Thin-Layer Chromatography (TLC) in the Screening of Botanicals-Its Versatile Potential and Selected Applications.

The aim of this paper is to present a comprehensive overview of the main aims and scopes in screening of botanicals, a task of which thin-layer chromatography (TLC) is, on an everyday basis, confronted with and engaged in. Stunning omnipresence of this modest analytical technique (both in its standard format (TLC) and the high-performance one (HPTLC), either hyphenated or not) for many analysts might at a first glance appear chaotic and random, with an auxiliary rather than leading role in research, and not capable of issuing meaningful final statements. Based on these reflections, our purpose is not to present a general review paper on TLC in screening of botanicals, but a blueprint rather (illustrated with a selection of practical examples), which highlights a sovereign and important role of TLC in accomplishing the following analytical tasks: (i) solving puzzles related to chemotaxonomy of plants, (ii) screening a wide spectrum of biological properties of plants, (iii) providing quality control of herbal medicines and alimentary and cosmetic products of biological origin, and (iv) tracing psychoactive plants under forensic surveillance.

Decolorization and biodegradation of melanoidin contained in beet molasses by an anamorphic strain of Bjerkandera adusta CCBAS930 and its mutants.

We used a ligninolytic strain of the white-rot fungus B. adusta CCBAS 930 and its mutants with modified ligninolytic activity to assess their potential to remove of molasses. The analyzed strains have been shown to be able to decolorize 1% or 2% molasses solutions containing brown-colored toxic melanoidins. It was found that the decolorization process was determined by the transition to the stage of production of sporulating aerial mycelium (liquid and agar cultures) coupled with an increase in peroxidase activity, which was accompanied by a decrease in the level of melanoidin, free radicals, and phenolic compounds. Four different peroxidase activities were detected in post-culture liquids, i.e. horseradish-like (HRP-like), manganese-dependent (MnP), lignin (LiP), and versatile (VP) peroxidase activities. The HRP-like peroxidase was characterized by the highest activity. The efficiency of removal of melanoidins from a 1% molasses solution by the parental strain and the mutants was dependent on the culture method. The highest efficiency was noted in immobilized cultures (threefold higher than in the mycelium-free cultures), which was accompanied by stimulation of HRP-like peroxidase activity. Mutant 930-5 was found to be the most effective in the decolorization and decomposition of melanoidin. The HRP-like activity in the immobilized cultures of B. adusta 930-5 was 640-fold higher than in the mycelium-free cultures of the fungus. Moreover, decolorization and biodegradation of melanoidin by B. adusta CCBAS 930 and 930-5 was coupled with detoxification.

Thin-layer chromatographic quantification of magnolol and honokiol in dietary supplements and selected biological properties of these preparations.

Two isomeric biphenyl neolignans, magnolol and honokiol, are considered as constituents responsible for the healing effect of magnolia bark, a traditional Oriental medicine. To survey the increasing number of dietary supplements that contain magnolia bark or its extract, an affordable quantitative thin-layer chromatography (TLC) - densitometry method was developed. The methanol extracts were analyzed on the silica gel plates after manual sample application using n-hexane - ethyl acetate - ethanol (16:3:1, v/v/v) as a mobile phase. For quantitation, the chromatograms were scanned in the absorbance mode at the wavelength λ = 290 nm. The limits of detection and quantitation were 90 and 280 ng/zone for magnolol and 70 and 200 ng/zone for honokiol, respectively. None of the two targeted neolignans were detected in two of the six analyzed supplements. In the other four samples, the measured amounts were between 0.95-114.69 mg g-1 for magnolol and 4.88-84.86 mg g-1 for honokiol. Moreover, separations of these two neolignans on the TLC and high-performance TLC (HPTLC) layers were compared and HPTLC was combined with antioxidant (DPPH) and antibacterial (Bacillus subtilis and Aliivibrio fischeri) assays and mass spectrometry (MS), using the elution-based interface. Both magnolol and honokiol exhibited effects in all bioactivity assays. The HPTLC-MS tests confirmed purity of neolignan zones in the extracts of dietary supplements and supported tentative identification of the alkaloid piperine and the isoflavone daidzein as additional bioactive components of the investigated dietary supplements. Using the same mobile phase in the orthogonal directions 2D-HPTLC-MS experiments proved degradation, i.e., instability of magnolol and honokiol on the silica gel adsorbent.

