Professional Documents
Culture Documents
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 HPV infection and cervical cancer development. High risk
second most frequent cancer in women worldwide, HPV, such as HPV16 and HPV18, has been detected in
accounting for 15% of all cancer related deaths in women 94 - 100% of cervical precancerous lesions and cancer
(Boyle and Ferlay, 2005). Each year 470,000 women are (Castellsagué et al., 2006).
diagnosed with invasive cervical cancer worldwide, Though the cervical cancer therapy is in advance, side
230,000 women die of this disease and 80% of these effects due to the non-specific cytotoxicity of drugs and
occur in developing countries (Bosch and de Sanjosé, resistance to treatment represent a great problem in the
2003). In Morocco, cervical cancer is the second most cervical cancer management. Therefore, development
frequent female cancer after breast cancer and and search of novel and effective anticancer agents,
represents a major public health problem. The diagnosis which in addition should overcome resistance, have
is usually made in advanced stages and mortality is high become very important issues (Cameron and Bell, 2004).
(Amrani et al., 2003). Natural compounds have provided many effective
Human Papillomavirus (HPV) is considered as the anticancer agents in current use. Currently, over 50% of
etiologic agent of cervical cancer. Epidemiological and drugs used in clinical trials for anticancer activity were
biological studies have shown close relationship between 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
*Corresponding author. E-mail: elmzibri@cnesten.org.ma, substances have so far been isolated from higher plants,
mzibri@yahoo.com. Tel: +212.37.71.27.51, +212.66.83.59.11. algae and lichens (Abonyi et al., 2009).
Fax: +212.37.71.18.46. To our knowledge, few studies described the use of
1046 J. Med. Plant. Res.
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)
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 Among the 7 medicinal plants tested methanolic
respectively HPV 16 and 18, the high oncogenic human extracts from Inula viscosa (L.) Ait., Retama monosperma
papillomavirus (Pater and Pater, 1985). (L.) Bois. and Ormenis eriolepis Coss. showed significant
The cytotoxic effect of 7 methanolic plant extracts on growth inhibitory effects in both SiHa and HeLa cells
SiHa and HeLa cell lines was determined using the MTT compared to the control. Their IC50 were 54, 99 and 94
assay. The MTT assays data are presented respectively µg/ml in SiHa cells respectively. In HeLa cells, the IC50 of
in Figures 1(A and B) and the corresponding IC50 are the same plant extracts were 60, 112 and 96 µg/ml
summarized in Table 2. respectively. To be a good drug candidate, the IC50 value
Merghoub et al. 1049
of such agent should be sufficiently low to avoid any medicinal plants. We also plan to carry more biological
possible unspecific effects. The American National activities, including the in vivo studies and the statute of
Cancer Institute assigns a significant cytotoxic effect of inhibition of HPV. Thus, these plants could be as a
promising anticancer product for future bioguided studies source for new lead structures in drug design to combat
if it exerts an IC50 value 30 µg/ml (Suffness and cancer.
Pezzuto, 1999). In this preliminary study, we have Experiments were performed in quadruplicate (n = 4)
focused our interest on crude plant extracts, the cytotoxic and data were expressed as means ± SDs. IC50 (inhibi-
activity could be due to the presence in the methanolic tory concentration 50%) and SD (standard deviation for
extracts of active products that could probably have 95% confidence) were determined by interpolation from
highly anti-growth effects. the viability curves of SiHa and HeLa cells versus metha-
I. viscosa (L.) Ait. extract had the greatest activity with nolic plant extract concentrations. Mitomycin C was used
lowest IC50 values. Several studies have been reported as a positive control.
on the phytochemical and other biological properties of Cells were incubated with different concentrations of
Inula viscosa (L.) Ait. It has been described to exhibit the plant extracts (ranged from 15.6 to 500 µg/ml) for 48
several biological activities such as anti-inflammatory h. Cell viability was determined by the MTT assay (n = 4).
