Bioassay-guided methods

A1 Dabbas et al. [163] reported the antibacterial activity of the ethyl acetate extract of the whole aerial part of Varthemia iphinoides L. The bioassay-guided fractionation led to the isolation and identification of an antibacterial eudesmane sesquiterpene, selina-4,ll(13)-dien-3-on-12 oic acid. Fig. (10). This compound exhibited potent antimicrobial activity against 6 bacterial species Staphylococcus aureus, Bacillus subtilis. Micrococcus luteus, Escherichia coli. Bacillus cereus and Salmonella enter Hides ). The MIC values of this sesquiterpene, which was determined by the agar dilution method, ranged between 250 and 500 ig/ml. 

Batista et al. (58) used an overlay method in the bioassay-guided fractionation of an acetone extract of the roots of Plectranthus hereroensis (Labiatae) to isolate the antibacterial diterpene (Compound 14). Staphylococcus aureus was used as the test organism. 

During bioassay-guided purifications, never discard a fraction until an analytical method is available that distinguishes the active compound(s) from the other components of the mixture. The compounds may be present but their effects masked by other components of the mixture. This is especially important in instances where the supply of organism is limited and recollection is difficult if not impossible. It is important not to waste any of the specimen. 

Using an affinity probe technique it has become possible to identify natural products that bond to DNA (79). Extracts derived from Albizia amara (Leguminosae) were found to demonstrate activity in a recently developed HPLC system designed to detect compounds capable of interacting with DNA. Further investigation led to the procurement of four sets of alkaloid isolates X1-X4 that were found to be macrocyclic pithecolobine alkaloids. Isolate Xi found to inhibit the catalytic activity of DNA polymerase, RNA polymerase, and HIV-1 reverse transcriptase. It has been identified as a mixture of budmunchiamines A (89), B (90), and C (91), in the ratio 4 1 1 80), whose structures were determined by spectroscopic methods 81). In further detailed bioassay-guided isolation, six more spermine macrocyclic alkaloids, budmunchiamines D-I (92-97) were obtained 82). The structures of these substances were confirmed by spectral analysis and comparison with the related alkaloids budmunchiamines A (89)-C (91). (See Fig. 10.)... 

A systematic approach to profiling active metabolites using a 96-well plate format was recently described (Shu et al., 2002). The approach is based on a rapid bioassay-guided metabolite detection and characterization. Drug metabolite mixtures (generated by various methods described below) are separated and fractions collected into microtiter plates such as 96-well plates. The fractions are then subjected to one or more relevant activity (e.g., receptor ligand binding) assays. 

Isolation of the active constituents was done using a bioassay-guided procedure. Depending on the extract, various combinations of chromatographic methods and solvent systems were used. Basic procedures included thin layer chromatography (analytical and preparative), as well as standard column chromatography. Structures were elucidated by a combination of NMR and MS spectrometric techniques. Details of the analyses will be reported elsewhere. 

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