244 Notes Biol. Pharm. Bull. 25(2) 244—246 (2002) Vol. 25, No.

Studies on Cancer Chemoprevention by Traditional Folk Medicines XXIV.1)

Inhibitory Effect of a Coumarin Derivative, 7-Isopentenyloxycoumarin,
against Tumor-Promotion
Masaki BABA,a Yongri JIN,a Atsuo MIZUNO,a Hisaya SUZUKI,a Yoshihito OKADA,a Nobuo TAKASUKA,b
Harukuni TOKUDA,c Hoyoku NISHINO,c and Toru OKUYAMA*, a
Department of Natural Medicine and Phytochemistry, Meiji Pharmaceutical University,a 2–522–1 Noshio, Kiyose, Tokyo
204–8588, Japan, Experimental Pathology and Chemotherapy Division, National Cancer Center Reseach Institute,b 5–1–1
Tsukiji, Chuo-ku, Tokyo 104–0045, Japan, and Department of Biochemistry, Kyoto Prefectural University of Medicine,c
Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602–0841, Japan. Received July 3, 2001; accepted November 1, 2001

7-Isopentenyloxycoumarin (1) was isolated from Heracleum lanatum MICHX. (Umbelliferae). Compound 1
inhibited phospholipid metabolism and Epstein–Barr virus activation caused by a potent tumor promoter. In an
in vivo experiment, topical application of 1 suppressed skin-tumor-formation induced by 12-O-tetradecanoyl-
phorbol-13-acetate (TPA) in 7,12-dimethylbenz[a]anthracene (DMBA) initiated mice. And it also suppressed or-
nithine decarboxylase activity stimulated by TPA on mouse skin. These results indicated that 7-isopentenyloxy-
coumarin is one of the effective compounds from natural resources for treating skin tumor formation.
Key words Heracleum lanatum; anti-tumor promoting activity; ornithine decarboxylase (ODC); 7-isopentenyloxycoumarin

The umbellifelous plants have been used not only as food-
stuff and spice, but also as traditional folk medicine. In our
previous studies, umbeliferous, liliaceous, and araliaceous
vegetables have been investigated for functional foods. Some
of these vegetables exhibited anti-carcinogenic effects.1—4)
Along this line, we also investigated the constituents of Her-
acleum lanatum MICHX. (Hana-udo in Japanese) and isolated
some known coumarin-derivatives from the active fractions
in a 32Pi-incorporation assay. However, the anti-carcinogenic
effects of these compounds were not examined. In our previ-
ous studies, some coumarin-derivatives exhibited the anti-
tumor promoting activity.4)
In this report, the cancer chemopreventive activity of 7-
isopentenyloxycoumarin, one of the known compounds in H.
lanatum, was studied.
MATERIALS AND METHODS
Materials 7-Isopentenyloxycoumarin (1) was isolated
from Heracleum lanatum MICHX. (Umbelliferae, Hana-Udo
in Japanese), and characterized by comparison of the spectral
data with that in the literature.5)
Animals Six-weeks ICR female mice were obtained
from Japan SLC Co., Ltd.
Chemicals 12-O-Tetradecanoyl-phorbol-13-acetate
(TPA) was purchased from SIGMA Chem. Co. Ltd. All other
chemicals were biological reagent grade.
Methods. TPA-Induced Epstein–Barr Virus Activation
in Raji Cells The inhibitory effect of 1 on TPA-induced
Epstein–Barr virus early antigen expression was examined
by the same methods of our previous report.2,3) The sample
was dissolved in dimethyl sulfoxide (DMSO). Vehicle con-
trol was treated with DMSO alone. Vehicle control cells did
not show any difference compared with non-treated cells in
this experiment.
TPA-Induced 32Pi-Incorporation into Phospholipids of
HeLa Cells The effect on TPA-induced radioactive-inor-
ganic phosphate incorporation into phospholipids of cultured
cells was determined by the methods described previously.6)
∗ To whom correspondence should be addressed. e-mail: okuyama@my-pharm.ac.jp
Sample and TPA were dissolved in DMSO. Vehicle control
was treated with DMSO alone.
Two-Stage Mouse Skin Carcinogenesis The experi-
ment was performed by the same methods of our previous re-
port.3) Initiation of skin carcinogenesis was carried out by a
single application of 100 m g 7,12-dimethylbenz[a]anthracene
(DMBA) (390 nmol) on the back of 7-week-old female ICR
mice (purchased from Japan CLEA Ind.). For promotion,
TPA (1.0 m g, 1.62 nmol) was applied on the same place twice
a week from 1 week after the initiation. Compound 1 (162
nmol and 325 nmol; molar ratio to TPA was 100 : 1 and 200 :
1, respectively) was applied simultaneously with TPA. TPA
or/and 1 were dissolved in 100 m l of acetone. The number of
tumors was determined once a week. Each experimental
group consisted of 15 mice. The experiment was continued
for 20 weeks.
TPA-Induced Ornithine Decarboxylase Activity The
activity of ornithine decarboxylase (ODC) was determined
by the same methods of our previous paper,3) that was based
on the method of Djurhuus7) by measuring the level of
[3H]putrescine derived from [3H]ornithine. Compound 1 was
applied with or without TPA at the dose of 1620, 325 nmol.
RESULTS AND DISCUSSION
In this study, all of the screening tests were to evaluate the
inhibitory potency of test samples on phorbol ester induced
various phenomena. These tests in vitro might be correlated
closely with two-stage skin carcinogenesis test in vivo.8—10)
Inhibitory Effect of 1 on TPA-Induced Epstein–Barr
Virus Activation in Raji Cells A coumarin-derivative, 1
(see Fig. 1), isolated from Heracleum lanatum MICHX., was
evaluated for its anti-tumor promoter activity. At first, we
examined the inhibitory activity of this compound on TPA-
induced Epstein–Barr virus early antigen expression. As
shown in Table 1, 1 exhibited an inhibitory effect in a dose
dependent manner. At the dose of 16 m M (500 fold to
TPA), this material did not show any cytotoxicity in this
assay.
© 2002 Pharmaceutical Society of Japan
February 2002 245

