Purified novel and new diferuloyl glycerate related phenolic acid from Pandanus odoratissimus flowers shows antioxidant, invertase inhibition and control against diabetic foot ulcer (DFU) causing bacterial pathogens - An in vitro study to establish an effective regulation over type 2 diabetes mellitus

Abstract Chronic type 2 diabetes mellitus (T2DM) and its associated diseases are major concern among human population and also responsible for significant mortality rate. Hence, the present study aims to evaluate and correlate the invertase inhibition, antioxidant activity and control against DFU causing bacterial pathogens by Pandanus odoratissimus flowers. Two dimensional preparative thin layer chromatography (2D PTLC) was adopted to purify the phenolic acid component and LC-MS2 was done to predict the phenolic acid structures. Standard spectrophotometry methods were adopted to investigate the in vitro invertase inhibitory and antioxidant (CUPRAC and ABTS) activities. Agar well diffusion and broth dilution assays were used to record the antibacterial property against DFU causing pathogens isolated from clinical samples. Statistical analyses were used to validate the experiments. A new and novel diferuloyl glycerate related phenolic acid (m/z 442) purified from PTLC eluate has recorded satisfactory cupric ion reducing power (ED50= 441.4±2.5 µg), moderate ABTS radical scavenging activity (IC50= 450.3±10 µg; 32.5±1.5%), and a near moderate, in vitro, invertase mixed type inhibition (24.5±4.5%; Ki: 400 µg). Similarly, bacterial growth inhibitory kinetics has showed a significant inhibition against E. coli and S. aureus.

Mass spectrometry imaging of thin-layer chromatography plates using laser desorption/low-temperature plasma ionisation.

Thin-layer chromatography (TLC) is a classic method for the separation and analysis of complex mixtures. Biological assays, chemical derivatisation and spectroscopy techniques are compatible with TLC and provide extra information about isolated compounds. However, coupling TLC to mass spectrometry is hampered by the difficulty to desorb the analytes from the silica surfaces. In this study, we used a multimodal ion source for laser desorption (LD) and low-temperature plasma (LTP) post-ionisation. Efficient desorption was reached by covering the TLC plates with activated carbon. Regions of interest can be analysed by spots, by lines or by area. We show the separation of methylxanthines from coffee, tea and cocoa preparations by TLC, with subsequent mass spectrometry imaging (MSI). Using a lateral resolution of 400 µm × 400 µm, allowed the acquisition of 21 895 spectra in 2.4 h (2.5 pixels per s). Further, we demonstrate the possibility of direct mass fragmentation studies and quantification. We mounted the system on an Open LabBot with a theoretical lateral resolution of 12.5 µm and performed the visualisation of ions of interest and the pixel-wise review of mass spectra with our free software RmsiGUI (). This non-proprietary and modular platform enables the cost-efficient adaption of the system and further development by the community.

Optimization of the procedure of solvent front position extraction for preparation of multi-component sample for instrumental analysis.

Solvent Front Position Extraction (SFPE) procedure has been recently introduced as a novel concept for multi-component sample preparation. According to the procedure, thin-layer chromatography (TLC) is used to separate the compounds of interest from matrix components, and to focus them into a common zone from which the compounds are extracted and transferred to apparatus for instrumental analysis. In the paper, we investigate different adsorbent types of the chromatographic plates and various mobile phases, including pH of their buffers, in respect of optimization conditions of the SFPE procedure. The research was carried out using a test sample containing 9 compounds characterised by different chemical properties, hence the conclusions from the obtained results can be applied to other multi-component samples. Under the optimal conditions, all target compounds are separated from other compounds (matrix), and evenly distributed along a narrow strip, which is advantageous for their quantitation. The determination results are good, the percentage values of relative error and relative standard deviation do not exceed 6%.

[Primary study on computer-aided qualitative and quantitative radial thin-layer chromatography methods for analysis of Yiqing tablets].

