Original Paper

147

Author

Mehrdad Iranshahi1, Farhad Kalategi1, Ramin Rezaee1, Ahmad Reza Shahverdi2, Chihiro Ito3, Hiroshi Furukawa3,
Harukuni Tokuda4, Masataka Itoigawa5

Affiliation

Affiliation addresses are listed at the end of the article

Key words
" Apiaceae

" Ferula

" anti-tumor promoting effect

" terpenoid coumarins

" Epstein-Barr virus activation

assay
" cancer chemoprevention

Abstract
!

Several natural products have been found to have

anti-tumor promoting activity. In the present
study, we carried out a primary screening of ten
terpenoid coumarins isolated from plants of the
Ferula species, examining their possible inhibitory effects on Epstein-Barr virus early antigen
(EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in Raji cells. Auraptene (7-geranyloxycoumarin, 1) and umbelliprenin (7-farnesyloxycoumarin, 2) were found to
significantly inhibit EBV-EA activation and pre-

Introduction
!

received August 6, 2007

revised December 25, 2007
accepted January 3, 2008
Bibliography
DOI 10.1055/s-2008-1034293
Planta Med 2008; 74: 147150
Georg Thieme Verlag KG
Stuttgart New York
Published online January 31,
2008
ISSN 0032-0943
Correspondence
Dr. Mehrdad Iranshahi
Department of Pharmacognosy
and Biotechnology
Biotechnology Research Center
Faculty of Pharmacy
Mashhad University of Medical
Sciences (MUMS)
P.O.Box: 917751365
Mashhad
Iran
Tel.: +98-511-882-3255-66
Fax: +98-511-882-3251
Iranshahim@mums.ac.ir

The exclusively old world genus Ferula belongs to

the family Apiaceae with about 130 species distributed throughout the Mediterranean area and
central Asia, specifically in the former USSR and
neighbouring countries such as Iran. This genus is
well documented as a good source of biologically
active compounds such as terpenoid coumarins
and sesquiterpene derivatives [1], [2], [3], [4].
In a recent study, it was found that asafoetida
(the oleogum resin obtained from some Ferula
species, especially Ferula foetida) could inhibit
early events of carcinogenesis [5]. Many compounds have been isolated from asafoetida including sulfur-containing compounds and sesquiterpene coumarins such as farnesiferols A, B,
C, kamolonol, ferocolicin, assacoumarin A, assacoumarin B, umbelliprenin, conferone and others
[6], [7]. However, sesquiterpene coumarins are
also major compounds of asafoetida.
Synthesis of the Epstein-Barr Virus (EBV) associated early antigen (EA) can be induced by the inducer combination TPA/n-BA (12-O-tetradecanoylphorbol 13-acetate plus n-butyric acid) in
Raji cells, a latently EBV-infected human lymphoblastoid cell line. The activated cells (or induced

served the high viability of Raji cells, suggesting

that they might be valuable anti-tumor-promoting agents (IC50 8.3 and 9.1 nM, respectively).
Our findings revealed that the presence of a prenyl moiety in the terpenoid coumarins plays an
important role in anti-tumor promoting activity
as previously reported for xanthones, coumarins,
flavonoids and phenylpropanoids.
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica

cells) are stained by high titer EBV-EA positive

sera from nasopharyngeal carcinoma (NPC) patients and detected by a conventional indirect
immunofluorescence technique. In positive controls (induced by TPA/n-BA) EA induction is ordinarily about 30 %. If there is a cancer chemopreventive agent (or anti-tumor promoting agent)
in the medium of the cells, EA induction will be
inhibited by that, resulting in a decrease of the
activated cells. Therefore, the inhibition rate can
be calculated by counting live activated and
non-activated Raji cells in the presence of a test
sample as compared with the positive control.
It has been recently found that some coumarins
including murrangatin, 6,8-di(3-methyl-2-butenyl)-7-methoxycoumarin, osthol, minumicrolin,
chloticol, sibiricol and murraol can significantly
inhibit Epstein-Barr virus early antigen (EBV-EA)
activation [8]. The above findings encouraged us
to investigate the anti-tumor promoting effects
of terpenoid coumarins as major chemical constituents of the genus Ferula. In the present work,
we tested the inhibitory effects on EBV-EA activation of some terpenoid coumarins with different
structures, isolated from plants of Ferula species.
Three of the tested terpenoid coumarins (2, 3, 7)
are the same ones as found in asafoetida.

Iranshahi M et al. Cancer Chemopreventive Activity Planta Med 2008; 74: 147 150

Downloaded by: University of Karachi. Copyrighted material.

