Isolation of some potent anti-tumor compounds from medicinal plants has motivated
researchers to screen different parts of plant for their anti-tumor effects. It has been reported
that several species of conifers posses’ cytotoxic activities on some tumor cell lines. Here
branchlets and berries of Juniperus foetidissima and J. sabina were collected, dried and
ethanol extracts of them obtained using percolation. Extracts were dried in reduced pressure
and cytotoxic effects of different concentrations (5, 10, 20 ?g/ml) were evaluated by MTT
assay against three tumor cell lines (Hela, KB, MDA-MB-468), using ELISA at 540 nm. The
extracts of the branchlets of male and female of J.foetidissima and berries extract of J. sabina
showed inhibitory activities against KB cells. Extracts of male branchlets of J. foetidissima
and berries extract of J. sabina were cytotoxic (cell survival less than 50%) against Hela cell
line. Regarding to MDA-MB-468, only the extract of male branchlets of J. foetidissima was
cytotoxic. Extracts of J. sabina were not cytotoxic at tested concentrations. According to the
results obtained by MTT assay, KB cells seem to be much more sensitive than the other cell
Acute and Subchronic Toxicity?of Teucrium polium Total Extract in Rats
Iranian Journal of Pharmaceutical Research
(2009), 8 (4): 281-286
Received: November 2008
Accepted: January 2009
Copyright ? 2009 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services
Evaluation of In Vitro
Cytotoxic Effects of Juniperus foetidissima and
Juniperus sabina Extracts
against a Panel of Cancer Cells
Hojjat Sadeghi-aliabadia,c*, Ahmad Emamib,
Babak Sadeghia and Abbas Jafarianc
aDepartment of Pharmaceutical Chemistry, Faculty of
Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences,
Isfahan, Iran. bDepartment of Pharmacognosy, Faculty of Pharmacy, Mashhad
University of Medical Sciences, Mashhad, Iran. cIsfahan Pharmaceutical Sciences
Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.
Isolation of some potent anti-tumor compounds from medicinal plants has
motivated researchers to screen different parts of plant for their anti-tumor
effects. It has been reported that several species of conifers posses? cytotoxic
activities on some tumor cell lines. Here branchlets and berries of Juniperus
foetidissima and J. sabina were collected, dried and ethanol extracts of them
obtained using percolation. Extracts were dried in reduced pressure and
cytotoxic effects of different concentrations (5, 10, 20 ?g/ml) were evaluated
by MTT assay against three tumor cell lines (Hela, KB, MDA-MB-468), using ELISA
at 540 nm. The extracts of the branchlets of male and female of J.foetidissima
and berries extract of J. sabina showed inhibitory activities against KB cells.
Extracts of male branchlets of J. foetidissima and berries extract of J. sabina
were cytotoxic (cell survival less than 50%) against Hela cell line. Regarding
to MDA-MB-468, only the extract of male branchlets of J. foetidissima was
cytotoxic. Extracts of J. sabina were not cytotoxic at tested concentrations.
According to the results obtained by MTT assay, KB cells seem to be much more
sensitive than the other cell lines.
Keywords:J. foetidissima; J. sabina; MTT assay; Hela, KB, MDA-MB-468.
Conifers are a small group of the flora of Iran (8 species from 8000 species).
All aromatic Iranian conifers belong to Cupressaceae family. In Iran this family
consists of one species of Platycladus, one species of Cupressus and five
species of Juniperus.
Juniperus species are the second most diverse genus of conifers. The genus
Juniperus consist of approximately 67 species and 28 varieties. This genus is
divided into three sections: Caryocedrus Edlicher(with only one species) ;
Juniperus (syn: Oxycedrus Spach with 12 species) and Sabina (Miller) Spach (with
55 species) (1).
Two examined species of Iranian Juniperus,
J. sabina and J. foetidissima, belong
to later section. J. sabina L. (Cupressaceae) is normally low shrub with
procumbent or obliquely ascending branches, or rarely a small tree to about 4m,
monoecious or dioecious. J. sabina, named ?Maymarz? in Persian, generally
distributed in central and southern Europe, Anatolia, the Caucasus, the southern
mountains of Asian Russia, Siberia, Mongolia and southwest of Asia (2, 3). J. sabina is a medicinal plant which is used in folk medicine as an abortive (4).
It?s lignanes have antineoplastic and antiviral activity (5). J. sabina?s
essential oil has shown antibacterial (6) and antifungal (7) activity.
J. foetidissima Willd. (Cupressaceae) is a tree with 5-15 m high, crown slender
conical, branched to the ground. This species, named ?Arduj? in persian, found
in mountains of Greece, Albania, Yugoslavia, Asia Minor to Transcaucasus (2,3).
