis, Ostrina furnacalis, and
Tribollum castaneum have been reported (Tu et al.,
MATERIALS AND METHODS
Ch em ica l An a lysis. 1H and 13C NMR spectra were re-
corded on a J EOL J NM GSX 270 NMR spectrometer with
1
CDCl
3
as solvent. H NMR was measured with TMS as
internal standard. Electron impact mass spectra (EI-MS) were
obtained at 70 eV by GC-MS on a Hewlett-Packard 5972 series
mass spectrometer interfaced with a Hewlett-Packard 5890
gas chromatograph fitted with a column (HP-5MS, 30 m × 0.25
mm i.d., temperature ) 140 °C, 4 °C/min). IR spectra were
determined with a Perkin-Elmer 1760-X infrared Fourier
transform spectrometer with an ordinated scale for the region
-1
4000-450 cm . Specific rotation was determined with a J asco
DIP-140 digital polarimeter.
Ma ter ia ls. Commercially available air-dried fruits of A.
oxyphylla MIQ. were obtained from Takasago Yakugyou Co.
(Osaka, J apan). D. melanogaster Meigen., used in bioassays
for insecticidal activity, was provided by Professor Ishikawa
of the University of Tokyo. Rotenone was purchased from
Sigma Chemical Co. (St. Louis, MO).
Extr a ction a n d Isola tion (F igu r e 1). Insecticidal com-
pound was isolated from fruit of A. oxyphylla by bioassay-
guided fractionation as outlined in Figure 1. Air-dried fruits
of A. oxyphylla (5 kg) were extracted under reflux with MeOH
for 12 h. The solvent was removed in vacuo to give 333.6 g of
the crude extract, which was successively reextracted with
CH Cl , EtOAc, BuOH, and water. The active CH Cl extract
2
2
2
2
2
after concentration (202.9 g) was fractionated by SiO
column
1
990; Liu et al., 1990; Wang et al., 1991; Gonz a´ lez et
chromatography with hexane-EtOAc. Fraction 3 (18.7 g) had
the most potent activity against larvae of D. melanogaster.
Furthermore, fraction 3 eluted with hexane-ether (4:1) was
al., 1993; Dadang et al., 1996).
fractionated by SiO
ether.
2
column chromatography with hexane-
*
Author to whom correspondence should be addressed
(
telephone +81-6-6721-2332; fax +81-6-6727-4301; e-mail
miyazawa@apch.kindai.ac.jp).
Rechromatography of fraction 7 (1.7 g) eluted with hexane-
ether (9:1) was rechromatographed on an SiO column with
2
hexane-ether to yield 298 mg of compound 1.
†
Kinki University.
University of Tokyo.
‡
1
0.1021/jf000325z CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/15/2000

3640 J. Agric. Food Chem., Vol. 48, No. 8, 2000
Miyazawa et al.
the transplantation, larvae were hatched and fed each test
compound with the artificial diet. At 25 °C, larvae generally
change to pupae for 7 days. The developmental stage was
observed, and the numbers of pupae were recorded and
compared with those of a control. Ten new eggs were used in
each of the three replicates. LC50 is the lethal concentration
for 50% mortality and was determined by log-probit analysis
(Litchfield and Wilcoxon, 1949).
Bioa ssa y for Acu t e Toxicit y a ga in st Ad u lt s of D.
m ela n oga ster . Acute toxicity was determined by topical
application to adults of D. melanogaster (Miyazawa et al.,
1
996a,b). Adults of D. melanogaster from culture bottles were
iced to stop their mobility and treated with each of the test
compounds at doses of 300, 200, 100, and 50 µg in 0.5 µL of
acetone placed on their abdomens with a 10 µL microsyringe.
Controls were treated with 0.5 µL of acetone only. After a set
time interval, survival of the adults was recorded. Fifty adults
were used in all assays. The LD50 was determined using log-
plobit analysis (Litchfield and Wilcoxon, 1949).
