The n butanol fraction was subjected to Medium Pressure Liquid Chromatography hepatocellular carcinoma using 5% acetone for washes and 15% acetone for elution. The fraction obtained from the 15% acetone elution was subjected to a polyamide column using 15% ethanol to wash, then 25% ethanol to elute. The fraction obtained from the 25% ethanol elution was subjected to a Sephadex LH 20 column to yield Corilagin. The purity of Corilagin reached 98. 7%, which was confirmed by High Performance Liquid Chromatography. Cell proliferation assay Sulforhodamine B was used to detect the effect of drugs on the proliferation of ovarian cancer cell lines and OSE cells. Cancer cells and OSE cells were seeded in 96 well plates and incu bated with Corilagin starting the following day and continuing for 3 days.

After 72 hours, 50 ul of 30% trichloroacetic acid was added and incubated for 60 min at 4 C. After washing and drying the plate, 100 ul of 0. 4% SRB was added for 30 min. The plates were rinsed with 0. 1% acetic acid and air dried, after which 100 ul of Tris base was added, and the plates were shaken for 5 min. The SRB value was measured at a wavelength of 490 nm. The experiment was performed in quintuplicate and repeated three times. Cell cycle analysis SKOv3ip and Hey cells were seeded in 60 mm plates and incubated with Corilagin or DMSO as a control the next day. Control and treated cells were trypsinized at 24 or 48 hours after treatment, collected in PBS and fixed on ice, followed by washing with 70% cold ethanol. After treatment with 10 ug/ml RNase, cells were stained with 50 ug/ml propidium iodide for 15 min at room temperature in preparation for cell cycle analysis.

Stained cells were analyzed by flow cytometry. The cell cycle information was analyzed using ModFit3. 0 software. Apoptosis analysis Hey cells were seeded in a 60 mm dish and incubated with Corilagin or DMSO as a control. Control and treated cells were trypsinized at 24 and 48 hours, collected in PBS and stained with Annexin V and PI according to the manufacturers instructions for the Vybrant W Apoptosis Assay Kit. The stained cells were analyzed by flow cytometry. Reverse phase protein array analysis Untreated and Corilagin treated HO8910PM cells were used for RPPA analysis at The University of Texas, M. D. Anderson Cancer Center RPPA Core Facility.

We followed the methods described at the following web address for professionals/scientific resources/core facilities and services/functional proteomics rppa core/index. html. Western blot analysis SKOv3ip cells and Hey cells were seeded in 60 mm plates and incubated with Corilagin or DMSO, as a control, for 24, 48 or 72 hours. Cell lysates were harvested with lysis buffer. HO8910PM snail cells were Drug_discovery seeded in a 60 mm plate and treated with TGF B1 alone or in combination with Corilagin. DMSO was used as the control.