Session 3

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With the commercial introduction of plates coated with fine particles by MERCK in the late 1970s, the foundation for the evolution of HPTLC was laid. Over the last four decades, significant progress has been seen in the development of instruments. Most recently, even fully automatic systems became available. The available tools - instrument and software - have supported scientific research of all aspects of planar chromatography, stimulated curiosity, and expanded the range of what is possible.
Yet, it seems that it was the discussion of a rigorous concept of standardization, its subsequent implementation into pharmacopoeias, and the resulting enforcement by authorities, that made HPTLC a distinct and accepted technique for routine analysis, particularly in quality control of herbal materials. What will be next?
This presentation connects fundamental concepts, such as standardization and comprehensive HPTLC fingerprinting, with current ideas about the evaluation of system suitability, comparison of data across multiple plates, and the use of complementary developing solvents, leading to HPTLC 4.0. This vision may be developed into a global platform for collaboration of researchers and routine users of HPTLC. It has the potential of taking the inherent advantages of planar chromatography into a bright future.

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The plant kingdom is an inexhaustible source of biologically active compounds used as components of supplements and pharmaceutical preparations. Effect directed detection (EDD), i.e. thin-layer chromatography combined with direct bioautography (TLC-DB), and TLC-screening are convenient tools for bio-profiling of active constituents of plants. In TLC-DB, the effect observed and measured directly on a TLC plate results from an action of biologically active constituents of a sample, e.g., inhibition of bacteria growth related to antibacterial activity of separated plant constituents. The detected bioactive components can be further identified using spectroscopic techniques such as GC-MS, LC-MS, IR, NMR. TLC–bio-profiling can also be used to distinguish among plants from various species and origin, find the most active ones, and point to possible adulterations. We focused on Schisandraceae family and Rhodiola rosea L. (various species, supplements, and pharmaceutical preparations) [1-3]. The most interesting findings were the strong antibacterial properties of S. chinensis and S. rubriflora fruits, as well as acetylcholinesterase, glucosidase, tyrosinase, and lipase inhibition of all tested species. Strong glucosidase inhibition was observed for Kadsura japonica fruits, which generally did not reveal biological activities. NP-PEG assay and TLC-DPPH test showed that leaves of various Schisandra species are rich in polyphenolic compounds and have strong antioxidant properties (especially leaves of S. rubriflora and K. japonica). Strong antioxidant, antibacterial and enzyme-inhibiting properties were found for the root and rhizome of R. rosea L.. Various R.rosea pharmaceutical preparations and supplements differ in their biological properties as well as in the content of marker compounds: rosavin, salidroside, and tyrosol.

References
[1]  E. Sobstyl, A. Szopa, H. Ekiert, S. Gnat, R. Typek, I.M. Choma, J. Chromatogr. A, 1618 (2020) 460942.
[2]  H. Nikolaichuk, M. Studziński, I.M. Choma, J. Liq. Chromatogr. Rel. Technol., 43 (2020) 361-366.
[3]  H. Nikolaichuk, R. Typek, S. Gnat, M. Studziński, I.M. Choma, J. Chromatogr. A, 1649 (2021) 462217.

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High-performance thin-layer chromatography (HPTLC) is a suitable method for separating, searching, and evaluating biological samples with complicated matrices such as plant extracts. Moreover, the HPTLC is compatible with detecting samples' biological and biochemical activity. The great advantage of HPTLC is the possibility of analyzing many samples and comparing the composition of the samples in one run. The detection of antibiotics, antioxidants, enzyme inhibitors, genotoxic compounds, and estrogen and androgen activity can be performed directly on the HPTLC plate. Additionally, the advantage of HPTLC is the direct possibility of isolating and identifying the target substances using high-resolution mass spectrometry (HRMS) [1,2].
In our present study, HPTLC hyphenated HRMS was applied to determine bioactive substances in 15 supplement preparations of Rhodiola rosea L. root, in fruits and leaves of Akebia quinata Decaisne, and flower of Clitoria ternatea L.. For this purpose, the enzyme inhibition assays such as acetylcholinesterase, butyrylcholinesterase, tyrosinase, lipase, α/β-glucosidase, α-amylase, and β-glucuronidase were used. Antibacterial properties were investigated using Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Avilibrio fischeri assays. Additionally, the plants were tested for estrogen, antiestrogen, androgen, and antiandrogen activity. Moreover, samples were tested for genotoxic activity. The active bands detected by bioassays directly on the HPTLC plate were identified using the TLC-MS interface.

References
[1] Nikolaichuk, H.; Typek, R.; Gnat, S.; Studziński, M.; Choma, I.M., J. Chromatogr. A 1649, (2021).
[2] Morlock, G.E., In Encyclopedia of Analytical Science, (2019).

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Assam, a state in India, belongs to the eastern Himalayan biodiversity region, wherein the local population uses various herbs to treat different ailments; but because of the indiscriminate use, these herbs have become endangered in their natural habitat. Two such herbs are Coptis teeta and Homalomena aromatica; the high polarities of protoberberine alkaloids have made it difficult to quantify the alkaloids in C. teeta whereas, it is a tedious stuff to separate the various terpenoids in the volatile oil of H. aromatica. Two distinct TLC based densitometric methods were developed for identification, quantification and validation of linalool in the volatile oil of rhizomes of H. aromatica and berberine in the methanol extract of rhizomes of C. teeta. The separation for quantification of berberine in C. teeta was achieved in a solvent system consisting of Butanol: Ethyl acetate: Formic acid: Water on TLC aluminum plates precoated with silica gel 60 F₂₅₄. For quantification of linalool, the chromatographic plates were developed in Toluene: Ethyl acetate solvent system at a ratio of 9.5:0.5 and visualized with p-anisaldehyde reagent. The validated method gave compact bands of berberine at R_F of 0.70 whereas, compact bands of linalool was observed at Rf of 0.35. Both the methods developed were precise, accurate and reproducible with regards to the International Conference on Harmonization guidelines. Linear results were obtained for both berberine and linalool with correlation coefficients of 0.997±0.09 % (R±SD) in the concentration range of 90-210 ng/band and 0.980±0.05 % (R±SD) in the concentration range of 2-20 nL/band respectively. The concentration of berberine in C. teeta was found to be 30.97±0.55 mg in 100 mg of the crude drug whereas, the concentration of linalool in the volatile oil of H. aromatica was found to be 5.839±0.42 μL in 10.000 μL of the H. aromatica volatile oil. The method developed here in can be implemented in the analysis and routine quality control of herbal materials and formulations containing C. teeta and berberine as well as volatile oils containing linalool and quality control of rhizomes of H. aromatica.