Radial thin-layer chromatography (RTLC) is a form of chromatography in which the sample and developing agent/solvent are applied at the edge of a circle on the silica-layered TLC plate. The sample components are radially transported from the center of the circle toward the periphery by the solvent flow. RTLC is an efficient separation and semi-preparation method, with the advantages of fast separation, low diffusion, and absence of tailing at a certain distance. Although RTLC isolation has been well documented in the literature, there are only a few qualitative and quantitative reports on this technique. In this study, a self-programming module combined with image processing software was adopted for digital processing of RTLC images, and a digital atlas was created. Qualitative and quantitative verification of RTLC was then conducted. With the aid of computer technology, qualitative and quantitative methods of radially expanded TLC for Yiqing tablets were investigated. The results showed that the qualitative RTLC method has good reliability, but the quantitative RTLC method has inherent drawbacks, which may be associated with the experimental environment. We strongly believe that better separation and data acquisition equipment, as well as the combination of computer image processing and programming methods, will open up infinite possibilities of analysis/separation using RTLC-based techniques. We also believe that TLC will take a new step in the future with the assistance of computer technology.

Analysis and Enantioseparation of Amino Acids by Liquid Chromatography.

Enantioseparation studies of proteinogenic, non-proteinogenic, and dansyl amino acids are described herein by using liquid chromatographic techniques, i.e., HPLC and TLC. A researcher who wants to perform amino acid (AA) analysis or separate enantiomers of AAs by HPLC or TLC can follow the method. Figures included represent the actual experiments.Synthesis and application of chiral derivatizing reagents (CDRs) based on cyanuric chloride (CC) and difluorodinitrobenzene (DFDNB) have been described for AA analysis and enantioseparation by indirect approach. The methods represent pre-column derivatization of AAs and represent a good and less expensive substitute of AA analyzer. The application of commercial "Chiralplate" and use of erythromycin and L-tartaric acid have been described as chiral selector either as impregnating reagent in the stationary phase or as an additive in the mobile phase for direct enantioseparation by TLC. Application of the homemade TLC plates has also been described; the methods are successful in obtaining the native enantiomer as well.

Simple interface for scanning chemical compounds on developed thin layer chromatography plates using electrospray ionization mass spectrometry.

A simple and cheap design for interfacing thin layer chromatography (TLC) with electrospray ionization mass spectrometry (ESI/MS) was developed to scan and characterize compounds on TLC plate. The developed TLC plate was rapidly and easily modified into two sawtooth-edged pieces that were positioned on an XYZ stage so that one of the triangular tips was pointed toward the MS inlet. A drop of methanol and high DC voltage was applied at the tip to induce ESI. After the analytes in the first tip were analyzed, the TLC piece was moved so that the second triangular tip was pointed toward the MS inlet for analysis. The process was repeated until all the triangular tips on the piece were analyzed. In this manner, the analytes, no matter visible or non-visible bands, were scanned and characterized. Since a 4.8 cm long TLC track were cut to 32 triangles on two sawtooth pieces for analysis, the spatial resolution of using the sawtooth TLC-ESI/MS for analysis is 1.5 mm/band. A mixture of dye standards and Datura metel flower extract was analyzed to demonstrate the capability of sawtooth TLC-ESI/MS on scanning and characterizing chemical compounds on the TLC plates. The limits of detection of the dye standards were between 0.25 and 2.5 ng/band. TLC bands containing alkaloids such as scopolamine and norscopolamine from the Datura metel flower extract were not visualized on the developed TLC track, but were successfully detected at different triangular tips using sawtooth TLC-ESI/MS. Based on these results, the Rf values of scopolamine and norscopolamine were determined.

Classification of Romanian medicinal plant extracts according to the therapeutic effects using thin layer chromatography and robust chemometrics.

