Application of Optimized and Validated Agar Overlay TLC–Bioautography Assay for Detecting the Antimicrobial Metabolites of Pharmaceutical Interest.

The agar overlay TLC–bioautography is one of the crucial methods for simultaneous in situ
detection and separation of antimicrobial metabolites of pharmaceutical interest. The main focus
of this research relies on the dereplication of an antimicrobial metabolite coriloxin derived from
mycoendophytic Xylaria sp. NBRTSB-20 with a validation of agar overlay TLC–bioautography technique. This polyketide metabolite coriloxin was purified by column chromatography, and its purity
was assessed by HPLC, UPLC-ESI-QTOF-MS, FT-IR and NMR spectral analysis. The antimicrobial
capability of ethyl acetate extract and the purified compound coriloxin was determined by disc
diffusion, minimal inhibitory concentration and agar overlay TLC–bioautography assay. The visible
LOD of coriloxin antimicrobial activity was found at 10 µg for Escherichia coli and 20 µg for both
Staphylococcus aureus and Fusarium oxysporum. Inter- and intra-day precision was determined as
the relative standard deviation is less than 6.56%, which proved that this method was precise. The
accuracy was expressed as recovery, and the values were found ranging from 91.18 to 108.73% with
RSD values 0.94–2.30%, respectively. The overall findings of this investigation suggest that agar
overlay TLC–bioautography assay is a suitable and acceptable method for the in situ determination
of antimicrobial pharmaceuticals