Biological activity of α-Thujaplicin, the minor component of Thujopsis dolabrata SIEB. et ZUCC. var. hondai MAKINO

Article abstract of DOI:10.1248/bpb.24.607

α-Thujaplicin, a minor component of Thujopsis dolabrata Sieb. et Zucc. var. hondai Makino, which was synthesized, showed the antibacterial activity, phytogrowth-inhibitory effect, inhibition of carboxypeptidase A and cytotoxic effect. Antibacterial activity of α-thujaplicin on Enterococcus faecalis IFO-12965 [minimum inhibitory concentration (MIC): 1.56 μg/ml] was higher than that of gentamicin (MIC: 6.25 μg/ml) used as a positive control. Inhibitory activity of α-thujaplicin on carboxypeptidase A [50% inhibitory concentration (IC₅₀): 3.24×10^-5 M] was higher than that of 1,10-phenanthroline used as a positive control. α-Thujaplicin showed germination inhibition toward the seed of Echinochloa utilis Ohwi et Yabuno even at the low concentration of 10 ppm and its growth inhibitory effect was stronger than that of sodium 2,4-dichlorophenoxyacetate used as a standard. α-Thujaplicin at 1.25 μg/ml inhibited cell growth of human stomach cancer KATO-III by 86%, and Ehrlich's ascites carcinoma by 87%, respectively. This compound even at the low concentration of 0.32 μg/ml also inhibited cell growth of the former by 66%, and the latter by 75%, respectively. The acute toxicity of α-thujaplicin [50% lethal dose (LD₅₀) value: 256 mg/kg] in mice was as strong as those of β-dolabrin (LD₅₀ value: 232 mg/kg) and γ-thujaplicin (LD₅₀ value: 277 mg/kg).

Full text of DOI:10.1248/bpb.24.607

June 2001
Biol. Pharm. Bull. 24(6) 607—611 (2001)
607
a
Biological Activity of -Thujaplicin, the Minor Component of Thujopsis
dolabrata SIEB. et ZUCC. var. hondai MAKINO
Yasuhiro MORITA,a Eiko MATSUMURA,b Hiroshi TSUJIBO,b Masahide YASUDA,b Yoshikazu SAKAGAMI,c
d
e
,b
*
Toshihiro OKABE, Nakao ISHIDA, and Yoshihiko INAMORI
Osaka Organic Chemical Industry, Ltd.,a 18–8 Katayama-cho, Kashiwara, Osaka 582–0020, Japan, Osaka University of
Pharmaceutical Sciences,b 4–20–1 Nasahara, Takatsuki, Osaka 569–1094, Osaka Prefectural Institute of Public Health,c
1–3–69 Nakamichi, Higashinari-ku, Osaka 537–0025, Japan Industrial Research Institute of Aomori Prefecture,d 80
Fukuromachi, Hirosaki, Aomori 036–8363, Japan, and The Sendai Institute of Microbiology,e ICR Building 2F, 6–6–3
Minamiyoshinari, Aoba-ku, Sendai 989–3204, Japan. Received December 14, 2000; accepted March 7, 2001
a-Thujaplicin, a minor component of Thujopsis dolabrata SIEB. et ZUCC. var. hondai MAKINO, which was syn-
thesized, showed the antibacterial activity, phytogrowth-inhibitory effect, inhibition of carboxypeptidase A and
cytotoxic effect. Antibacterial activity of a-thujaplicin on Enterococcus faecalis IFO-12965 [minimum inhibitory
concentration (MIC): 1.56 mg/ml] was higher than that of gentamicin (MIC: 6.25 mg/ml) used as a positive con-
trol. Inhibitory activity of a-thujaplicin on carboxypeptidase A [50% inhibitory concentration (IC50): 3.24؋10؊5
M] was higher than that of 1,10-phenanthroline used as a positive control. a-Thujaplicin showed germination in-
hibition toward the seed of Echinochloa utilis Ohwi et Yabuno even at the low concentration of 10 ppm and its
growth inhibitory effect was stronger than that of sodium 2,4-dichlorophenoxyacetate used as a standard. a-Thu-
japlicin at 1.25 mg/ml inhibited cell growth of human stomach cancer KATO-III by 86%, and Ehrlich’s ascites
carcinoma by 87%, respectively. This compound even at the low concentration of 0.32 mg/ml also inhibited cell
growth of the former by 66%, and the latter by 75%, respectively. The acute toxicity of a-thujaplicin [50% lethal
dose (LD50) value: 256 mg/kg] in mice was as strong as those of b-dolabrin (LD50 value: 232 mg/kg) and g-thu-
japlicin (LD50 value: 277 mg/kg).
