Reagents and cell lines
The solvent used for plant extraction (95% ethanol) was commercial grade and obtained from Labscan Asia Co. Ltd. (Bangkok, Thailand). The cell culture reagents including RPMI 1640, DMEM, fetal bovine serum (FBS), phosphate buffered saline (PBS), penicillin and streptomycin antibiotics were obtained from Gibco Life Technologies (NY, USA). CCA cell line (CL-6) used for both in vitro and in vivo study was kindly supplied by Professor Kesara Na-Bangchang, Chullabhorn International College of Medicine, Bangkok, Thailand. Methyl thiazol diphenyl tetrazolium (MTT) reagent was obtained from Life Technologies (CA, USA) and dimethyl sulfoxide (DMSO) was obtained from MP Biomedicals (CA, USA). 5-FU was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and tween-80 was obtained from Sigma-Aldrich (MO, USA).
Plant material collection and crude extract preparation
The dried leaves of the plant E.camaldulensis was obtained from the study areas (Northwest Ethiopia) in consultation with the traditional herbalists. Botanical identification and authentication of the plant material was carried out at the National herbarium, Addis Ababa University, Department of Plant Biology and Biodiversity, where voucher specimen was deposited with voucher number 0012 (E. camaldulensis). The plant leaves were collected from the study areas (Western Gojjam and Southern Gondar), Ethiopia, by informed personnel for collection along with the two investigator close supervision to obtain ample amount of leaves from several trees of the specific plant. Then the leaves were washed under running tap water to remove adhering dust, air dried under shade for7 days and then powdered using a grinding mill. Extraction techniques including sonication, heating under reflux and maceration (1 day, 3 days and 7 days) were conducted by using the solvent 95% ethanol and the plant powder. An appropriate solvent (300ml) was mixed with a sample powder of about 10gm and sonicated with the ultrasonicator (Elma™, Meditop) for about 75 minutes. The plant rhizome of about 10gm in 300ml of ethanol was also used for heating under reflux by using water bath (Memmert, Germany) for about 50 minutes according to the previous methods with modification [12]. For maceration technique, about 10gm of the powdered leaves of E.camaldulensis were also mixed with in 300ml of ethanol with small flasks and allowed to wait for 1 day, 3 days and 7 days. Later on, about 1,000gm powdered plant material (rhizome) was added in each three large clean flask of containing 5 liters of 95% ethanol and allowed to wait for 7 days at 25–30°C by occasional shaking and stirring. The extracts were separated first (filtered) with gauze and then through Whatman no. 1 filter paper (GE HealthcareR, England). The residues were re-macerated for another 24 h and then filtered as indicated above, followed by extract evaporation to remove the solvent by using a rotary evaporator (Heidolph, Germany) and then further concentration by water bath. The crude extract obtained by using the different methods was weighed for percentage yield calculations and stored at -20°C until used (as shown below) to ensure the apparent stability and activity of the extract according to the previous methods [12, 13].
In vitro cytotoxicity evaluation of E. camaldulensis leaves extract
The crude ethanolic extract from E. camaldulensis was initially dissolved in 50% ethanol. The concentrated stock solution of about 5mgextract was prepared by adding in a known volume (500 µl) of solvent (50% ethanol) to prepare the initial highest concentration (500 µg/ml) of extract preparation in fresh medium, and then serially diluted (1:2) with the media to obtain the working solutions at eight final concentrations (i.e.500, 250, 125, 62.5, 31.25, 15.6, 7.8, and 3.9 µg/ml) according to the previous methods used [14]. Positive control agent 5-FU was also prepared as a stock solution by dissolving in 50% ethanol (500 µl) and then serially diluted with fresh medium. The concentration of 5-FU to be used also started from 500 µg/ml in fresh medium as an initial highest concentration and then serial dilutions of it was prepared as 250, 125, 62.5, 31.25, 15.6, 7.8, and 3.9 µg/ml) according to according to the previous methods used with modification [14, 15].
The cholangiocarcinoma cell line (CL-6) was used for cytotoxic screening of E. camalduensis leaf extract. CL-6 cell line was cultured in T25 and T75 cm3 culture flasks (Corning Inc., NY, USA) with RPMI 1640 medium (Gibco Life Technologies, NY, USA) supplemented with 10% heated fetal bovine serum (FBS) and 100 IU/ml of penicillin-streptomycin solution. The cell cultures were maintained at 37°C in a 5% CO2 atmosphere with 95% humidity according to the previous methods used [14]. When the optimum confluency of cultured cells was reached (90%), cells were removed from the culture flask by trypsinization, collected in a 15ml conical tube, centrifuged at 100 × g for 5 min at 25 oC, the supernatant was removed and cells were resuspended in 2 ml of complete media and the cell number was counted using a hemocytometer chamber.
The MTT colorimetric assay with some modification was used to screen for cytotoxic activity of the plant extract and standard drug preparations on cultured CL-6 cells in 96-well plates (Corning Inc., NY,USA). The cells were seeded at a density of 104 cells/well in 100 µl culture medium and after 24-h incubation and attachment, the cells were treated with different concentrations (serial dilutions) of the plant extract and 5-FU prepared (as mentioned above). After 48h incubation, MTT solution (20 µl of 5 mg/ml in PBS) was added and then incubated at 37°C for 3 h, followed by removal of the media and then cells were lyzed with dimethyl sulfoxide (DMSO). Absorbance (OD) was measured at 570 nm using a Varioscan™ flash microplate reader machine (Thermoschientific, MA,USA) and finally the percentage of cytotoxicity compared to the untreated cells was determined with the equation according to the previous methods [16] .
Where, A is absorbance (OD) of control cells and B is absorbance of treated cells.
The results were evaluated from three independent experiments (triplicates) and CalcusynRversion 1.1 software (Biosoft, Cambridge, UK) was used to calculate the IC50 value (concentration that inhibits cell growth by 50%). Similarly, another cultured normal fibroblast cell lines (OUMS) were cultured in Dulbecco's modified eagle's medium (DMEM) and treated with the E. camaldulensis extract, and 5-FU for MTT cell proliferation assay and estimation of selectivity index [14, 15]. Selectivity index was calculated from IC50 ratio of normal fibroblast cell lines (OUMS) and CCA cell lines (CL-6).