Dorota Staszek

Development of chromatographic and free radical scavenging activity fingerprints by thin-layer chromatography for selected Salvia species.

INTRODUCTION Plant-derived free radical scavengers have become the subject of intensive scientific interest. Recently, the concept of coupling chromatographic fingerprints with biological fingerprinting analysis has gained much attention for the quality control of plant extracts. However, identification of free radical scavenging activity of each single compound in a complex mixture is a difficult task. Thin-layer chromatography with post-chromatographic derivatisation with the methanol solution of DPPH can be a valuable tool in such analyses. OBJECTIVE Development of chromatographic and free radical scavenging fingerprints of nineteen Salvia species grown and cultivated in Poland. METHODOLOGY Chromatography was performed on the silica gel layers with use of two eluents, one for the resolution of the less polar compounds, and the other one for the resolution of the medium and highly polar ones. The plates were sprayed with the vanillin-sulfuric acid reagent to produce chemical fingerprints, and with DPPH solution to generate free radical scavenging fingerprints. RESULTS With four Salvia species, it was revealed that their strong free radical scavenging properties are not only due to the presence of polar flavonoids and phenolic acids, but also due to the presence of several free radical scavengers in the less polar fraction. Because of the similarities in both the chromatographic and the free radical scavenging fingerprints, S. triloba can be introduced as a possible equivalent of the pharmacopoeial species, S. officinalis. CONCLUSION Fingerprints developed in the experiments proved useful for the analysis of complex extracts of the different Salvia species.

TLC-MS VERSUS TLC-LC-MS FINGERPRINTS OF HERBAL EXTRACTS. PART II. PHENOLIC ACIDS AND FLAVONOIDS

In the previous paper from this series, we proposed mass spectrometric fingerprinting of a complex and volatile botanical sample upon an example of the essential oil derived from Salvia lavandulifolia. In that paper, we compared two variants of fractionation of such a mixture. A simpler one-dimensional variant consisted of the low-temperature thin-layer chromatographic fractionation coupled with mass spectrometric fingerprinting of each separated fraction (1D LT TLC-MS). A more sophisticated variant was the two-dimensional liquid chromatographic system composed of the low-temperature thin-layer chromatography, high-performance liquid chromatography, and mass spectrometric detection (2D LT TLC-LC-MS). In this study, we present an analogous approach to the non-volatile botanical mixtures upon an example of the pharmacologically important phenolic acids and flavonoids selectively extracted from Salvia lavandulifolia. With these non-volatile fractions, the thin-layer chromatographic separations were carried out at ambient temperature (21 ± 0.5°C). Once again, we compared two variants of fractionation. A simpler one-dimensional variant consisted of the thin-layer chromatographic mode coupled with mass spectrometric fingerprinting of each separated fraction (1D TLC-MS). A more sophisticated variant was the two-dimensional liquid chromatographic system composed of the thin-layer chromatography and high-performance liquid chromatography, with mass spectrometric detection (2D TLC-LC-MS). As expected, the two-dimensional mode proved better performing than the one-dimensional mode (1D TLC-MS). It was concluded that thin-layer chromatography directly or indirectly coupled with mass spectrometric detection can prove very useful in the analysis of the phenolic acid and flavonoid fraction selectively extracted from botanical material.

Experimental Investigation of the Oscillatory Transenantiomerization of L-Tyrosine