Cancer Chemopreventive Activity of Terpenoid

Coumarins from Ferula Species

Original Paper

Materials and Methods

Anti-tumor-promoting activity

The inhibition of EBV-EA activation was assayed using Raji cells

(EBV non-producer type), the EBV genome-carrying human lymphoblastoid cells, which were cultivated in 10 % FBS RPMI1640 medium solution. The indicator cells (Raji) (1 106/mL)
were incubated with 1 mL of the medium containing n-butyric
acid (4 mM, inducer), 32 pmol of TPA (20 ng/mL in DMSO) and a
known amount of test compound at 37 8C in a CO2 incubator. After 48 h, reacted cell solutions were centrifuged at 1000 rpm for
10 min, and the reacted medium was removed. Residues were
redissolved with 0.1 mL of phosphate buffer solution and this
cell suspension was used for smears. The activated cells were
stained by high titer EBV-EA positive sera from nasopharyngeal
carcinoma (NPC) patients and detected by a conventional indirect immunofluorescence technique. In each assay, at least 500
cells were counted and the experiments were repeated three
times. The average EA induction was compared with that of positive control experiments with n-butyric acid plus TPA in which
EA induction was ordinarily around 30 %. Cell number and viability were determined after 48 h by means of a hemocytometer
(Trypan blue staining method).

Cell lines, instruments and chemicals

Original Raji cells have been obtained from Prof. Gerge Klain at
the Karolinska Institute in Sweden. RPMI-1640 and other reagents were purchased from Wako Pure Chemical Co.. Immunofluorescence technique was carried out using an Ultraviolet Microscope (Nikon Co. Ltd.). Melting points were determined on an
Electrothermal 9100 apparatus and are uncorrected. NMR spectra were measured on a Bruker DRX 500 (Bruker Biospin). The
operating frequencies were 500.13 MHz for acquiring 1H-NMR
and 125.75 MHz for 13C-NMR spectra. Column chromatography
was conducted with silica gel 230 400 mesh from Merck. Preparative TLC was performed on silica gel GF254 s plates (20 20 cm,
Merck) and observation of plates was carried out under a UV CAMAG spectrometer (254 nm).

Plant materials
Roots of Ferula flabelliloba were collected in the Binalood Mountains, north east of Iran, in April 2005, and identified by Mohammadreza Joharchi, Ferdowsi University of Mashhad Herbarium
(FUMH). A voucher specimen (1004) is deposited at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy,
Mashhad University of Medical Sciences. Fruits of Ferula
badrakema were collected in the Hezarmasjed Mountains, north
east of Iran, in August 2005, and identified by Mohammadreza
Joharchi, Ferdowsi University of Mashhad Herbarium (FUMH).
A voucher specimen (1002) is deposited at the Herbarium of the
Department of Pharmacognosy, Faculty of Pharmacy, Mashhad
University of Medical Sciences.

Statistics
The results were expressed as mean SD of three experiments.
IC50 was calculated with the PRISMA software.

Supporting information
The NMR spectral data for compounds 7 9 are available as Supporting Information.

Test products

Results and Discussion

The compounds 1 6 and 10 tested in this study have been

isolated and described in previous papers [4], [9]. For isolating
the compounds 7 and 9, 500 g of the powdered roots of Ferula
flabelliloba were extracted by dichloromethane (3 L) by maceration (24 h), yielding a brown viscous residue (40 g). Part of the
extract (20 g) was subjected to column chromatography on silica
gel (5 50 cm, mesh 230 400) using petroleum ether-ethyl acetate as solvent [petroleum ether 360 mL, petroleum ether-ethyl
acetate (20 : 1) 1050 mL, (15 : 1) 960 mL, (10 : 1) 990 mL, (9 : 1)
1900 mL, (8 : 1) 2700 mL, (7: 1) 800 mL, (6 : 1) 3710 mL, (5 : 1)
1800 mL, (4 : 1) 2500 mL, (3 : 1) 6267 mL, (2 : 1) 3600 mL and ethyl acetate 2000 mL]. The fractions were compared by TLC (silica
gel using petroleum ether-ethyl acetate as solvent), and those
giving similar spots were combined. 48 fractions were finally obtained. Fraction 23 and 41 afforded white crystals of compounds
7 (437 mg) and 9 (772 mg), respectively. The structures of the
compounds were confirmed by comparison of 1H- and 13C-NMR
spectra as well as melting point values with those of previous reports [10], [11].
Similarly, compound 8 was isolated and purified from the fruits
(500 g) of Ferula badrakema according to the above conditions.
Six fractions were finally obtained. Fraction 3 afforded white
crystals of compound 8 (770 mg). The structure of compound 8
was also confirmed by comparison of 1H- and 13C -MR spectra
as well as melting point value with those of previous reports
[10], [11]. The purity of the tested compounds was checked by
TLC and 13C NMR experiments. Copies of the original spectra are
obtainable from the author of correspondence.

