Bcr-abl ncentrated hydrochloric acid and extracted with an equal volume of ethyl acetate thrice. The ethyl acetate layers were combined, dried over anhydrous sodium sulfate and concentrated at 30C using a rotary flash evaporator. The oily yellow residue obtained after drying was then loaded on a silica gel column previously equilibrated with hexane and eluted successively with 200 ml of 100% hexane, 200 ml of linear gradient hexane : dichloromethane, 200 ml of 100% dichloromethane, 200 ml of linear gradient dichloromethane : ethyl acetate, 200 ml of 100% ethyl acetate and finally with 200 ml of 100% methanol. Two fractions were collected from each jak stat combination. Fifty two fractions of 100 ml were collected, evaporated and analysed to check the purity by TLC in hexane benzene, 5 : 5, hexane benzene, 25 : 75, benzene acetone, 2 : 8, benzene acetone, 3 : 7, benzene acetone, 4 : 6, benzene acetone, 5 : 5, benzene acetone, 7 : 3, chloroform methanol, 8 : 2 as mobile phase.
Components were visualized by viewing under UV254 366 nm and under iodine fumes. Based on TLC profile, fractions 50 and 51 52 were then combined, respectively, into sixteen STAT signaling pathway fractions and the antibacterial activity of these fractions were determined by well diffusion assay against B. subtilis, which was selected as initial test microorganism. The methanol fraction showed high antibacterial activity and was selected for further purification. About 28 g of methanol fraction was loaded onto a silica gel column and eluted successively with 100 ml of 100% chloroform, 100 ml of linear gradient chloroform acetone, 100 ml of 100% acetone, 100 ml of linear gradient acetone methanol and finally with 100 ml of 100% methanol. The collected fractions were tested for antibacterial activity against B. subtilis and analysed using TLC in chloroform methanol, 8 : 2. Active fractions giving singlespot in TLC and single peak in HPLC 1 were purified by crystallization using appropriate solvents. Spectroscopic measurements UV spectrophotometer. UV visible spectrum of the pure daunorubicin compounds was recorded on a Systronics double beam spectrophotometer 2201, India, at room temperature.
Fourier transform infrared spectroscopy. FTIR spectrum of the isolated compounds was recorded on a Perkin Elmer FTIR Spectrometer at room temperature 1. Liquid chromatography mass spectrophotometer. LC MS was recorded using Applied Biosystems API 4000 QTrap manufactured by ABSCIEX. Mass spectra were recorded by electro spray ionization technique using photomultiplier detector with a flow rate of 01 ml min1 on C 18 column. The mobile phase was methanol, and the total run time was 15 min. Diode array was used as a detector. Nuclear magnetic resonance. NMR spectra were recorded on a Bruker DRX 500 NMR instrument operating at 500 MHz for 1H and 125 MHz for 13C at room temperature, and chemical shifts were recorded as ppm values and coupling constants, J, in Hertz. Signals were referred to internal standard tetramethylsilane. Differential scanning calorimetry. The melting point of the pure compounds was measured with a differential scanning calorimeter in a Mettler Toledo DSC 822e instrument. Temperature ranges from 30 to 300C were employed. Determination of antibacterial activity Agar well diffusion method. In vitro antibacterial.