ty. These fractions were pooled and further purified with silica column chromatography packed with silica gel 60 F254, 0.25 mm thickness . Elution began with 100% hexane and polarity of eluting solvent was gradually increased using Me2CO and MeOH. Eluents of 25 ml volume were collected in numbered vials. The separation of each eluent was monitored on pre-coated thin layer chromatography silica gel 60 F254 plate using n-hexane/acetone as developing solvent and the TLC zones were detected after spraying with p-anisaldehyde reagent and heating at 105uC for 5 10 min. The eluents were pooled according to the similarity of the chemical composition detected on TLC and the excess solvent was evaporated under reduced pressure using a rotary evaporator to yield a total of 8 sub-fractions designated as PPWH-1, PPWH2,…..,PPWH-8. The sub-fractions were subjected to Neutral Red Uptake assay as described later. The solvent system profile and the yield of each fractions and sub-fractions obtained are summarized in LCMS-MS Analysis The most active sub-fraction, PPWH-7 was analyzed by LCMS/MS system equipped with the 3200 QTrap mass spectrometer and Shidmazu UHPLC system. The chromatographic separation was performed on a 50 mm 62.0 mm 65 mM Aqua C18 column, eluted with a mobile phase consisting of water and acetonitrile containing 0.2% formic acid and 2 mM ammonium formate. A gradient elution was used to separate the compounds of interest prior to mass spectral analysis. The mass spectrometer analysis was performed in a positive ion mode for detection of secondary compounds. Identities of the compounds were obtained by matching their molecular ions obtained by LC-MS/MS with reference 3 Apoptosis Induction of Phyllanthus watsonii Fractions/Subfractions a Solvent system for CC hexane 100% hexane/Me2CO hexane/Me2CO hexane/Me2CO hexane/Me2CO hexane/Me2CO hexane/Me2CO hexane/Me2CO; hexane/ Me2CO hexane/Me2CO; Me2CO/ MeOH Me2CO/MeOH; Me2CO/ MeOH hexane 100% hexane/Me2CO hexane/Me2CO hexane/Me2CO hexane/Me2CO; hexane/ Me2CO hexane/Me2CO hexane/Me2CO hexane/Me2CO; hexane/ Me2CO Ratio 100 40: 60 40: 60 40: 60 40: PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189787 60 40: 60 40: 60 Yield 5.49 4.67 60.64 5.13 0.30 3.10 0.83 PWH-1 PWH-2 PWH-3 PWH-4 PWH-5 PWH-6 PWH-7 PWH-8 PWH-9 PWH-10 b 40: 60; 50: 50 0.16 50: 50; 40: 60 8.83 40: 60; 90: 10 41.71 100 90: 10 90: 10 90: 10 10.26 14.56 3.34 2.79 PPWH-1 PPWH-2 PPWH-3 PPWH-4 PPWH-5 PPWH-6 PPWH-7 PPWH-8 90: 10; 60: 40 0.91 60: 40 50: 50 20.71 10.61 50: 50; 40: 60 1.27 PWH-1, PWH-2,…….,PWH-10: fractions of PW-H of P. watsonii. PPWH-1, PPWH-2,….,PPWH-8: sub-fractions from pooled fractions. doi:10.1371/journal.pone.0034793.t001 b a at a concentration of 30,000 cells/mL and placed in a CO2 incubator at 37uC to allow the cells to adhere before addition of the the extracts, fractions and sub-fractions of P. watsonii. After 3 hours, cells were treated 
with extracts and fractions of P. watsonii at six different concentrations i.e., 1, 10, 25, 50, 75 and 100 mg/mL and incubated for 72 hours. Wells containing untreated cells were regarded as a negative control, whereas cells treated with doxorubicin PAK4-IN-1 site served as a positive control. At the end of the incubation period, the medium was replaced with medium containing 50 mg/mL Neutral Red solution and incubated for further 3 hours to allow for uptake of the vital dye into the lysosomes of viable and uninjured cells. The medium was then removed and cells were rapidly washed with the calcium chlorideformaldehyde mixture. The dye with