J. foetidissima is also a medicinal plant with antifungal activity (8).
Following our main project to screen Iranian medicinal plant for their cytotoxic
effects, these species were evaluated. Several investigators have shown that the
leaves of several genera of conifers (Taxus, Platycladus, Libocedrus,
Chaemacyparis and Callitris) possess cytotoxic compounds or tumor necrosing
substances (9). Also, cytotoxic effects of Juniperus sabina and Platycladusorientalis extracts against Hela cells (7), ethanol extracts of J. phenicea,
J. bermudiana, J. communis and Libocedrusdecrrens against KB cell lines (10) have
been previously reported. Our previous studies revealed that different parts of
Iranian conifers possess cytotoxic effects on some tumor cell lines (11-13). It
is believed that some lignans such as podophyllotoxin and desoxypodophyllotoxin
are responsible for this effect. In this study we sought to evaluate the
cytotoxic effects of different parts of J. excelsa and J. polycarpos on Hela, KB
and MDA-MB-468 cell lines.
The branchlets of male and female of and berries of
J. sabina were collected
from Aliabad Katol (2000 m, Golestan province, north of Iran); J. foetidissima
was collected from Vinaq in Arasbaran (1950 m, East Azarbayejan province,
northwest of Iran) in September 2001. The plants were identified by the
Department of Forestry, University of Tehran, Iran. The plant materials were
stored at -20 ?C before use. Voucher specimens of the plants (No. 1414-1415)
were deposited in the herbarium of School of Pharmacy and Pharmaceutical
Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Extraction and isolation
Fifty g of each plant part was crushed and soaked in 75 ml of ethanol (80% V/V)
for 24 h and then percolated (5 h, 30 drops/min) (14). The extracts were
concentrated by a rotary evaporator and dried in an oven at 40 ?C to give
0.5-0.8 g of solid residue. Twenty mg of solid residues were dissolved in one ml
of ethanol and diluted to 100 ml with distilled water and filtered through 0.22
? microbiological filters. Dilution was continued so that the final
concentrations of extracts were 5, 10 and 20 ?g/ml (12).
Hela (human cervix carcinoma), KB (human Caucasian/epidermal carcinoma) and
MDA-MB-468 (human breast adenocarcinoma) cell lines were purchased from Pasture
Institute, Tehran, Iran.
Maintenance of human cell lines
Cells were grown in RPMI-1640 [supplemented with 10% of fetal calf serum,
penicillin/ streptomycin (50 IU ml-1 and 500 ?g ml-1 respectively), sodium pyruvate (1 mM), NaHCO3 and l-glutamine (2 mM)]. Completed media was sterilized
using 0.22 ? microbiological filters and kept at 4 ?C prior to use. Cells were
maintained and grown in RPMI 1640 up to 15 subcultures. A sample of each cell
lines was frozen and kept under liquid nitrogen for future studies.
MTT-based cytotoxicity assay
The cytotoxic effect of obtained extracts against previously mentioned human
tumor cell lines was determined by a rapid colorimetric assay, using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and compared
with untreated controls (15). This assay is based on the metabolic reduction of
soluble MTT by mitochondrial enzyme activity of viable tumor cells, into an
insoluble colored formazan product, which can be measured spectrophotometrically
after dissolving in dimethyl sulfoxide (DMSO). Briefly, 200 ?l of cells (5 ? 104
cells per ml of media) were seeded in 96-well microplates and incubated for 24 h
(37 ?C, 5% CO2 air humidified). Then 20 ?l of prepared concentrations of each
extract was added and microplates containing cells and extracts were incubated
for another 72 h in the same condition. Doxorubicin was used as a positive
control. The first column of each microplate was assumed as negative control
(containing no extracts or doxorubicin). To evaluate cell survival, 20 ?l of MTT
solution (5 mg/ml in phosphate buffer solution) was added to each well and
incubated for 3 h. Then gently 150 ?l of old medium containing MTT was replaced
by DMSO and pipetted to dissolve any formed formazan crystals. Absorbance was
then determined at 540 nm by ELISA plate reader. Each extract concentration was
assayed in 8 wells and repeated 6-times. Standard curves (absorbance against
number of cells) for each cell line were plotted. Intraday and interday
variations were determined. Based on standard curves percent cell survival was
calculated. Percent of cell survival in ethanol treated cells (1% as negative
control) was assumed 100.
SIGMASTATTM (Jandel Software, San Raphael, CA) was used to perform statistical
tests. Analyze-of-variance followed by Dunkan test was used to see the
differences among groups. Significance was assumed at the 5% level.