Abstract In our earlier studies, we demonstrated an ability of selected enantiomeric profen drugs (e.g., S-(+)-ibuprofen, S-(+)-naproxen, and S-(+)- and R-(−)-flurbiprofen) and one amino acid (i.e., L-α-phenylalanine) to undergo oscillatory transenantiomerization when dissolved in simple, low molecular weight solvents (e.g., water, ethanol, dichloromethane, acetonitrile, etc.) and stored for a longer period of time at ambient temperature or in a refrigerator. Experimental evidence of this process originates from a number of analytical techniques, with thin layer chromatography (TLC) and polarimetry among the best performing ones. There are two common structural features of all these compounds, namely: (i) they are 2-arylpropionic acids (2-APAs), and (ii) their chirality center is located on the α-carbon atom of the respective molecules. It has also been established that the basic and the amphiprotic environment catalyzes the oscillatory transenantiomerization of the investigated compounds, while the acidic environment tends to hamper this process. Moreover, it has been established that all the aforementioned compounds can organize molecules present in the solution in such a manner as to produce the density anisotropy of the liquids considered. Model explanation of the oscillatory transenantiomerization of profens and L-α -phenylalanine was also developed as a starting point, adapting an earlier established oscillator known as Templator. The new model comprises two linked Templators. The quintessence of the Templator model adapted to the demands of the oscillatory transenantiomerization of profens and amino acids was based on an assumption that the H-bonded 2-APA dimer is a template, able to generate the new dimers having the same steric configuration of their respective monomeric units. From our earlier studies, it clearly comes out that in spite of common traits of the oscillatory transenantiomerization of the selected profens and L-α-phenylalanine, the dynamics of this process can significantly differ from one compound to another, due to their differentiated molecular structure and, hence, to the different electron density distribution. Thus, in this study, we investigated the ability of L-tyrosine (another 2-APA and the amino acid regarded as essential for the humans) to undergo oscillatory transenantiomerization. Solubility of L-tyrosine in the amphiprotic binary mixture (70{%} aqueous ethanol solution) widely used in our earlier studies proved too low to use it as a solvent in the present investigation. Instead, we traced the behavior of L-tyrosine when stored for over one week in the following mixed solvents: ethanol–1M NaOH (7:3, v/v) and ethanol-1M HCl (7:3, v/v). The results of our experiments clearly confirm the ability of L-tyrosine to undergo the oscillatory transenantiomerization, similar to that of the previously studied profens and L-α-phenylalanine, although the individual dynamics of the oscillatory transenantiomerization with this particular enantiomer is also evident and discussed. It is apparent that the model of the two linked Templators applies to L-tyrosine as well, as an adequate explanation of the mechanism of its oscillatory transenantiomerization.

TLC-MS VERSUS TLC-LC-MS FINGERPRINTS OF HERBAL EXTRACTS. PART I. ESSENTIAL OILS

In our earlier studies, we proposed the low-temperature thin-layer chromatography with densitometric and mass spectrometric detection for fingerprinting of essential oils derived from several different species from the Salvia genus. Development of the proposed LT-TLC-MS approach was only possible with use of the TLC-MS interface able to couple the chromatographic plate with mass spectrometer. The goal of this study was to develop for the same purpose the two-dimensional liquid chromatographic system, which was only possible with use of the TLC-MS interface. It included the low-temperature thin-layer chromatography coupled with high-performance liquid chromatography, to build an overall LT TLC-LC-MS system. We compared the fingerprinting results for the essential oil of Salvia lavandulifolia obtained with use of the one-dimensional system (1D LT TLC-MS) with those obtained with use of the two-dimensional system (2D LT TLC-LC-MS). It was shown that the 2D approach provides more fingerprints for further investigations than the 1D approach. Moreover, it is also clear that the 2D approach is better suited for identification of individual chemical species contained in a given mixture, due to the simpler patterns of mass spectra obtained from the 2D analytical mode than from the 1D one. However, with the constituents of the essential oil derived from Salvia lavandulifolia, a straightforward identification was not possible, basically due to numerous groups of isomers among the terpenes and terpenoids contained in the examined sample that have the same molecular weights and hence, can give identical m/z signals.

