Journal of Medicinal Plants Research Vol. 3(12), pp.

1045-1050, December, 2009

Available online at http://www.academicjournals.org/jmpr
ISSN 1996-0875© 2009 Academic Journals

Full Length Research Paper

Cytotoxic effect of some Moroccan medicinal plant

extracts on human cervical cell lines
Nawal Merghoub1,2, Laïla Benbacer1, Saaid Amzazi2, Hamid Morjani3 and Mohamed El mzibri1*
1
Unité de Biologie and Recherche Médicale CNESTEN, Rabat, Marocco or CNESTEN BP 1382 RP, 10001 Rabat,
Marocco.
2
Laboratoire de Biochimie-Immunologie. Faculté des sciences de Rabat, Agdal, Marocco.
3
MEDyC CNRS UMR 6237, UFR Sciences et UFR Pharmacie, Reims, France.
Accepted 16 October, 2009

Organic extracts of 7 selected plant species, used by Moroccan traditional healers to treat cancer or
non-cancer diseases, were tested for their anti-cancerous activities. The plant selection was based on
ethnobotanic information and interviews with local healers. Extracts from Inula viscosa (L.) Ait., Retama
monosperma (L.) Bois., Ormenis mixta (L.) Dumont., Ormenis eriolepis Coss., Rhamnus lycioides (L.),
Berberis hispanica Bois and Reut. and Urginea maritima (L.) Baker. were tested for their potential
cytotoxic effects on the human cervical cancer cell lines SiHa and HeLa, harbouring HPV16 and HPV 18
respectively. MTT (Tetrazolium blue) colorimetric assay was used to evaluate the viability of cell
cultures in the presence of the extracts. The extract from Inula viscosa (L.) Ait., Retama monosperma
(L.) Bois., Ormenis eriolepis Coss. exhibited marked cytotoxic effect on the two cell lines. These
species could be considered as potential sources of anticancer compounds. Further studies are
necessary for chemical characterization of the active principles and more extensive biological
evaluations.

Key words: Moroccan medicinal plants, cervical cancer cells, cytotoxic activity.

