Distinction and valorization of 30 root extracts of five goldenrod (Solidago) species.

A high-performance thin-layer chromatography (HPTLC) method was developed for rapid and easy-to-perform discrimination between five goldenrod species present in Europe: the native Solidago virgaurea and the four invasive aliens, S. canadensis, S. gigantea, S. rugosa and S. graminifolia. The chemotaxonomic distinction was based on the chemical profile of their root extracts, confirmed by principal component analysis. This allowed the distinction of the goldenrods in wintertime, when classical morphological methods are not applicable. Their enzyme inhibitory profiles were determined by HPTLC combined with α-glucosidase, ß-glucosidase, α-amylase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) assays. Two compounds of S. canadensis showed the most intense enzyme inhibition in all assays, having also antibacterial activity against Bacillus subtilis, Xanthomonas euvesicatoria and Aliivibrio fischeri strains. HPTLC-high-resolution mass spectrometry (HRMS), bioassay-guided isolation, NMR spectroscopy and literature data led to the characterization and identification of the labdane diterpenes solidagenone and presolidagenone as the active S. canadensis root components. The previously identified polyacetylenes (2Z,8Z and 2E,8Z matricaria esters) of S. virgaurea, also inhibited all enzymes. Except for the known anti-AChE effect of the 2Z,8Z-matricaria ester, this is the first report on the α-glucosidase, ß-glucosidase, α-amylase, AChE and BChE inhibitory activity of these potent compounds. The anti-hyperglycemic effects of the S. canadensis labdanoids were also observed for the first time. Combined with effect-directed assays and HRMS, hyphenated HPTLC allowed an effect-directed high-throughput screening and a fast characterization of multipotent compounds. The investigation of botanicals by fast, hyphenated, bioanalytical tools substantially increased the information gain with regard to active principles (bioprofiling) and efficiently pointed to potent candidates for drug development.

Effect-directed screening of Bacillus lipopeptide extracts via hyphenated high-performance thin-layer chromatography.

Bacillus species produce a wide array of biologically active metabolites, including nonribosomaly synthesized lipopeptides (LPs). The high-performance thin-layer chromatography (HPTLC) technique hyphenated with different bioassays and mass spectrometry was demonstrated as a valuable tool for effect-directed analysis of iturins, surfactins, fengycins and kurstakins homologues from complex mixtures of LPs. As proof of this straightforward strategy, the found surfactin and iturin A homologues were characterized and compared with reference substances. This study considered two different extraction methods for LPs produced by five Bacillus strains. The ethyl acetate extraction (Ex-1), and the acidic precipitation followed by methanol extraction (Ex-2) were investigated. Diverse enzyme inhibitions and antimicrobial potentials of LPs were analyzed, and in parallel, high-resolution mass spectra (HRMS) were online recorded from the HPTLC zones of interest. No antimicrobial effect against Gram-positive B. subtilis was evident for iturin, whereas a response was detected for surfactin. The nonpolar kurstakin compounds showed a pronounced B. subtilis antimicrobial activity in Ex-1 of almost all strains, whereas the fengycin homologues were detected in Ex-2 of SS-10.7 and SS-27.2. Iturin had also no activity against Gram-negative Aliivibrio fischeri, while again surfactin showed an enhancing luminescent activity. Contrary, kurstakin compounds caused a decrease in the luminescence in Ex-1 of all strains, particularly for SS-13.1. Both, iturin and surfactin showed a strong acetylcholinesterase (AChE) and α-glucosidase inhibition, but surfactin caused a much stronger inhibition. This was evident in all bacterial strains, except for SS-13.1 in Ex-1 and for SS-38.4 in Ex-2. Although, iturin and surfactin exhibited no DPPH˙ scavenging activity, Ex-1 of all strains contained more intense DPPH˙ scavenging compounds compared to Ex-2, and surfactin methyl esters showed a pronounced DPPH˙ activity, particularly in SS-12.6 in Ex-1. This study pointed to active metabolites of strains that can be used as therapeutics and biocontrol agents with beneficial effects on human health. The straightforward HPTLC profiling served as an excellent bioanalytical tool to control the formed bioactive metabolites. As the fermentation process is very sensitive to external influences, it could be a helpful control tool for standardization of the biotechnological processing.

Fast Equivalency Estimation of Unknown Enzyme Inhibitors in Situ the Effect-Directed Fingerprint, Shown for Bacillus Lipopeptide Extracts.

