as used. Briefly, U937 cells after being treated with a near IC50 concentration of MAL-A were washed with PBS, labeled with NAO, acquired and analyzed 
in a flow cytometer. Measurement of Annexin V positivity Double staining for Annexin V-FITC and propidium iodide was performed as previously described. Translocation of phosphatidylserine from the inner aspect to the outer leaflet of the plasma membrane occurs during apoptosis which can be ascertained by exploiting the high binding affinity of Annexin V, a Ca+2 14937-32-7 web dependent phospholipid binding protein to phosphatidyl serine. To examine whether cell death occurred via apoptosis or necrosis, PI was used, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189963 which being a non permeable stain having affinity towards nucleic acids, selectively enters necrotic or late apoptotic cells. Therefore, co-staining of Annexin V and PI helps discriminate between live cells, cells in early apoptosis, cells In situ detection of DNA fragmentation by terminal deoxynucleotidyl Transferase mediated dUTP nick end labeling In situ detection of DNA fragments was measured by terminal deoxyribonucleotide transferase mediated dUTP nick end labeling according to the manufacturer’s instructions. Briefly, U937 cells were treated with a near IC50 concentration of MAL-A for 24 and 48 h at 37uC; cells were washed, fixed with paraformaldehyde and after being kept on ice for 1 h, cells were centrifuged MAL-A Causes ROS Induced Apoptosis , resuspended in PBS, and spotted on glass slides. The air dried slides were washed with PBS, placed on ice and permeabilized with freshly prepared, chilled Na-Citrate in Triton X-100 solution for 2 minutes. Cells were washed twice with PBS following which a reaction mixture containing enzyme and nucleotide mixture was added. The slides were then incubated in a humidified chamber at 37uC for 1 h, washed with PBS and convertor POD was added and incubated for 30 minutes at 37uC. Finally, the substrate diaminobenzidine was added, slides were kept at 4uC for 10 minutes, washed with deionised water and observed microscopically under oil immersion objective. At least 20 randomly selected microscopic fields were examined. Images were taken using a digital compact camera with a 46 zoom and modified using Adobe Photoshop 7.0. using either histogram or quadrant plot with CellQuest Pro software. Statistical Analysis Each experiment was performed at least thrice in duplicates and results expressed as mean 6 SEM/SD. Statistical analysis was evaluated by Students t-test and one way ANOVA followed by Tukey multiple comparison test, using Graph Pad Prism software, version 4; p,0.05 was considered as statistically significant. Results Cytotoxicity of Rampatri and its derived compounds Diarylnonanoids isolated from a methanolic extract of the fruit rind of Myristica malabarica namely malabaricone-A, malabaricone-B, malabaricone-C and malabaricone-D, MAL-D were tested for their cytotoxic potential in leukemic cell lines, U937 and MOLT-3. The crude extract and the malabaricones, except MAL-C, showed an IC50 ranging from 12.760.46 to 24.560.44 mg/ml in U937 and 12.361.67 to 20.461.72 mg/ml in MOLT-3 cell lines; DMSO, representative of the highest concentration present in malabaricones showed no effect on cell viability, confirming its biological inertness. As MAL-A and MAL-D were comparable as regards their cytotoxicity, we proceeded to study MAL-A as a representative compound. Measurement of DNA laddering To determine DNA laddering, total cellular DNA was isolated