INTRODUCTION

Cervical cancer is a major cause of death. It is the
second most frequent cancer in women worldwide,
accounting for 15% of all cancer related deaths in women
(Boyle and Ferlay, 2005). Each year 470,000 women are
diagnosed with invasive cervical cancer worldwide,
230,000 women die of this disease and 80% of these
occur in developing countries (Bosch and de Sanjosé,
2003). In Morocco, cervical cancer is the second most
frequent female cancer after breast cancer and
represents a major public health problem. The diagnosis
is usually made in advanced stages and mortality is high
(Amrani et al., 2003).
Human Papillomavirus (HPV) is considered as the
etiologic agent of cervical cancer. Epidemiological and
biological studies have shown close relationship between
*Corresponding author. E-mail: elmzibri@cnesten.org.ma,
mzibri@yahoo.com. Tel: +212.37.71.27.51, +212.66.83.59.11.
Fax: +212.37.71.18.46.
HPV infection and cervical cancer development. High risk
HPV, such as HPV16 and HPV18, has been detected in
94 - 100% of cervical precancerous lesions and cancer
(Castellsagué et al., 2006).
Though the cervical cancer therapy is in advance, side
effects due to the non-specific cytotoxicity of drugs and
resistance to treatment represent a great problem in the
cervical cancer management. Therefore, development
and search of novel and effective anticancer agents,
which in addition should overcome resistance, have
become very important issues (Cameron and Bell, 2004).
Natural compounds have provided many effective
anticancer agents in current use. Currently, over 50% of
drugs used in clinical trials for anticancer activity were
isolated from natural sources or are related to them
(Newman and Gragg, 2007). The use of plants or plants
products, traditionally, as antiviral agents is relatively
wider than their use in modern medicine. Some antiviral
substances have so far been isolated from higher plants,
algae and lichens (Abonyi et al., 2009).
To our knowledge, few studies described the use of
1046 J. Med. Plant. Res.
medicinal plants in treatment of infections by Human
Papilloma Virus, HPV. Craigo et al. (2000) have shown
that, some methylated derivatives of plant lignan, nordi-
hydroguaiaretic acid, which were found to be a potent
antiviral agent against the Human Immunodeficiency
Virus (HIV) and Herpes Simplex Virus (HSV), inhibit the
gene expression from the early promoter P97 of HPV 16
and can be used in the therapy of papillomavirus infec-
tions. Likewise, Deng et al. (2004) have also found that
water extracts from Asarum heterotropoides are effective
on anti-human papillomavirus.
There are two main strategies for the selection of plants
species in anticancer drug discovery: random screening
and ethnomedical knowledge. The second approach
includes plants used in organize traditional medical
systems like herbalism and folklore (Pieters and Vlietnick,
2005).
Uncontrolled proliferation is a universal property of
tumour cells. Investigation of the cellular growth control
mechanisms has contributed to the understanding of
carcinogenesis and identification of compounds with
specific antitumoral activities. Thus, cytotoxicity screening
models provide important preliminary data to help select
plant extracts with potential antitumoral properties for
future studies (Cardellina et al., 1999).
In Morocco, the use of traditional medicine is
widespread practice. The ethnobotanical and ethnophar-
macological surveys conducted in different areas allowed
the compilation of an inventory of 360 species and more
than 500 prescriptions are recorded (Bellakhdar, 1997).
In the Moroccan traditional medicine, the use of plants in
the form of infusions or decoctions is a common practice
among people of rural communities and their use is
increasing in urban populations. Moroccan medicinal
plants were already studied for their use in different
human diseases (Gonzalez-Tejero et al., 2008).
In the course of our screening strategy for the
anticancer compounds from plants, we undertook the
present study to evaluate the in vitro cytotoxic activity of
seven plants used in the Moroccan traditional medicine
for various diseases such as cancer, inflammation or
infectious diseases. The selection of plants was made on
the basis of their reputation as folk medicines in the
treatment of tumours and related diseases. Table 1
shows the ethnobotanical data of the investigated plant
species, including botanical names, local names,
ethnomedical uses, as well as the plant parts employed
in this study. The cytotoxic activity of selected plants was
studied on the human cervical carcinoma cells lines, SiHa
and HeLa, harbouring HPV16 and HPV18 respectively.
MATERIALS AND METHODS
Plant material
The selected plants were collected in different areas of Morocco in
May, 2007 and were identified by Dr. M. Fennane from the
Scientific Institute of Rabat. Voucher specimens are kept in the
“Herbarium” of the Institute.
Preparation of extract
Plant materials were dried at room temperature in the dark and
ground finely using blender. Exactly 20 g of each powdered sample
(aerial plants, leaves, root bark or bulbs) were extracted by abso-
lute methanol (100 ml, three times) for 72 h at room temperature
with constant shaking. The extracts were pooled and evaporated to
dryness under reduced pressure at 40°C. A total of 40 mg of
obtained extract were dissolved in dimethyl sulfoxide (DMSO) to
give a solution stock to 40 mg/ml. Extracts were sterilized by
filtration using sterile 0.22 µm pore size filters.
In vitro cytotoxic activity assay
Cell lines and culture medium
Cancer cell lines were kindly provided by Dr. P. Coursaget,
INSERM U618, University François Rabelais, Tours, France. Two
Human cervical cancer cell lines were used in this study, SiHa
harbouring the HPV16 and HeLa harbouring the HPV18. Cells were
grown as monolayers in Minimum Essential Medium (MEM)
supplemented with 10% heat-inactivated fetal calf serum and 1%
Penicillin-Spreptomycin mixture. Cultures were maintained at 37°C
in 5% CO2 and 100% relative humidity atmosphere.
Cytotoxicity assay
Cytotoxicity of sample on tumor cells was measured by microculture
tetrazolium (MTT) assay (Mosmann, 1983). For the assays, 96-well
microplates were seeded with 100 µl medium containing 10, 000
cells in suspension. After 24 h incubation and attachment, the cells
were treated with 6 fourfold dilution of crude extracts. Exactly from
the stock solution (40 mg/ml), each extract sample was applied in a
series of 6 dilutions (final concentrations ranging from 15.6 to 500
µg/ml) with a final DMSO concentration of 0.1% and was tested in
quadruplicate.
After 48 h incubation, cell viability was determined by adding
(Sigma) tetrazolim salt as cytotoxicity indicator and by reading
absorbance at 590 nm with a scanning multiwell spectrophotome-
ter. Tetrazolium salts are cleaved to formazan dye by cellular
enzymes (only in the viable cells). The level of absorbance directly
correlates to the metabolically active cells. Mitomycin C (~ 95 %
HPLC, sigma-Aldrich) was used as a positive control.
RESULTS AND DISCUSSION
Using the ethnomedical data approach, some Moroccan
plants that are used in the Moroccan traditional medicine
for various diseases, including cancer, were collected
and evaluated for their cytotoxic activities. The search for
new anti-cancer drugs is one of the most prominent
research areas of natural products. To investigate the
cytotoxic potential of 7 extracts from Morrocan plants
used in traditional medicine for the treatment of various
diseases such as cancer, inflammation or infectious
diseases. We collected a selection of seven plants in
order to screen them for possible cytotoxic activity
cytotoxic activity against cervical cancer cell lines.
The cytotoxic activity was evaluated on two human cer-
 Merghoub et al. 1047