Key words a-thujaplicin; antibacterial activity; phytogrowth-inhibitory effect; carboxypeptidase A; inhibition; cytotoxic activ-
ity
It has already been reported that the hinokitiol1)-related
was also examined.
compounds hinokitiol (b-thujaplicin), b-dolabrin and g-thu-
japlicin are among the constituents of Thujopsis dolabrata
SIEB. et ZUCC. var. hondai MAKINO (Chart 1).2) Among them,
hinokitiol, the major component of this plant has been found
to show a broad spectrum of biological activities, including
antimicrobial activity,3—5) insecticidal effect,6) phytogrowth-
inhibitory activity,7) cytotoxic effect on mammalian tumor
cells8) and inhibitory activity on catechol-O-methyltrans-
ferase,9) and metalloproteases such as carboxypeptidase A,10)
collagenase10) and thermolysin.10) At the same time, we have
reported that b-dolabrin and g-thujaplicin, like hinokitiol,7)
showed a phytogrowth-inhibitory effect,11) and inhibitory ac-
tivities on metalloproteases.10) We recently reported that hi-
nokitiol, b-dolabrin and g-thujaplicin had antifungal activi-
ties against wood-rotting fungi and insecticidal effects on Ty-
rophagus putrescentiae and Coptotermes formosanus.12)
Strong cytotoxic effects on two kinds of tumor cell lines were
also found in b-dolabrin and g-thujaplicin13) as well as hi-
nokitiol.8) On the other hand, because a-thujaplicin is a
minor component (0.2%) of the wood of T. dolabrata SIEB. et
ZUCC. var. hondai MAKINO, no work has yet been done on its
above-mentioned biological activities.
MATERIALS AND METHODS
Synthesis of a-thujaplicin was performed according to the
1
following method. Measurement: The H-NMR spectra were
measured with a JEOL GSX-270 spectrometer using tetram-
ethylsilane as an internal standard. HR-MS spectrum was
measured with JEOL JMS-HX-100.
Synthesis of 7,7-Dichloro-4-isopropylidene-bicyclo[3.2.0]-
hept-2-en-6-ones To a stirred solution of freshly distilled
6,6-dimethylfilvene (70.0 g, 74.9%, 0.49 mol)14) and di-
chloroacetylchloride (106.6 g, 0.72 mol) in n-hexane (500 ml)
was added dropwise Et3N (72.8 g, 0.72 mol) over a period of
In this work, to expand our knowledge of the biological
activity of hinokitiol-related compounds, the synthesis of a-
thujaplicin (Chart 1) and its antibacterial activity, phy-
togrowth-inhibitory effect and inhibitory activity on metallo-
proteases were investigated in comparison with those of
other hinokitiol-related compounds. As basic research on the
cytotoxic effect of hinokitiol-related compounds, this effect
of a-thujaplicin on two kinds of tumor cell lines of human
stomach cancer, KATO-III and Ehrlich’s ascites carcinoma,
Chart 1
To whom correspondence should be addressed.
