Michał Hajnos

Two-dimensional thin-layer chromatography with adsorbent gradient as a method of chromatographic fingerprinting of furanocoumarins for distinguishing selected varieties and forms of Heracleum spp.

There are a lot of taxonomic classifications of the genus Heracleum, and many authors indicate they need revision. Morphological identification is difficult to perform, as there are only few characteristic differences between each Heracleum species, varieties and forms. Furanocoumarins are characteristic compounds for the Apiaceae family, and they can be found in the whole genus in large quantities. Despite this fact, it is difficult to use the furanocoumarin profiles of plants, for their discrimination, as furanocoumarins are difficult to separate, due to their similar chemical structures and physicochemical properties. In this paper, a new, simple method is proposed for the discrimination of selected species, varieties and forms of the genus Heracleum. Thin-layer chromatography (TLC) with an adsorbent gradient (unmodified silica gel+octadecylsilica wettable with water) enables complete separation of the structural analogues. The proposed method gives the possibility to distinguish selected species, varieties and forms of the Heracleum genus, as they produce distinctive furanocoumarin fingerprints. The method is characterised by high specificity, precision, reproducibility and stability values. It is for the first time that graft TLC is used for constructing fingerprints of herbs. The complete separation of ten structural analogues, by combining gradient TLC with the unidimensional multiple development technique, has not been reported yet.

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.

Two-Dimensional thin-layer chromatography of structural analogs. Part I: Graft TLC of selected coumarins

Coumarins are natural, biologically active substances, normally found in complex mixtures. Unfortunately their separation causes many difficulties, because of to their similar chemical structure and physicochemical properties. A new, reliable method has been established for analysis of coumarin fractions present in selected fruit extracts. The substances were separated in chromatographic systems that enabled use of orthogonal separation mechanisms (i.e. characterized by different selectivity). The greatest selectivity differences were obtained by use of two chromatographic systems — first dimension CN-silica with 30% ACN in H2O as mobile phase (triple developed) and second dimension SiO2 with 35% AcOEt in n -heptane as mobile phase (triple developed), or first dimension SiO2 with 35% AcOEt in n -heptane as mobile phase (triple developed) and second dimension RP-18 with 55% MeOH in H2O as mobile phase. The aforementioned two-dimensional TLC systems were used for separation of coumarin fractions present in extracts from Archangelica officinalis, Pastinaca sativa and Heracleum sphondylium fruits.

Application of Hydrophilic Interaction TLC Systems for Separation of Highly Polar Glycosidic Compounds from the Flowers of Selected Verbascum Species

Separation of highly polar compounds, for example, iridoids and triterpene saponins, present in natural samples is a challenging task. Because of their nonvolatility and the lack of chromophores, their analysis by means of gas chromatography or high-performance liquid chromatography (HPLC) is difficult. The use of normal as well as reversed-phase systems fails to resolve highly polar compounds that are strongly adsorbed on polar stationary phases and poorly retained on alkyl-bonded stationary phases usually used in HPLC. Because of these facts, separation has been performed in hydrophilic interaction systems by means of thin-layer chromatography (TLC). Planar chromatography offers also a possibility to use different derivatizing agents to visualize the resolved compounds. Another problem encountered in the analysis of such compounds present in plant extracts is that they occur as multicomponent mixtures. In this article, two-dimensional (2D) TLC systems were used for the resolution of compounds present in the polar fractions of the different Verbascum spp. flower extracts. TLC separations were performed on silica gel plates, with two different mobile phases used in the perpendicular directions. First, the analyzed samples were developed with AcOEt-MeOH-H2 O-25% aqueous NH3 (55:35:9:1, v/v/v/v) and redeveloped with MeOH-AcOEt-H2 O-HCOOH (10:90:26:22, v/v/v/v) in a perpendicular direction. The resolved compounds were visualized using the vanillin-sulfuric acid reagent. The obtained videoscans were further used for preliminary comparative studies of the investigated species. It is for the first time that 2D hydrophilic interaction systems, characterized with different pH values, are being applied for the analysis of highly polar compounds present in selected Verbascum spp. flower extracts. Anew approach of using an image-processing program for the comparative studies and for method validation is also presented.

Low-temperature TLC-MS of essential oils from five different sage (Salvia) species

In a previous paper we discussed the possibility of fractionating the essential oils of different sage species by low-temperature preparative layer chromatography (PLC), followed by preparative isolation of the contents of each fraction and further analysis by GC-MS. In that way we attempted to emphasize the practical usefulness of lowtemperature planar chromatography for investigation of volatile compounds. In this study, we explore a possibility of fractionating essential oils contained in the different sage species by low-temperature analytical TLC followed by direct mass spectrometric analysis of the separated fractions. This objective can be achieved by TLC-MS with on-line transfer of the eluted fractions. The densitograms obtained from five different sage species (i.e., S. lavandulifolia, S. staminea, S. hians, S. triloba, and S. nemorosa) are compared. Each densitogram is accompanied by mass spectra recorded for each peak. Videoscans of the chromatograms are also presented. In this way multiple fingerprints of the analyzed plant material, each comprising a densitogram and a selection of mass spectra, were obtained. Advanced chemometric treatment of these multiple fingerprints can be used to reveal statistically significant differences between the plant species. Analytical and chemotaxonomic advantages and further aspects for this kind of approach are discussed.

