cancer cells were treated with 100 g/ml of PRE, EA-fraction and Hex-fraction of the methanol extract of the roots of P. fulgens. In case of mixture of three compounds which were isolated from the EA-fraction i.e MedChemExpress INK1117 epicatechin, ursolic acid and gallic acid, each was taken 10 g/ml. These three compounds were taken from three different EA-subfractions and their selection was based on their higher yield. In all these experiments the cells were exposed for 24h. Cell killing ability by PRE and EA-fraction to normal and cancer cells This was determined by Trypan blue exclusion assay. Freshly collected human peripheral blood lymphocytes was used as normal cells and MCF-7 and U87 cells were used as cancer cells. Heparinized peripheral blood from two healthy male donors was obtained after having their written consent for participation and used soon after its collection. Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation for 30 min at 400g from these freshly drawn heparinized whole blood. Lymphocytes cultures were set up in RPMI 1640 medium supplemented with 10% heat inactivated FCS. Penicillin and streptomycin and 2 mM L-Glutamine were added to the medium. Lymphocytes were stimulated with PHA and after 24 h lymphocytes were treated with PRE and EA-fraction for 24 h. These cultures were incubated at 37 C and were harvested soon after the treatment. This study was 
approved by the Institutional and Human Ethics Committee. In case of cancer cells, MCF-7 and U87 cells were used and treated with PRE and EA-fraction for 24 h. Cells were washed with fresh medium and then incubated for 10 min at room temperature with 0.4% final trypan blue. Dead cells were stained blue, and live ones were unstained. Experiments were repeated three times. Cell killing ability by PRE and EA-fraction was also evaluated by flow-cytometric analysis. Effect on cell proliferation in MCF-7 and U87 cells The cellular proliferation assay was performed in MCF-7 and U87 cells after treating the cells with PRE, EA and Hex-fractions. Cells at a density of 5 x 105 were grown in 25 cm2 flasks in DMEM medium at 37C and 5% CO2. After 24 h of seeding, the cells were exposed to PRE, EA or Hex-fraction at a final concentration of 100 g/ml for 24 h and finally fixed in 70% ethanol. All the above fixed cells were washed in PBS and resuspended in 500 l of propidium iodide solution for 1 h incubation at room temperature in the dark. 10,000 cells were acquired for each sample and analyzed them in a FACS Calibur using Cellquest software in order to quantify cell cycle compartments to estimate the percentage of cells distributed in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19730234 the different cell cycle phases. Flow cytometric analysis of mitochondrial membrane potential The dissipation of the mitochondrial membrane potential is considered to be a mitochondrial disruption event before apoptosis. It is caused PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19729642 by a sudden increase in the inner mitochondrial 4 / 16 Anticarcinogenic Potential of Potentilla fulgens Roots membrane permeability, known as the mitochondrial permeability transition. Mitochondrial damage was evaluated using the lipophilic fluorescent probe 5,50,6,60 -tetrachloro-1,10,3,30 tetra-ethyl-benzimidazol-carbocyanine iodide according to the manufacturer’s protocol. Apoptotic cell death was evaluated in MCF-7 and U87 cells treated with and without PRE, EA-fraction, Hex-fraction and mixture of EC, UA and GA using JC-1 stain. In brief, JC-1 working solution was prepared in 1xAssay buffer
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