Guenther Stecher

Proanthocyanidins: target compounds as antibacterial agents.

Grape seeds accumulate in huge quantities as byproduct during wine production and are therefore a cheap source for pharmacologically active agents. However, studies prove poor antibacterial activity, and results of analyses are sometimes contradictory. The aim of this study was, thus, to determine the antibacterial activity of grape seed extracts with special focus on the chromatographic characterization of active fractions. In the course of these investigations, extraction protocols were optimized so that microwave-assisted extraction (MAE) guaranteed highest preconcentration efficiency. Proanthocyanidins, monomeric flavonoid aglycones, as well as some of their glycosides could be identified within yielded extracts via high-performance liquid chromatography-mass spectrometry (HPLC-MS). By that means the coherence number of possible isomers of procyanidins was approximated by a newly developed equation. As far as antibacterial activity determined via screening tests is concerned, the extracts generally have been found to be positively responsive toward 10 different gram-positive and gram-negative bacteria strains. After fractionation of the raw extracts, proanthocyanidins P2, P3, P4 and gallate esters P2G and P3G (P = proanthocyanidin consisting of catechin and epicatechin units, n = oligomerization degree, G = gallate ester) were determined as active antibacterial agents toward 10 different pathogens. Only moderate activity was found for monomeric flavonoid fractions.

Quality assessment and quantitative analysis of flavonoids from tea samples of different origins by HPLC-DAD-ESI-MS.

Components of green tea ( Camellia sinensis) have been of considerable interest in recent years because of their potential utility as pharmaceutical agents, particularly for their antioxidant and anticarcinogenic activity. Responding to the increasing scientific validation of numerous health benefits of tea, a comprehensive approach was adopted to carry out analysis for the quality assessment of flavonoids in tea samples of different origins. For this purpose, extraction, separation, and mass spectrometric parameters were optimized. Extraction methods evaluated include reflux extraction, a modified accelerated solvent extraction (ASE), namely, Aquasolv extraction, and microwave-assisted extraction (MAE) using different percentages of solvents. Separation was performed by a specifically developed reversed phase high-performance liquid chromatography (RP-HPLC) method using different C18 and C8 stationary phases. Optimization of extraction techniques clearly proved the performance of MAE, which delivered highest yields in a very short time. Additionally, the comparison with Aquasolv extraction provided new insights, as variations in quantified amounts of target compounds between the extracts could be explained on the basis of thermal degradation and epimerization phenomena. Especially the epimerization phenomenon for catechin/epicatechin oligomers, that is, of procyanidins P 2 and P 3, was observed for the first time. Finally, an optimized extraction and separation system was used for qualitative and quantitative investigations of compounds from different green tea samples from Ceylon (cultivated under biologically controlled conditions), Japan, India, and China as well as from one black tea sample from India.

Determination of carbohydrates in medicinal plants-comparison between TLC, mf-MELDI-MS and GC-MS

INTRODUCTION Quality control in the pharmaceutical and phytopharmaceutical industries requires fast and reliable methods for the analysis of raw materials and final products. OBJECTIVE This study evaluates different analytical approaches in order to recognise the most suitable technique for the analysis of carbohydrates in herbal drug preparations. METHODOLOGY The specific focus of the study is on thin-layer chromatography (TLC), gas chromatography (GC), and a newly developed mass spectrometric method, i.e. matrix free material enhanced laser desorption/ionisation time of flight mass spectrometry (mf-MELDI-MS). Samples employed in the study were standards and microwave-assisted water extracts from Quercus. RESULTS TLC analysis proved the presence of mono-, di- and trisaccharides within the biological sample and hinted at the existence of an unknown carbohydrate of higher oligomerisation degree. After evaluation of different derivatisation techniques, GC-MS confirmed data obtained via TLC for mono- to trisaccharides, delivering additionally quantified values under a considerable amount of time. A carbohydrate of higher oligomerisation degree could not be found. The application of mf-MELDI-MS further confirmed the presence of carbohydrates up to trisaccharides, also hinting at the presence of a form of tetrasaccharide. Besides this information, mf-MELDI-MS delivered further data about other substances present in the extract. Quantitative determination resulted in 1.750, 1.736 and 0.336 mg/mL for glucose, sucrose and raffinose respectively. CONCLUSION Evaluation of all three techniques employed, clearly proved the heightened performance of mf-MELDI-MS for the qualitative analysis of complex mixtures, as targets do not need modification and analysis requires only a few minutes. In addition, GC-MS is suitable for quantitative analysis.