Thin layer chromatography in combination with image analysis and advanced chemometric methods were successfully used to classify the medicinal herbs according to their therapeutic effects and usage. The investigations were conducted using two types of plates (HPTLC Silica gel 60 and HPTLC Silica gel 60 F254) which were evaluated in UV light at 254 and 365 nm. The holistic evaluation of the numerical data corresponding different image processing channels (blue, grey, red, green) was performed by employing appropriate multivariate methods: hierarchical cluster analysis (HCA), principal component analysis (PCA), fuzzy principal component analysis (FPCA) and linear discriminant analysis (LDA) applied to the first relevant principal components. The results obtained by applying LDA method indicate a highly accurate separation of the medicinal herbs within the four groups, in good agreement with therapeutic effects and usage. According to this classification, the best image processing channels were identified for each of the investigated HPTLC plates: blue channel for HPTLC Silica gel 60 F254 (with 92.9% percent of discrimination in case of PCA and FPCA) and respectively red channel for HPTLC Silica gel 60 (with 93.9% percent of discrimination in case of FPCA). The 2D and 3D score scatterplots illustrate also the accurate and reliable discrimination between the four distinct groups.

Biennial Review of Planar Chromatography: 2015-2017.

The most important advances in planar chromatography published between November 1, 2015, and November 1, 2017, are reviewed in this paper. Included are an introduction to the current status of the field; student experiments and reviews; apparatus and techniques for sample preparation and TLC separations; detection and identification of separated zones; quantitative analysis; preparative layer chromatography; and thin-layer radiochromatography. Selected applications are given in the various sections of the review.

Kinetic Analysis of the Exonuclease Activity of the Bacteriophage T4 Mre11-Rad50 Complex.

Bacteriophage T4 encodes orthologs of the proteins Rad50 (gp46) and Mre11 (gp47), which form a heterotetrameric complex (MR) that is responsible for host genome degradation and the processing of DNA ends for recombination-dependent DNA repair. In this chapter, we describe the ensemble methods currently employed by our laboratory to characterize the exonuclease activity of the T4 MR complex. DNA exonucleases play a vital role in maintaining the integrity of DNA through their participation in DNA repair pathways and as proofreaders for DNA polymerases. Methods for quantifying the general features of the exonuclease, and for determining steady-state kinetic parameters (Km, kcat), the polarity of exonuclease activity, and processivity are presented. These methods should be applicable to all DNA exonucleases, and to some extent endonucleases.

Centrifugal-driven, reduced-dimension, planar chromatography.

A fundamental problem with efficiency in capillary action driven planar chromatography results from diminishing flow rates as development proceeds, giving rise to molecular diffusion related band dispersion for most sample types. Overpressure and electrokinetic means to speed flow have been used successfully in TLC. We explore the use of centrifugal force (CF) to drive flow for reduced-dimension planar platforms (ultra-TLC, low micrometer features, and nano-TLC, nanoscale features). The silicon wafer platforms have two forms of continuous 2D arrays created by either photolithography or metal dewetting followed by deep reactive ion etching and coated with porous SiO2 . The flow pattern is unusual with co-planar flows above and within the arrays. The effects of parameters such as spin rate, solvent type, and surface character on flow rates is established and can be substantially greater than capillary action flow. Using fluorescent dyes, we investigate retardation factors and chromatographic plate height; the latter falls in the low to sub-micrometer range. To the best of our knowledge, we demonstrate the first analytical separations performed in pillar arrays using CF to augment solvent flow.

Logit-log evaluation of planar yeast estrogen screens.

Receptor assays like the yeast estrogen screen (YES) performed in microtiter plates normally provide dose-response curves with a sigmoidal shape in semi-log plots. Such sigmoidal plots can be linearized by the logit function resulting in logit-log plots, as mainly known for the evaluation of enzyme-linked immunosorbent assays and radioimmunoassays. Since the planar yeast estrogen screen (pYES) represents the transfer of the receptor assay YES to high-performance thin-layer chromatography (HPTLC), it was assumed to obtain sigmoidal shaped dose-response curves from the measured signals, which subsequently could be used to generate logit-log plots. However, it was observed that typical sigmoidal curves were not obtained, when peak areas were plotted against the applied amount on a logarithmic scale (log amount). Therefore, peak heights were examined in the present study, which revealed proper dose-response curves when plotted against the log amount. The presence of sigmoidal dose-response curves from HPTLC-pYES made it possible to transform the signals into logits and, therefore, to create logit-log plots with linear correlations. The logit-log plots for the estrogen active compounds (EAC) 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2) provided a working range up to 500pg/zone. Applying logit-log plots, mean recovery rates for E2 and EE2 from spiked water samples (2-20ng/L) were determined to 90% and 108%, respectively, with ≤24% RSD. Moreover, the linear graphs allowed an easy determination of the half maximal effect dose (ED50) of EAC, since the intersection of the graph with the abscissa represents the ED50. Additionally, with the knowledge of the ED50 values, the estrogenic potential of EAC in terms of estradiol equivalent factors (EEF) could be determined, resulting in 0.64 for EE2.