Table 1. Ethnobotanical data and some reported pharmacological activities of plants species used in this study.

Plants species (Family) Trivial name Place of collection Part plant collected Traditional use pharmacological activities
Inflammatory effects
(Hernandez et al., 2007)
Skin diseases, treats cutaneous
abcesses, Antimicrobial activity
Inula viscosa L. Ait Magramane- (Maoz and Neeman, 1998)
Ain atik Temara Leaves wound healing,
(Asteraceae) Terhala Antifungal activity (Cafarchia et
Tuberculosis, bronchial infections
al., 2002)
(Bellakhdar, 1997)
Antitumoral activity
(Rozenblat et al., 2008)
Purgative, vermifuge,
Retama monosperma Sidi-Boughaba antihelmintic, abortive and
R’tm Leaves disinfectant No information available.
L. Bois (Fabaceae) Mahdia
(Benrahmoune, 2003)

Blood pressure, digestive Antimicrobial activity

disorders, anorexia, urinary (Li et al., 2007 ; Singh et al.,
Berberis hispanica system, nephritic, liver and 2007)
Bois and Reut. Argis - Azargnat Tamahdit Bark roots gastrointestinal disorders, ocular Antitumor activities
(Berberidaceae) affections, febrifuge. (Fukuda et al., 1999)
Antileishmania, antitumoral.
(Bellakhdar, 1997)

Stomachic, Antibactrial activities

Ormenis eriolepis Coss.
Hellala Ouarzazat Aerial part anthelmintic and antidiabetic Antileishmania activities
(Asteraceae) (Antifungic activity
(Bellakhdar, 1997)
anxiolytic, nervous breakdown,
Ormenis mixta Sidi-Boughaba
Hellala Aerial part hepatic and gastric insufficiencies Antimicrobial activity
(Asteraceae) Mahdia
(Haddad et al., 2003)
Rhamnus lycioides ssp. Oleoides
Sidi-Boughaba Hypotensive activity
(Rhamnaceae) S’afira El- harcha Leaves Laxative and diuretic
Mahdia Terencio et al., 1990)

Cardiac failures, whooping-cough,

pneumonia, abortive, vipers bites, Cytotoxic and antimalarial
and aphrodisiac activities (Sathiyamoorthy et al.,
Urginea maritima L. Baker Ansal-Baslet el Sidi-Boughaba Bulbs
1999)
(Lemnaceae) dib Mahdia Cough, bronchitis, the jaundice,
diuretic, and internal tumours Anti-insect activity (Pascual-
Villalobos and Fernandez, 1999)
(Bellakhdar, 1997)
1048 J. Med. Plant. Res.