The hyphenation of high-performance thin-layer chromatography (HPTLC) with enzyme inhibition assays followed by high-resolution mass spectrometry (HRMS) represents a targeted profiling of complex natural samples required in the development of new natural pharmaceuticals, functional foods, and cosmetics. This direct combination of a chromatogram with an enzymatic assay substantially extents the understanding of inhibitor properties in vitro. For the first time, a straightforward workflow was established for estimating the equivalency of unknown inhibitors directly in the autogram. Exemplarily, lipopeptides produced as secondary metabolites by five different Bacillus strains were analyzed by HPTLC hyphenated with the tyrosinase and acetylcholinesterase (AChE) assays. Lipopeptides that showed an inhibition were characterized by HPTLC-HRMS. Among the many reports about the biological properties of lipopeptides, their enzyme inhibitory properties are new. The most intense inhibitors were identified as surfactin and iturin A according to reference substances and exact masses. Three further inhibitors were supposedly assigned as fengycin, iturin C, and surfactin methyl ester according to their exact masses. The inhibitory activities of surfactin and iturin A were quantitatively compared with kojic acid and piperine, as references for common natural inhibitors. Their equivalently calculated tyrosinase inhibition showed that 1 µg kojic acid was equal to 1.8 µg and 3.2 µg of iturin A and surfactin standards, respectively; regarding to AChE inhibition, 1 µg piperine was equal to 1.7 µg and 0.6 µg of iturin A and surfactin, respectively. Further unknown enzyme inhibitors found in the sample were exemplarily calculated as surfactin, iturin A, kojic acid, and piperine equivalents to estimate their importance.

Direct bioautography hyphenated to direct analysis in real time mass spectrometry: Chromatographic separation, bioassay and mass spectra, all in the same sample run.

Mass spectra were recorded directly in situ the bioautogram, i.e., in the presence of microorganisms, bioassay medium and substrate reagent. The desorption-based direct analysis in real time mass spectrometry (DART-MS) was applied immediately after direct bioautography (DB). It turned out to be an advantageous combination, as it offered the possibility of a straightforward mass spectrometric detection of bioactive analytes within the bioautogram, and at the same time, it was discriminating microorganism cells and highly polar bioassay medium ingredients which could otherwise stress the MS system. DB-DART-MS was investigated for bioactive compounds in cosmetics using the Bacillus subtilis and Aliivibrio fischeri bioassays for detection of Gram-positive and Gram-negative antimicrobials, respectively, and the planar yeast estrogen screen for detection of estrogen-effective compounds. The influences of the three different bioassay matrices on the analyte response and DB-DART-MS performance on different layers were studied on the example of parabens in hand creams. It was shown that with increasing culture medium complexity, the ion suppression increased. As proof-of-principle, the mass spectrometric quantification at the nanogram level in situ the bioautogram was verified by comparison to HPTLC-DART-MS. The total paraben contents of hand creams 1 and 2 were 0.17-0.20% and 0.30-0.34%, respectively, depending on the method used (DB-DART-MS with two different bioassays or HPTLC-DART-MS as well as on RPW or NP plate). In contrast to the current practice of applying the sample twice and subjecting one track to the bioassay and another to MS, the introduced hyphenation DB-DART-MS is straightforward and highly efficient.

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

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.

Bioprofiling of unknown antibiotics in herbal extracts: Development of a streamlined direct bioautography using Bacillus subtilis linked to mass spectrometry.

Working in the field of profiling and identification of bioactive compounds in herbal extracts is faced with the challenge that common chromatographic methods do not directly link to bioactive compounds. Direct bioautography, the combination of TLC/HPTLC with bioassays, linked to structure elucidating techniques is demonstrated to overcome this challenge. The combination of TLC and Bacillus subtilis bioassay was already demonstrated to detect the antibiotics in samples. However, previous studies in this field were faced with some challenges, like being time-consuming, leading not to a homogenous plate background or being restricted to a non-acidic mobile phase. In this study, these aspects were investigated and a streamlined HPTLC-B. subtilis bioassay was developed that generated a homogenous plate background, which was crucial to yield a good baseline for biodensitometry. Two commonly used broths for B. subtilis and a self-designed medium were compared with regard to their capability of detection and baseline noise. The workflow developed allowed the use of acidic mobile phases for the first time. To prove this, 20 herbal extracts were screened for antimicrobial substances developed in parallel with an acidic mobile phase. The main antimicrobial substance in Salvia officinalis tincture detected was further characterized by microchemical reactions, Aliivibrio fischeri, ß-glucosidase and acetylcholinesterase (bio)assays as well as mass spectrometry. Scientists looking for new herbal-based medicine may benefit from this time-saving and streamlined bioactivity profiling.