TLC-Direct Bioautography as a High Throughput Method for Detection of Antimicrobials in Plants

Abstract

The richness of bioactive compounds in plant materials encourages continuous development of separation methods and bioassays for their isolation and identification. Thin-layer chromatography-direct bioautography links separation on the adsorbent layer with biological tests performed directly on it. Therefore, the method is very convenient for searching plant constituents with biological activity, such as antibiotics. Test bacteria grow directly on a plate surface excluding places where antibacterials are located. They can be detected with reagents converted by living bacteria. TLC-DB is a high throughput method enabling analyses of many samples in parallel and the comparison of their activity. Both screening and semi-quantitative analysis is possible. The targeted compounds can be identified using spectroscopic methods, mostly mass spectrometry, that can be performed directly on a TLC plate. This paper discusses all above mentioned aspects of TLC-DB, illustrating them with literature, schemes and our own results.

Antibacterial Activity of Fractions of Ethyl Acetate Extract of Garcinia Lattissima Miq. Fruits

“…Tetrazolium salt used in this MIC microdilution assay is a (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), which is converted to purple formazan by the dehydrogenases enzyme of living microorganisms [15]. This reaction is a reduction reaction, and the purple to show the bacteria growth [15].…”

“…This reaction is a reduction reaction, and the purple to show the bacteria growth [15]. Makes this assay reliable for visual observation.…”

“…Many antibiotics are known to be powerful, but no longer effective against these and other infections. The situation is mostly related to increasing bacterial resistance to commonly used antibiotics [15]. Chromatography is the perfect tool for analyzing plant constituents.…”

Establishment of an effective TLC bioautographic method for the detection ofMycobacterium tuberculosisH37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate

“…In mycobacteria, PGI is engaged through glucose-6P with L-rhamnose and is involved in the biosynthesis of the galactan residue of arabinogalactan, the components of the cell wall of these microorganisms [6][7][8] . The major application of bioautography is screening of the antimicrobial and antifungal properties of various plant samples with Thin Layer Chromatography (TLC)-plates 9 . The term ''autography'' is used to detect other biological processes, e.g.…”

“…enzyme inhibition 10 . However, the definition of the bioautographic method is often broadened and covers the determination of enzyme inhibition, antioxidant agents as well as oestrogenic compounds 9,11 . In our opinion, there is a clear trend towards the use of the TLC bioautography technique as a method for screening active compounds against microorganisms rather than a method for the detection of enzyme activities or inhibitors.…”