Antibacterial potential of the Cistus incanus L. phenolics as studied with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis.

The main aim of this study was to detect and identify antibacterial components of fraction I derived from eleven commercial C. incanus herbal teas. Fraction I obtained by a well-established phytochemical protocol of a multi-step extraction was expected to contain flavonoid aglycons alone. Antibacterial profile of fraction I was demonstrated by means of thin-layer chromatography - direct bioautography (TLC-DB) using a Gram positive B. subtilis and a Gram negative A. fischeri strain. Six chromatographic zones of fraction I exhibited a well pronounced antibacterial potential. In qualitative terms, a good agreement was observed among chromatographic fingerprints and the corresponding bioautograms of the eleven samples. The compounds isolated from the six zones were analyzed by HPLC- diode array detector (DAD)-electrospray ionization (ESI)-MS. High numerical m/z values valid for certain constituents of these isolates suggested that some selected antibacterial components are, unexpectedly, flavonoid glycosides. In order to confirm this suggestion, three independent HPTLC methods (multi-development on amino phase and two two-dimensional developments on silica gel phase) were devised to in situ hydrolyze flavonoid glycosides and then separate and visualize the liberated glucose and some other building blocks of the zones' components. Additionally, the sensitivity of glucose detection with p-aminobenzoic acid reagent was enhanced by paraffin. In that way, the presence of the kaempferol glycosides (and not only the aglycones alone) in fraction I was confirmed. Beside kaempferol, p-coumaric acid as a building block unit was shown by HPLC-DAD-MS analysis of the hydrolyzed isolates. Results proved apigenin, kaempferide and acylated kaempferol glycosides (cis- and trans-tiliroside and their conjugates with p-coumaric acid) to be antibacterial components of fraction I. Because isomers of the coumaric acid conjugated tiliroside were detected only in fraction I and not in the crude C. incanus extract, they are regarded as artifacts produced through fractionation.

A Comparison of Antibacterial Activity of Selected Thyme (Thymus) Species by Means of the Dot Blot Test with Direct Bioautographic Detection.

Bioautography carried out with the aid of thin-layer chromatographic adsorbents can be used to assess antibacterial activity in samples of different origin. It can either be used as a simple and cost-effective detection method applied to a developed chromatogram, or to the dot blot test performed on a chromatographic plate, where total antibacterial activity of a sample is scrutinized. It was an aim of this study to compare antibacterial activity of 18 thyme (Thymus) specimens and species (originating from the same gardening plot and harvested in the same period of time) by means of a dot blot test with direct bioautography. A two-step extraction of herbal material was applied, and at step two the polar fraction of secondary metabolites was obtained under the earlier optimized extraction conditions [methanol-water (27+73, v/v), 130°C]. This fraction was then tested for its antibacterial activity against Bacillus subtilis bacteria. It was established that all investigated extracts exhibited antibacterial activity, yet distinct differences were perceived in the size of the bacterial growth inhibition zones among the compared thyme species. Based on the results obtained, T. citriodorus "golden dwarf" (sample No. 5) and T. marschallianus (sample No. 6) were selected as promising targets for further investigations and possible inclusion in a herbal pharmacopeia, which is an essential scientific novelty of this study.

Antioxidant Activity of Selected Thyme (Thymus L.) Species and Study of the Equivalence of Different Measuring Methodologies.

