Biologically active components against Drosophila melanogaster from Podophyllum hexandrum

Article abstract of DOI:10.1021/jf9903509

In the course of screening for novel naturally occurring insecticides from Chinese crude drugs, a dichloromethane extract of Podophyllum hexandrum was found to give an insecticidal activity against larvae of Drosophila melanogaster Meigen. From the extrac

Full text of DOI:10.1021/jf9903509

5108
J. Agric. Food Chem. 1999, 47, 5108−5110
Biologica lly Active Com p on en ts a ga in st Dr osoph ila m ela n oga ster
fr om P od oph yllu m h exa n d r u m
Mitsuo Miyazawa,*,† Mami Fukuyama,† Kimio Yoshio,† Takenori Kato,† 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,
Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, J apan
In the course of screening for novel naturally occurring insecticides from Chinese crude drugs, a
dichloromethane extract of Podophyllum hexandrum was found to give an insecticidal activity against
larvae of Drosophila melanogaster Meigen. From the extract, an insecticidal compound was isolated
by bioassay-guided fractionation. The compound was identified as podophyllotoxin (1) by comparison
of its spectroscopic characteristics with literature data. In bioassays for insecticidal activity, 1 showed
a LC50 value of 0.24 µmol/mL diet against larvae of D. melanogaster and a LD50 value of 22 µg/
adult against adults. Acetylpodophyllotoxin (1A), however showed slight insecticidal activity in both
assays, indicating that the 4-hydroxyl group was an important function for enhanced activity of 1.
Keyw or d s: Podophyllum hexandrum; Podophyllaceae; lignan; Drosophila melanogaster Meigen;
podophyllotoxin; insecticidal activity; structure-activity relationship
INTRODUCTION
of plants and in the processes of plant symbioses and
plant parasitism.
In our search for new naturally occurring insecticidal
compounds, we have used Chinese crude drugs having
a history of safe use as medicine (Miyazawa et al., 1991,
1992, 1993, 1996a,b). A CH2Cl2 extract of Podophyllum
hexandrum was found to exhibit insecticidal activity
against larvae of Drosophila melanogaster Meigen.
The genus Podophyllum has four well-known species,
one in the Himalayas, one in America, and two of
Chinese origin. Several other less-known Chinese spe-
cies have also been identified and chemically investi-
gated. The genus was the subject of vigorous phytochem-
ical investigations in the early 1960s after the isolation
of several lignans (Atta-ur-Rahman et al., 1995).
Among the plant polyphenols, of which over 8000 are
known, the flavonoids such as quercetin form the largest
group. However, phenolic quinones, lignans, xanthones,
coumarins, and other classes exist in considerable
numbers. In addition to monomeric and dimeric struc-
tures, there are three important groups of phenolic
polymerssthe lignins of the plant cell wall, the black
melanin pigments of plants, and the tannins of woody
plants.
Plant polyphenols are economically important because
they make major contributions to the taste and color of
our food and drinks. The flavor and taste of tea is
related to the fact that the tea leaf contains up to 30%
of its dry weight as polyphenol. Likewise, the bitterness
of beer is due to the content of the phloroglucinol
derivative, humulone, while the color of red wine is
imparted by anthocyanins, such as the pigment malvin.
In nature, phenolics have a significant role in protecting
plants from being overeaten by herbivores. They also
act as chemical signals in the flowering and pollination
A number of lignans isolated from Podophyllum
species have shown a wide range of biological activities,
such as antitumor, antimitotic, and antiviral activities.
Some of them have also shown toxicity to fungi, insects,
and vertebrates (Macrea et al., 1984). In a previous
paper, the successful chemical conversion of the podo-
phyllotoxin into the clinically useful anticancer drugs
Etoposide and Teniposide has also triggered further
research in this area (Issel et al., 1984). The insecticidal
activity of podophyllotoxins and congeners against Blat-
tella germanica, Epilachna sparsa orientalis, and Plu-
tella xylostella has been reported (Inamori et al., 1986).
However, there is no detailed report about the insecti-
cidal activity of podophyllotoxin against insects of
Diptera order. In this paper, we describe the isolation
and identification of the insecticidal compounds against
D. melanogaster Meigen from P. hexandrum.
EXPERIMENTAL PROCEDURES
Ch em ica l An a lysis. 1H and 13C NMR spectra were re-
corded on a J EOL GSX 270 NMR spectrometer with CDCl3 as
the solvent. 1H NMR spectra were measured with TMS as the
internal standard. Electron impact mass spectra (EI-MS) were
obtained at 70 eV by GC-MS on a Hewlett-Packard 5890. IR
spectra were determined with a Perkin-Elmer 1760-X infrared
Fourier transform spectrometer with an ordinated scale for
the region 4000-450 cm-1. Specific rotation was determined
with a J ASCO DIP-140 digital polarimeter.
Ma t er ia ls. A commercially available air-dried root of P.
hexandrum was obtained from Takasago Yakugyou Co.
(Osaka). D. melanogaster Meigen., used in the bioassay for
insecticidal activity, was provided by Professor Ishikawa of
University of Tokyo.
* To whom correspondence should be addressed (telephone
+81-6-6721-2332; fax +81-6-6727-4301).
† Kinki University.
Extr a ction a n d Isola tion . Air-dried rhizomes of P. hex-
andrum (800 g) were extracted with CH2Cl2 under reflux for
‡ University of Tokyo.
10.1021/jf9903509 CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/18/1999

