Upon observing the results of TLC, 1 g dried chloroform non-small-cell lung carcinoma fraction of the petroleum ether extract was subjected to column chromatography and loaded on a glass column (60 �� 3 cm) packed with silica gel G (40 g, 60�C120#, Spectrochem Pvt. Ltd.) as the stationary phase. Gradient elution was performed using toluene: methanol (10:0, 9.5:0.5, 9:1 up to 0:10) as the mobile phase. A total of 200 fractions were collected in test tubes. Upon evaporation of the mobile phase from the test tubes, pure, white crystals of a compound were obtained in test tubes of toluene: methanol (9:1) fraction. A single spot resolved at Rf 0.65 using the mobile phase toluene: methanol (9:1).
Spectral analysis and structure elucidation This compound was subjected to spectral analysis: ultraviolet (UV; Labtronic, RKCP), infrared (IR; KBr; CSMCRI, Bhavnagar), gas chromatography-mass spectroscopy (GC-MS; CSMCRI, Bhavnagar) and 1H-nuclear magnetic resonance (1H-NMR) (CDCl3; CSMCRI, Bhavnagar). Upon the analysis of melting point and spectral data, the compound was suspected to be a sterol and triterpenoid. This was confirmed when the compound gave Salkowski test and Liebermann-Burchard test positive. The structure of the compound was elucidated on the basis of the spectra. Method development for estimation by HPTLC A novel HPTLC method for estimation of the isolated compound was developed . The instrument used was Camag Linomat V (semi-automatic spotting device) with Hamilton 100 ��l HPTLC syringe, Camag twin trough chambers (20 �� 10 cm), Camag TLC Scanner 3, Camag CATS 4 Integration software and Camag Reprostar-3.
Stationary phase used was pre-coated silica gel 60 F254 plate (E. Merck; methanol-washed, thickness 0.2 mm, 20 �� 20 cm) and the mobile phase used was toluene: methanol (9:1). The spotting parameters included start position of 15 mm from bottom edge, band width of 6 mm, space between two bands 12 mm and spraying rate of 6 sec/��l. The chromatographic conditions included ascending separation technique, twin trough chamber for plate development, chamber saturation time 4 min and migration distance 10 cm at a temperature of 25 �� 2��C. Detection was done in UV�Cvisible range. The spotting volume for calibration curve was 4�C20 ��l and for chloroform fraction of petroleum ether extract was 40 ��l. The amount sprayed for standard curve was 160�C800 ng. The mobile phase used was toluene: methanol (9:1). Densitometric scanning was carried Entinostat out in absorbance/reflectance mode at 254 nm using mercury lamp and slit dimension of 4 �� 3 mm.