Except for compound 1 (auraptene), all compounds (2 10) tested in this study belong to the class of sesquiterpene coumarins
" Fig. 1). The sesquiterpene unit of these compounds is linked
(
to the hydroxy group of the umbelliferone (7-hydroxycoumarin)
nucleus. These coumarins were tested for their anti-tumor promoting activity by using a short-term in vitro assay of 12-O-tetradecanoylphorbol 13-acetate (TPA) induced Epstein-Barr virus
early antigen (EBV-EA) activation in Raji cells. Their inhibitory
effects on activation of the virus and their effects on the viability
" Table 1.
of the Raji cells are shown in
All compounds tested in this study were more effective than the
previously tested umbelliferone [8]. Furthermore, all compounds tested with the Raji cells showed only weak cytotoxicity
at 32 nM. In other words, the cell viability of all tested compounds was in an acceptable range (> 60 %) at different concentrations. The coumarins having a prenyloxy unit at C-7, auraptene (1) and umbelliprenin (2) showed a significant inhibitory
effect (92.8 97.4 % inhibition of activation at 32 nM, and 22.1
24.2 % and 1.4 3.6 % inhibition of activation at 3.2 and 0.32 nM,
respectively). The effects of the other terpenoid coumarins (3
10) were weaker than that of 7-prenyloxycoumarins (1 and 2),
-carotene (a vitamin A precursor commonly used in cancer prevention studies as a standard reference) and curcumin. The IC50
values of these compounds, except for two prenyloxy coumarins
(1, IC50 = 8.3 nM and 2, IC50 = 9.1 nM) were comparable to reference compounds, -carotene (IC50 = 10.4 nM) and curcumin
" Table 1).
(IC50 = 8.3 nM) (
These results suggest that the compounds auraptene (1) and
umbelliprenin (2) might be more valuable anti-tumor promot-

Iranshahi M et al. Cancer Chemopreventive Activity Planta Med 2008; 74: 147 150

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148

Original Paper

Table 1

The structures of the tested compounds.

Inhibitory effects of terpenoid coumarins on TPA-induced EBV-EA activation

Compound

32 a

16

7-Methoxycoumarin

100 (30)

28.3

3.2

0.32

0.0

IC 50 (nM)

0.0

plant
Citrus jamnhiri [8]

Auraptene (1)

97.4b 0.3 (60)

71.4 1.4 (>80)

24.2 2.0 (>80)

3.6 0.4 (>80)

8.3

F. szowitsiana

Umbelliprenin (2)

92.8 0.5 (60)

68.6 1.4 (>80)

22.1 2.1 (>80)

1.4 0.3 (>80)

9.1

F. szowitsiana

Farnesiferol A (3)

84.7 0.6 (60)

55.7 1.5 (>80)

18.6 2.2 (>80)

0.0 0.2 (>80)

13.1

F. persica

Badrakemone (4)

83.1 0.7 (60)

54.2 1.5 (>80)

16.8 2.2 (>80)

0.0 0.1 (>80)

13.3

F. persica

Methyl galbanate (5)

86.1 0.5 (60)

57.2 1.4 (>80)

20.3 2.0 (>80)

0.0 0.3 (>80)

11.7

F. szowitsiana

Galbanic acid (6)

87.5 0.5 (60)

58.4 1.3 (>80)

20.7 2.1 (>80)

0.0 0.3 (>80)

11.8

F. szowitsiana

Feselol (7)

88.7 0.4 (60)

52.7 1.6 (>80)

27.9 1.6 (>80)

1.7 0.3 (>80)

13.0

F. flabelliloba

Mogoltacin (8)

88.9 0.4 (60)

53.9 1.6 (>80)

28.1 1.5 (>80)

2.0 0.4 (>80)

12.6

F. badrakema

Conferone (9)

87.0 0.5 (60)

48.9 1.9 (>80)

27.0 1.7 (>80)

0.0 0.3 (>80)

14.3

F. flabelliloba

Ferukrinone (10)

87.9 0.5 (60)

50.1 1.9 (>80)

27.2 1.6 (>80)

0.0 0.4 (>80)

13.7

F. persica

Carotene c
Curcuminc

90.9 0.5 (60)

65.7 1.1 (>80)

17.3 1.8 (>80)

0.0 0.2 (>80)

10.4

77.2 1.8 (>80)

18.3 1.9 (>80)

0.0 0.2 (>80)

8.3

100

0.5 (60)

Compound concentration in nM.