Podophyllum hexandrum
J. Agric. Food Chem., Vol. 47, No. 12, 1999 5109
were used. LD50 is the lethal dose for 50% mortality, as was
determined from log-probit analysis (Litchfield and Wilcoxon,
1949).
23
P od op h yllotoxin (1): [R]D -96.4°; mp 103-109 °C; IR
1
γ
max, cm-1 1774, 1593, 1481, 1230, 940, 1428, 1381, 1126; H
NMR (270, 1 MHz, CDCl3) δ 2.04 (1H, s, OH), 2.80 (2H, m,
H-2,3), 3.74 (6H, s, OCH3-3′,5′), 3.80 (3H, s, OCH3-4′), 4.02 (H,
t, J ) 9.5 Hz, H-3aâ), 4.58 (2H, m, H-1,3aR), 4.73 (1H, d, J )
8.6 Hz, H-4b), 5.95 (1H, d, J ) 1.6 Hz, OCH2O), 5.97 (1H, d,
J ) 1.6 Hz, OCH2O), 6.37 (2H, s, H-2′,6′), 6.49 (1H, s, H-8),
7.11 (1H, s, H-5).
RESULTS AND DISCUSSION
Isola tion of Active P r in cip le. The insecticidal
compounds were isolated from P. hexandrum by bio-
assay-guided fractionation. P. hexandrum was extracted
with CH2Cl2 under reflex for 10 h. The CH2Cl2 extract
was fractionated by silica gel column chromatography
with hexane-ether. The isolation scheme for 1 by
bioassay-guide is shown in Figure 1. Fraction 3 had the
most potent activity against larvae of D. melanogaster.
Furthermore, the fraction 3 was fractionated by silica
gel column chromatography with CH2Cl2-MeOH. The
fraction 7 was repeatedly chromatgraphied on silica gel
by which 1 was isolated as the active principle. Com-
F igu r e 1. Isolation scheme of compound 1 from Podophyllum
hexandrum.
10 h. The solvent was removed under reduced pressure to give
a crude extract. The crude extract with CH2Cl2 (21.6 g) was
fractionated by silica gel column chromatography with a
hexane-ether mixture. Furthermore, fraction 3 eluated with
ether fractionated by silica gel column chromatography with
CH2Cl2-MeOH. Rechromatography of fraction 7 eluting with
CH2Cl2-MeOH (5:5) gave 9.21 g of compound 1.
Acetyla tion of 1. The acetate (compound 1A) of 1 was
obtained by reaction with acetic anhydride and pyridine.
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 described
previously (Miyazawa et al., 1991, 1992, 1993, 1996a,b). Five
concentrations (0, 0.1, 0.5, 1, and 2 µmol/mL diet) were used
for determining the LC50 value. Test compounds were dissolved
in 50 µL of EtOH and mixed in 1 mL of artificial diet (Brewers’
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; in which artificial diet
poured into the bottom of a Petri dish was placed, and allowed
to oviposit at 25 °C and RH >60% for 3 h. The diet was taken
out of the bottle; 10 newly laid eggs were collected and
transplanted onto each diet in a 1 mL glass tube and reared
at 25 °C and RH >90% for 8 days. The developmental stage
was observed, and the number of pupae was recorded and
compared with those of a control. Ten newly laid eggs were
used in each of the three replicates. LC50 is the lethal
concentration for 50% mortality, as was determined by log-
probit analysis (Litchfield and Wilcoxon, 1949).
pound 1 was identified as podophyllotoxin by spectral
data compared with previous reports (Sibata et al., 1961;
J ackson et al., 1984; Ayres and Lim, 1972).
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 larvae fed with artificial diet contain-
ing test compound. When larvae were fed with the diet
containing 2.0 µmol/mL diet, 1 and 1A killed 100% and
93.3% of the larvae. At 0.5 µmol/mL diet, 1 killed 76.6%
of larvae whereas 1A caused only 40.0% mortality.
Therefore, the concentration of compounds 1 and 1A at
50% lethal concentration (LC50) of larvae as 0.24 and
0.64 µg/mL diet, respectively. Furthermore, compounds
1 and 1A had a more powerful insecticidal activity than
(E)-anethole (Miyazawa et al., 1993), safrole (Miyazawa
et al., 1991), (-)-tetrahydroberberine (Miyazawa et al.,
1996a), asaricin (Miyazawa et al., 1991), methyleugenol
(Miyazawa et al., 1992), elemicine (Miyazawa et al.,
1992), and γ-asarone (Miyazawa et al., 1992).
Bioa ssa y for Acu te Toxicity a ga in st Ad u lts of D.
m ela n oga ster . Acute toxicity was determined by topical
application to adults of D. melanogaster (Miyazawa et al.,
1996a,b). Adults of D. melanogaster from a culture bottle were
frozen to stop their movement, and treated with each of the
test compounds at doses of 100, 50, 30, 20, 15, 10, and 5 µg in
0.5 µL of acetone 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
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. Acute toxicities of these lignans were shown in
Table 2. Application at 30 µg/adult of 1 per adult
resulted in 75% mortality. At 15 µg/adult, 1 killed 30%

