**5.1.5 Identification and isolation of antibacterial compounds from MeOH extract**

100 g MeOH extract was loaded onto column (10 x 50 cm) packed with silica gel 60 particle size 0.063-0.2 mm (70-230 mesh) (Fluka Chemika) as stationery phase, the extracts as the mobile phase and the column was eluted with ten different concentrations of hexane:ethylacetate (9:1-1:9) and finally with ethylacetate:methanol (9:1-1:9) solvent systems (with gradual increase in polarity), 190 fractions were obtained and pooled to give a total of 20 (M1-M20) similar fractions based on their R.f. values as indicated by TLC plate analysis in chloroform:methanol:ethylacetate (CHCl3:MeOH:EtOAc; 16:0.8:1.2) solvent system. Antibacterial active fractions M9-M13 and M16-M18 as indicated by bioautography of the extract afforded two crystallized compounds which were further purified by using preparative column chromatography on silica gel 60 and eluted with hexane:ethylacetate (9:1-1:9) to produce 76 (P1- P76) fractions. Eluents in test tubes P1- P10, P12- P21, and P22- P35 upon crystallization with absolute ethanol afforded pure antibacterial compound AB-1 (white crystals, 29 mg, R.f. 0.70-CHCl3:MeOH:EtOAc, 16: 0.8: 1.2). Fractions P41- P58 upon crystallization with absolute ethanol afforded pure antibacterial compound AB-2 (colourless crystals, 35 mg, R.f. 0.64 (CHCl3:MeOH:EtOAc; 16: 0.8: 1.2), respectively. The purity of both antibacterial compounds were further determined through high performance liquid chromatography (HPLC) and running TLC plates in different binary and ternary solvent systems. Structures of both antibacterial compounds were elucidated through the integration of 1H- and 13C NMR spectra and comparison of their physical, chemical and spectral data was made with the previous reported data of the same compounds.

*3-O-β-D-glucosyl-14-deoxyandrographolides* (AB-1): M.P. 242-244°C, UV λmax MeOH nm: 202. IR (cm-1) ν: 3351, 1732, 165, 899. 1H NMR (600 MHz, in DMSO-*d6*), δ (ppm): 1.25 (o, 1H, C1- CH2), 1.71 (o, 1H, C1-CH2), 2.10 (m, 1H, C2-CH2), 1.98 (m, 1H, C2-CH2), 3.92 (o, 1H, C3-CH- ), 1.3 (m, 1H, C5-CH-), 1.85 (m, 2H, C6-CH2), 2.4 (m, 2H, C7-CH2), 3.35 (d, J=8.4 Hz, 1H, C9- CH-), 1.8 (m, 2H, C11-CH2), 2.6 (m, 1H, C12-CH2), 2.3 (m, 1H, C12-CH2), 7.10 (t, 1H, C14- CH-), 4.77 (brs, 2H, C15-CH2), 4.87 (brs, 1H, C17-CH2), 4.59 (brs, 1H, C17-CH2), 1.007 (brs, 3H, C18-CH3), 4.03 (d, J=9.6 Hz, 1H, C19-CH2), 3.22 (d, J=9.6, 1H, C19-CH2), 0.66 (brs, 3H, C20-CH3), 4.24 (d, J=7.2 Hz, 1H, C1'-CH-), 3.37 (o, 1H, C2'-CH-), 3.39 (o, 1H, C3'-CH-), 3.57 (o, 1H, C4'-CH-), 3.59 (o, 1H, C5'-CH-), 3.84 (dd, J=11.4, 4.8 Hz, 2H, C6'-CH2), *"o" denotes overlapping signals*; 13C NMR (125.76 MHz, in DMSO*d*6), δ (ppm): 38.19 (C1), 29.72 (C2), 75.04 (C3), 39.56 (C4), 56.44 (C5), 35.97 (C6), 38.44 (C7), 147.30 (C8), 56.18 (C9), 38.89 (C10), 21.72 (C11), 24.46 (C12), 136.02 (C13), 143.84 (C14), 70.11 (C15), 174.33 (C16), 107.08 (C17), 18.93 (C18), 62.61 (C19), 15.41 (C20), 103.10 (C1'), 71.72 (C2'), 72.73 (C3'), 74.03 (C4'), 76.23 (C5'), 70.72 (C6'). From this spectral data and their direct comparison with the previously published spectral data (Zhou *et al*., 2008) of the same compound, AB-1 was unambiguously identified as 3-*O*-β-D-glucosyl-14-deoxyandrographolide (Fig. 3).

*14-deoxyandrographolide* (AB-2): M.P. 172-174°C, UV λmax MeOH nm: 223. IR (cm-1) ν: 3367, 1736, 1646, 896. 1H NMR (600 MHz, in DMSO-*d6*), δ (ppm): 1.32 (m, 2H, C1-CH2), 2.04 (brs, 1H, C2-CH2), 1.99 (o, 1H, C2-CH2), 3.35 (o, 1H, C3-CH-), 1.46 (o, 1H, C5-CH-), 2.18 (brs, 1H, C6-CH2), 1.98 (brd, J=6 Hz, 1H, C6-CH2), 2.45 (t, 2H, C7-CH2), 3.50 (o, 1H, C9-CH-), 2.43 (dd, J= 4.2, 2.4 Hz, 2H, C11-CH2), 2.55 (dd, J= 12, 7.2 Hz, 2H, C12-CH2), 7.10 (brs, 1H, C14-CH-), 4.91 (brs, 2H, C15-CH2), 4.59 (brs, 1H, C17-CH2), 4.45 (brs, 1H, C17-CH2), 1.59 (o, 3H, C18- CH3), 4.20 (brs, 1H, C19-CH2), 4.18 (brs, 1H, C19-CH2), 0.71 (s, 3H, C20-CH3), *"o" denotes overlapping signals*; 13C NMR (125.76 MHz, in DMSO*d*6), δ (ppm): 38.93 (C1), 37.74 (C2), 80.50 (C3), 64.13 (C4), 55.21 (C5), 28.22 (C6), 37.06 (C7), 148.94 (C8), 55.96 (C9), 42.96 (C10), 24.88 (C11), 23.76 (C12), 146.64 (C13), 127.83 (C14), 66.31 (C15), 172.17 (C16), 108.86 (C17), 22.67 (C18), 74.61 (C19), 15.16 (C20). From this spectral data and their direct comparison with the previously published data (Poonam *et al*., 2010) of the same compound, AB-2 was unambiguously identified as 14-deoxyandrographolide (Fig. 4).
