From Bioprofiling and Characterization to Bioquantification of Natural Antibiotics by Direct Bioautography Linked to High-Resolution Mass Spectrometry: Exemplarily Shown for Salvia miltiorrhiza Root

Abstract

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.

Effect-directed classification of biological, biochemical and chemical profiles of 50 German beers

“…For example using direct bioautography, antimicrobials were detected by changes in the microbial growth directly on the HPTLC chromatogram (Morlock, 2013). Due to its simplicity, speed and cost-efficiency, bioautographic methods were developed in academic and industrial laboratories to discover and characterize, e.g., antimicrobial, antifungal, anti-tumour, anti-protozoa and oestrogen-effective compounds in complex food, botanicals and environmental samples (Morlock, 2013;i e e & Morlock, 2015a;Jamshidi-Aidji & Morlock, 2016).…”

HI-HPTLC-UV/Vis/FLD-HESI-HRMS and bioprofiling of steviol glycosides, steviol, and isosteviol in Stevia leaves and foods

“…As demonstrated in previous studies, the individual active compounds can further be characterized by HPTLC-HESI-HRMS and elucidated in its structure [ 64 , 70 ]. Quantification of the discovered activities can be performed by external standards or a well-known reference (equivalency calculation), when a standard compound is not available or the bioactive compound is unknown/unidentified [ 63 , 71 ].…”

“… For the Gram-positive B. subtilis bioassay, 3.5 mL bacteria suspension (100 μL bacterial cryostock per 20 mL 2.3% Müeller-Hinton broth, optical density of ca. 0.8 at 600 nm [ 63 ]) was sprayed, followed by incubation at 37 °C for 2 h. For generation of the colorless (white) antibacterial bands on a purple background, the plate was sprayed with 500 μL 0.2% phosphate-buffered saline (0.8% sodium chloride, 0.02% potassium chloride, 0.14% disodium hydrogen phosphate, and 0.02% potassium dihydrogen phosphate)-buffered MTT solution and incubated at 37 °C for 60 min, followed by drying (50 °C, 5 min, Plate Heater, CAMAG). The PC was tetracycline (0.5, 1.5, and 3 μL/band, 0.004 mg/mL in ethanol).…”

Coding Recognition of the Dose-Effect Interdependence of Small Biomolecules Encrypted on Paired Chromatographic-Based Microassay Arrays

“…With this method, the chromatographic spots of bioactive compounds among diverse compounds can be color developed in situ by a bioassay. Recently, Morlock et al immersed the HPTLC plates with the separated sample extracts in the bacterial culture medium and extracted the components in the bioactive spots through a special TLC-MS interface (Camag) used for biotracing 9 . However, due to the interference of the chromatographic matrix with many bioassays, this method has not yet been applied to mammalian cells.…”