Two-Dimensional Thin-Layer Chromatography of Structural Analogs. Part II. Method for Quantitative Analysis of Selected Coumarins in Plant Material

Coumarins are interesting group of natural compounds, because of their biological and pharmacological activity, and are widely investigated. They are normally found in complex mixtures, e.g. plant extracts, and are difficult to separate in one chromatographic run. Mixtures of coumarins have been separated by two-dimensional thin-layer chromatography on CN-silica plates by use of aqueous and nonaqueous mobile phases. Complete separation was also achieved by use of graft thin-layer chromatography on connected layers — silica with RP-18W or CN-silica with silica. The systems characterized by the best efficiency and selectivity were used for separation of coumarin fractions from extracts of selected Apiaceae plants. These orthogonal systems were used for quantitative analysis of selected coumarins. The results obtained show two dimensional thin-layer chromatography is useful tool for quantification of some furanocoumarins in plant extracts. The best results were obtained on connected silica and octadecyl silica layers.

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.

New approach to mechanism of action of paclitaxel by means of BioArena studies

The accumulation of hydrogen peroxide (H2O2) is an early and crucial step in paclitaxel-induced cancer cell death before commitment of the cells to apoptosis. In these intracellular events formaldehyde (HCHO) as endogenous, indispensable component may be present mainly as hydroxymethyl groups and so there is a possibility of its endogenous interaction with H2O2 in which singlet oxygen (1O2) and excited HCHO (H*CHO) can be formed.1O2 can interact with H2O molecules and in this interaction dihydrogen trioxide (H2O3) is formed. The disproportion of this molecule–among others–results in ozone (O3). It is supposed that this reaction series is also valid for the conditions in layer chromatographic spots after inoculation. Results with paclitaxel support this idea. Using BioArena as a complex bioautographic system the HCHO molecules could be captured with well-known endogenous HCHO capture molecules (l-arginine, glutathione) in the spots of paclitaxel on the TLC/OPLC adsorbent layer after inoculation. Capture of HCHO resulted in a dose-dependent decrease of the antibacterial activity of paclitaxel. The antibacterial activity of paclitaxel in the chromatographic spots can be increased dramatically by using Cu(II) ions as HCHO-mobilizing and carrier ions in the culture medium. The HCHO molecule can N-hydroxymethylate the C3′ amide of paclitaxel. By applying an O3 scavenger (e.g. indigo carmine) this oxidant, as a key reaction product of HCHO, could be detected indirectly in chromatographic spots of paclitaxel. It seems that these small molecules–from HCHO to endogenous O3–may be crucial factors of the mechanism of antiproliferative action of the paclitaxel including killing of bystander cancer cells also. It seems that the basic molecule (paclitaxel) does not have a direct effect on the bacterial cells; its induction of the formation of H2O2 molecules (and indirectly HCHO molecules) may, however, be resulting in this complicated process.

Use of reversed-phase and normal-phase preparative thin-layer chromatography for isolation and purification of coumarins from Peucedanum tauricum Bieb. leaves

Peucedanum tauricum, an endemic perennial plant of the Apiaceae family, has previously been reported to contain furocoumarins – peucedanin in the fruits, roots and leaves [1] and isoimperatorin and bergapten in the fruits [2]. Coumarins, very common secondary metabolites, have a variety of pharmacological activity, e.g. toxicity and phototoxicity [3, 4], antiproliferative activity [5], calcium antagonistic effect [6], and apoptosis induction activity [7]. Some coumarins, e.g. psoralens, intercalate readily into DNA and form light-induced mono and diadducts with pyrimidine bases [8]; these are therefore used in photochemotherapy of vitiligo and psoriasis [9, 10]. Isolation of natural coumarins from plant material is important for further investigation of their biological activity [11, 12]. In this work a methanolic extract of leaves of Peucedanum tauricum was examined for the presence of coumarins. Separation and isolation of the components was performed by use of polar and nonpolar adsorbents, binary mobile phases, and a combination of RP and NP TLC with densitometry and RP HPLC with diodearray detection (DAD) [2, 13].

Strategy for preparative separation of quaternary alkaloids from Chelidonium majus L. by thin-layer chromatography

A Chelidonium majus L. quaternary alkaloid fraction has been used as a model mixture to investigate the effect of the procedure used on the preparative separation of its components. The steps optimized were: the mode of application of the starting band to the layer; the effect on the separation of the bands of the number of developments in unidimensional multiple development (UMD); the effect of concentration overloading on the separation of the bands; and the effect of the volume of solution introduced to the adsorbent layer on the resolution of neighboring bands.