© 2001 Pharmaceutical Society of Japan

608
Vol. 24, No. 6
1 h. The mixture was stirred for an additional 6 h and allowed
Animals Male Slc:ddY mice aged 4 weeks were pur-
to stand overnight. The reaction mixture was washed with chased from Japan SLC (Shizuoka, Japan). The mice were
H2O (3ϫ300 g). After removal of the solvent, the residue was fed an autoclaved commercial diet (NMF: Oriental Yeast,
distilled under vacuum to give 53.6 g of cycloadduct.
Tokyo, Japan) and given drinking water ad libitum. After
prefeeding for 1 week, mice were used for the acute toxicity
15)
a
Synthesis of 3-Isopropenyl tropolone ( -Dolabrin)
To a solution of 50% NaOH aq (88 g) in AcOH (160 g) was test.
added the cycloadduct (50.0 g, 0.23 mol). The mixture was
Methods The antibacterial activity test was evaluated by
refluxed 18.5 h. After cooling to room temperature, toluene the agar dilution method. The phytogrowth–inhibitory activ-
(150 g) was added. The combined solution was washed with ity test was evaluated according to the method of Inamori et
H2O (50 g) and dried over sodium sulfate. After removal of al.7) Inhibition testing of the metalloproteases carboxypepti-
the solvent, the residue was distilled under reduced pressure dase A, collagenase and thermolysin was performed accord-
to give 18.7 g of a-dolabrin.
ing to the method reported previously.16) The cytotoxic effect
Synthesis of -Thujaplicin A solution of a-dolabrin was also determined by the method reported previously.13)
(18.7 g, 0.115 mol) in EtOH (100 g) was stirred for 11 h in The acute toxicity of a-thujaplicin was examined according
the presence of 5% Pd–C (4.0 g) under hydrogen atmosphere. to the following method. Mice were divided into groups of 5
After filtration of the catalyst, the residue from evaporation animals each. a-Thujaplicin was suspended in 5% gum ara-
of the filtrate was distilled under vacuum, and recrystallized bic saline solution and intraperitoneally administered. The
a
1
from iso-hexane to give a-thujaplicin (2.0 g). H-NMR (300 condition of mice and mortality were followed for 8 d after
MHz, CDCl3) 1.26 [d, Jϭ4.3 Hz, 6 H, 3-CH(CH3)2], 3.74 administration. The LD50 was calculated according to the
[seven lines, Jϭ4.3 Hz, 1H, 3-CH(CH3)2], 7.00—7.51 (m, 4 Van der Waerden method.17)
H, 4, 5, 6, 7-CH), 9.81 (br s, 1H, OH). HR-MS m/z:
164.0840 (Mϩ Calcd for C10H12O2: 164.0837).
RESULTS AND DISCUSSION
GC-MS of acid oil obtained by distillation of the wood of
T. dolabrata SIEB. et ZUCC. var. hondai MAKINO was mea-
Antibacterial Activity of a-Thujaplicin The antibacte-
sured. Analytical conditions of GC-MS (Shimadzu GC-MS rial activity of a-thujaplicin was investigated by the agar di-
QP-5000) were as follows. Column: DB 1701, 15 mϫI.D. lution method and compared with those of other hinokitiol-
0.25 mm; oven temperature: 60 to 250 °C at 8 °C/min; injec- related compounds (Table 1). a-Thujaplicin, like the other
tion temperature: 250 °C; detector temperature: 250 °C. Re- compounds, showed clear antibacterial activity against all of
tention time (tR) of a-thujaplicin, hinokitiol and b-dolabrin the bacteria examined. The activity on E. faecalis IFO-12965
were 9.703, 11.197 and 11.377 min, respectively. The con- was particularly strong, its minimum inhibitory concentra-
tents of the three compounds were 7.28, 63.59 and 26.3%, re- tion (MIC) being 1.56 mg/ml. The antibacterial activity of a-
spectively.