Figure 1. Cytotoxic activity of methanolic extracts from 7 medicinal plants against SiHa cells (A) and Hela cells
(B).
Cells were incubated with different concentrations of the plant extracts (ranged from 15.6 to 500 µg/ml) for 48 h.
Cell viability was determined by the MTT assay (n=4).
Viability curves: Percentage viability = absorbance of test wells/absorbance of control wells) × 100) plotted
against the concentration of extract.

vical cancer cell lines, SiHa and HeLa, harbouring
respectively HPV 16 and 18, the high oncogenic human
papillomavirus (Pater and Pater, 1985).
The cytotoxic effect of 7 methanolic plant extracts on
SiHa and HeLa cell lines was determined using the MTT
assay. The MTT assays data are presented respectively
in Figures 1(A and B) and the corresponding IC50 are
summarized in Table 2.
Among the 7 medicinal plants tested methanolic
extracts from Inula viscosa (L.) Ait., Retama monosperma
(L.) Bois. and Ormenis eriolepis Coss. showed significant
growth inhibitory effects in both SiHa and HeLa cells
compared to the control. Their IC50 were 54, 99 and 94
µg/ml in SiHa cells respectively. In HeLa cells, the IC50 of
the same plant extracts were 60, 112 and 96 µg/ml
respectively. To be a good drug candidate, the IC50 value

