Rapid Screening of Lipase Inhibitors from Ophiopogonis Radix Using High-Performance Thin Layer Chromatography by Two Step Gradient Elution Combined with Bioautographic Method

In this study, a high-performance thin layer chromatography (HPTLC) method by two step gradient elution with two mobile phases was developed for the simultaneous analysis of seven constituents in Ophiopogonis Radix. The chromatography was performed on silica gel 60 F₂₅₄ plate with dichloromethane-methanol-ethyl acetate-water (70:25:12:3, v/v/v/v) and dichloromethane-methanol (300:1, v/v) as the mobile phase for two step gradient elution. Then, the HPTLC profiles were observed after derivatization with 10% sulfuric acid in ethanol solution. The obtained HPTLC images were further analyzed by chemometric approaches and the samples could be clustered based on regions and/or growth years, which were two important factors affecting the constituents in Ophiopogonis Radix. Furthermore, five compounds including ophiopogonin D, ophiopojaponin C, ophiopogonin D’, ophiopogonin C’ and methylophiopogonanone B were screened as potential lipase inhibitors from Ophiopogonis Radix by the HPTLC-bioautographic method. The binding modes and interactions between the five compounds and lipase were further explored by molecular docking analysis. The developed HPTLC method could be used for quality control of Ophiopogonis Radix and screening of the potential lipase inhibitors.     

Antimicrobial,DPPH scavenging and tyrosinase inhibitory activities of Thymus vulgaris,Helichrysum arenarium and Rosa damascena mill. ethanol extracts by using TLC bioautography and chemical screening methods

Antimicrobial, DPPH scavenging and tyrosinase inhibitory activities of Thymus vulgaris, Helichrysum arenarium and Rosa damascena Mill. ethanol extracts by using TLC bioautography and chemical screening methods. The ethanol extracts of Thymus vulgaris (Tv), Helichrysum arenarium (Ha) and Rosa damascena Mill. (Rm) (red) were screened for their antimicrobial, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and tyrosinase inhibitory activities. The test microorganisms included bacteria of Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). Thin Layer Chromatography (TLC) - bioautography, disk diffusion and well diffusion methods were used for the antimicrobial activity assays. Rosa damascena Mill. extract was effective against E. coli and all plant extracts showed antimicrobial activity against S. aureus. The phenolic acids in the structure of the extracts were also identified by LC-MS analysis. Human blood agar well diffusion method and TLC-DPPH assays were used to identify the hemolytic and antioxidant activity of plant extracts, respectively, along with 10 compounds including phenolic acids as a standard. Among these compounds, caffeic acid (Rf = 0.68) was detected in all extracts while vanillic acid (Rf = 0.75), and gallic acid (Rf = 0.51) was found in Tv extract. Kojic acid (Rf = 0.36), on the other hand, was detected in Rm extract as a tyrosinase inhibitor. All plant extracts presented tyrosinase inhibitory activities on TLC-bioautography assay.     

An Evolving Technology That Integrates Classical Methods with Continuous Technological Developments: Thin-Layer Chromatography Bioautography

Thin-layer chromatography (TLC) bioautography is an evolving technology that integrates the separation and analysis technology of TLC with biological activity detection technology, which has shown a steep rise in popularity over the past few decades. It connects TLC with convenient, economic and intuitive features and bioautography with high levels of sensitivity and specificity. In this study, we discuss the research progress of TLC bioautography and then establish a definite timeline to introduce it. This review summarizes known TLC bioautography types and practical applications for determining antibacterial, antifungal, antitumor and antioxidant compounds and for inhibiting glucosidase, pancreatic lipase, tyrosinase and cholinesterase activity constitutes. Nowadays, especially during the COVID-19 pandemic, it is important to identify original, natural products with anti-COVID potential compounds from Chinese traditional medicine and natural medicinal plants. We also give an account of detection techniques, including in situ and ex situ techniques; even in situ ion sources represent a major reform. Considering the current technical innovations, we propose that the technology will make more progress in TLC plates with higher separation and detection technology with a more portable and extensive scope of application. We believe this technology will be diffusely applied in medicine, biology, agriculture, animal husbandry, garden forestry, environmental management and other fields in the future.     

