Fast Evaluation of Herbal Substance Class Composition by Relative Mass Defect Plots.

A holistic, nontargeted mass spectrometric analysis of any herbal material and preparation is intimately connected to fast chemical profiling and visualization of secondary plant metabolite classes or single compounds. High-resolution mass spectral data enable a broad variety of analytical possibilities. Often a fast and comprehensive overview on compound classes (phytochemical profiling) is needed before single-substance considerations. We present a fast approach for the initial characterization and substance class profiling using relative mass defect plots for the visualization of herbal compositions. From a dataset of 1160 common plant metabolites that represent a varied mixture of molecular classes in polarity, glycosylation, and alkylation, manually annotated for substance classes, the relative mass defects were calculated using theoretical molecular masses. For the calculation of the relative mass defect, a new approach incorporating two correction functions to obtain correct relative mass defect results also for large hydrocarbons, and a multitude of polyhalogenated molecules was developed. Using the Khachyan algorithm, elliptical areas clustering substance classes within the relative mass defect plots were calculated. The resulting novel relative mass defect plots provide a quick way of two-dimensional substance class mapping directly from high-resolution mass spectral data and may be considered as a unique fingerprint for herbals, part of them or herbal preparations. We show that adding the retention time as a third dimension improves the resolution power of the two-dimensional relative mass defect plot and offers the possibility for a more detailed substance class mapping.

Novel Natural Products for Healthy Ageing from the Mediterranean Diet and Food Plants of Other Global Sources-The MediHealth Project.

There is a rapid increase in the percentage of elderly people in Europe. Consequently, the prevalence of age-related diseases will also significantly increase. Therefore, the main goal of MediHealth, an international research project, is to introduce a novel approach for the discovery of active agents of food plants from the Mediterranean diet and other global sources that promote healthy ageing. To achieve this goal, a series of plants from the Mediterranean diet and food plants from other origins are carefully selected and subjected to in silico, cell-based, in vivo (fly and mouse models), and metabolism analyses. Advanced analytical techniques complement the bio-evaluation process for the efficient isolation and identification of the bioactive plant constituents. Furthermore, pharmacological profiling of bioactive natural products, as well as the identification and synthesis of their metabolites, is carried out. Finally, optimization studies are performed in order to proceed to the development of innovative nutraceuticals, dietary supplements or herbal medicinal products. The project is based on an exchange of researchers between nine universities and four companies from European and non-European countries, exploiting the existing complementary multidisciplinary expertise. Herein, the unique and novel approach of this interdisciplinary project is presented.

Near-infrared and Mid-infrared Spectroscopic Techniques for a Fast and Nondestructive Quality Control of Thymi herba.

In this study, novel near-infrared and attenuated total reflectance mid-infrared spectroscopic methods coupled with multivariate data analysis were established enabling the determination of thymol, rosmarinic acid, and the antioxidant capacity of Thymi herba. A new high-performance liquid chromatography method and UV-Vis spectroscopy were applied as reference methods. Partial least squares regressions were carried out as cross and test set validations. To reduce systematic errors, different data pretreatments, such as multiplicative scatter correction, 1st derivative, or 2nd derivative, were applied on the spectra. The performances of the two infrared spectroscopic techniques were evaluated and compared. In general, attenuated total reflectance mid-infrared spectroscopy demonstrated a slightly better predictive power (thymol: coefficient of determination = 0.93, factors = 3, ratio of performance to deviation = 3.94; rosmarinic acid: coefficient of determination = 0.91, factors = 3, ratio of performance to deviation = 3.35, antioxidant capacity: coefficient of determination = 0.87, factors = 2, ratio of performance to deviation = 2.80; test set validation) than near-infrared spectroscopy (thymol: coefficient of determination = 0.90, factors = 6, ratio of performance to deviation = 3.10; rosmarinic acid: coefficient of determination = 0.92, factors = 6, ratio of performance to deviation = 3.61, antioxidant capacity: coefficient of determination = 0.91, factors = 6, ratio of performance to deviation = 3.42; test set validation). The capability of infrared vibrational spectroscopy as a quick and simple analytical tool to replace conventional time and chemical consuming analyses for the quality control of T. herba could be demonstrated.

Application of benchtop and portable near-infrared spectrometers for predicting the optimum harvest time of Verbena officinalis.