of such agent should be sufficiently low to avoid any
possible unspecific effects. The American National
Cancer Institute assigns a significant cytotoxic effect of
promising anticancer product for future bioguided studies
if it exerts an IC50 value 30 µg/ml (Suffness and
Pezzuto, 1999). In this preliminary study, we have
focused our interest on crude plant extracts, the cytotoxic
activity could be due to the presence in the methanolic
extracts of active products that could probably have
highly anti-growth effects.
I. viscosa (L.) Ait. extract had the greatest activity with
lowest IC50 values. Several studies have been reported
on the phytochemical and other biological properties of
Inula viscosa (L.) Ait. It has been described to exhibit
several biological activities such as anti-inflammatory
(Hernandez et al., 2007), antimicrobial (Maoz and
Neeman, 1998) and antifungal effects (Cafarchia et al.,
2002). This plant is a source of a number of bioactives
compounds as well as flavonoids (Hernandez et al.,
2007) and sesquiterpene derivatives (Fontana et al.,
2007). Recently, Rozenblat et al. (2008) reported that
tomentosin and inuviscolide, a sesquiterpene lactones
isolated from I. viscosa (L.) Ait. were able to inhibit cell
growth of three different human melanoma cell lines.
R. monosperma (L.) Bois. and O. eriolepis Coss.
extracts showed also a significant growth inhibitory
effects in both SiHa and HeLa cells. Theses effects are
less marked than that obtained with I. viscosa (L.) Ait.
extract. These two plants were also reported in previous
studies. The Retama species have been reported to
contain alkaloids (Abdelhalim et al., 1997) and flavonoids
(Kassem et al., 2000). Fifteen quinolizidine and 3
dipiperidine alkaloids were isolated from the leaves of
flowering plants of R. monosperma subsp. Eumonosper-
ma collected from Morocco (Touati et al., 1996). On the
other hand, Retama genus has been described to contain
flavonoids, alkaloids and tannins. In addition, a variety of
plant flavonoids and alkaloids have also been shown to
be anti-carcinogenic in several animal models (Cassady
et al., 1990).
O. eriolepis Coss. has been described to have an
antibacterial, antileishmania and antifungic effects. To our
knowledge, no data relative to the chemical constituents
of O. eriolepis Coss. has being proposed. This plant has
not yet been assessed for in vitro cytotoxicity against
cancer cells.
The results obtained in this preliminary study indicate
that the methanolic extracts of the plants I. viscosa (L.)
Ait, R. monosperma (L.) Boiss and O. eriolepis Coss.
were shown to induce significant and dose-dependent
inhibitory activities against human cervical cancer cell
lines SiHa and HeLa. There remains interesting to eva-
luate the cytotoxic activity of selected plants in vitro on
other cancer cell lines.
This study provides an important basis for further
investigation into the isolation, characterization and
mechanism of cytotoxic compounds from the screened
Merghoub et al. 1049
medicinal plants. We also plan to carry more biological
activities, including the in vivo studies and the statute of
inhibition of HPV. Thus, these plants could be as a
source for new lead structures in drug design to combat
cancer.
Experiments were performed in quadruplicate (n = 4)
and data were expressed as means ± SDs. IC50 (inhibi-
tory concentration 50%) and SD (standard deviation for
95% confidence) were determined by interpolation from
the viability curves of SiHa and HeLa cells versus metha-
nolic plant extract concentrations. Mitomycin C was used
as a positive control.
Cells were incubated with different concentrations of
the plant extracts (ranged from 15.6 to 500 µg/ml) for 48
h. Cell viability was determined by the MTT assay (n = 4).
Viability curves: Percentage viability = absorbance of
test wells/absorbance of control wells) × 100) plotted
against the concentration of extract.
REFERENCES
Abdelhalim OB, Abdel Fattah H, Halim AF, Murakoshi I (1997).
Comparative chemical and biological studies of the alkaloid content
of Lygos species and varieties growing in Egypt. Acta Pharmaceutica
Hungarica 67: 241-247.
Abonyi DO, Adikwu MU, Esimone CO, Ibezim EC (2009). Plants as
sources of antiviral agents. Afr. J. Biotechnol. 8: 3989-3994.
Amrani M, Lalaoui K, El Mzibri M, Lazo P, Belabbas MA (2003).
Molecular detection of human papillomavirus in 594 uterine cervix
samples from Moroccan women (147 biopsies and 447 swabs). J.
Clin. virol. 27: 286-295.
Bellakhdar J (1997). La pharmacopée marocaine traditionnelle- Méde-
cine arabe ancienne et savoirs populairs, Paris, Editions Ibis Press.
Benrahmoune IZ (2003). Invitation à l’Amour des plantes– Réserve
biologique de Sidi-Boughaba ; Edition Scriptra pp. 114-228.
Bosch FX, de Sanjosé S (2003). Human papillomavirus and cervical
cancer-burden and assessment of causality. J. Natl. Cancer Inst.
Monogr. 31: 3-13.
Boyle P, Ferlay J (2005). Cancer incidence and mortality in Europe,