Sweet spot matching: A thin-layer chromatography-based countercurrent solvent system selection strategy.

TLC-based strategies were proposed in 1979 (Hostettmann et al.) and 2005 (Friesen & Pauli; GUESS method) to minimize the number of partitioning experiments required for countercurrent separation (CCS) solvent system selection. As semi-empirical approaches, both proposed that the K values defining the sweet spot of optimal CCS corresponded to a matching Rf value range from the silica gel TLC plate developed in the organic phase of a biphasic or a corresponding monophasic solvent system. Despite their simplicity, there has been an absence of theoretical support and a deficiency of reported experimental evidence. The present study explores the theory required to develop correlations between Rf and K. All theoretical models surmise that the optimal Rf value range should be centered at 0.5. In order to validate the feasibility of the concept of matching Rf and K values, 43 natural products and six solvent system families were investigated. Out of 62 correlations, 45 resulted in matched Rf and K values. Based on this study, practical guidelines for the TLC-based prediction strategy are provided. These approaches will equip CCS users with an updated understanding of how to apply the TLC-based solvent system selection strategy to accelerate a targeted selection of CCS conditions.

Lysergic acid amide as chemical marker for the total ergot alkaloids in rye flour - Determination by high-performance thin-layer chromatography-fluorescence detection.

Ergot alkaloids are generally determined by high-performance liquid chromatography (HPLC) coupled to fluorescence detection (FLD) or mass selective detection, analyzing the individual compounds. However, fast and easy screening methods for the determination of the total ergot alkaloid content are more suitable, since for monitoring only the sum of the alkaloids is relevant. The herein presented screening uses lysergic acid amide (LSA) as chemical marker, formed from ergopeptine alkaloids, and ergometrine for the determination of the total ergot alkaloids in rye with high-performance thin-layer chromatography-fluorescence detection (HPTLC-FLD). An ammonium acetate buffered extraction step was followed by liquid-liquid partition for clean-up before the ergopeptine alkaloids were selectively transformed to LSA and analyzed by HPTLC-FLD on silica gel with isopropyl acetate/methanol/water/25% ammonium hydroxide solution (80:10:3.8:1.1, v/v/v/v) as the mobile phase. The enhanced native fluorescence of LSA and unaffected ergometrine was used for quantitation without any interfering matrix. Limits of detection and quantitation were 8 and 26µg LSA/kg rye, which enables the determination of the total ergot alkaloids far below the applied quality criterion limit for rye. Close to 100% recoveries for different rye flours at relevant spiking levels were obtained. Thus, reliable results were guaranteed, and the fast and efficient screening for the total ergot alkaloids in rye offers a rapid alternative to the HPLC analysis of the individual compounds.

New simple and low-cost methods for periodic checks of Cyclone® Plus Storage Phosphor System.

The recent large use of the Cyclone® Plus Storage Phosphor System, especially in European countries, as imaging system for quantification of radiochemical purity of radiopharmaceuticals raised the problem of setting the periodic controls as required by European Legislation. We described simple, low-cost methods for Cyclone® Plus quality controls, which can be useful to evaluate the performance measurement of this imaging system.

A simple analytical platform based on thin-layer chromatography coupled with paper-based analytical device for determination of total capsaicinoids in chilli samples.

