Bioactivity profiles of six baobab fruit pulp powders via planar chromatography hyphenated with effect-directed analysis.

Baobab (Adansonia digitata) fruit pulp has a high nutrient content and has been traditionally used for medicinal purposes (e.g., as an anti-inflammatory and antioxidant agent) that may help protect against chronic diseases. Six different baobab fruit pulp powders were investigated using three different extractants and analyzed by high-performance thin-layer chromatography (HPTLC) hyphenated with antibacterial bioassays and enzyme inhibition assays. The developed non-target effect-directed screening was performed after extraction with pentyl acetate - ethanol 1:1 (V/V) on the HPTLC plate silica gel 60 using toluene - ethyl acetate - methanol 6:3:1 (V/V/V) as mobile phase system and derivatization via the anisaldehyde sulfuric acid reagent for detection. The physico-chemical profiles of the six baobab fruit pulp powder extracts were comparable, although the intensity of some zones was moderately different. The following effect-directed profiling via tyrosinase, α-glucosidase, and acetylcholinesterase inhibition assays as well as antibacterial Aliivibrio fischeri and Bacillus subtilis bioassays revealed one prominent multipotent bioactive compound zone in common, more or less active in all five studied (bio)assays. Via the recording of high-resolution mass spectra, this compound zone was tentatively assigned to coeluting saturated (palmitic acid 16:0 and stearic acid 18:0), monounsaturated (oleic acid 18:1), and polyunsaturated (linoleic acid 18:2 and linolenic acid 18:3) fatty acids. This finding was confirmed by other studies, which already proved individual activities of fatty acids. The first (bio)activity profiling of baobab fruit pulp powders via HPTLC-effect-directed analysis revealed that the baobab fruit could be considered as a functional food, however, further research is needed to study the impact on health and the influences on the bioactivity arising from different climates, years and soils or regions.

High-throughput enzyme inhibition screening of 44 Iranian medicinal plants via piezoelectric spraying of planar cholinesterase assays.

A rapid and straightforward approach was developed for screening the acetyl- and butyrylcholinesterase (ChE) inhibitory activity of 44 Iranian medicinal plant extracts at laboratory scale. After a fast ChE inhibitory pre-testing of samples applied as band pattern, 40 out of the 44 Iranian medicinal plant extracts were selected. These were adjusted in the application volume depending on their inhibition activity, applied on both plate sides and simultaneously developed in a horizontal developing chamber. Different mobile phases were studied to achieve maximum separation of ChE inhibitors and minimum co-elution with matrix. Contrary to immersion, the piezoelectric spraying reduced the consumption of assay solutions, prevented zone tailing, zone shift and cross-contamination, and homogeneously covered the entire plate surface with the assay solutions. The ChE inhibitors of the six most bioactive plant extracts were tentatively assigned by high-resolution mass spectrometry in combination with the spectral and chromatographic information obtained.

New incorporation of the S9 metabolizing system into methods for detecting acetylcholinesterase inhibition.

The activity of individual biotransformation products cannot be measured in multicomponent mixtures by the current status-quo assays. A prior separation and tedious isolation of compounds is required, and often in addition, a concentration step into a solvent suitable for the cell-/enzyme-based assay. Hence, the metabolizing S9 system, mimicking the complex biotransformation reactions in the liver, was aimed to be integrated into two orthogonal methods for analysis of the acetylcholinesterase (AChE) inhibition. For the microtiter plate assay method, the evaluation of the generated fluorescence signal was impaired by the incorporated S9 system. In contrast, the metabolic activator (S9 mixture) was successfully incorporated into the high-performance thin-layer chromatography (HPTLC) method. As proof of principle, four reference AChE inhibitors were studied in complex samples with and without metabolic activation. In addition to the neurotoxic carbamate eserine and the organophosphate insecticides chlorpyrifos, quinalphos and parathion, the tris(nonylphenyl) phosphite and nonylphenol, both originating from food contact materials, were tested in isolation but also in food packaging migrate and extract. A method comparison and benchmarking pointed to multifold advantages of using this newly developed bioanalytical tool for assessment of individual neurotoxins in complex samples. The sensitive HPTLC-S9-AChE assay allowed the detection of neurotoxic chemicals with and without metabolic activation, at levels consistent with the threshold of toxicological concern of organophosphates and carbamates. This new on surface metabolism system can be applied to other toxicities and samples.