This study presents the results of comparative evaluation of the antioxidant activity of the phenolic fraction exhaustively extracted with aqueous methanol from 18 different thyme (Thymus L.) specimens and species. This evaluation is made with use of the same free radical source (DPPH• radical), three different free radical scavenging models (gallic acid, ascorbic acid, and Trolox), and three different measuring techniques (the dot blot test, UV-Vis spectrophotometry, and electron paramagnetic resonance spectroscopy, EPR). A comparison of the equivalence of these three different measuring techniques (performed with use of hierarchical clustering with Euclidean distance as a similarity measure and Ward's linkage) is particularly important in view of the fact that different laboratories use different antioxidant activity measuring techniques, which makes any interlaboratory comparison hardly possible. The results obtained confirm a semiquantitative equivalence among the three compared methodologies, and a proposal is made of a simple and cost-effective dot blot test that uses the DPPH• radical and provides differentiation of antioxidant activity of herbal matter comparable with the results of the UV-Vis spectrophotometry and EPR.

Evaluation of the toxicity of anticancer chemotherapy in patients with colon cancer.

BACKGROUND: Modern anticancer chemotherapy can cause numerous adverse effects in the organism, whose functioning has already been disrupted by the neoplastic process itself. OBJECTIVES: The aim of the study was to evaluate and compare the frequency and severity of the toxicity of FOLFOX-4 and CLF-1 anticancer therapy in patients with colon cancer, and to analyze certain factors that might have increased the toxicity of the chemotherapy. MATERIAL AND METHODS: The study involved 64 patients suffering from generalized colon cancer, including 48 patients treated according to the FOLFOX-4 regimen and 16 patients treated according to the CLF-1 regimen. The toxicity of each regimen was analyzed on the basis of a confidential questionnaire formulated by the authors and laboratory research according to the extended WHO toxicity criteria. RESULTS: The analysis of the symptoms of toxicity symptoms associated with the use of the FOLFOX-4 and CLF-1 therapeutic regimens revealed that the most common side effects included nausea and vomiting, despite ondansetron premedication, and neurotoxicity. Disruption of the functioning of the nervous system under the FOLFOX-4 regimen statistically significant exacerbation that increased with the number of chemotherapy cycles administered; this was more common and more severe in women. Paresthesia was also revealed to be a neurotoxic effect of the FOLFOX-4 regimen after termination of therapy. A statistically significant relationship was observed between the use of vitamin supplements and the incidence and severity of the toxicity of the FOLFOX-4 regimen. CONCLUSIONS: The findings of the current study regarding the toxicity of the FOLFOX-4 and CLF-1 therapy regimens should be taken into consideration when monitoring chemotherapy safety in colon cancer. The patients' tolerance of the administered medication and the side effects reported by patients should be constantly evaluated, which will help prevent these side effects, apply appropriate therapy and contribute to the improvement of the patients' quality of life. The functioning of the central nervous system should be carefully evaluated when planning the anticancer therapy, especially if repeated administration of neurotoxic drugs is necessary in cases of a recurrence of the disease. Chemotherapy should be thoroughly monitored for safety, especially in women over 65 years of age suffering from coexisting diseases. Colon cancer patients and their families should be informed of the risks of nutritional supplements before the start of the anticancer chemotherapy, and may need to dispense with their use.

Fingerprinting of the volatile fraction from selected thyme species by means of headspace gas chromatography with mass spectrometric detection.

The Thymus genus has a firm position in Mediterranean folk medicine and culinary tradition, and yet certain confusion is observed in its botanical taxonomy. Therefore, chemical analysis of secondary metabolites and selection of reliable chemotaxonomic markers can prove helpful. In this study, the volatile fraction derived from 20 different thyme specimens was analyzed by means of headspace GC with MS detection. From the obtained chromatographic fingerprints, the diversity of the volatile fraction originating from the different thyme species clearly emerged. Identification of volatile components was based on a software library of mass spectra. There was only one common component derived from all 20 investigated thyme species, i.e., ß-linalool. For three different Thymus species, a comparison was made of the performance of the headspace extraction and hydrodistillation. Characteristic differences in composition of the volatile fraction obtained with use of these two techniques were discussed. Finally, the obtained chromatographic fingerprints of the volatile fraction were analyzed by means of a chemometric approach (principal component analysis), in order to trace similarities in their chemical composition.