Inhibitory Effect on TPA-Induced 32Pi-Incorporation promoter activity in vitro as shown above. Next, we exam-
into Phospholipids of HeLa Cells Compound 1 was also ined the two-stage skin carcinogenesis in vivo.
examined in other in vitro assays for anti-tumor promotion. The average number of tumors are summarized in Fig. 2a,
The compound strongly suppressed TPA-induced 32Pi-incor- and the incidence is shown in Fig. 2b. Compound 1 sup-
poration into phospholipids of HeLa cells. (see Table 2) pressed skin-tumor formation in a dose dependent manner.
Anti-tumor Promoting Effect on Two-Stage Mouse The group treated with 1 at the dose of 200 fold to TPA ex-
Skin Carcinogenesis Compound 1 exhibited anti-tumor hibited significant inhibition in tumor multiplicity at the end
of experimental period, the inhibitory ratio was about 75%.
(p.0.05; student’s t-test)
No significant difference was observed in histopathologi-
cal studies. Almost all the tumors were considered as papil-
loma. (Data not shown) The observation of this experiment,
Fig. 1. Structure of Compound 1 body weight gain and water intake and diet, suggested that
this material may not have any toxicity at the doses tested.
Table 1. Effect of 1 on EBV Activation Induced by Tumor Promoter
Inhibitory Effect on TPA-Induced Ornithine Decar-
boxylase Activity The effects of 1 on murine epidermal
Concentration of 1 EBV-EA ODC activity were examined. ODC is the rate-limiting en-
Condition
(molar ratio to TPA) induction (%) zyme of polyamine metabolism, and over induction of this
enzyme activity results in stimulation of tumor-promotion.11)
Vehicle 0
Control (132 nM TPA) 100
As shown in Table 3, 1 exhibited a suppressive effect in this
11 32 nM ( 1) 100 assay.
32310 nM ( 10) 100 Compound 1 have been studied for its biological activity,
323102 nM ( 100) 77.5 Ca11 blocking effect as hypotensive agent,12) and growth re-
163103 nM ( 500) 34.5 tarding effect against other plants (allelopathyc effect).5) Re-
323103 nM (1000) 0*
cently, Murakami et al. reported a related compound, au-
*: Ratio of viability to control was 70%. Raji cells, Epstein–Barr virus (EBV) rapten, isolated from Citrus fruits, that exhibited a potent
genome-carrying human lymphoblastoid cells, were incubated for 48 h in 1 ml of suppressing effect on two-stage mouse skin carcinogenesis
medium containing n-butyric acid (4 mM), TPA (32 nM) and various concentrations of
test compounds. After 48 h, the induction rate of early antigen (EA) of EBV was mea- model.13)
sured. Data are expressed as relative ratio of activation with respect to positive control. Compound 1 exhibited a potent suppressive activity on
TPA-induced phenomenon in vitro and in vivo.
These results indicate that 1 is a potent anti-cancer com-
Table 2. Effect of Coumarin Derivatives on 32Pi-Incorporation into Phos-
pholipids of HeLa Cells Stimulated by Tumor Promoter
Table 3. Effect of 1 on ODC-Activation Stimulated by Tumor Promoter in
Condition cpm/mg protein 3103 Inhibition % Mouse Epidermis

Vehicle 14.6 ODC activity

Condition % of control
Control (150 nM TPA) 148.0 (nmol putrescine/mg protein/h)
11 16 m M 133.4 11
162 m M 102.6 34 Vehicle 0.5
1620 m M 29.4 89 Control (TPA: 16.2 nmol) 25.4 100
11 325 nmol 21.1 83
HeLa cells were treated with various concentrations of 1. After 1 h, 32Pi (74 kBq/cul- 1620 nmol 17.9 70
ture) was added with or without TPA (50 nM). Sample and TPA were dissolved in
DMSO. Vehicle control was treated with DMSO alone. Incubation was continued for Mice were treated with each sample and/or 16.2 nmol of TPA in 200 m l acetone.
4 h, and then the radioactivity incorporated into the phospholipid fraction was mea- Control was treated with TPA and vehicle in acetone. Mice were saclificed 4 h after
sured. Data, expressed as percentage on TPA-enhanced 32Pi incorporation, are mean TPA treatment, and the epidermis cells were collected to determine ODC activity. Data
values of duplicate experiments. are mean value of three mice.

Fig. 2. Inhibitory Effect of 1 on Tumor Promotion in DMBA-Initiated Mice

Initiation was carried out by a single application of 100 m g DMBA (390 nmol) on the back skin of mice. TPA (1.0 m g, 1.62 nmol), as promoter, was applied on the same place
twice a week for 20 weeks from 1 week after the initiation. Compound 1 was applied simultaneously with TPA at the dose of 162 and 325 nmol (molar ratio to TPA was 100 : 1,
200 : 1, respectively). The number of tumors was determined once a week. Each experimental group consisted of 15 mice.
246 Vol. 25, No. 2

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