thujaplicin on E. faecalis IFO-12965 was higher than those
Chemicals a-Thujaplicin, which was synthesized ac- of other hinokitiol-related compounds and gentamicin used
cording to the above method, was used for various biological as a positive control. Although on S. epidermidis IFO-12993
activities. Hinokitiol,10) b-dolabrin10) and g-thujaplicin10) iso- the activity was lower than those of hinokitiol and b-
lated from T. dolabrata SIEB. et ZUCC. var. hondai MAKINO dolabrin, its growth-inhibitory activity on this bacterium was
were also used for these biological activities. The metallopro- higher than that of gentamicin used as a positive control. The
teases carboxypeptidase A, collagenase and thermolysin antibacterial activities of hinokitiol, b-dolabrin and g-thu-
were purchased from Sigma Chemical Co. (U.S.A.). 1,10- japlicin in the present work were almost equal to those re-
Phenanthroline (a positive control for inhibition of metallo- ported in the previous paper.10) In addition to these three
protease) was purchased from Nakalai Tesque, Inc., Kyoto, compounds,10) the strong antibacterial effect of a-thujaplicin
Japan. Gentamicin (a positive control for the antibacterial ac- on E. faecalis IFO-12965 and S. epidermidis IFO-12993
tivity test) was purchased from Schering-Plough Co., Ltd. seems to be a common biological activity of most hinokitiol-
Sodium 2,4-dichlorophenoxyacetate (2,4-D, a positive con- related compounds. Such a strong antibacterial activity of a-
trol for a phytogrowth-inhibitory activity test) was purchased thujaplicin, hinokitiol, b-dolabrin and g-thujaplicin on E.
from Tokyo Kasei Kogyo Co., Ltd.
faecalis IFO-12965, which is a nosocomial infectious bac-
Organisms Bacteria: Bacteria used for the antibacterial terium, is of considerable interest. Therefore, their antibacte-
activity test were as follows: Staphylococcus aureus IFO- rial activities on vancomycin-resistant Enterococci (VRE)
12732, methicillin-resistant S. aureus (MIC against methi- should be investigated. Considering that many application of
cillin: 1600 mg/ml), methicillin-resistant S. aureus (MIC hinokitiol to skin18—22) have already been reported and a-thu-
against methicillin: 12.5 mg/ml), S. epidermidis IFO-12993, japlicin, hinokitiol, b-dolabrin and g-thujaplicin showed
Enterococcus faecalis IFO-12965, Escherichia coli IFO- strong antibacterial activities against S. epidermidis IFO-
3806, Proteus vulgaris IFO-3988, Serratia marcescens IFO- 12993, one of the residents of skin, basic dermatological re-
12648, Enterobacter cloacae IFO-13535, Enterohaemor- search on hinokitiol-related compounds seems desirable.
a
Inhibitory Activity of -Thujaplicin on Metallopro-
rhagic E. coli, Klebsiella planticola IFO-3317, and Pseu-
domonas aeruginosa IFO-13275. Plants: Plants used for the teases The inhibitory activity of a-thujaplicin on car-
phytogrowth-inhibitory activity test were Brassica cam- boxypeptidase A was examined according to the method of
pestris L. and Echinochloae utilis Ohwi et Yabuno.
Petra (Table 2).16) The compound showed the inhibitory ac-
Cells Tumor cell lines used for the cytotoxic activity test tivity toward this enzyme, although this activity was slightly
were human stomach cancer KATO-III and Ehrlich’s ascites lower than those of other hinokitiol-related compounds10); it
carcinoma.