A good relationship between absorbance and the number of cells was observed for
Hela, KB and MDA-MB-468 cell lines, (r2 = 0.9789, 0.9852 and 0.9919,
respectively). Intraday and interday variations for all standard curves were
acceptable (%CV < 15). Doxorubicin (20 ?g/ml), a known cytotoxic antibiotic, as
a positive control significantly inhibited the proliferation of all tested cell
lines to less than 25% . Extracts were considered cytotoxic when cell viability
decreased to less than 50%.
Cytotoxic effect of extracts against Hela cells
As shown in Figure 1, hydroalcoholic extract of male branchlets of
J.foetidissima (20 ?g/ml), was cytotoxic against Hela cells; whereas other plant
samples were not cytotoxic at tested concentrations.
Cytotoxic effect of extracts against KB cells
Hydroalcoholic extracts of the terminal branchlets of male and female and
berries of J. foetidissima in all tested concentration showed an excellent
inhibitory effects against KB cells (IC50 < 5 ?g/ml; Figure 2). However, cytotoxicity was seen at highest concentration (20 ?g/ml) of berries extract of
J. sabina (Figure 2).
Cytotoxic effect of extracts against MDA-MB-468 cells
In the case of MDA-MB-468 cells only the highest concentration (20 ?g/ml) of
obtained extract from branchlets of J. foetidissima was cytotoxic (Figure 3);
whereas with extracts of J. sabina IC50 was not obtained.
Cytotoxic compounds are one of the most important classes of drugs used for
cancer treatment. There have been several researches to get new cytotoxic
agents. In this regard compounds such as colchicine, Vinca alkaloids and
paclitaxel isolated from medicinal plants showed considerable promises. Studies
on different genera of conifers showed the presence of cytotoxic compounds or
tumor necrosing substances (9). Furthermore, cytotoxicity of some Iranian
conifers has been shown in our previous studies (11-13). To evaluate the
cytotoxicity of the other genera of Iranian conifers, this study was conducted.
In the present study, MTT assay was used for evaluation of cytotoxic activity of
conifers. As seen in Figure 2, hydroalcoholic extracts of all tested parts of J. foetidissima showed cytotoxic effects comparable to that of doxorubicin
(positive control) against KB cells (IC50 < 5 ?g/ml). Lignans, neolignans and
flavonoid glycosides in juniperus species are responsible for their
antibacterial and cytotoxic properties (16, 17). Substances that induce
apoptosis toward human promyelocytic leukemia HL-60 cells have been extracted
from leaves of Juniperustaxifolia (18). The results of a study conducted by
Topcu and co-workers (19) showed that diterpenes and sesquiterpenes extracted
from the berries of J. excelsa had cytotoxic activity against a panel of cell
lines [human colon cancer cell line (LNCaP), KB-V (+VLB) and KB-V (-VLB)] and
Mycobactrium tuberculosis. Emami and colleagues (20) showed that extracts
obtained from leaves and fruits of Iranian J. foetidissima posses good
antioxidant activity. Also, it has been revealed that antioxidants have
beneficial effects in cancer (21). So it can be concluded that J. foetidissima
can be effective in cancer by different mechanisms.
Emami and Asili (22) found that flavonoids are the major components of
J. foetidissima extract while their presence in J. Sabina was not that much.
Sabina had no cytotoxic effect against all cell lines used in this study.
Comparing the cytotoxicity of J. foetidissima and J. sabina may lead to
conclusion that this effect is related to its flavonoid contents.
Our preliminary findings showed that KB cells were the most sensitive cells
which are consistence with the findings that susceptibility of cells to
cytotoxic compounds is variable (23). To evaluate the apoptosis mechanism of
these extracts more experiments such as TUNEL and Annexin V assays are underway.
This study was supported by a grant from the Research Council of Isfahan
University of Medical Sciences, Isfahan, Iran (No. 78303). The authors are
thankful to the late Dr. K. Javanshir, University of Tehran for identification
of the plant material.