Nootk a ton e (1) was obtained as bright yellow needles: mp
+
3
1
1
5
6-39 °C; [R]
D
+181° (c 1.0, CHCl
3
); MS (70 eV), m/z 218 ([M ],
9%), 147 (100), 121 (68), 91 (66), 41 (64); IR υmax, 2935, 1671,
-
1
1
456, 1456, 1286, 888 cm ; H NMR (270.1 MHz, CDCl
3
) δ
.77 (1H, s, H-1), 4.74 (2H, d, H-12), 1.74 (3H, s, H-13), 1.13
(
3H, s, H-15), 0.97 (3H, d, H-14); 13C NMR (67.8 MHz, CDCl
3
)
δ 14.87 (C-14), 16.82 (C-15), 20.80 (C-13), 31.60 (C-8), 33.00
(
(
C-6), 39.29 (C-5), 40.30 (C-4), 40.44 (C-7), 42.05 (C-3), 43.91
C-9), 109.22 (C-12), 124.65 (C-1), 149.05 (C-11), 170.47 (C-
1
0), 199.59 (C-2).
Ep in ootk a tol (1A) was obtained as a colorless oil: [R]
D
+
+
90° (c 1.0, CHCl
3
); MS (70 eV), m/z 220 ([M ], 75%), 177 (100),
1
8
21 (60), 41 (59); IR υmax, 3317, 2858, 1645, 1456, 1373, 1024,
-
1 1
88, 757 cm ; H NMR (270.1 MHz, CDCl ) δ 5.33 (1H, bd,
3
H-1), 4.68 (2H, m, H-12), 4.25 (1H, m, H-2), 1.00 (3H, s, H-15),
.89 (3H, d, H-14), 1.72 (3H, m, H-13); 13C NMR (67.8 MHz,
CDCl ) δ 15.36 (C-14), 18.20 (C-15), 20.80 (C-4), 32.32 (C-6),
32.86 (C-8), 37.20 (C-3), 38.14 (C-5), 39.23 (C-13), 40.73 (C-7),
4.56 (C-9), 67.99 (C-2), 108.52 (C-12), 124.24 (C-1), 146.01
C-11), 150.22 (C-10).
0
F igu r e 1. Scheme for the isolation of the insecticidal com-
pound from A. oxyphylla.
3
4
(
Red u ction of Nootk a ton e. To the ether solution (6 mL)
of nootkatone (1) (200 mg) was added 25 mg of LiAlH
4
, and
the mixture was stirred overnight at room temperature. The
RESULTS AND DISCUSSION
Isola tion of Active P r in cip le. An insecticidal com-
pound was isolated from fruit of A. oxyphylla by bioas-
say-guided fractionation as outlined in Figure 1. Air-
dried fruit of A. oxyphylla were extracted with MeOH
under reflux for 12 h. The crude extract was concen-
trated and partitioned with CH2Cl2, EtOAc, BuOH, and
water successively. The extract with CH2Cl2 was frac-
tionated by SiO2 column chromatography with hexane-
EtOAc. Fraction 3 had the most potent activity against
larvae of D. melanogaster. Furthermore, fraction 3 was
fractionated by SiO2 column chromatography with hex-
ane-ether. Fraction 7 was repeatedly chromatographed
on an SiO2 column, by which 1 was isolated as the active
principle. Compound 1 was identified as nootkatone by
suspension was extracted with ether. The organic phase was
dried over anhydrous Na SO , and the solvent was removed
under reduced pressure. The residue was chromatographed
on silica gel [hexane-acetone (97:3)]. Nootkatol (7 mg) was
eluted, followed by epinootkatol (1A) (oily, 185 mg).
Bioa ssa y for In secticid a l Activity a ga in st La r va e of
D. m ela n oga ster . The bioassay for insecticidal activity
against larvae of D. melanogaster was carried out as follows
2
4
1
13
GC, GC-MS, and H and C NMR spectroscopy, respec-
tively.
In secticid a l Effects of 1 a n d 1A a ga in st La r va e.
The insecticidal effect of 1 and 1A against larvae of D.
melanogaster is shown in Table 1. The insecticidal effect
against larvae of D. melanogaster was determined by
our own system of feeing larvae with artificial diet
containing test compound. When larvae were fed with
the diet containing 36.7 µmol of 1 or 1A/mL of diet, 1
and 1A killed 100 and 73.3% of larvae, respectively. At
(
Miyazawa et al., 1991, 1992, 1993, 1996a,b). Four concentra-
tions (4.6, 9.2, 18.3, and 36.7 µmol/mL of diet) were used for
determining LC50 values. Test compounds were dissolved in
5
yeast (60 g), glucose (80 g), agar (12 g), and propionic acid (8
mL) in water (1000 mL)]. A control diet was treated with 50
µL of EtOH only.
