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Phytochemicals are promising agents in the development of new antibiotics. A streamlined strategy for rapid screening and reliable characterization of antibiotics in botanicals was demonstrated in contrast to the commonly applied chromatographic column fractionation followed by microtiter plate assay. Modern direct bioautography hyphenated to structure elucidation techniques is a straightforward bioanalytical tool, especially if microbiological assays were taken into account. At one go, lipophilic antimicrobials in Salvia miltiorrhiza root samples were analyzed using high-performance thin-layer chromatography (HPTLC) in direct combination with Aliivibrio fischeri and Bacillus subtilis bioassays. The most intense antimicrobials were characterized via HPTLC-high-resolution mass spectrometry. As proof of this streamlined strategy, dihydrotanshinone, cryptotanshinone, and tanshinone IIA were identified and also compared with a reference. Two further antimicrobial zones in the bioautograms were tentatively assigned to be methylenetanshinquinone and tetrahydrotanshinone I (or its structural isomer methylenedihydrotanshinquinone). In another run, a validation study was performed for the bioquantification of ciprofloxacin and marbofloxacin via HPTLC-Bacillus subtilis. This pointed out the improved quality of the performance that was reached. Cryptotanshinone was biologically quantified in two S. miltiorrhiza root samples. Antimicrobials without an available reference standard were calculated as cryptotanshinone-bioequivalents. The results were of relevance, as 1 ng of cryptotanshinone was calculated to be bioequivalent to 0.6 ng and 2 ng of the synthetic antibiotics, ciprofloxacin and marbofloxacin, respectively. For the first time, quantitative direct bioautography via HPTLC-B. subtilis was shown as a reliable tool for streamlined bioprofiling of complex samples.

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