Raman spectroscopic evaluation of the influence of Pseudomonas bacteria on aflatoxin B1 in the BioArena complex bioautographic system

The mechanism of the antibacterial‐toxic effect of aflatoxin B1 (AFB1) was investigated in the BioArena complex bioautographic system that consists of planar liquid chromatographic development and biological detection. In this system, the killing‐inhibiting activity of AFB1 against Pseudomonas savastanoi (Psm) bacterial cells was visualised. The role of formaldehyde (HCHO) was suggested in the antibacterial‐toxic action of AFB1. Raman spectroscopy (RS) was used to investigate whether the excess HCHO found earlier in the AFB1 spot (comparing with background) originated partly from the demethylation of the toxin, or only from the enhanced demethylation of the normal cell ingredients because of the stress situation. Fourier transform (FT) Raman and surface‐enhanced FT‐Raman spectra were obtained in situ about the AFB1 chromatographic and background spots in bacteria‐free and inoculated thin layer chromatography (TLC) layers, and ex situ about their dried methanolic extract. The reduction of the δ CH₃ band of AFB1 (1386 cm⁻¹) in the presence of Psm can indicate the demethylation of aflatoxin at its methoxy‐group in BioArena. It seems from the Raman spectra that, apart from demethylation, aflatoxin does not suffer other structural changes. However, Psm can reduce the Raman activity of νCO bond of the pyran ring of AFB1, causing lower intensity and a broader band (1747 and 1754 cm⁻¹). Psm can also disrupt the strong interactions between the AFB1 and the adsorbent layer through the νCO bond of the cyclopentene ring causing a blue‐shift (1667 → 1686 cm⁻¹). The very reactive HCHO that originated from exogenous sources, e.g. from the demethylation of AFB1, has no determined biochemical pathway, so it means higher risk. Copyright © 2008 John Wiley & Sons, Ltd.     

Fractionation of Lycopodiaceae Alkaloids and Evaluation of Their Anticholinesterase and Cytotoxic Activities

In view of the abundant evidence that Lycopodiaceae alkaloids, including the well-known huperzine A (HupA), are among the potent acetylcholinesterase (AChE) inhibitors, an attempt was made to search for new compounds responsible for this property. For this purpose, three plant species belonging to the Lycopodiaceae family, commonly found in the Euro-Asia region, were subjected to the isolation of bioactive compounds, their identification and subsequent evaluation of their anticholinesterase and cytotoxic activities. Methanolic extracts of two Lycopodium and one Hupezia species were obtained via optimized pressurized liquid extraction (PLE) and then pre-purified using innovative gradient vacuum liquid chromatography (gVLC). For the first time, three sorbents of different porosity packed in polypropylene cartridges and mobile phase systems of different polarity were used to elute the target compounds. This technique proved to be a rapid tool for the obtainment of alkaloid fractions and allowed one to select the appropriate process conditions to yield potent AChE inhibitors in each of the species studied. More than 100 collected fractions were analyzed via HPLC/ESI-QTOF-MS, which enabled one to detect more than 50 compounds, including several new ones previously unreported. Some of them were present in high purity fractions (60–90% of the established purity). TLC bioautography assays proved that the analyzed species are rich sources of AChE inhibitors, but H. selago showed the highest anti-AChE activity. Additionally, the modified silanized silica gel sorbent used allowed one to isolate L. clavatum alkaloids more efficiently using an aqueous reversed-phase solvent system. Furthermore, the tested extracts from the three plant extracts were found to be safe, as they did not exhibit cytotoxicity to skin fibroblasts.     

Effect-Directed Profiling of Monofloral Honeys from Ethiopia by High-Performance Thin-Layer Chromatography and High-Resolution Mass Spectrometry

Ethiopian honey is used not only as food but also for treatment in traditional medicine. For its valorization, bioactive compounds were analyzed in nine types of monofloral Ethiopian honey. Therefore, a non-target effect-directed profiling was developed via high-performance thin-layer chromatography combined with multi-imaging and planar effect-directed assays. Characteristic bioactivity profiles of the different honeys were determined in terms of antibacterial, free-radical scavenging, and various enzyme inhibitory activities. Honeys from Hypoestes spp. and Leucas abyssinica showed low activity in all assays. In contrast, others from Acacia spp., Becium grandiflorum, Croton macrostachyus, Eucalyptus globulus, Schefflera abyssinica, Vernonia amygdalina, and Coffea arabica showed more intense activity profiles, but these differed depending on the assay. In particular, the radical scavenging activity of Croton macrostachyus and Coffea arabica honeys, the acetylcholinesterase-inhibiting activity of Eucalyptus globulus and Coffea arabica honeys, and the antibacterial activity of Schefflera abyssinica honey are highlighted. Bioactive compounds of interest were further characterized by high-resolution mass spectrometry. Identifying differences in bioactivity between mono-floral honey types affects quality designation and branding. Effect-directed profiling provides new insights that are valuable for food science and nutrition as well as for the market, and contributes to honey differentiation, categorization, and authentication.     

Preliminary results of layer chromatography combined with bilateral band compression to search active minor components

Abstract

Bilateral band compression (BBC) is a process where a solvent flow perpendicular to the direction of development squeezes bands into a smaller area. BBC of the 10 mm wide lanes of high-performance thin-layer chromatography (HPTLC) separation resulted in more than 6 times increase in peak height and peak area of Canadian goldenrod labdane diterpenes solidagenone and presolidagenone. Simultaneously, the signal-to-noise ratio increased only up to 2.2–2.4 times due to the higher noise level. The BBC increased the relative standard deviations (RSDs) of peak height and area reached three to four times of the original as well. It is known that overpressured layer chromatography (OPLC) separates more efficiently than conventional TLC/HPTLC techniques. In order to increase the bio-detection sensitivity, we combined bioautography, BBC, and fully offline infusion-OPLC using band-shaped sample application/separation. Thyme essential oil has been applied as a multi-component object to show the potential of this process. It is demonstrated that biologically active minor components, which may be missed in case of a conventional process, can also be detected by means of this combined method.     