was higher, however, than that of 1,10-phenanthroline used

June 2001
609
Table 1. Antibacterial Activity of a-Thujaplicin, Hinokitiol, g-Thujaplicin and b-Dolabrin
MIC (mg/ml)a)
g-Thujaplicin
Pathogenic bacteria
a-Thujaplicin
Hinokitiol
b-Dolabrin
Gentamicin
Gram-positive bacteria
Staphylococcus aureus IFO-12732
MRSA IID-1677
12.5
12.5
12.5
25
25
12.5
25
25
25
25
25
25
3.13
12.5
50
Methicillin-resistant S. aureus
(MIC against methicillin: 1600 mg/ml)
Methicillin-resistant S. aureus
(MIC against methicillin: 12.5 mg/ml)
Staphylococcus epidermidis IFO-12993
Enterococcus faecalis IFO-1296
Gram-negative bacteria
12.5
12.5
12.5
12.5
12.5
0.78
1.56
0.39
6.25
12.5
3.13
0.39
3.13
1.56
6.25
Escherichia coli IFO-3806
Proteus vulgaris IFO-3988
12.5
12.5
12.5
12.5
12.5
12.5
100
12.5
12.5
12.5
12.5
12.5
12.5
100
12.5
12.5
12.5
12.5
12.5
12.5
100
12.5
12.5
12.5
12.5
12.5
12.5
100
1.56
1.56
1.56
1.56
1.56
1.56
6.25
Serratia marcescens IFO-12648
Enterobacter cloacae IFO-13535
Enterohaemorrhagic Escherichia coli
Klebsiella planticola IFO-3317
Pseudomonas aeruginosa IFO-13275
a) Minimum inhibitory concentration value was determined by the agar dilution method on a Muller–Hinton medium, which was incubated at 37 °C for 24 h.
Table 2. Inhibitory Activity of a-Thujaplicin on Carboxypeptidase A,
Table 3. Inhibitory Activity of a-Thujaplicin on Plant Growth
Collagenase and Thermolysin
Growth (ratio)a)
IC50 (M)a)
Concentration
(ppm)
Plant
Compound
Compound
Metalloprotease
Echinochloa utilis
OHWI et YABUNO
Brassica campestris L.
Carboxypeptidase Ab) Collagenasec) Thermolysind)
a-Thujaplicin
Hinokitiol
g-Thujaplicin
b-Dolabrin
1,10-Phenanthroline
3.24ϫ10Ϫ5
2.76ϫ10Ϫ6
1.95ϫ10Ϫ5
1.06ϫ10Ϫ5
4.21ϫ10Ϫ4
Ͼ1.00ϫ10Ϫ3 Ͼ1.00ϫ10Ϫ3
2.40ϫ10Ϫ5 6.10ϫ10Ϫ5
a-Thujaplicin
Hinokitiol
g-Thujaplicin
b-Dolabrin
2,4-Dc)
50
30
10
50
30
10
50
30
10
50
30
10
50
30
10
0b)
0.01
0.36
0
0.06
0.44
0
0.04
0.42
0
0
0
0
0
0.03
0.20
0
0.02
0.19
0
0.05
0.32
0
8.90ϫ10Ϫ5 Ͼ1.00ϫ10Ϫ3
1.89ϫ10Ϫ5
1.83ϫ10Ϫ4
6.85ϫ10Ϫ5
3.40ϫ10Ϫ4
a) 50% Inhibitory concentration. b) Carboxypeptidase A was isolated from bovine
pancrease(Sigma Chemical Co.). Carboxypeptidase A solution was incubated at 37 °C
and pH 7.5 for 15 min. Each value represents the mean of duplicate assays. c) Colla-
genase was isolated from Clostridium histolyticum and was incubated at 37 °C and pH
7.5 for 15 min. Each value represents the mean of duplicate assays. d) Thermolysin
was isolated from Bacillus thermoproteolucum. Thermolysin solution was incubated at
37 °C and pH 7.5 for 15 min. Each value represents the mean of duplicate assays.
0
0.27
0.05
0.07
0.09
0
0.02
as a positive control. Unlike other hinokitiol-related com-
pounds, it did not show the inhibitory activity toward colla-
genase or thermolysin.10) However, it is not clear whether the
difference in inhibitory activity between a-thujaplicin and
other hinokitiol-related compounds is due to 1) the difference
in enzyme specificity or 2) the difference in steric effect
a) Growth (length of radicle) in control experiments after 7 d was taken as 1.00.