A new analytical platform based on the use of thin-layer chromatography (TLC) coupled with paper-based analytical device (PAD) was developed for the determination of total capsaicinoids in chilli samples. This newly developed TLC-PAD is simple and low-cost without any requirement of special instrument or skillful person. The analysis consisted of two steps, i.e., extraction of capsaicinoids from chilli samples by using ethanol as solvent and separation of capsaicinoids by thin-layer chromatography (TLC) and elution of capsaicinoids from the TLC plate with in situ colorimetric detection of capsaicinoids on the PAD. For colorimetric detection, Folin-Ciocalteu reagent was used to detect phenolic functional group of capsaicinoids yielding the blue color. The blue color on the PAD was imaged by a scanner followed by evaluation of its grayscale intensity value by ImageJ program. This newly developed TLC-PAD method provided a linear range from 50 to 1000mgL-1 capsaicinoids with the limit of detection as low as 50mgL-1 capsaicinoids. The proposed method was applied to determine capsaicinoids in dried chilli and seasoning powder samples and the results were in good agreement with those obtained by HPLC method.

Comparative study of different approaches for multivariate image analysis in HPTLC fingerprinting of natural products such as plant resin.

Considering the introduction of phytochemical fingerprint analysis, as a method of screening the complex natural products for the presence of most bioactive compounds, use of chemometric classification methods, application of powerful scanning and image capturing and processing devices and algorithms, advancement in development of novel stationary phases as well as various separation modalities, high-performance thin-layer chromatography (HPTLC) fingerprinting is becoming attractive and fruitful field of separation science. Multivariate image analysis is crucial in the light of proper data acquisition. In a current study, different image processing procedures were studied and compared in detail on the example of HPTLC chromatograms of plant resins. In that sense, obtained variables such as gray intensities of pixels along the solvent front, peak area and mean values of peak were used as input data and compared to obtained best classification models. Important steps in image analysis, baseline removal, denoising, target peak alignment and normalization were pointed out. Numerical data set based on mean value of selected bands and intensities of pixels along the solvent front proved to be the most convenient for planar-chromatographic profiling, although required at least the basic knowledge on image processing methodology, and could be proposed for further investigation in HPLTC fingerprinting.

Ultra-thin layer chromatography with integrated silver colloid-based SERS detection.

Simplified lab-on-a-chip techniques are desirable for quick and efficient detection of analytes of interest in the field. The following work involves the use of deterministic pillar arrays on the micro-scale as a platform to separate compounds, and the use of Ag colloid within the arrays as a source of increased signal via surface enhanced Raman spectroscopy (SERS). One problem traditionally seen with SERS surfaces containing Ag colloid is oxidation; however, our platforms are superhydrophobic, reducing the amount of oxidation taking place on the surface of the Ag colloid. This work includes the successful separation and SERS detection of a fluorescent dye compounds (resorufin and sulforhodamine 640), fluorescent anti-tumor drugs (Adriamycin and Daunomycin), and purine and pyrimidine bases (adenine, cytosine, guanine, hypoxanthine, and thymine).

High performance thin-layer chromatography-mass spectrometry of Japanese knotweed flavan-3-ols and proanthocyanidins on silica gel plates.