LOW TEMPERATURE PLANAR CHROMATOGRAPHY–DENSITOMETRY AND GAS CHROMATOGRAPHY OF ESSENTIAL OILS FROM DIFFERENT SAGE (SALVIA) SPECIES

Essential oils of plant origin are the multicomponent mixtures of mono-, di-, tri-, and sesquiterpenes. Due to their recognized curative, cosmetic, and nutritional properties on the one hand and an outstanding modern analytical potential on the other, qualitative and quantitative composition of essential oils currently is in the focus of interest for phytochemistry and pharmacognosy. Due to the recognized volatility of the essential oil components, in their case the analytical method of choice is gas chromatography with mass spectrometric detection (GC-MS). However, great versatility of planar chromatography has resulted in a number of successful applications of this relatively simple and inexpensive separation technique to the investigations on composition of the volatile plant constituents as well. Generally, the low temperature preparative layer chromatography (PLC) is used for preliminary fractionation of the essential oils, and the separated fractions are further analyzed by means of GC-MS. In this study, we scrutinized a possibility of using the low temperature analytical thin-layer chromatography (TLC) to fingerprinting of the essential oils originating from the five different sage (Salvia) species, i.e., S. lavandulifolia, S. staminea, S. hians, S. triloba, and S. nemorosa. We also used the low temperature PLC for the preliminary fractionation of these essential oils prior to the GC-MS analysis. It was shown that the low temperature TLC can successfully be applied to fingerprinting the different sage (Salvia) species. Fractionation of the essential oils from the sage species by means of the low temperature PLC prior to the GC-MS analysis is also possible, although individual stages of the approach still need an additional optimization.

COMPARISON OF TLC AND HPLC FINGERPRINTS OF PHENOLIC ACIDS AND FLAVONOIDS FRACTIONS DERIVED FROM SELECTED SAGE (SALVIA) SPECIES

This study is part of a larger project on the contents and composition of essential oils, phenolic acids, and flavonoids originating from over twenty species belonging to the Salvia genus. These herbs are recognized in the traditional European pharmacy and cuisine for their curative and aromatic properties, and yet they have not attracted sufficient attention from the side of phytochemists. Thus, it seemed inevitable to focus on the contents of the phenolics in the individual sage species, basically due to the recognized antioxidant properties of this class of compounds. In this paper, we compared the results of a spectrophotometric assessment of the overall contents of phenolic acids and flavonoids (selectively extracted from twenty three different Salvia species) with those of a more detailed analysis carried out with use of HPLC/DAD and the TLC-based video imaging. Six species with the highest contents of phenolic acids and five species with the highest contents of flavonoids, and additionally Salvia officinalis, were chosen for the chromatographic fingerprinting of the selectively extracted fractions of the free and bonded phenolic acids, and the flavonoid aglycons and glycosides. As Salvia officinalis is the only representative of the discussed genus recognized as a medicinal plant by Polish Pharmacopoeia, our goal was to compare its phenolic contents with those of the other phenolic-rich sage species. This part of our sage investigation project has methodological importance and shows how thin-layer chromatographic video imaging can be used in a phytochemical study in combination with other analytical techniques.

Optimization of extraction based on the thin-layer chromatographic fingerprints of common thyme.

The main goal of this study was to establish the optimum accelerated solvent extraction (ASE) conditions for the isolation of the total phenolics contained in common thyme (Thymus vulgaris L.) based on the results of TLC. Additionally, two different extraction methods, i.e., Soxhlet extraction and ASE, were compared in terms of their relative performance. Binary methanol-water mixtures in different volume proportions were used as the extraction solvents. Thirteen experiments utilizing different methanol concentrations and different extraction temperatures were designed using response surface methodology. Additionally, the temperature dependence of rosmarinic and caffeic acid extraction yields was analyzed by applying HPLC. Finally, the optimum working conditions established within the framework of the assumed central composite experimental design were determined to be 27% methanol at 130°C. It was found that the extraction carried out by ASE provided higher total phenolic yields than that done in the Soxhlet apparatus. The final aim of this study was a comparison of the six different Thymus species in terms of their total phenolic content and their TLC fingerprints. It was found that the phenolic content of four species, T. pulegioides, T. kosteleckyanus, T. citriodorus, and T. marschallianus, differs from the phenolic content of T. vulgaris and T. serpyllum.