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.

The use of TLC-DPPH* test with image processing to study direct antioxidant activity of phenolic acid fractions of selected Lamiaceae family species.

TLC coupled with 2,2-diphenyl-1-picrylhydrazyl staining was used to analyze phenolic acid fractions of selected Salvia and Thymus species. Documented videoscans were processed by means of an image processing program. This is the first time that free phenolic acids fractions, as well as fractions containing phenolic acids derived from basic and acidic hydrolysis, have been analyzed and compared for selected sage and thyme species. The analyzed samples along with caffeic acid (CA; standard) were chromatographed on silica gel plates with toluene-ethyl acetate-formic acid (60 + 40 + 1, v/v/v) mobile phase. The extracts were investigated with respect to the activity of CA. It was found that CA was most abundant in the fractions derived from basic hydrolysis. This compound was not detected in any of the fractions obtained after acidic hydrolysis. S. officinalis and S. triloba have similar free radical scavenging activity fingerprints obtained for all the analyzed fractions.

TLC-MS versus TLC-LC-MS fingerprints of herbal extracts. Part III. Application of the reversed-phase liquid chromatography systems with C18 stationary phase.

In the previous paper from this series, we proposed mass spectrometric fingerprinting of complex botanical samples upon the examples of the pharmacologically important phenolic acids and flavonoids selectively extracted from Salvia lavandulifolia. In this study, we explore fingerprinting efficiency with a novel two-dimensional analytical system composed of the reversed-phase thin-layer chromatography and the reversed-phase high performance liquid chromatography with mass spectrometric detection (2D RP-TLC-RP-LC-MS). We also compare its efficiency with that of the one-dimensional analytical system (the reversed-phase thin-layer chromatography with mass spectrometric detection; 1D RP-TLC-MS). As our present study is basically focused on the method development, we considered it as justified to carry out our comparison with the phenolic acid extracts selectively derived from the Salvia lavandulifolia species, similar as it was done in Part II from this series. Upon the results obtained, it was established that the 1D RP-TLC-MS mode and the 2D RP-TLC-RP-LC-MS mode can be applied to fingerprinting of herbal extracts, and that the 2D RP-TLC-RP-LC mode can provide a more abundant information than that originating from the 1D RP-TLC mode.

Binary HPLC-diode array detector and HPLC-evaporative light-scattering detector fingerprints of methanol extracts from the selected sage (Salvia) species.

This study is focused on an important family of the sage (Salvia) species, with Salvia officinalis L. having a long-established position in European traditional medicine. Binary fingerprints (chromatographic profiles) of six different sage species were compared using HPLC coupled with two different detectors: the diode-array detector and the evaporative light-scattering detector. Advantages of using binary fingerprinting over single-detector fingerprinting are demonstrated and discussed, with selected examples. Experimental data are provided for a comparison of the chemical composition of sage samples originating from two harvesting seasons (2007 and 2008). A number of phytochemical standards (i.e., certain phenolic acids, flavonoids, and coumarin) were used that allowed identification and semiquantitative estimation of these particular compounds in the analyzed methanol extracts.

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.

Validated binary high-performance thin-layer chromatographic fingerprints of polyphenolics for distinguishing different Salvia species.

Salvia (sage) is the largest plant genus in the family Lamiaceae, embracing ca. 900 species. There is a growing interest in investigating chemical contents of different Salvia species, as some of them have been reported to exhibit a wide spectrum of biological activity. In this paper, conjugated (i.e., binary) chromatographic fingerprints have been introduced for the twenty Salvia species that are grown and cultivated in Poland. Apart from videoscans traditionally used for a comparison of the high-performance thin-layer chromatography fingerprints, digital scanning profiles and images obtained with use of the image processing program have also been employed. It is the first time that binary chromatographic fingerprints are used for the investigation of chemical contents of the Salvia species. The proposed procedure is rapid when compared with the similar ones presented in the literature, and moreover, it is easy to handle. The proposed method offers a possibility to discern the investigated species. It can be applied not only for chemotaxonomic purposes but also for finding new plant species that can be used as medical herbs (as it has been proposed, with S. triloba having a similar profile to S. officinalis). Validation of the method reveals that it is specific, reproducible, precise, and robust.