On-line elution based TLC-MS is now a well-established technique, but the quality of the data obtained can sometimes be hampered by a severe spectral background or by strong ion suppression, especially when silica gel plates are used in combination with an acidic modifier in the developing solvent. We solved this issue simply and efficiently using two pre-developments of the plates, firstly with methanol-formic acid (10:1, v/v) and secondly with acetonitrile-methanol (2:1, v/v). This solution resulted in significant improvement in the sensitivity of HPTLC-MS methods. The applicability of this approach was proven on analysis of flavan-3-ols and proanthocyanidins in crude extracts of Japanese knotweed (Fallopia japonica Houtt.) rhizomes. Separations on HPTLC silica gel and HPTLC silica gel MS grade plates using developing solvents toluene-acetone-formic acid (3:3:1, 6:6:1, 3:6:1, v/v) and dichloromethane-acetone-formic acid (1:1:0.1, v/v) were followed by post-chromatographic derivatization with 4-dimethylaminocinnamaldehyde (DMACA) detection reagent. Examination of the stability of the analytes on the start confirmed that the plates should be developed immediately after the application of standards and sample test solutions. In a five hours stability testing after development we discovered an unexpected phenomenon of enhanced absorption at 280nm. However, based on an experiment with post-chromatographic derivatization with DMACA detection reagent, the analytes were proven to be sufficiently stable in the time frame of an HPTLC-MS analysis. This was important for development of the first HPTLC-MS and HPTLC-MSn methods for identification of flavan-3-ols and B-type proanthocyanidins from monomers up to decamers. For the first time, based on this research methodology, trimers, trimer gallates, tetramer gallates, pentamers, pentamer gallates, hexamers, hexamer gallates, heptamers, octamers, nonamers and decamers were tentatively identified in Japanese knotweed rhizomes. Additionally, all developed HPTLC-MS methods have enabled simultaneous identification of stilbenes (resveratrol, piceatannol hexoside, piceid) and anthraquinones (emodin, emodin-O-hexoside, emodin-O-(acetyl)-hexoside and emodin-O-(6'-O-malonyl)-hexoside).

The bridge between thin layer chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry: The realization of liquid thin layer chromatography-mass spectrometry (LTLC-MS).

The combination of thin layer chromatography (TLC) and mass spectrometry (MS) has been studied for decades, but for most cases MS detection is done after TLC separation is finished. Here, an online simultaneous TLC-MS analysis system, liquid thin layer chromatography-mass spectrometry (LTLC-MS), is developed which successfully synchronize TLC separation process and MS detection process like GC-MS and HPLC-MS do. And there's no need to use specially designed TLC, just regular TLC plates are enough. LTLC-MS method is composed of a newly developed ambient ionization method, glow discharge-matrix assisted infrared desorption ionization (GD-MAIRDI), and forced-flow TLC (FFTLC) technique, which guarantees the MS detection process does not disturb the TLC separation process throughout the whole analysis. The whole LTLC-MS analysis only need two steps and less than 15min. Mixtures as well as the two main components of a pain relief pills have been successfully analyzed by LTLC-MS. This proof of concept study opens up new possibilities of combining TLC with MS, and will further broaden the application abilities of TLC.

Use of thin-layer chromatography to detect counterfeit sulfadoxine/pyrimethamine tablets with the wrong active ingredient in Malawi.

BACKGROUND: Substandard and falsified anti-malarial medicines pose a serious threat to public health, especially in low-income countries. Appropriate technologies for drug quality analysis in resource-limited settings are important for the surveillance of the formal and informal drug market. The feasibility of thin-layer chromatography (TLC) with different solvent systems was tested using the GPHF Minilab in a study of the quality of sulfadoxine/pyrimethamine tablets in Malawi. METHODS: Twenty eight samples of sulfadoxine/pyrimethamine tablets were collected from randomly selected health facilities of four districts of southern Malawi. A mystery shopper approach was used when collecting samples from illegal street vendors, and an overt approach for the other facilities. Samples were subjected to visual inspection, disintegration testing and TLC analysis. 10 samples were further investigated according to the methods of the US Pharmacopeia using high performance liquid chromatography (HPLC). RESULTS: One sample was found to be falsified, containing a mixture of paracetamol tablets and co-trimoxazole tablets. These had been repackaged into paper strip packs labelled as a brand of sulfadoxine/pyrimethamine. TLC with different solvent systems readily proved that these tablets did not comply with their declaration, and provided strong evidence for the active pharmaceutical ingredients which were actually contained. Full pharmacopeial analysis by HPLC confirmed the results suggested by TLC for this sample, and showed two further samples to be of substandard quality. CONCLUSIONS: Due to the absence of the declared anti-malarial ingredients and due to the presence of other pharmaceutical ingredients, the identified falsified medicine represents a serious health risk for the population. Thin-layer chromatography (TLC) using different solvent systems proved to be a powerful method for the identification of this type of counterfeiting, presenting a simple and affordable technology for use in resource-limited settings.