TLC-based start-to-end method of analysis of selected biologically active compounds contained in common sage (Salvia officinalis L.).

Abstract Common sage (Salvia officinalis L.) is an herb native to the Mediterranean region and nowadays is cultivated in many parts of the world. This plant has been known and utilized for hundreds of years in natural medicine, due to its curative properties and good performance in combating various diseases. In spite of its well recognized curative potential, the chemical composition of common sage is far from being sufficiently explored, and research is needed in the areas of phytochemistry and pharmacognosy to better scrutinize its constituents and therapeutic properties. It was the aim of this study to elaborate a start-to-end approach to extraction and chromatographic separation of the compounds contained in common sage, based on thin layer chromatography with densitometric detection and preparative layer chromatography, additionally supported by high performance liquid chromatography with diode-array detection and head-space gas chromatography with mass spectrometry detection. This approach was meant to enable an effective fingerprint analysis, focused mainly on flavonoids, phenolic acids, and terpenes, of the common sage samples, and its performance was demonstrated by analysis of a commercially available common sage tea preparation.

The HPLC/DAD Fingerprints and Chemometric Analysis of Flavonoid Extracts from the Selected Sage (Salvia) Species

The results of spectrophotometric and HPLC/DAD analysis are discussed, and a comparison is made of selectively extracted flavonoid fractions derived from twenty six sage species belonging to the Salvia genus. The sage samples were harvested in the vegetation seasons 2007, 2008, and 2009. It was a goal of this study to find out which species contain the highest yields of flavonoids (recognized for their free-radical-scavenging activity), as those with the highest yields could be applied in official medicine. It was spectrophotometrically established that the four sage species can be recognized for their highest flavonoid levels, while the HPLC/DAD analysis pointed out to the four other species. The source of the discrepancy between the two evaluation approaches was discussed. Moreover, the HPLC/DAD fingerprints of the flavonoid fraction underwent a chemometric pre-treatment, and then the purified fingerprints were analyzed by means of Principal Component Analysis (PCA) for the differences in the harvesting period. A difference was revealed between the herbs harvested in the 2007 season, and those harvested in 2008 and 2009. The main source of this difference could be the seasonal weather variation and the relatively longest storage period with the plants harvested in 2007.

MARKER FINGERPRINTS ORIGINATING FROM TLC AND HPLC FOR SELECTED PLANTS FROM THE LAMIACEAE FAMILY

Phytochemical analysis of plant extracts is an increasingly important field of analytical chemistry, and for several reasons. In the case of medicinal plants usually traded in a dried and crumbled form, phytochemical analysis serves the purpose of authentication of herbal material and protects patients against adulteration. In botanical sciences, phytochemical analysis is employed for the development of plant systematics known as chemotaxonomy. Due to an unknown composition of plant extracts and an inherent difficulty in selecting proper phytochemical standards, a phytochemical approach has been developed known as fingerprinting. This approach circumvents tedious identification of individual constituents of plant extracts and instead, focuses on a comparison of the whole chromatograms in order to trace similarities and dissimilarities among the plants. In this study, we present the results of a comparison of six plant species belonging to three genera of the Lamiaceae family (i.e., Salvia, Thymus, and Dracocephalum). This comparison was purposely carried out for the Lamiaceae plants, as many of them have been recognized for their curative properties by traditional medicines in many regions of the world (and in the first instance, in the Mediterranean zone). As phenolic acids and flavonoids play crucial role in many curative processes, in our study we employed TLC and HPLC to fingerprint the selectively extracted six phenolics fractions for the six plants belonging to the three aforementioned genera. The obtained results point out to the flavonoid aglycons (FA) fraction which most distinctly discriminates among the individual plant species, while the remaining five fractions are the carriers of less vital information. Finally, an assumption was made that the chromatograms derived by means of TLC and HPLC for the fraction of flavonoid aglycons can be considered as marker fingerprints, able to distinguish among closely related plants belonging to different genera of the Lamiaceae family.