Bioactive compounds and antioxidant and antiproliferative activities of Korean white lotus cultivars.

In traditional Korean medicine, lotus (Nelumbo nucifera Gaertn) roots have been used as an antidiabetic and an antiproliferative remedy. However, scientific publications on lotus properties are very limited. Therefore, it was decided to investigate the Korean white lotus cultivars in order to find out their bioactivity. It was found that all lotus cultivars (Inchisa, Muan, Garam, and Chungyang) possess high amounts of bioactive compounds: total phenols, between 7.95 +/- 0.8 and 4.21 +/- 0.3 mg of gallic acid equivalents (GAE)/g dry weight (DW); ascorbic acid, between 15.8 +/- 1.1 and 22.3 +/- 1.7 mg of ascorbic acid/g DW; and amino acids, between 15.05 +/- 0.82% and 16.62 +/- 0.90% DW. The highest contents of polyphenols (7.95 +/- 0.8 mg of GAE/g DW) and the highest levels of antioxidant [by 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl assays, 54.27 +/- 6.1 and 21.98 +/- 2.5 microM Trolox equivalents/g DW, respectively] and antiproliferative activities on both human cancer cell lines (Calu-6 for human pulmonary carcinoma and SMU-601 for human gastric carcinoma, 59.75 +/- 3.99% and 71.21 +/- 2.79% cell viability, respectively) were found in the Chungyang cultivar. Fluorometry and Fourier transform infrared spectroscopy can be applied as rapid methods for determination of bioactive compounds, such as polyphenols. The correlation between the bioactive compounds and the antioxidant activity was high. In conclusion, all Korean white lotus cultivars are valuable medicinal foods, and in order to receive the best results a combination of lotus cultivars has to be consumed.

Comparative study of health properties and nutritional value of durian, mangosteen, and snake fruit: experiments in vitro and in vivo.

In vitro and in vivo studies of the health and nutritional properties of durian (Durio zibethinus Murr.) were compared with snake fruit (Salacca edulis Reinw.) and mangosteen (Garcinia mangostana). Dietary fibers, minerals, and trace metals were comparable. Total polyphenols (mg of GAE/100 g of FW) and flavonoids (85.1+/-6.1) were significantly higher (p<0.05) than in snake fruit (217.1+/-13.2 (mg of CE/100 g of FW)), durian (309.7+/-19.3 and 61.2+/-4.9), and mangosteen (190.3+/-12.1 and 54.1+/-3.8). Antioxidant activity (microM TE/100 g of FW) of durian measured by DPPH and ABTS assays (228.2+/-13.4 and 2016.3+/-81.1) was significantly higher (p<0.05) than in snake fruit (110.4+/-7.9 and 1507.5+/-70.1) and mangosteen (79.1+/-5.9 and 1268.6+/-62.3). HPLC/DAD analysis of durian (microg/100 g of FW) showed that quercetin (1214.23+/-116.7) was present at levels three times that of caffeic acid, and twice as high as p-coumaric and cinnamic acids. The correlation coefficients between the bioactive compounds of fruits and their antioxidant activities were high (R2=0.99). Male Wistar rats (25) were divided into five dietary groups: the control group was fed the basal diet (BD); in addition to BD, the cholesterol (Chol) group was supplemented with 1% of Chol; the diets of the Chol/Durian, Chol/Snake, and Chol/Mangosteen groups were supplemanted with 5% of these fruits, respectively. It was found that diets supplemented with durian, and to a lesser degree with snake fruit and mangosteen, significantly hindered the rise in plasma lipids and the decrease in antioxidant activity. The nutritional values were comparably high. In conclusion, it could be suggested that inclusion of studied tropical fruits, especially durian, in known disease-preventing diets could be beneficial.