This study examined the applicability of near-infrared (NIR) spectroscopy coupled with multivariate data analysis (MVA) to determine the ideal harvest time of Verbena officinalis. NIR analyses were performed non-invasively on the fresh plant material based on the quantification of the key constituents verbenalin and verbascoside. Vibrational spectroscopic measurements were performed applying a conventional NIR benchtop device as well as a laboratory independent handheld NIR spectrometer. A novel high performance liquid chromatography (HPLC) method was applied as a reference method. For both instruments partial least squares (PLS) regression models were established performing cross validations (CV) and test-set validations (TSV). Quality parameters obtained for the benchtop device revealed that the newly established NIR method enabled reliable quantifications of the main compounds verbenalin and verbascoside related to the dried and fresh plant material. The results of the miniaturised spectrometer revealed that accurate quantitative calibration models could be developed for verbascoside achieving a comparable prediction power to the benchtop device. PLS models for verbenalin were less precise suggesting the application of portable devices including a different spectral range and resolution. The work demonstrated the feasibility of NIR vibrational spectroscopy performing direct measurements on pharmaceutically relevant fresh plant material enabling a quick and simple determination of the ideal harvest time of Verbena officinalis.

Solid-phase extraction of galloyl- and caffeoylquinic acids from natural sources (Galphimia glauca and Arnicae flos) using pure zirconium silicate and bismuth citrate powders as sorbents inside micro spin columns.

Galloyl- and caffeoylquinic acids are among the most important pharmacological active groups of natural compounds. This study describes a pre-step in isolation of some selected representatives of these groups from biological samples. A selective solid-phase extraction (SPE) method for these compounds may help assign classes and isomer designations within complex mixtures. Pure zirconium silicate and bismuth citrate powders (325 mesh) were employed as two new sorbents for optimized SPE of phenolic acids. These sorbents possess electrostatic interaction sites which accounts for additional interactions for carbon acid moieties as compared to hydrophilic and hydrophobic sorbents alone. Based on this principle, a selective SPE method for 1,3,4,5-tetragalloylquinic acid (an anti-HIV and anti-asthamatic agent) as a starting compound was developed and then deployed upon other phenolic acids with success. The recoveries and selectivities of both sorbents were compared to most commonly applied and commercially available sorbents by using high performance liquid chromatography. The nature of interaction between the carrier sorbent and the acidic target molecules was investigated by studying hydrophilic (silica), hydrophobic (C18), mixed-mode (ionic and hydrophobic: Oasis(®) MAX) and predominantly electrostatic (zirconium silicate) materials. The newly developed zirconium silicate and bismuth citrate stationary phases revealed promising results for the selective extraction of galloyl- and caffeoylquinic acids from natural sources. It was observed that zirconium silicate exhibited maximum recovery and selectivity for tetragalloylquinic acid (84%), chlorogenic acid (82%) and dicaffeoylquinic acid (94%) among all the tested sorbents.

Solid-phase extraction method for the isolation of plant thionins from European mistletoe, wheat and barley using zirconium silicate embedded in poly(styrene-co-divinylbenzene) hollow-monoliths.

Thionins are cysteine-rich, biologically active small (∼5 kDa) and basic proteins occurring ubiquitously in the plant kingdom. This study describes an efficient solid-phase extraction (SPE) method for the selective isolation of these pharmacologically active proteins. Hollow-monolithic extraction tips based on poly(styrene-co-divinylbenzene) with embedded zirconium silicate nano-powder were designed, which showed an excellent selectivity for sulphur-rich proteins owing to strong co-ordination between zirconium and the sulphur atoms from the thiol-group of cysteine. The sorbent provides a combination of strong hydrophobic and electrostatic interactions which may help in targeted separation of certain classes of proteins in a complex mixture based upon the binding strength of different proteins. European mistletoe, wheat and barley samples were used for selective isolation of viscotoxins, purothionins and hordothionins, respectively. The enriched fractions were subjected to analysis by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometer to prove the selectivity of the SPE method towards thionins. For peptide mass-fingerprint analysis, tryptic digests of SPE eluates were examined. Reversed-phase high-performance liquid chromatography hyphenated to diode-array detection was employed for the purification of individual isoforms. The developed method was found to be highly specific for the isolation and purification of thionins.

Synthesis and in vitro characterization of a preactivated thiomer via polymerization reaction.

The objective of this study was to synthesize 6-(2-acryloylamino-ethyldisulfanyl)-nicotinic acid (ACENA) for subsequent copolymerization with acrylic acid (AA) as a new method for synthesis of preactivated thiomers. Copolymerization reactions of ACENA and AA with different molar ratios were performed and the molecular weight (M(w)) values of the resulting copolymers were calculated and reported from 3046 to 3271 Da. The disulfide bond content values in the polymer chain were determined from 400 to 544 µmol disulfide bond per gram polymer. The transport enhancement ratio for 0.5% (m/v) solution of poly(acrylic acid) (PAA) was 1.1 using sodium fluorescein (Na-Flu) as model drug, in Ussing-type chambers, whereas it was over 1.9 for 0.5% (m/v) solution of ACENA and AA copolymers. Resazurin cell-viability test showed no significant toxicity for the polymers. Copolymerization of AA and disulfide-bond-containing monomers can open new horizons for the preparation of preactivated thiomers taking the better controllability and the huge variety of available monomers and combinations thereof into account.