Inhibition rate on EBV-EA activation in percentage.

Positive control substance. Values are EBV-EA activation ( %) SD in the presence of the test compound relative to the positive control (100 %). Values in parentheses
represent the surviving Raji cells measured by Trypan blue staining. A minimum 60 % survival rate of Raji cells 2 days after treatment with the compounds is required
for an accurate result. IC50 represents the concentration in nM that inhibits 50 % of positive control (100 %) activated with 32 pmol TPA. The experiments were
repeated three times.

ing agents in case of chemical carcinogenesis than farnesiferol A

(3), badrakemone (4), methyl galbanate (5), galbanic acid (6),
feselol (7), mogoltacin (8) conferone (9) and ferukrinone (10).
In addition, these results suggest that the presence of a prenyl
moiety on the structure of umbelliferone nucleus plays an important role in anti-tumor promoting activity as has been previously reported for xanthone, coumarins, flavonoids and phenyl" Fig. 2, the compropanoids [8], [12], [13], [14]. As shown in
pound 6,8-di(3-methyl-2-butenyl)-7-methoxycoumarin, containing two prenyl on its structure, was the most potent (21.8 %

inhibition of activation at 0.32 nM) among thirty-four tested

coumarin derivatives in a previous study [8].
In a previous study, it has been found that some coumarins including murrangatin, 6,8-di(3-methyl-2-butenyl)-7-methoxycoumarin, osthol, minumicrolin, chloticol, sibiricol and murraol
can more significantly inhibit Epstein-Barr virus early antigen
(EBV-EA) activation than our compounds. The former compounds showed 18.2 22.4 % inhibition of activation at a 1 10
mol ratio (0.32 nM) [8].

Iranshahi M et al. Cancer Chemopreventive Activity Planta Med 2008; 74: 147 150

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Fig. 1

149

150

Original Paper

Many investigations, in particular by Tanaka and coworkers,

have shown that auraptene is one of the most promising natural
chemopreventive agents against cancer of the liver, skin, tongue,
oesophagus and colon in rodents [15]. It exerts a dose-dependent chemoprevention of tumorogenesis in the large bowel of
the rat, the effect being correlated with the inhibition of cell proliferation and lipid peroxidation, and with the induction phase
drug-metabolizing enzyme. However, auraptene showed the
most potent activity among the compounds tested in this study,
confirming the valuable bioactivity of the compound. Auraptene
is also present in some edible fruits, especially Citrus species. Epidemiological surveys and animal experiments have indicated
that adding to the diet some constituents of vegetables and
fruits may contribute to a decreased incidence of cancer in humans [16]. Galbanic acid (6) and umbelliprenin (2) have been
also reported as hepatoprotective agents [17] and matrix metalloproteinase (gelatinase) inhibitors [18] in previous studies, respectively.
In conclusion, our studies showed that the prenylated coumarins, auraptene (1) and umbelliprenin (2), might be valuable
anti-tumor-promoting agents in several carcinogenesis test systems. Consequently, some foods (Citrus species) and medicinal
plants (Ferula species) which contain these compounds, can be
considered as valuable sources of chemopreventive agents.

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Acknowledgements
!

This research was partially supported by a grant (85 082) from

the Mashhad University of Medical Sciences Research Council.
Affiliation
1

3
4

Department of Pharmacognosy and Biotechnology, Biotechnology Research

Center, Faculty of Pharmacy, Mashhad University of Medical Sciences
(MUMS), Mashhad, Iran
Department of Pharmaceutical Biotechnology and Pharmaceutical Science
Research Center, Faculty of Pharmacy, Medical Sciences/University of Tehran, Tehran, Iran
Faculty of Pharmacy, Meijo University, Tempaku, Nagoya, Japan
Department of Molecular Biochemistry, Kyoto Prefectural University of
Medicine, Kamigyo-ku, Kyoto, Japan
Tokai Gakuen University, Nagoya, Japan

Iranshahi M et al. Cancer Chemopreventive Activity Planta Med 2008; 74: 147 150

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Fig. 2 The structure

of 6,8-di(3-methyl-2butenyl)-7-methoxycoumarin as the most
potent coumarin in a
previous study [8]. Two
prenyl parts are seen in
the structure.