Experimental size: quality of light, 9000 m2. cd. sr; 20 seeds/group, 2 groups; observa-
tion time, 7 d; temperature, 27 °C; illumination time, 12 h/d. b) No germination.
c) Sodium 2,4-dichlorophenoxyacetate.
caused by the difference in position of the isopropyl group in utilis OHWI et YABUNO it was higher than that of these other
their molecules. Considering that a-thujaplicin did not show compounds. It completely inhibited the germination of the
the inhibitory activity toward collagenase or thermolysin, the seeds of E. utilis OHWI et YABUNO even at the low concentra-
cause of metalloprotease inhibition seems not always due to tion of 10 ppm. Considering that carboxypeptidase A has al-
metal chelation between the carbonyl group at C-1 and the ready been found to play an important role in the early stage
hydroxyl group at C-2 in molecules of hinokitiol-related of germination in the seeds of plants,23) this activity of a-thu-
compounds. However, the reason for this is not clear at pre- japlicin is supported from the above inhibition on car-
sent.
Phytogrowth-Inhibitory Activity of
boxypeptidase A. These findings suggest that phytogrowth-
-Thujaplicin inhibitory action is a common biological activity of hinoki-
a
The phytogrowth-inhibitory activity of a-thujaplicin was in- tiol-related compounds.
vestigated according to the method of Inamori et al.7) and, as
As the color of cotyledons of B. campestris L. treated with
shown in Table 3, was seen to be strong. Its growth-in- a-thujaplicin changed to yellowish white, its chlorophyll
hibitory activity on B. campestris L. was as high as those of content was measured by the A.O.A.C. method24); the results
other hinokitiol-related compounds, while this activity on E. are summarized in Table 4. Like other hinokitiol-related

610
Vol. 24, No. 6
Table 4. Chlorophyll Contents of Brassica campestris Treated with a-Thujaplicin and Other Hinokitiol-Related Compounds
Compound
Concentration (ppm)
Total chlorophyll (%)a)
Chlorophyll a (%)
Chlorophyll b (%)
a-Thujaplicin
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30
8.50
0
0
6.73
0
0
1.77
0
0
Hinokitiol
g-Thujaplicin
b-Dolabrin
2,4-Db)
3.81
2.71
0.38
5.38
4.23
0
6.80
5.28
4.29
5.86
4.98
3.18
19.81
2.64
1.91
0.25
4.25
3.92
0
5.66
4.26
3.40
4.51
3.11
2.87
13.32
1.17
0.80
0.13
1.13
0.31
0
1.14
1.02
0.89
1.35
1.87
0.31
6.49
Control
a) % (wet/wt). Analytical method, A.O.A.C. method; observation time, 7 d; temperature, 27 °C; illumination, 9000 lux. b) Sodium 2,4-dichlorophenoxyacetate.
compounds, the chlorophyll content in cotyledons of this
plant treated with a-thujaplicin was decreased as compared
with the control. However, this decrease by a-thujaplicin
treatment was weaker than those of other hinokitiol-related
compounds. Based on the findings that the chlorophyll con-
tent in the cotyledons of B. campestris L. treated with all of
the hinokitiol-related compounds examined was greatly de-
creased compared with the control, at least part of the phy-
togrowth-inhibitory actions of these compounds also seems
to cause this decrease.
Cytotoxic Activity of a-Thujaplicin on Cell Lines of
Human Stomach Cancer KATO-III and Ehrlich’s Ascites
Carcinoma The cytotoxic activity of a-thujaplicin was ex-
amined with two kinds of cell lines of human stomach cancer
KATO-III and Ehrlich’s ascites carcinoma and compared
Fig. 1. Inhibitory Activity of a-Thujaplicin on Growth of Cell Lines of
Human Stomach Cancer KATO-III in Comparison with Other Hinokitiol-
Related Compounds
with those of other hinokitiol-related compounds. a-Thu-
japlicin was dissolved in DMSO and was diluted in complete
medium at 0.32—20 mg/ml. Final concentration (0.0012—
0.08% in complete medium) of DMSO at this time did not
influence the cell growth of the two cell lines (data not
shown), while a-thujaplicin showed strong cytotoxic effect
on both cell lines in vitro. As shown in Figs. 1 and 2, the
growth of both tumor cell lines by a-thujaplicin was sup-
pressed in concentration-dependent fashion. a-Thujaplicin at
1.25 mg/ml inhibited cell growth of human stomach cancer
KATO-III by 86%, and Ehrlich’s ascites carcinoma by 87%.