About 100 adults from the colonies of D. melanogaster were
introduced into a new culture bottle, into which artificial diet
had been poured into a Petri dish, and allowed to oviposit at
0 µL of EtOH and mixed in 1 mL of artificial diet [brewers’
4
.6 µmol/mL of diet, 1 killed 26.7% of larvae. At the
same concentration 1A did not kill larvae. Therefore,
0% lethal concentrations (LC50) of larvae were 11.5 and
2
5 °C and relative humidity >60% for 3 h. The diet was taken
5
out of the bottle, and 10 new eggs were collected and
transplanted onto each diet in 1 mL glass tubes and reared at
2
25.6 µmol/mL of diet for 1 and 1A, respectively. All of
these compounds were, however, less active than ro-
5 °C and relative humidity >90% for 8 days. One day after

Insecticidal Compound against Drosophila melanogaster
J. Agric. Food Chem., Vol. 48, No. 8, 2000 3641
LITERATURE CITED
Ta ble 1. In secticid a l Activities of Com p ou n d s 1 a n d 1A
a ga in st La r va e of D. m ela n oga ster
Bhattacharyya, P. R.; Nath, S. C.; Bordoloi, D. N. Insecticidal
activity of Ranunculus sceleratus (L.) against Drosophila
melanogaster and Tribolium castaneum. Indian J . Exp. Biol.
1993, 31, 85-86.
concentration (µmol/mL of diet)a
control 36.7 18.3 9.2 4.6
c
LC50 (µmol/
compd
mL of diet)
10, 10, 10b 0, 0, 0 1, 2, 2 6, 5, 5 8, 7, 7
1
11.5
25.6
1
A
3, 3, 2 7, 7, 6 8, 8, 9 10, 10, 10
Dadang; Ohsawa, K.; Kato, S.; Yamamoto, I. Insecticidal
compound in tuber of Cyperus rotundus L. against the
diamondback moth larvae. J . Pestic. Sci. 1996, 21, 444-
concentration (µmol/mL of diet)a
c
LC50 (µmol/
4
46.
compd
0.65
0.13
0.05
0.01
0.005 mL of diet)
Gonz a´ lez, A. G.; J im e´ nez, I. A.; Ravelo, A. G.; Bazzocchi, I. L.
Minor sesquiterpenes from Maytenus canariensis with
insecticidal and antifeedant activity. Tetrahedron 1993, 49,
6637-6644.
Itokawa, H.; Aiyama, R.; Ikuta, A. A pungent diarylheptanoid
from Alpinia oxyphylla. Phytochemistry 1981, 20, 769-771.
Itokawa, H.; Aiyama, R.; Ikuta, A. A pungent principle from
Alpinia oxyphylla. Phytochemistry 1982, 21, 241-243.
Litchfield, J . T., J r.; Wilcoxon, F. Simplified method of
evaluating dose-effect experiments. J . Pharmacol. Exp.
Ther. 1949, 96, 99-113.
Liu, J . K.; J ia, Z. J .; Wu, D. G.; Zhou, J .; Wang, Q. G. Insect
antifeeding agents: sesquiterpene alkaloids from Celastrus
angulatus. Phytochemistry 1990, 29, 2503-2506.
Miyazawa, M.; Ishikawa, Y.; Toshikura, M.; Kameoka, H.
Insecticidal compounds from Asiasarum heterotropoides
Maek. var mandshuricum Maek. Chem. Express 1991, 6,
703-706.
Miyazawa, M.; Ishikawa, Y.; Toshikura, M.; Kameoka, H.;
Insecticidal allylbenzenes from Asiasarum heterotropoides
Maek. var mandshuricum Maek. Chem. Express 1992, 7,
69-72.
rotenone 0, 0, 0 0, 0, 0 3, 2, 2 7, 8, 7 9, 9 ,9
0.022
a
Test compounds of each concentration were dissolved in 50
µL of EtOH and mixed in 1 mL of artificial diet. b Numbers of
pupae: After 8 days from transplantation, the 10 newly laid eggs
c
on the diet, and three replicates. LC50 (the lethal concentration
for 50% mortality) determined by log-probit analysis.