Same analytical method for both (bio)assay and zone isolation to identify/quantify bioactive compounds by quantitative nuclear magnetic resonance spectroscopy.

Differing sensitivity is the main obstacle for a direct combination of HPTLC with NMR spectroscopy. A sufficient amount of the isolated compound zone must be provided by HPTLC for subsequent offline NMR detection (HPTLC//NMR). To fill the gap, a straightforward procedure was developed using the same analytical HPTLC system for both bioprofiling and isolation of bioactive zones from multicomponent mixtures. The HPTLC-effect-directed analysis (EDA) revealed several bioactive compounds in five botanical extracts, i.e. Salvia officinalis, Thymus vulgaris and Origanum vulgare, all Lamiaceae, and peels of red and green apples (Jonagored and Granny Smith, respectively), both Rosaceae. A tricky case study was designed to show how to deal with potentially coeluting bioactive structural isomers, e.g., ursolic (UA), oleanolic (OA) and betulinic acids (all C30H48O3), which are most difficult to identify and assign. A multipotent bioactive HPTLC zone showed the same hRF value and mass signal in HPTLCHRMS, though containing the coeluting structural isomers UA and OA. After zone isolation from the HPTLC plate, first the 1H NMR spectrum allowed to distinguish distinct allylic H-18 protons, i.e. 2.20 ppm for UA and 2.85 ppm for OA, and at the same time, to quantify the two isomers by using the PUlse Length-based CONcentration methodology (HPTLC//1H qNMR-PULCON). In case of a partial overlap of the diagnostic signal with that of the matrix, results were corroborated with those obtained by using the 1H deconvoluted or 2D 1H-13C Heteronuclear Single Quantum Coherence spectra. The comparison of the quantitative results showed a good correlation (R2 = 0.9718) between the two orthogonal methods HPTLC-Vis and HPTLC//1H qNMR-PULCON. A sufficient zone isolation from the HPTLC plate (mean isolation rate of 82%) for both UA and OA (0.27 - 4.67 mM) was achieved for HPTLC//qNMR, comparing the isolated bioactive compound zone with the respective zone in the botanical extract via HPTLC-Vis densitometry. The HPTLC-EDA-Vis//1H qNMR-PULCON procedure for bioprofiling and quantification/identification/confirmation of bioactive compounds in botanical extracts is considered as straightforward, eco-friendly (only 16 mL solvent required), simple (NMR calibration used over weeks) and reliable new alternative to the status quo of bioactivity-guided fractionation.

Automated piezoelectric spraying of biological and enzymatic assays for effect-directed analysis of planar chromatograms.

Bioanalytical questions are more and more solved by bioassays directly in situ the planar separation. If compared to chemical derivatization in situ, several reagent applications on the same chromatogram make the workflow for enzymatic and biological assays more complex. Hence, if compared to piezoelectric spraying of chemical derivatization reagents, an assay transfer to the piezoelectric spraying technique was much more challenging. Important aspects were investigated, i.e., plate pre-wetting, spraying nozzle type and applied volumes for microorganism suspension as well as enzyme and substrate-chromogenic solutions. Finally, with the newly developed piezoelectric spraying procedures for the application of biological (Aliivibrio fischeri) and enzymatic (acetyl- and butyrylcholinesterase) assays, several obstacles of the state-of-the-art automated immersion were avoided such as the (1) required high volumes of solutions, (2) tailing of highly water-soluble zones upon slow plate withdrawal, (3) zone distortion or shift observed after previous buffer salt applications or long/slow immersion times/speeds, (4) gradual inactivation of the enzyme solution along with its ongoing re-use, and (5) lack of covering the whole plate surface. The benchmarking of both techniques also showed that simplicity remains the key argument for immersion. As proof of concept, piezoelectrically sprayed autograms were compared with those of immersion, by taking the example of Peganum harmala (P. h.) seed extract. The plate background and thus homogeneity of the applied solutions were found to be almost comparable. Three bands among the pronounced fluorescent bands were responsible for the most antibacterial activity of P. h. seed extract in the A. fischeri bioassay and were also inhibiting the AChE. These AChE and three further BChE inhibitors were detected, whereby the AChE inhibition was twice as strong as the BChE inhibition. By their in situ HRMS spectra, the active zones in the P. h. seed extract were assigned to be the AChE-inhibiting ß-carboline alkaloids, harmine, harmaline and ruine, as well as the BChE-inhibiting quinazoline alkaloids, vasicine and deoxyvasicine, and the ß-carboline alkaloid harmol. For the first time, the found inhibitors were calculated equivalently to the well-known ChE-inhibitor physostigmine, and thus, piezoelectric spraying was proven to be suited for quantifications.