(Hernandez et al., 2007), antimicrobial (Maoz and Viability curves: Percentage viability = absorbance of
Neeman, 1998) and antifungal effects (Cafarchia et al., test wells/absorbance of control wells) × 100) plotted
2002). This plant is a source of a number of bioactives against the concentration of extract.
compounds as well as flavonoids (Hernandez et al.,
2007) and sesquiterpene derivatives (Fontana et al., REFERENCES
2007). Recently, Rozenblat et al. (2008) reported that
tomentosin and inuviscolide, a sesquiterpene lactones Abdelhalim OB, Abdel Fattah H, Halim AF, Murakoshi I (1997).
isolated from I. viscosa (L.) Ait. were able to inhibit cell Comparative chemical and biological studies of the alkaloid content
of Lygos species and varieties growing in Egypt. Acta Pharmaceutica
growth of three different human melanoma cell lines.
Hungarica 67: 241-247.
R. monosperma (L.) Bois. and O. eriolepis Coss. Abonyi DO, Adikwu MU, Esimone CO, Ibezim EC (2009). Plants as
extracts showed also a significant growth inhibitory sources of antiviral agents. Afr. J. Biotechnol. 8: 3989-3994.
effects in both SiHa and HeLa cells. Theses effects are Amrani M, Lalaoui K, El Mzibri M, Lazo P, Belabbas MA (2003).
Molecular detection of human papillomavirus in 594 uterine cervix
less marked than that obtained with I. viscosa (L.) Ait.
samples from Moroccan women (147 biopsies and 447 swabs). J.
extract. These two plants were also reported in previous Clin. virol. 27: 286-295.
studies. The Retama species have been reported to Bellakhdar J (1997). La pharmacopée marocaine traditionnelle- Méde-
contain alkaloids (Abdelhalim et al., 1997) and flavonoids cine arabe ancienne et savoirs populairs, Paris, Editions Ibis Press.
Benrahmoune IZ (2003). Invitation à l’Amour des plantes– Réserve
(Kassem et al., 2000). Fifteen quinolizidine and 3 biologique de Sidi-Boughaba ; Edition Scriptra pp. 114-228.
dipiperidine alkaloids were isolated from the leaves of Bosch FX, de Sanjosé S (2003). Human papillomavirus and cervical
flowering plants of R. monosperma subsp. Eumonosper- cancer-burden and assessment of causality. J. Natl. Cancer Inst.
ma collected from Morocco (Touati et al., 1996). On the Monogr. 31: 3-13.
Boyle P, Ferlay J (2005). Cancer incidence and mortality in Europe,
other hand, Retama genus has been described to contain
2004. Ann. Oncol. 16: 481-488.
flavonoids, alkaloids and tannins. In addition, a variety of Cafarchia C, De Laurentis N, Milillo MA, Losacco V, Puccini V (2002).
plant flavonoids and alkaloids have also been shown to Antifungal activity of essential oils from leaves and flowers of Inula
be anti-carcinogenic in several animal models (Cassady viscosa (Asteraceae) by Apulian region. Parassitologia. 44: 153-156.
Cameron D, Bell R (2004). Optimizing treatment for patients with breast
et al., 1990).
cancer. Semin. Oncol. 31: 4–5.
O. eriolepis Coss. has been described to have an Cardellina JH, Fuller RW, Gamble WR, Westergaard C, Boswell J,
antibacterial, antileishmania and antifungic effects. To our Munro MHG, Currens M, Boyd MP (1999). Evolving strategies for the
knowledge, no data relative to the chemical constituents selection dereplication and prioritization of antitumor and HIV-
inhibitory natural products extracts. In: Bohlin, L., Bruhn, J.G. (Eds),
of O. eriolepis Coss. has being proposed. This plant has
Bioassay Methods in Natural Product Research and Development.
not yet been assessed for in vitro cytotoxicity against Kluwer Academic Publishers, Dordrecht pp. 25-36.
cancer cells. Cassady JM, Baird WM, Chang CJ (1990). Natural products as a
The results obtained in this preliminary study indicate source of potential cancer chemotherapeutic and chemopreventive
agents. J. Nat. Prod. 53: 23-41.
that the methanolic extracts of the plants I. viscosa (L.)