2004. Ann. Oncol. 16: 481-488.
Cafarchia C, De Laurentis N, Milillo MA, Losacco V, Puccini V (2002).
Antifungal activity of essential oils from leaves and flowers of Inula
viscosa (Asteraceae) by Apulian region. Parassitologia. 44: 153-156.
Cameron D, Bell R (2004). Optimizing treatment for patients with breast
cancer. Semin. Oncol. 31: 4–5.
Cardellina JH, Fuller RW, Gamble WR, Westergaard C, Boswell J,
Munro MHG, Currens M, Boyd MP (1999). Evolving strategies for the
selection dereplication and prioritization of antitumor and HIV-
inhibitory natural products extracts. In: Bohlin, L., Bruhn, J.G. (Eds),
Bioassay Methods in Natural Product Research and Development.
Kluwer Academic Publishers, Dordrecht pp. 25-36.
Cassady JM, Baird WM, Chang CJ (1990). Natural products as a
source of potential cancer chemotherapeutic and chemopreventive
agents. J. Nat. Prod. 53: 23-41.
Castellsagué X, Diaz M, de Sanjosé S, Munoz N, Herrero R, Franceschi
S, Peeling RW, Ashley R, Smith JS, Snijiders PJ, Meijer CJ, Bosch
FX (2006). Woldwide human papillomavirus etiology of cervical
adenocarcinoma and its cofactors: implications for screening and
prevention. J. Natl. Cancer Inst. 98: 303-315.
Craigo J, Callahan M, Huang RC, DeLucia AL (2000). Inhibition of
human papillomavirus type16 gene expression by nordihydro-
guaiaretic acid plant lignan derivatives. Antiviral Res. 47: 19-28
Deng Y, Feng Y, Sun J, Zhou D, Yang L, Lai J (2004). Study on anti-
HPV activity of Asarum heterotropoides. Zhong Yao Cai. 27: 665-
667.
Fontana G, La Rocca S, Passannanti S, Paternostro MP (2007). Ses-
1050 J. Med. Plant. Res.
quiterpene compounds from Inula viscose. Nat. Prod. Res. 20: 824-
827.
Fukuda K, Hibiya Y, Mutoh M, Koshiji M, Akao S, Fujiwara H (1999).
Inhibition by berberine of cyclooxygenase-2 transcriptional activity in
human colon cancer cells. J. Ethnopharmacol. 66 : 227-233.
Gonzalez-Tejero MR, Casares-Porcel M, Sanchez-Rojas CP, Ramiro-
Gutierrez JM, Molero-Mesa J, Pieroni A, Giusti ME, Censorii, E, de
Pasquale C, Della A, Paraskeva-hadijchambi D, Hadjichambis A,
Houmani Z, EL-Demerdash M, El-Zayat M, Hmamouchi M, Eljohriq S
(2008). Medicinal plants in the Mediterranean Area: synthesis of the
results of the project Rubia. J. Ethnopharmacol. 116: 341-357.
Haddad PS, Depot M, Settaf A, Chabli A, Cherrah Y (2003).
Comparative study on the medicinal plants most recommended by
traditional practitioners in Morocco and Canada. J. Herbs Spices
Med. Plants 10: 25-45.
Hernández V, Recio MC, Manez S, Giner RM, Rios J.L (2007). Effects
of naturally occurring dihydroflavonols from Inula viscosa on inflam-
mation and enzymes involved in the arachidonic acid metabolism.
Life Sci. 80 : 480–488.
Kassem M, Mosharrafa SA, Saleh NA, Abdel-Wahab S-M (2000). Two
new flavonoids from Retama raetam. Fitoterapia. 71: 649-654.
Li AR, Zhu Y, Li XN, Tian XJ (2007). Antimicrobial activity of four
species of Berberidaceae. Fitoterapia. Short report. 78 : 379–381.
Maoz M, Neeman I (1998). Antimicrobial effects of aqueous plant
extracts on the fungi Microsporum canis and Trichophyton rubum and
on three bacterial species. Lett. Appl. Microbiol. 26: 61-63.
Mosmann T (1983). Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J.
Immunol. Methods 16: 55-63.
Newman DJ, Gragg GM (2007). Natural Products as Sources of New
Drugs over the Last 25 Years. J. Nat. Prod. 70: 461-477.
Pascual-Villalobos MJ, Fernandez M (1999). Insecticidal activity of
ethanolic extracts of Urginea maritima (L.) Baker bulbs. Industrial
Crops and Products. 10: 115-120.
Pater MM, Pater A (1985). Human papillomavirus types 16 and 18
sequences in carcinoma cell lines of the cervix. Virol. 145: 313-318.
Pieters L, Vlietnick AJ (2005). Bioguided isolation of pharmacologically
active plant components, still a valuable strategy for the finding of
new lead compounds?. J. Ethnopharmacol. 100: 57-60.
Rozenblat S, Grossman S, Bergman M, Gottlieb H, Cohen Y, Dovrat S
(2008). Induction of G2/M arrest and apoptosis by sesquiterpene
lactones in human melanoma cell lines. Biochem. Pharmacol. 75:
369-382.
Sathiyamoorthy P, Lugasi-Evgi H, Schlesinger P, Kedar I, Gopas
J, Pollack Y, Golan-Goldhirsh A (1999). Screening for Cytotoxic and
Antimalarial Activities in Desert Plants of the Negev and Bedouin
Market Plant Products. Pharm. Biol (Formerly Int. J. Pharmacogn.).
37: 188-195.
Singh M, Srivastava S, Rawat AK (2007). Antimicrobial activities of
Indian Berberis species. Fitoterapia 78: 574-576.
Suffness M, Pezzuto J.M (1990). Assays related to cancer drug
discovery. In: Hostettmann, K. (Ed). Methods in Plant Biochemistry:
Assays for Bioactivity, vol. 6. Academic Press, London, 71-133.
Terencio MC, Sanz MJ, Paya M (1990). A hypotensive procyanidin-
glycoside from Rhamnus lycioides ssp. Lycioides. J. Ethnophar-
macol. 30: 205-214.
Touati D, Allain P, Pellecuer J, Fkih-Tétouani, S, Agoumi (1996). A
Alkaloids from Retama monosperma ssp. eumonosperma.
Fitoterapia. 67: 49-52.