Investigation of antibacterial and cytotoxic potential of phenolics derived from Cistus incanus L. by means of thin-layer chromatography-direct bioautography and cytotoxicity assay

Cistus incanus L. (hairy rockrose) is a medicinal plant which belongs to the Cistaceae family and the Cistus genus, with a well established position in traditional medicine of the Mediterranean basin and the Middle East. It was the aim of this study to compare antibacterial activity of the phenolics derived from fourteen C. incanus samples of different origin (Turkey, Albania, Greece, and an unknown geographical location) obtained as herbal teas from a local market of diet supplements. This activity was assessed with the use of thin-layer chromatography–direct bioautography (TLC-DB) applied to crude extracts against the Gram negative naturally luminescent marine bacterium Aliivibrio fischeri and the Gram positive soil bacterium Bacillus subtilis as the test microorganisms. It was established that in spite of different origin of the investigated herbal samples, in qualitative terms their antibacterial activity was closely comparable and more strongly pronounced against the Gram positive than the Gram negative bacterium. Crude extract originating from one herbal specimen labelled A3 (sample no. 3 from Albania) underwent selective multi-step extraction of the phenolics, dividing them into six fractions (I to VI) that expectedly contain flavonoid aglycons, free phenolic acids, non-polar flavonoid glycosides, polar flavonoid glycosides, and phenolic acids obtained through the acidic and basic hydrolysis from the respective glycosides. Antibacterial activity of each A3 fraction was then assessed with the use of the same TLC-DB approach and it was established that the strongest effect was exerted by fractions I and II (flavonoid aglycons and free phenolic acids). Moreover, cytotoxic assay was performed for the crude C. incanus extracts against the human colon adenocarcinoma cells and a moderate yet well measurable cytotoxic effect was observed with all investigated C. incanus samples. In analogy to antibacterial activity, also in this case cytotoxic potential of all investigated crude C. incanus extracts was similar.     

Quick identification of xanthine oxidase inhibitor and antioxidant from Erycibe obtusifolia by a drug discovery platform composed of multiple mass spectrometric platforms and thin‐layer chromatography bioautography

As a final step of the purine metabolism process, xanthine oxidase catalyzes the oxidation of hypoxanthine and xanthine into uric acid. Our research has demonstrated that Erycibe obtusifolia has xanthine oxidase inhibitory properties. The purpose of this paper is to describe a new strategy based on a combination of multiple mass spectrometric platforms and thin‐layer chromatography bioautography for effectively screening the xanthine oxidase inhibitory and antioxidant properties of E. obtusifolia. This strategy was accomplished through the following steps. (i) Separate the extract of E. obtusifolia into fractions by an autopurification system controlled by liquid chromatography with mass spectrometry. (ii) Determine the active fractions of E. obtusifolia by thin‐layer chromatography bioautography. (iii) Identify the structure of the main active compounds with the information provided by direct analysis in real time mass spectrometry. (iv) Calculate the IC₅₀ value of each compound against xanthine oxidase using high‐performance liquid chromatography. Using the caulis of E. obtusifolia as the experimental material, seven target peaks were screened out as xanthine oxidase inhibitors or antioxidants. Our screening strategy allows for rapid analysis of small molecules with almost no sample preparation and can be completed within a week, making it a useful assay to identify unstable compounds and provide the empirical foundation for E. obtusifolia as a natural remedy for gout and oxidative‐stress‐related diseases.     

TLC–direct bioautography as a method for evaluation of antibacterial properties of Thymus vulgaris L. and Salvia officinalis L. essential oils of different origin

ABSTRACT

The dot-blot bioautography was used to evaluate the antibacterial properties of Thymus vulgaris L. and Salvia officinalis L. essential oils (EOs) produced by three different manufacturers. The whole samples were applied at three concentrations on thin-layer chromatography (TLC) plates which were then subjected to bioautography against Bacillus subtilis. The samples of the highest activity were found. Then, they were separated using TLC and once again subjected to bioautography against B. subtilis. As was proved, only the essential oils of T. vulgaris L. possessed strong antibacterial properties for which mostly thymol and carvacrol were responsible. Their contents were calculated using TLC–UV densitometry. The highest contents were found in the essential oils of the highest antibacterial activity revealed in the dot-blot test. It means that a dot-blot test can be used for simple and fast evaluation of antibacterial properties of essential oils.