Preparation of polymer based sorbents for solid phase extraction of polyphenolic compounds

This article discusses the preparation of different polymeric sorbents for solid phase extraction. Various monomers like acrylamide, methacrylic acid and 4-vinylpyridine (VP), cross-linkers such as divinylbenzene (DVB) and ethyleneglycoldimethacrylate (EDMA), porogens like tetrahydrofuran (THF) and dimethylsulphoxide (DMSO) / acetonitrile (ACN) with different ratios were investigated in order to optimize recoveries. Resulting polymers were characterized through scanning electron microscope (SEM) and compared with Oasis HLB (Waters, MA, USA) and Strata-X (Phenomenex, Torrance, USA) on the basis of extraction performance, recovery efficiency and loading capacity. Sample applied was a mixture of flavonoid standards (rutin, myricetin, luteolin, quercetin, apigenin and kaempferol). HPLC hyphenated with PDA was used for the analysis of samples. Results showed that among the prepared SPE materials, 4-VP-co-EDMA produced best results. Comparison of the produced polymers with of Oasis HLB and Strata-X resulted in comparable efficiencies; especially the polymer 4-VP-co-EDMA gave almost similar results for all analytes to those of commercially available SPE materials. A general trend of decrease in retention efficiency with increase in polarity has been observed in both synthesized and already available SPE materials. The newly synthesized polymeric materials can be employed as SPE sorbents for efficient extraction of polyphenolic compounds especially for flavonoid aglycons.

Online coupling of thin layer chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: synthesis and application of a new material for the identification of carbohydrates by thin layer chromatography/matrix free material enhanced laser desorption/ionization mass spectrometry

This article describes the online hyphenation of thin layer chromatography with matrix free material enhanced laser desorption/ionization mass spectrometry (mf-MELDI-MS), the preparation of new material for MELDI and application of this newly synthesized material using TLC/MELDI-MS for the analysis of carbohydrate reference standards and plant extracts. Samples included within these analyses are standard solutions of glucose, sucrose, raffinose and a plant extract of Quercus robur, which is used for its anti-inflammatory, anti-viral and anthelminitc properties in phytomedicine. A new material for mf-MELDI-MS is prepared by immobilizing bradykinin--a peptide, on silica gel coupled to 4-(3-triethoxysilylpropylureido)azobenzene. This modification enables the absorption of laser energy sufficient for desorption and ionization of low molecular weight molecules like carbohydrates and amino acids. The newly synthesized material delivered excellent results in respect to signal-to-noise (S/N) ratio (S/N ratio: >9/1) and sensitivity (limit of detection (LOD): lower to ng/microL). Hyphenation of TLC to MELDI-MS employing the novel developed material simultaneously as chromatographic and mass spectrometric sorbent was shown for the first time for the analysis of low molecular weight molecules like mono- and oligosaccharides.

Matrix Free Material Enhanced Laser Desorption Ionization Mass Spectrometric Analysis of Amino Acids in Althaea Officinalis, Matricaria Chamomilla, and Taraxacum Officinale

The standardization of natural product drugs needs reliable methods for the analysis of raw materials and final products. In this study, qualitative analysis of amino acids in herbal extracts was performed using mass spectrometric method, that is, matrix- free material enhanced laser desorption ionization time of flight mass spectrometry (mf-MELDI-MS). Samples employed in the study were standards and water extracts from Althaea officinalis, Taraxacum officinale, and Matricaria chamomilla. These analyses confirmed the presence of amino acids and clearly proved the adequate performance of mf-MELDI-MS for the qualitative analysis of complex mixtures, as targets do not need modification and analysis requires only a few minutes. Determination of amino acids in these plants were performed and reported for the first time using the modified silica gel (4,4′-azodianiline modified silica gel) and mf-MELDI-MS technique.