Antiproliferative activity of selected medicinal plants of Jordan against a breast adenocarcinoma cell line ( MCF 7 )

76 ethanolic extracts of medicinal herbs from the Jordanian flora, belonging to 67 species and 34 families, were evaluated for their antiproliferative activity on a breast cancer cell line (MCF7). The cells were cultured in RPMI 1640 medium and incubated with the extracts for 72 hours. Sulphorhodamine B (SRB) assay was used to test cytotoxicity. From the tested crude extracts, Inula graveolens, Salvia dominica, Conyza canadiensis and Achillea santolina showed potent antiproliferative activity and the activity resided in the chloroform/ethanolic extracts. The most active plant was I. graveolens with an IC50 of 3.83 g/ml. Phytochemical screening indicated the presence of flavonoids, terpenoids, and phenolics in all active extracts. These results indicate the possible potential use of medicinal plants from the Jordanian flora as antineoplastic agents.


Introduction
Since medieval times, plants have been the source of medicines for the treatment of diseases.Regardless of the availability of a wealth of synthetic drugs, plants remain -even in the 21 st century -an integral part of the health care in different countries, especially the developing ones.In the late 90's, the WHO stated that a big percentage of the world's population depends on plant based therapies to cover the needs of the primary health care (WHO 1999) [1].Moreover, towards the end of the 20 th century, plant based OTC products, nutroceuticals and food supplements comprising the complementary and alternative therapies have gained a big share in the drug market in the developed countries.
Medicinal plants -either through systematic screening programs or by serendipity -possess an important position in the drug discovery and many modern drugs have their origin in traditional medicine of different cultures.Hence, despite the advantages of the synthetic and combinatorial chemistry as well as molecular modeling, medicinal plants remain an important source of new drugs, new drug leads and new chemical entities [2,3].The latter study reported that of the 877 small molecule new chemical entities (NCEs) introduced between 1981 and 2002 nearly the half (49%) were natural products, semi-synthetic natural products, semi-synthetic natural products analogues or synthetic compounds based on natural products.
The areas of cancer and infectious diseases have a leading position in utilization of medicinal plants as a source of drug discovery.Among FDA approved anticancer and anti-infectious preparations drugs of natural origin have a share of 60% and 75% respectively [3].It is worthy to mention the vivid current interest in discovery of natural drugs for cancer treatment and chemoprevention [4,5].Huge number of plant species is screened and bioassayed for this purpose worldwide [6][7][8][9][10][11][12][13][14][15][16][17].
In many countries, cancer is the second leading cause of death after heart diseases [15,18].The estimated worldwide incidence of different carcinomas is about 10 million; half of these are in developed countries [13].Among the cancer patients in the USA, the use of complementary and alternative medicine, represented mainly by plants, ranges between 30-75% [6].This in turn justifies the interest in search of possible anticancer agents from the flora of different countries.
In accordance with this worldwide trend, the current study was undertaken to screen the ethanolic extracts of 67 plant species found in the Jordanian flora or sold by the local herbalist shops.Among the screened plants there are only few plants recommended by the traditional healers for the treatment of cancer (i.e Arum palestinum) while some tested plants are belonging to the genera with reported anticancer activities (i.e.Salvia dominica).

Plant extracts' preparation
Plant samples collected during spring/summer 2005 or purchased in the same period were dried at room temperature and finely ground with a hammer mill.Each 2.5 g powdered plant material was extracted by refluxing with 25 ml ethanol for 30 min and kept overnight at room temperature before filtration.After filtration, ethanol was evaporated until dryness and the crude extracts were weighed.0.1 g of the crude extract was dissolved in dimethyl sulphoxide (DMSO) to a final stock concentration of 10 mg/ml.All extracts were kept at 20 °C until cytotoxicity tests were carried out.

Phytochemical screening
Phytochemical screening using thin layer chromatography (TLC) was carried out only for plant species (Achillea santolina, Conyza canadiensis, Inula graveolens and Salvia dominica) indicating promising anticancer activity using MCF7 cell line.The ethanolic extracts were subjected to TLC examination for group determination of the secondary metabolites.Modified Dragendroff's reagent for alkaloids, ferric chloride reagent for phenolics, Naturstoff reagent for flavonoids, ethanolic KOH for coumarins and vanilline/sulfuric acid reagent for terpenoids were used.Solvent systems for the development of ready coated analytical TLC plates (Merck) were selected according to Wagner and Bladt [19].

Cell culture
The cell line under investigation was human breast adenocarcinoma (MCF7).It was purchased from the European Collection of Animal Cell Culture (ECACC No. 86012803).The cells were cultured in RPMI 1640 medium supplement with 10% heated foetal bovine serum, 1% of 2 mM l-glutamine, 50 IU/ml penicillin and 50 g/ml streptomycin.
According to the cells growth profile, cells were seeded with a density of 5000 cell/well.This number was sufficient to give a reliable reading with the SRB assay, which corresponded well with the cell number and was the one that gave exponential growth throughout the incubation period with the plant extracts.

