Immunofluorescence Cellular microtubules in interphase or mitotic HeLa cells were visualized using indirect immunofluorescence techniques as previously described. LC/MS was done on a Waters Alliance 2695 HPLC element, 996 photodiode array detector, and Micromass Quattro triple quadrupole mass spectrometer equipped with ESI. The purities of all compounds Dabrafenib 1195768-06-9 were determined to be greater than 95-pound by NMR and LC/MS. The rhizomes and roots were collected from living plants and stored at 80 C until lyophilized. Pulverized and dried rhizomes of T. chantrieri were produced in several groups using supercritical CO2 with MeOH. The crude extracts were cleaned with hexanes and extracted with CH2Cl2. The extracts were put through silica gel flash chromatography and eluted with hexances:isopropanol to acquire the taccalonolide enriched fraction. This fraction was further purified on a silica-gel HPLC column and eluted with isooctane:isopropanol to produce fragments 1 8. E and taccalonolides A were obtained from fragments 2 Organism and 4 respectively. Fraction 1 was divided on a C 18 HPLC column, eluting with a gradient of acetonitrile:H2O from thirty days to 800-854 over 40 minutes, to yield 1. 2 mg of 0 and taccalonolide AA. 8 mg of taccalonolide T. Fraction 3 was purified on silica-gel flash column and eluted with CH2Cl2:acetone 85:15 to generate taccalonolide Page1=46. The roots and rhizomes of T. integrifolia were taken to yield 11. 7 grams of CH2Cl2 extract utilizing the same method as T. chantrieri. The extract was purified by silica gel flash chromatograph followed by repeated normal phase HPLC to provide 13. 1 mg of taccalonolide Z. Taccalonolide A was dissolved in 4 mL of methanol and to this solution 8 mL of 0. 05 M sodium bicarbonate was added. The perfect solution is was stirred at room temperature for 44 hours. The reaction solution was extracted with EtOAc and filtered on natural compound library HPLC to provide 25. 8 mg of taccalonolide T. Taccalonolides N and AB were made by hydrolysis of taccalonolides E and Z, respectively, using the same approach. Cell culture The HeLa cervical cancer cell line was obtained from American Type Tissue Culture Collection and developed in Basal Media Eagle medium supplemented with 50 ug/ ml gentamicin sulfate and one hundred thousand fetal bovine serum. Inhibition of cellular growth The effects of the taccalonolides were assessed using the SRB assay20 as previously described. 16 The concentration of drug that causes a 500-milligram inhibition of cellular proliferation was calculated from the linear part of the log dose response curve. Each IC50 value represents the mean and standard deviation of 3 5 impartial experiments, each performed in triplicate. Paclitaxel is roofed as a reference compound. The determination of IC50 values was performed on substance after NMR analysis and future lyophilization. Ethanol was used as the car for all cellular studies.