Coleon A lactone was isolated from leaves of Plectranthus barbatus collected in Handeni, Tanga Region, Tanzania. Leaves were dried at ambient temperature under sunlight, homogenized, and extracted 3 times with chloroform . This chloroform extract was concentrated 100:1 on a rotary evaporator and subjected to chromatographic separation on a LaFlash chromatography apparatus from VWR using VWR SuperVarioFlash silica cartridges , with chloroform:acetic acid 200:1 as the solvent. Coleon AL was isolated as the second major peak showing absorbance at 254 nm. Thin layer chromatography TLC plates were obtained from Macherey Nagel . For the experiments described here, 20620 cm aluminum plates coated with TLC silica gel 60 containing a UV254 fluorescence indicator were used . Plates were loaded manually, using a finely tapered micropipette tip, with 10 mg of crude extract , dried for 15 seconds with a hair dryer at low heat, and placed in an enclosed, upright 25625610 cm glass chamber containing 100 ml toluene ethyl formate formic acid 5:4:1 .
High resolution electrospray ionization mass spectrometry Electrospray ionization mass spectra were recorded in positive and negativemode on an orthogonal acceleration quadrupole time offlight mass spectrometer . The electrospray needle voltage was set to 3000 V or 22850 Vandetanib selleck chemicals V for the positive and negative mode respectively. Fragment ion spectra were obtained by selecting the precursor ion in the quadrupole and collisional activation with argon gas in the collision cell. Accurate mass measurements were performed at a resolution of 9000 using the protonated leucine enkephaline ion as lock mass. NMR spectroscopy 1H and 13C NMR spectra were recorded on a Bruker Avance II 500 spectrometer operating at 500.130 MHz for 1H and at 125.758 MHz for 13C, and using a gradient equipped inverse 5 mmtriple probe with p 2 pulses of 6.5, and 14.5 ms respectively. The standard Bruker Topspin 2.1 software under Windows XP was used throughout. All experiments were performed at 22 uC in deuterochloroform solution with the solvent peak as internal standard set at 7.
27 ppm or 77.0 vs.TMS respectively. First order analysis was applied throughout, and firstorder multiplets or apparent first order multiplets were denoted as follows: s singlet, screening compounds selleck chemicals d doublet, dd double doublet, t triplet. J values were extracted directly from the splittings in the spectrum, and are not optimised. Spectral assignments were based not only on the usual chemical shift rules and coupling patterns, but especially on routine 2D correlations such as COSY45 , GHSQC and GHMBC experiments . The data for coleon AL are summarized in Fig. 4 and compared with previously reported values . Imaging Zebrafish were screened for GFP fluorescence using an Axiovert 40 CFL microscope from Zeiss equipped with an MBQ 52 AC fluorescence lamp from LEJ .