This compound even at the low concentration of 0.32 mg/ml
also inhibited cell growth of the former by 66%, and the lat-
ter by 75%. Cytotoxic activity of a-thujaplicin was almost
equal to that of b-dolabrin.13) Judging from the results of cy-
totoxic activity of a-thujaplicin in the present work and other
hinokitiol-related compounds in the previous paper,8,13) these
effects seem to be common biological activities of hinokitiol-
related compounds.
Toxicity Profile of Mice and Mortality Following the
Fig. 2. Inhibitory Activity of a-Thujaplicin on Growth of Cell Lines of
Ehrlich’s Ascites Carcinoma in Comparison with Other Hinokitiol-Related
Compounds
a
Administration of -Thujaplicin The toxicity profile of
mice and mortality following the intraperitoneal administra-
tion of a-thujaplicin was investigated, and the mortality is
compared with other hinokitiol-related compounds in Table
In each group given 300 mg/kg of a-thujaplicin, the reduc-
5. When 320 mg/kg of a-thujaplicin was administered, con- tion in voluntary movement began about 18 h after adminis-
vulsion developed at approximately 30 min after administra- tration and crouching accompanied with eye-closing devel-
tion and all the mice died within 2 d.
oped. The animals began to die approximately 2 d later.

June 2001
611
nokitiol, b-dolabrin and g-thujaplicin was reported earlier,13)
this toxicity of a-thujaplicin is reported for the first time
here.
Considering that similar activities were found not only in
a-thujaplicin, but also in other hinokitiol-related com-
pounds,7,8,10—13) other biological activities of these com-
pounds should be further investigated.
Studies on the synthesis of 4-acetyltropolone, one of the
minor components of T. dolabrata Sieb. et ZUCC. var. hondai
MAKINO and its biological activities are in progress.
Table 5. Toxicity Profile of Mice Injected with a-Thujaplicin, Hinokitiol,
b-Dolabrin and g-Thujaplicin
Mortality (%)
Dose
(mg/kg)
day
1
2
3
4
5
6
7
8
320
300
280
260
240
220
200
0
0
0
0
0
0
0
100 100 100 100 100 100 100
20 40 60 60 60 60
80
60
40
40
20
0
0
0
20 60 60 60 60
20 40 40 40 40
a-Thujaplicin
40 40 40 40 40 40
0
0
0
0
20 20 20 20
Acknowledgement The authors wish to express their
gratitude to Takasago Int. Corp. for providing g-thujaplicin.
0
0
0
0
LD50: 256 mg/kg
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240
225
210
195
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60 60 60 80 60 60
20 20 40 40 40 40
80
60
60
40
20
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5) Shibasaki I., Terui G., J. Ferment. Technol., 33, 216—223 (1955).
6) Hara H., Japan. Kokai Tokkyo Koho, JP 01242508 (Oct. 16, 1989);
Fukada M., Japan. Kokai Tokkyo Koho, JP 100696 (Oct. 9, 1972).
7) Inamori Y., Nishiguchi K., Matsuo N., Tsujibo H., Baba K., Ishida N.,
Chem. Pharm. Bull., 39, 2378—2381 (1991).
Hinokitiol
0
0
20 20 20 20 20
0
0
0
0
0
0
LD50: 191 mg/kg
310
295
280
265
250
235
220
205
190
175
100 100 100 100 100 100 100 100
8) Inamori Y., Tsujibo H., Ohishi H., Ishii F., Mizugaki M., Aso H.,
Ishida N., Biol. Pharm. Bull., 16, 521—523 (1993).
