Page 19 - Demo

SECTION I: MOLECULAR GENETIC ENGINEERING AND BIOCHEMICAL TECHNOLOGY 2330%u00b0C and 280 rpm for 6 days, each culture broth was centrifuged to separate supernatant and cell mass. The supernatant was extracted with ethyl acetate and the cell pellet was extracted with methanol. The extracts were concentrated and adistributed into 96-well plates for aliquoting. Each actinomycete isolate consequently generated nine different extracts. 2.2. Screening, isolation and structure determination of anti-tuberculosis lead compounds from ECUMEach extract distributed in 96 well plate was dissolved in dimethyl sulfoxide (DMSO) and screened for inhibition of replicating M. tuberculosis by using microplate alamar blue assay (MABA)[6] .A large-scale mycelial methanolic extract of hit strain was obtained from a 20 L fermenter culture. Isolation of active constituents involved three sequential steps of orthogonal separation methods: adsorption, gel permeation, and countercurrent chromatography. Vacuum liquid chromatography of the extract (128.7 g) on POLYGOPREP 100%u201350 C18 silica gel using a H2O/MeOH gradient in 20% steps. After recombination (8.47 g), gel permeation on a Sephadex LH-20 open column with 100% MeOH as eluent. Finally, single compounds was purified by Using high speed countercurrent chromatography (HSCCC).Among the most active fractions, H14 was the purest (91%) by qHNMR (100% method)7 and, therefore, selected for structural elucidation. The structure was determined by high resolution ESI-MS, IR, 1H NMR, 13C NMR and extensive analysis of the 2D NMR spectra, particularly COSY, TOCSY, HSQC, and HMBC. 2.3. Activity of ecumicin against replication and latent M. tuberculosisMinimal bactericidal concentration (MBCs) for replicating cultures were determined by subculture from the MABA plates