(1) Adams RP. Juniperus of the world. Trafford
Publishing Co. Vancouver (2004) P. 1
Assadi M. Cupressaceae In: Assadi M, Khatamsaz M, Maassoumi AA and Mozaffarian
V. (eds.) Flora of Iran. Vol. 21, Research Institute of Forests and Rangelands,
Tehran (1998) 19-28
Sabeti H. Forests, Trees and Shrubs of Iran. Ministry of Information and Tourism
Press, Tehran (1975) 420-5
Alm T. Juniperus sabina in Norwegian folk tradition. Sevenbom Juniperus sabina i
folketradisjonen i Norge (2003) 61: 186-9
San Feliciano A, Gordaliza M, Miguel Del Corral JM, Castro MA, Garcia-Gravalos
MD and Ruiz-Lazaro P. Antineoplastic and antiviral activities of some
cyclolignans. Planta Medica (1993) 59: 246-9
Akimov Yu A, Kharchenko GI, Krylova AP and Belova NN. Antimicrobe action of
terpenes from savin (Juniperus sabina L.). Appl. Biochem. Microbiol. (1977) 13:
San Feliciano A, Miguel Del Corral JM, Gordaliza M and Castro A. Lignans from
Juniperus sabina. Phytochemistry (1990) 29: 1135-8
Balaban M, Atik C and Ucar G. Fungal growth inhibition by wood extracts from Juniperus foetidissima and J.
oxycedrus. Holz als Roh - und Werkstoff (2003) 61:
Van Uden W, Homan B, Woerdenbag HJ, Pras N, Malingr? TM, Wichers HJ and Harkes
M. Isolation, purification, and cytotoxicity of 5-methoxypodophyllotoxin, a
lignan from a root culture of Linum flavum. J. Nat. Prod. (1992) 55: 102-10
Cairnes DA, Ekundayo O and Kingston DG. Plant anticancer agents. X. Lignans from
Juniperus phoenicea. J. Nat. Prod. (1980) 43: 495-7
Sadeghi-aliabadi H, Emami SA, Saeidi M and Jafarian A. Cytotoxic effects of the
extracts of Iranian Taxus baccata and Cupressus horizentalis on cancer cells.
Iranian J. Pharm. Res. (2003) 2: 107-10
Jafarian-Dehkordi A, Emami SA, Saeidi M and Sadeghi H. Cytotoxicologic studies
of the extracts of Iranian Juniperus sabina and Platycladusorientalis on cancer
cells. J. Res. Med. Sci. (2004) 5: 7-11
Emami SA, Sadeghi-aliabadi H, Saeidi M and Jafarian A. Cytotoxic evaluation of
Iranian conifers on cancer cells. Pharmaceutical Biology (2005) 43: 299-304
List H and Schmidt P. Technologie Pflanzlicher Arzneizuberitungen.
Wissenschafliche Verlagsgesellschaft mbH Stuttgart, (1984) 140
Mosmann T. Rapid colorimetric assay for cellular growth and survival:
Application to proliferation and cytotoxicity assays. J. Immunol. Methods (1983)
San Feliciano A, Gordaliza M, Miguel del Corral JM, Castro MA, Garcia-Gravalos
MD and Ruiz-Lazaro P. Antineoplastic and antiviral activities of some
cyclolignans. Planta Medica (1993) 59:246-9
Gao R, Gao C, Tian X, Yu X, Di X, Xiao H and Zhang X. Insecticidal activity of
deoxypodophyllotoxin, isolated from Juniperus sabina L, and related lignans
against larvae of Pieris rapae L. Pest Manag. Sci. (2004) 60:1131-6
Muto N, Tomokuni T, Haramoto M, Tatemoto H, Nakanishi T, Inatomi Y, Murata H and
Inada A. Isolation of apoptosis- and differentiation-inducing substances toward
human promyelocytic leukemia HL-60 cells from leaves of Juniperustaxifolia.
Biosci. Biotechnol. Biochem. (2008) 72:477-84
Topcu G, Erenler R, Cakmak O, Johansson CB, Celik C, Chai HB and Pezzuto JM.
Diterpenes from the berries of Juniperus excelsa. Phytochemistry (1999)
Emami SA, Asili J, Mohagheghi Z and Hassanzadeh MK. Antioxidant activity of
leaves and fruits of Iranian conifers. eCAM (2007) 4: 313-9
Jeong JM, Choi CH, Kang SK, Lee IH, Lee JY and Jung H. Antioxidant and
chemosensitizing effects of flavonoids with hydroxy and/or methoxy groups and
structure-activity relationship. J. Pharm. Pharm. Sci. (2007) 10:537-46
Emami SA and Asili J. phytochemical evaluation and antimicrobial activity of
essential oils of Iranian conifers. Pharm D thesis, School of Pharmacy, Mashhad
University of Medical Sciences (1997) 33-95
Suzuki F, Hashimoto K, Ishihara M, Westman G, Samuelsson K, Kawase M, Motohashi
N and Sakagami H. Tumor-specificity and type of cell death induced by
phenoxazines. Anticancer Res. (2007) 27: 4233-8