80
80
60
20
20
0
0
0
0
80 80 80 80 80 80
80 80 80 80 80 80
60 60 60 60 60 60
60 80 80 80 80 80
40 40 40 40 40 40
40 40 40 40 40 40
20 20 40 40 40 40
20 20 20 20 20 20
80
80
60
80
40
40
40
20
0
9) Borchardt R. T., J. Med. Chem., 16, 377—382 (1973).
10) Inamori Y., Shinohara S., Tsujibo H., Okabe T., Morita Y., Sakagami
Y., Kumeda Y., Ishida N., Biol. Pharm. Bull., 22, 990—993 (1999).
11) Sakagami Y., Inamori Y., Isoyama N., Tsujibo H., Okabe T., Morita Y.,
Ishida N., Biol. Pharm. Bull., 23, 645—648 (2000).
12) Inamori Y., Sakagami Y., Morita Y., Shibata M., Sugiura M., Kumeda
Y., Okabe T., Tsujibo H., Ishida N., Biol. Pharm. Bull., 23, 995—997
(2000).
b-Dolabrin
0
0
0
0
0
0
13) Matsumura E., Morita Y., Date T., Tsujibo H., Yasuda M., Okabe T.,
Ishida N., Inamori Y., Biol. Pharm. Bull., 24, 299—302 (2001).
14) Yates P., David Kronis J., Can. J. Chem., 62, 1751—1766 (1984).
15) Asao A., Machiguchi T., Kitamura T., Chem. Commun., 1970, 89—90.
16) Petra E., Methods Enzymol., 19, 460—503 (1970).
17) Van der Waerden B. L., “Mathematisch Statistik,” Springer-Verlag,
Berlin, Gottingen, and Heidelberg, 1957.
LD50: 232 mg/kg
325
310
295
280
265
250
235
100 100 100 100 100 100 100 100
80
60
60
40
20
0
80 80 80 80 80 80
60 60 60 60 60 60
60 60 60 60 60 60
40 40 40 40 40 40
20 20 20 20 20 20
80
60
60
40
20
0
g-Thujaplicin
18) Baba T., Nakano H., Tamai K., Sawamura D., Hanada K., Hashimoto
I., Arima Y., J. Invest. Dermatol., 110, 24—28 (1998).
0
0
0
0
0
0
19) Sakai I., Tamura U., Jpn. Kokai Tokkyo Koho, JP 63 188 69 [Chem.
Abstr., 111, 102562n (1989)].
20) Oreal Cosmetic K. K., Jpn. Kokai Tokkyo Koho, JP 81 147 704
[Chem. Abstr., 111, 148977y (1982)].
LD50: 277 mg/kg
Animals: ddY strain mice (male) 26—29 g (body weight). Route: intraperitoneal in-
jection. Calculation of LD50: Van der Waerden method.
21) Oreal Cosmetic K. K., Jpn. Kokai Tokkyo Koho, JP 81 147 705
[Chem. Abstr., 111, 148977y (1982)].
Comparison of the 50% lethal dose (LD50) value of a-thu-
japlicin to that of other hinokitiol-related compounds in mice
is also summarized in Table 5. The LD50 value of a-thu-
japlicin was found to be 256 mg/kg. Its acute toxicity was
lower than that of hinokitiol, but was almost equal to those of
b-dolabrin and g-thujaplicin. Though the acute toxicity of hi-
22) Akiyama H., Torigoe R., Kanzaki H., Arata J., Chemotherapy (Tokyo),
42, 1202—1211 (1994).
23) Ranki H., Mendez-Lozano J., Sopanen T., Physiol. Plant., 91, 90—96
(1994).
24) “Official Methods of Analysis of the Association of Agricultural
Chemistry,” 8 th ed., The Association, Washington D.C., 1955, p.122.