5110 J. Agric. Food Chem., Vol. 47, No. 12, 1999
Miyazawa et al.
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
insecticidal activity against D. melanogaster similar to
the recent paper, the activity of 1A was weaker than
that of 1. Furthermore, investigation of structure-
activity relationship suggested that the 4-hydroxyl
group was an important function for enhanced insecti-
cidal activity of 1. The difference between our results
and a previous report is considered to be due to the
difference in the metabolic pathways of test insects.
concentration
(µmol/mL diet)a
LC50 (µmol/
compound
control
2
1
0.5
0.1
mL diet)c
1
1A
10, 10, 10b 1, 0, 0 1, 1, 1 2, 3, 2 6, 4, 6
1, 1, 0 2, 3, 2 7, 5, 6 8, 7, 9
0.24
0.64
concentration
(µmol/mL diet)a
LITERATURE CITED
LC50 (µmol/
1.30
0.65
0.13
0.05
0.01 0.005 mL diet)c
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Rotenone 0, 0, 0 0, 0, 0 0, 0, 0 2, 3, 2 7, 8, 6 9, 9, 9
0.022
a
Test compounds of each concentrations were dissolved in 50
µL of EtOH and mixed in 1 mL of artificial diet. Numbers of
pupae: After 8 days from transplantation the newly 10 eggs laid
on the diet, and three replicates. c LC50 (the lethal concentration
for 50% mortality) determined by log-probit analysis.
b
Ta ble 2. Acu te Toxicities of Com p ou n d s 1 a n d 1A a ga in st
Ad u lt of D. m ela n oga ster
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survivala (in % to controls)
at doseb (µg/adult)
LD50
compound
100
50
30
20
15
5
(µg/adult)c
1
1A
0
45
0
60
25
63
50
70
70
85
100
100
22
80
survivala (in % relative to controls)
at doseb (µg/adult)
LD50
10
0
7.0
10
5.0
3.0
1.0
0.5 (µg/adult)c
Rotenone
30
70
90
95
3.7
a
Test compounds of each doses were dissolved in 0.5 µL of
acetone and treated on the abdomen of an adult with a 10 µL-
microsyringe. Controls were treated with 50 µL of acetone only.
b
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of adults. The LD50 of adults was found to be 22 µg/
adult. At 100 and 30 µg/adult, 1A killed 55% and 37%
of adults, respectively. Thus, 1A had a lower activity
and the LD50 value was 80 µg/adult.
In this research, only 1 was detected as an insecticidal
component from P. hexandrum. In previous paper, this
species was shown to contain at least 10 aryltetralin:
4′-demethylpodophyllotoxin, â-peltatin, R-peltatin, des-
oxypodophyllotoxin, isopicropodophyllone, podophyllo-
toxone, 4′-demethylpodophyllotoxone, 4′-demethyldes-
oxypodophyllotoxin and 4′-demethylisopicropodophyllone
(J ackson and Dewick, 1985). These compounds were
useful to cathartics in America. Also, they had a were
reported antitumor action but cannot adapt to the whole
body because of their high toxicity (Karime, 1967). With
regard to insecticidal activity, it was previously reported
that against Blattella germanica, Epilachna sparsa
orientalis, Plutella xylostella, and Culex pipiens moles-
tus, 1 and deoxypodophyllotoxin interestingly gave
contrasting results in that the insecticidal activity of 1
was weaker than that of deoxypodophyllotoxin except
for this action on Epilachna sparsa orientalis (Inamori
et al., 1986). Although 1 and 1A demonstrated an
Received for review April 9, 1999. Revised manuscript received
September 1, 1999. Accepted September 14, 1999.
J F9903509