Bioprofiling of Salvia miltiorrhiza via planar chromatography linked to (bio)assays, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

An affordable bioanalytical workflow supports the collection of data on active ingredients, required for the understanding of health-related food, superfood and traditional medicines. Targeted effect-directed responses of single compounds in a complex sample highlight this powerful bioanalytical hyphenation of planar chromatography with (bio)assays. Among many reports about biological properties of Salvia miltiorrhiza Bunge root (Danshen) and their analytical methods, the highly efficient direct bioautography (DB) workflow has not been considered so far. There was just one TLC-acetylcholinesterase (AChE) method with a poor zone resolution apart from our two HPTLC-DB studies, however, all methods were focused on the nonpolar extracts of Danshen (tanshinones) only. The current study on HPTLC-UV/Vis/FLD-(bio)assay-HRMS, followed by streamlined scale-up to preparative layer chromatography (PLC)-1H-NMR, aimed at an even more streamlined, yet comprehensive bioanalytical workflow. It comprised effect-directed screening of both, its polar (containing phenolics) and nonpolar extracts (containing tanshinones) on the same HPTLC plate, the biochemical and biological profiling with four different (bio)assays and elucidation of structures of known and unidentified active compounds. The five AChE inhibitors, salvianolic acid B (SAB), lithiospermic acid (LSA) and rosmarinic acid (RA) as well as cryptotanshinone (CT) and 15,16-dihydrotanshinone I (DHTI) were confirmed, but also unidentified inhibitors were observed. In the polar extracts, SAB, LSA and RA exhibited free radical scavenging properties in the 2,2-diphenyl-1-picrylhydrazyl assay. CT, DHTI and some unidentified nonpolar compounds were found active against Gram-positive Bacillus subtilis and Gram-negative Aliivibrio fischeri (LOD 12 ng/band for CT, and 5 ng/band for DHTI). For the first time, the most multipotent unidentified active compound zone in the B. subtilis, A. fischeri and AChE fingerprints of the nonpolar Danshen extract was identified as co-eluted band of 1,2-dihydrotanshinone and methylenetanshinquinone in the ratio of 2:1.

Simultaneous determination of sorbic and benzoic acids in milk products using an optimised microextraction technique followed by gas chromatography.

A rapid and reliable method for direct determination of sorbic and benzoic acids in milk products was developed by dispersive liquid-liquid microextraction (DLLME) and gas chromatography with flame ionisation detector (GC-FID). A response surface methodology (RSM) based on a central composite design (CCD) was applied for optimisation of the main variables, such as volume of extraction and dispersive solvents, pH and salt effect. The primary extraction of sorbic and benzoic acids were performed in 8 mL NaOH (0.1 M) in a closed-vessel system. Carrez solutions (potassium hexaferrocyanide and zinc acetate) were used for protein sedimentation. The best simultaneous extraction efficiency was identified using acetone and 1-octanal as dispersive and extraction solvents, respectively. For DLLME, central composite design resulted in the optimised values of microextraction parameters as follows: 475 µL of dispersive and 60 µL of extraction solvents, 2 g NaCl at pH 2.5. Under optimum conditions, the calibration curve was linear over the range 0.1-50 µg mL(-1) and the square of correlation coefficient (R(2)) was 0.9992 for sorbic acid and 0.9994 for benzoic acid. Relative standard deviation (RSD %) was 6.1% and 3.1% (n = 5) for sorbic and benzoic acids, respectively. Limits of detection were 150 ng g(-1) for sorbic acid and 140 ng g(-1) for benzoic acid and recoveries were 88% and 103.7% respectively. Good reproducibility (RSD %), short extraction time and no matrix interference were advantages of the proposed method which was successfully applied to the determination of sorbic and benzoic acids in milk products.