M. Popp

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.

Analysis of vitamin E in food and phytopharmaceutical preparations by HPLC and HPLC-APCI-MS-MS

SummaryThe analysis of α, β, γ, δ-tocopherols, trienols, α-tocopheryl acetate and nicotinate (vitamin E) in complex matrices was carried out using a new liquid chromatographic (HPLC) method giving better separation efficiency, selectivity and sensitivity than that described in the literature. The use of normal-phase (NP)-HPLC on silica gel with issoctane-diisopropylether-1,4-dioxane as optimized mobilepphase yielded higher resolution than conventional reversed-phase (RP)-HPLC using methanol mobile phase. Identification of peaks was by UV-absorbance at 295 nm, diode array, or fluorescence detection (λex = 295 nm,λex = 330 nm). The latter was found to be more selective and ten times more sensitive than UV-absorbance detection. A quadrupole, ion-trap mass spectrometer with an atmospheric-pressure ionization (APCl) interface was used to detect vitamin E constituents in the femtomole range. With collision-induced dissociation (CID) in the ion source, which gave characteristic fragmentation, the identity of the investigated compounds could be confirmed. Plots of peak area versus amount injected allowed quantitation of α, β, γ, δ-tocopherols and-trienols, α-tocopheryl acetate and nicotinate in real samples such as peanut, almond, spinach, spelt grain bran, latex and tablets. The method described offers fast identification and quantitation of vitamin E constituents of complex biological origin.

Isotachophoretic analysis of flavonoids and phenolcarboxylic acids of relevance to phytopharmaceutical industry

Abstract Using capillary isotachophoresis, the rapid analysis of flavonoids and phenolcarboxylic acids was accomplished in the nanomole range. Optimum separations were achieved at pH 9.5 with a leading electrolyte containing 15 mM hydrochloric acid, 30% methanol and 0.2% hydroxypropylmethylcellulose. Under these conditions, the content of rutin could be determined in a methanolic extract of Sambuci flos, which is a common constituent of phytopharmaceutical products.

Simultaneous quantification of verbenalin and verbascoside in Verbena officinalis by ATR-IR and NIR spectroscopy

Attenuated-total-reflectance infrared spectroscopy (ATR-IR) and near-infrared diffuse reflectance spectroscopy (NIR) in hyphenation with multivariate analysis was utilized to quantify verbenalin and verbascoside in Verbena officinalis. A new high performance liquid chromatography (HPLC) method as a reference was established and validated. For both vibrational spectroscopic methods test-set and cross validation were performed. Different data-pre-treatments like SNV, 1st and 2nd derivative were applied to remove systematic errors and were evaluated. Quality parameters obtained for the test-set validation revealed that ATR-IR (verbenalin: R(2)=0.94, RPD=4.23; verbascoside: R(2)=0.93, RPD=3.63) has advantages over NIR (verbenalin: R(2)=0.91, RPD=3.75; verbascoside: R(2)=0.80, RPD=2.35) in the given application.

Comparison of NIR and ATR-IR spectroscopy for the determination of the antioxidant capacity of Primulae flos cum calycibus

In this study, near-infrared (NIR) and attenuated-total-reflectance infrared (ATR-IR) spectroscopy techniques in hyphenation with partial least squares (PLS) regression were utilized to determine the antioxidant capacity of Primulae flos cum calycibus samples. Folin–Ciocalteu (FC), ferric ion reducing antioxidant power (FRAP), 2,2-diphenyl-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and cupric reducing antioxidant capacity (CUPRAC) assays were performed as reference methods. Different spectral pretreatments such as standard normal variate (SNV), 1st or 2nd derivative, were applied to remove scattering effects. For all assays, cross and test-set validations were performed. The ability of the two spectroscopic techniques to replace the five assays was evaluated and compared. The standard error of prediction (SEP) and the ratio performance deviation (RPD) were determined and corrected for the imprecision of the reference data to obtain the respective SEPcorr and RPDcorr values. In general, NIR demonstrated advantages over ATR-IR spectroscopy and resulted best for the ABTS assay (R2: 0.94, RPDcorr: 4.66; test-set validation). Also with ATR-IR spectroscopy, the best prediction power was obtained for the ABTS assay (R2: 0.94, RPDcorr: 4.10; test-set validation). The feasibility of vibrational spectroscopy as a fast and simple tool to replace wet chemistry assays for the measurement of the antioxidant capacity of Primulae flos cum calycibus samples was demonstrated.