Castellsagué X, Diaz M, de Sanjosé S, Munoz N, Herrero R, Franceschi
Ait, R. monosperma (L.) Boiss and O. eriolepis Coss. S, Peeling RW, Ashley R, Smith JS, Snijiders PJ, Meijer CJ, Bosch
were shown to induce significant and dose-dependent FX (2006). Woldwide human papillomavirus etiology of cervical
inhibitory activities against human cervical cancer cell adenocarcinoma and its cofactors: implications for screening and
prevention. J. Natl. Cancer Inst. 98: 303-315.
lines SiHa and HeLa. There remains interesting to eva-
Craigo J, Callahan M, Huang RC, DeLucia AL (2000). Inhibition of
luate the cytotoxic activity of selected plants in vitro on human papillomavirus type16 gene expression by nordihydro-
other cancer cell lines. guaiaretic acid plant lignan derivatives. Antiviral Res. 47: 19-28
This study provides an important basis for further 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-
investigation into the isolation, characterization and 667.
mechanism of cytotoxic compounds from the screened 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- Pater MM, Pater A (1985). Human papillomavirus types 16 and 18
827. sequences in carcinoma cell lines of the cervix. Virol. 145: 313-318.
Fukuda K, Hibiya Y, Mutoh M, Koshiji M, Akao S, Fujiwara H (1999). Pieters L, Vlietnick AJ (2005). Bioguided isolation of pharmacologically
Inhibition by berberine of cyclooxygenase-2 transcriptional activity in active plant components, still a valuable strategy for the finding of
human colon cancer cells. J. Ethnopharmacol. 66 : 227-233. new lead compounds?. J. Ethnopharmacol. 100: 57-60.
Gonzalez-Tejero MR, Casares-Porcel M, Sanchez-Rojas CP, Ramiro- Rozenblat S, Grossman S, Bergman M, Gottlieb H, Cohen Y, Dovrat S
Gutierrez JM, Molero-Mesa J, Pieroni A, Giusti ME, Censorii, E, de (2008). Induction of G2/M arrest and apoptosis by sesquiterpene
Pasquale C, Della A, Paraskeva-hadijchambi D, Hadjichambis A, lactones in human melanoma cell lines. Biochem. Pharmacol. 75:
Houmani Z, EL-Demerdash M, El-Zayat M, Hmamouchi M, Eljohriq S 369-382.
(2008). Medicinal plants in the Mediterranean Area: synthesis of the Sathiyamoorthy P, Lugasi-Evgi H, Schlesinger P, Kedar I, Gopas
results of the project Rubia. J. Ethnopharmacol. 116: 341-357. J, Pollack Y, Golan-Goldhirsh A (1999). Screening for Cytotoxic and
Haddad PS, Depot M, Settaf A, Chabli A, Cherrah Y (2003). Antimalarial Activities in Desert Plants of the Negev and Bedouin
Comparative study on the medicinal plants most recommended by Market Plant Products. Pharm. Biol (Formerly Int. J. Pharmacogn.).
traditional practitioners in Morocco and Canada. J. Herbs Spices 37: 188-195.
Med. Plants 10: 25-45. Singh M, Srivastava S, Rawat AK (2007). Antimicrobial activities of
Hernández V, Recio MC, Manez S, Giner RM, Rios J.L (2007). Effects Indian Berberis species. Fitoterapia 78: 574-576.
of naturally occurring dihydroflavonols from Inula viscosa on inflam- Suffness M, Pezzuto J.M (1990). Assays related to cancer drug
mation and enzymes involved in the arachidonic acid metabolism. discovery. In: Hostettmann, K. (Ed). Methods in Plant Biochemistry:
Life Sci. 80 : 480–488. Assays for Bioactivity, vol. 6. Academic Press, London, 71-133.
Kassem M, Mosharrafa SA, Saleh NA, Abdel-Wahab S-M (2000). Two Terencio MC, Sanz MJ, Paya M (1990). A hypotensive procyanidin-
new flavonoids from Retama raetam. Fitoterapia. 71: 649-654. glycoside from Rhamnus lycioides ssp. Lycioides. J. Ethnophar-
Li AR, Zhu Y, Li XN, Tian XJ (2007). Antimicrobial activity of four macol. 30: 205-214.
species of Berberidaceae. Fitoterapia. Short report. 78 : 379–381. Touati D, Allain P, Pellecuer J, Fkih-Tétouani, S, Agoumi (1996). A
Maoz M, Neeman I (1998). Antimicrobial effects of aqueous plant Alkaloids from Retama monosperma ssp. eumonosperma.
extracts on the fungi Microsporum canis and Trichophyton rubum and Fitoterapia. 67: 49-52.
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.