Cytotoxicity assay
For the assay, cells were washed three times with phosphate buffer saline (PBS).
PBS was decanted and cells detached with 0.025% trypsin-EDTA (Sigma).RPMI 1640 was added to a volume of 10 ml.The cell suspension was centrifuged at 1000 X g for 10 minutes and the pellet was resuspended in 10 ml of medium to make a single cell suspension.Viability of the cells was determined by trypan blue exclusion and it exceeded 90% as counted in a haemocytometer.The cell suspension was diluted afterwards to give the optimal seeding density and 100 l of the cell suspension was plated in a 96 well plate and incubated at 37 °C in a humidified atmosphere containing 5% CO2.After 24 hours the cells were treated with the extracts or pure compounds.
Each extract (initially dissolved in DMSO), was diluted with the medium and passed through a 0.2 m filter.50 g/ml of each extract was tested initially, and, from the results, the active extracts were considered to be those which gave less than 50% survival at exposure time 72 hours.The active extracts were further diluted in medium to produce eight concentrations (0.1, 0.5, 1, 5, 10, 25, 50, 100 g/ml) of each extract.100 l/well of each concentration was added to the plates in six replicates.The final dilution used for treating the cells contained not more than 1% of the initial solvent, this concentration being used in the solvent control wells.The plates were incubated for 72 hours.At the end of the exposure time, cell growth was analyzed using the SRB assay.Two replicate plates were used to determine the cytotoxicity of each extract.

Sulphorhodamine B assay
After incubation for 72 hours, adherent cell cultures were fixed in situ by adding 50 l of cold 40% (w/v) trichloroacetic acid (TCA) and incubated for 60 min at 4 °C.
The supernatant was then discarded and the plates were washed five times with demonized water and dried.50 l of SRB solution (0.4% w/v in 1% acetic acid) was added to each well and incubated for 30 min at room temperature.Unbound SRB was removed by washing five times with 1% acetic acid.Then, the plates were air-dried and 100 l of 10 mM Tris base pH 10.5 (Sigma) were added to each well to solubilize the dye.The plates were shaken gently for 20 minutes on a plate shaker and the absorbance (OD) of each well was read on an ELISA reader at 570 nm.Cell survival was measured as the percentage absorbance compared to that of the control (nontreated cells).

Results and Discussion
In the present study, the cytotoxic effect of 76 ethanolic plant extracts that belong to 34 families on MCF7 cells were characterized by conducting cell viability assay stained with sulphorohdamine B. Cultures of MCF7 cells were treated with the extracts first at one concentration of 50 g/ml and the results are shown in Table 1.Control assays were carried out for samples containing only the appropriate volumes of blank solutions and those showed no effect on cell growth.
For plant extracts that showed less than 50% survival rate, further dilutions were made to calculate the exact IC 50 values (Figure 1 and Table 2).For the most potent extracts, their antiproliferative activity was studied again using chloforom and water extracts (Table 3).
In the US NCI plant screening program, a crude extract is generally considered to have in vitro cytotoxic activity if the IC 50 value (concentration that causes a 50% cell kill) in carcinoma cells, following incubation between 48 and 72 hours, is less than 20 g/ml, while it is less than 4 g/ml for pure compounds [20].Tab. 1. Percentage cell survival of MCF7 cells following 72 hours exposure to 50 g/ml fractions from ethanolic plant extracts.L: leaves, S: seeds, St: stems, F: flowers, Fr: fruits, R: Roots, H: herb, *: fresh plant used.
The data presented in Table 1 show that the ethanolic extracts of five plants that belong to the families of Compositae and Labiatae exhibited high cytotoxic activity.
Calculation of the IC 50 values for these extracts confirmed that the most potent plant extract was I. graveolens (IC 50 3.83 g/ml) followed by S. dominica (IC 50 7.28 g/ml), C. canadiensis (IC 50 12.76 g/ml) and A. santolina (IC 50 24.12g/ml).Among these five plant species the least active was T. leucocladum (IC 50 39.242g/ml).The cytotoxic activity was further studied using chloroform and water extracts of the potent plants and for these, the activity was mainly in the chloroform and/or ethanolic extracts (Table 3).

Tab. 4. Phytochemical screening of plant extracts with high cytotoxic activity
Cytotoxicity screening models provide important preliminary data to help selecting plant extracts with potential antineoplastic properties for future work [21].Sulforhodamine B assay is a well-established in vitro method for cytotoxicity against cancer cell lines and non-cancer cell lines, and here it was utilized to determine the selective activity of the extracts [15].
Several plant species rich in flavonoids are reported having disease preventive and therapeutic properties.This observation is of particular importance since flavonoids are ingredients of many vegetables and fruits and the association of vegetable and fruit consumption with reduced cancer risk has been reported [22,23].
Antiproliferative activity recorded in the present study is in accordance with this finding, since the phytochemical evaluation indicated the presence of flavonoids in all of the four plant species with promising activity.Among these active plant species the genus

Fig. 1 .
Fig. 1.Growth inhibition of MCF7 cell line by active plant extracts.Results present the average and standard deviation of 6 replicates.

Table 2
The results of the phytochemical screening of the plant species with potent anticancer activity are given in Table4.Flavonoids, terpenoids and phenolics were identified in all tested plants while the presence of alkaloids could be weakly detected only in one species (A.santolina).