Identification of Verbena officinalis Based on ITS Sequence Analysis and RAPD-Derived Molecular Markers

Verbenae herba is a widely used drug and consists of the aerial parts of Verbena officinalis (Verbenaceae). Until now, the identification has been performed based on morphological and phytochemical analyses, which are not reliable enough to distinguish Verbena officinalis from other relevant species of the genus Verbena. Hence, impurities and adulterants, negatively influencing the therapeutic effect of the drug, may remain undetected. In an attempt to generate an accurate authentication method we used two different DNA-based approaches: comparison of ITS sequences and molecular markers (RAPD). Both approaches generally enabled discrimination of V. officinalis from the rest of the genus despite the intraspecific variation existing within V. officinalis. The application of the two independent methods, supporting each other, increases the security of identification. For better reproducibility and faster analysis, however, a SCAR marker and primers for HRM were derived from the RAPD results. The SCAR marker could distinguish V. officinalis from all other verbena species except its closest relative V. hastata, while discrimination of V. officinalis even from V. hastata was unproblematic with HRM.

Fourier transform infrared imaging analysis in discrimination studies of St. John's wort (Hypericum perforatum)

In the present study, Fourier transform infrared (FTIR) imaging and data analysis methods were combined to study morphological and molecular patterns of St. Johns wort (Hypericum perforatum) in detail. For interpretation, FTIR imaging results were correlated with histological information gained from light microscopy (LM). Additionally, we tested several evaluation processes and optimized the methodology for use of complex FTIR microscopic images to monitor molecular patterns. It is demonstrated that the combination of the used spectroscopic method with LM enables a more distinct picture, concerning morphology and distribution of active ingredients, to be gained. We were able to obtain high-quality FTIR microscopic imaging results and to distinguish different tissue types with their chemical ingredients.

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.

Comparison of near-infrared diffuse reflectance (NIR) and attenuated-total-reflectance mid-infrared (ATR-IR) spectroscopic determination of the antioxidant capacity of Sambuci flos with classic wet chemical methods (assays)

Near-infrared diffuse reflectance (NIR) and attenuated-total-reflectance mid-infrared (ATR-IR) spectroscopy techniques in hyphenation with multivariate analysis were utilized to determine the antioxidant capacity of ground Sambuci flos samples. Folin–Ciocalteu (FC), ferric ion reducing antioxidant power (FRAP), cupric reducing antioxidant capacity (CUPRAC), 2,2-diphenyl-picrylhydrazyl (DPPH) and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were optimized and performed as reference methods. To remove systematic errors several spectral pretreatments like 1st and 2nd derivative Savitzky–Golay, standard normal variate (SNV) or multiplicative scatter correction (MSC) were applied. Cross-validations and test-set validations were performed for all assays. The quality parameters, standard error of prediction (SEP) and the ratio performance deviation (RPD), were calculated. An acceptable quality of the calibration can be confirmed for ATR-IR spectroscopy (e.g. for the CUPRAC assay: R2: 0.85, RPDcorr: 2.68, SECV: 0.13% GAE for cross-validation; R2: 0.81, RPDcorr: 2.20, SEP: 0.15% GAE for test-set validation). Surprisingly all models calculated for NIR spectroscopy were of poor quality and point to unpredictability of the antioxidative capacity. Further investigations of extracts by high performance liquid chromatography (HPLC) with a diode array detector (DAD) coupled to mass spectroscopy (MS) were performed to analyze the principal compounds. Thus, rutin and chlorogenic acid were confirmed to be the main components in the samples. This study demonstrates that ATR-IR spectroscopy is suitable to determine the antioxidative capacity in ground Sambuci flos samples and can be used for quality control.