Ta ble 2. Acu te Toxicities of Com p ou n d s 1 a n d 1A a ga in st
Ad u lts of D. m ela n oga ster
a
survival (% relative to controls)
b
at dose (µg/adult)
c
LD50 (µg/
compd
300
200
100
50
73
100
adult)
1
0
27
17
57
47
87
96
222
1
A
a
survival (% relative to controls)
b
at dose (µg/adult)
c
LD50 (µg/
compd
10
0
7.0
10
5.0
3.0
1.0
0.5
95
adult)
rotenone
30
70
90
3.7
a
After 3 h, survival of the adults was recorded and compared
b
with controls. Test compounds of each dose were dissolved in 0.5
Miyazawa, M.; Ota, H.; Ishikawa, Y.; Kameoka, H. An
insecticidal compound from Illicium verum. Chem. Express
µL of acetone and treated on the abdomen of adult with a 10 µL
microsyringe. Controls were treated with 0.5 µL of acetone only.
1
993, 8, 437-440.
c
LD50 is the lethal dose for 50% mortality determined by log-probit
Miyazawa, M.; Yoshio, K.; Ishikawa, Y.; Kameoka, H. Insec-
ticidal alkaloid against Drosophila melanogaster from tubers
of Corydalis bulbosa. Nat. Prod. Lett. 1996a , 8, 299-302.
Miyazawa, M.; Yoshio, K.; Ishikawa, Y.; Kameoka, H. An
insecticidal alkaloid against Drosophila melanogaster form
rhizomes of Nuphar japonicum DC. Nat. Prod. Lett. 1996b,
8, 307-310.
Muraoka, O.; Fujimoto, M.; Tei, H.; Tanabe, G.; Kubo, M.;
Yoshikawa, M. Study on pharmacologically active sesquit-
erpenoids from the fruits of Alpinia oxyphylla (Kainan
Island). 42nd Symposium on the Chemistry of Terpenes,
Essential Oils, and Aromatics; 1998; pp 180-182.
Namba, T. Coloured Illustrations of Wakan-Yaku; Hoikusha
Publishing Co. Ltd.: Osaka, J apan, 1980; p 242.
Shoji, N.; Umeyama, A.; Takemoto, T.; Ohizumi, Y. Isolation
of a cardiotonic principle from Alpinia oxyphylla. Planta
Med. 1984, 50 (2), 186-187.
analysis.
tenone, a naturally occurring poison that killed all of
the larvae at 0.13 µmol/mL of diet.
Acu te Toxicities of 1 a n d 1A a ga in st Ad u lts.
Acute toxicity against adults of D. melanogaster was
determined by topical application on the abdomen of
adults (Table 2).
Acute toxicities of these sesquiterpenes are shown in
Table 2. At a dose of 100 µg/adult of 1, 53% of adults
were dead. At 50 µg/adult, 1 killed 27% of adults. The
5
0% lethal dose (LD50) of adults was found to be 96 µg/
adult. At 300 and 200 µg/adult, 1A killed 73 and 43%
of adults, respectively. However, 1A had slight activity,
and the LD50 value was 222 µg/adult.
In this research, 1 was only detected as an insecticidal
component from A. oxyphylla. However, it was previ-
ously reported to contain at least nine sesquiterpe-
noids: zingiberene, zingiberol, nootkatone, nootkatol,
oxyphyllenonic acid A and B, oxyphyllenone A and B,
and oxyphyllenodiol (Shoji et al., 1984; Muraoka et al.,
Tu, Y. Q.; Wu, D. G.; Zhou, J .; Chen, Y. Z.; Pan, X. F. Bioactive
sesquiterpene polyolesters from Euonymus bungeanus. J .
Nat. Prod. 1990, 53, 603-608.
Yamahara, J .; Yu, H. L.; Tamai, Y. Anti-ulcer effect in rats of
bitter cardamon constituents. Chem. Pharm. Bull. 1990, 38,
3053-3054.
1
998). However, the insecticidal activity was not re-
Wang, M.; Qin, H.; Kong, M.; Li, Y. Insecticidal sesquiterpene
polyol ester from Celastrus angulatus. Phytochemistry 1991,
ported. Although 1 and 1A demonstrated insecticidal
activity against D. melanogaster in this study, the
activity of 1A was weaker than that of 1. Furthermore,
investigation of the structure-activity relationship sug-
gested that the carbonyl group at the 2-position was an
important structural feature for the enhanced insecti-
cidal activity of 1.
3
0, 3931-3933.
Received for review March 13, 2000. Revised manuscript
received May 24, 2000. Accepted May 25, 2000.
J F000325Z