Invited speakers

Irena Maria Choma, Hanna Nikolaichuk, Ewelina Sobstyl
Faculty of Chemistry, Maria Curie-Skłodowska University, Maria Curie-Skłodowska Sq.3, 20 031 Lublin, Poland
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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.

Chayanis Sutcharitchan1, Worakorn Wiwatcharakornkul1, Somchai Keawwangchai2, Tasawan Kaewwangchai2, Sornkanok Vimolmangkang1, Suchada Sukrong1, Wanchai De-Eknamkul1
1Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
2Department of Chemistry, Faculty of Science, Mahasarakham University, Mahasarakham 44150, Thailand
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Thailand is known to be one of the world’s best sources of cannabis strains, and thus has a potential to be a cannabis hub in Asia. Thai strains of cannabis commonly refer to 100% sativa strains native to Southeast Asia. Each strain contains different ratios of the psychoactive substances, tetrahydrocannabinol (THC) and cannabidiol (CBD), which are known to have medical benefits. Three major cannabis chemotypes (THC dominant, intermediate, and CBD dominant) and two minor ones (CBG dominant and cannabinoid-free) have been categorized for chemotaxonomic discrimination. So far, very little has been known about the chemotypes and genetic backgrounds of the diversified Thai cannabis strains and hybrids. Therefore, the techniques of HPTLC and DNA profilings were introduced to observe the relationship between their metabolic and genetic levels. Almost 100 samples of dried flowers obtained from various strains of Thai cannabis and their hybrids were collected and prepared for HPTLC and DNA analyses. The solvent system used for the HPTLC separation of cannabis extracts was modified from the previous report (1). The results showed that there was a high variation in the chemotypes among the Thai cannabis samples. For the DNA analysis, tetrahydrocannabinolic acid-synthase (THCA-synthase) and cannabidiolic acid-synthase (CBDA-synthase) genes (2,3) were used as the chemotype markers of various cannabis samples. The preliminary results obtained by PCR and DNA sequencing techniques suggested that there was a good correlation between the HPTLC chemical profiles and the presence of functional forms of THCA-synthase (drug-type) and CBDA-synthase (fiber-type). Therefore, the sequence of both genes can be used for early prediction of the cannabinoid profiled in Thai cannabis plants.

References:
[1] CAMAG Application Note A-108.1 (2017).
[2] C. Onofri, E. de Meijer, G. Mandolino, Phytochemistry 116 (2015) 57–68.
[3] A. R. Garfinkel, M. Otten, S. Crawford, Genes 12 (2021) 228. https://doi.org/10.3390/genes12020228

Acknowledgements:
This work was supported by Chulalongkorn University's Natural Product Biotechnology Research Unit (for WD) and Center of Excellence in DNA Barcoding of Thai Medicinal Plants (for SS).

Eliza Łata, Agnieszka Fulczyk, Mieczysław Sajewicz, Teresa Kowalska
Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland
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Effective diffusion is one of these phenomena which on an everyday basis take place in chromatographic systems, yet usually tend to be overlooked in an everyday analytical practice. Unlike in the column chromatography, in planar chromatography effective diffusion is two-dimensional [1].
On some rare occasions, horizontal direction of effective diffusion can proof beneficial. It happens, when certain compounds of a very similar chemical structure (and/or molecular weight) cannot be separated vertically without applying special separation conditions. So far, however, an exact physicochemical mechanism of horizontal separations remains rather obscure. It can only be assumed that the chirality of silica gel (as confirmed by the circular dichroism (CD) spectroscopy [2]) is one reason of horizontal separations. In combination with horizontal effective diffusion, it can result in spectacular yet rare horizontal separations. Moreover, it was documented upon numerous examples that horizontal separations in the TLC/HPTLC systems most likely occur with the rod-shaped molecules additionally equipped with asymmetry centers [3] and with the pairs of enantiomers [4]. The mechanism of such separations can be by the auto-driven propeller-like motions of the analytes themselves, the Magnus forces-induced motion, or by still another yet unknown mechanism [3].
In this talk, a very spectacular example of horizontal separation is presented of the rod-shaped red betacyanin pigments equipped with asymmetry centers, which are derived from the red beet (Beta vulgaris L.) juice [5].

References
[1] T. Kowalska, K. Kaczmarski, W. Prus, Theory and mechanism of thin-layer chromatography. In: J. Sherma and B. Fried (Eds.), Handbook of Thin-Layer Chromatography, 3rd Ed. Revised and Expanded, Marcel Dekker, New York, 2003; pp. 47-80.
[2] M. Sajewicz, H.-E. Hauck, G. Drabik, E. Namysło, B. Głód, T. Kowalska, J. Planar Chromatogr. 19 (2006) 278-281.
[3] J. Polanski, M. Sajewicz, M. Knas, A. Zywocinski, M. Weloe, T. Kowalska, Chemistry Today, Monographic Supplement “Chiral Technologies” 30 (2012) 18-21.
[4] M. Sajewicz, R. Piętka, A. Pieniak, T. Kowalska, Acta Chromatogr. 15 (2005) 131-149.
[5] E. Łata, A. Fulczyk, T. Kowalska, M. Sajewicz, J. Chromatogr. Sci. 58 (2020) 5-15.

Ágnes M. Móricz
Plant Protection Institute, Centre for Agricultural Research, ELKH, Herman O. Str. 15, 1022, Budapest, Hungary
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HPTLC hyphenations enable fast and high-throughput screening for bioactive compounds of complex matrices as well as their characterization. However, in several cases the identification of the effective compounds are impossible due to their insufficient separation or low abundance. Here are three solutions. Overpressured layer chromatography (OPLC) as a pump-driven forced flow technique enabled a longer development distance, thus the partial separation of two tansy root compounds, which were not separable by conventional HPTLC [1]. The OPLC-bioassays and OPLC-DART-HRMS showed six compounds responsible for different bioactivity and allowed the identification of four polyacetylenes. Moreover, using irregular particle layers, the separation time was approximately halved by OPLC, if compared to HPTLC. Two closely related constitutional isomers, oleanolic acid and ursolic acid are co-migrated in normal phase HPTLC. These compounds are not distinguishable by HRMS and give a common inhibition zone in the HPTLC-bioassays. The two-dimensional liquid chromatographic hyphenation, the heart-cutting HPTLC-HPLC-DAD-MS via an elution head-based interface enabled the separation and assignment of both isomers in the lemon balm leaf [2]. Ursolic acid was more abundant and is mainly responsible for the bioeffects. In bilateral band compression (BBC) the samples are applied in band, and after development, the lanes are compressed in a perpendicular direction from both sides with appropriate solvent. HPTLC-BBC resulted in more than six times higher peak height and area, however, the noise level and the RSDs were increased as well [3]. OPLC-BBC-bioassay allowed the bio-detection of the minor antibacterial components of thyme essential oil, which were not detectable by a conventional OPLC-bioassay process. This work was partially funded by the National Research, Development and Innovation Office of Hungary (NKFIH K128921).

[1] Á.M. Móricz, T.T. Häbe, P.G. Ott, G.E. Morlock, J. Chromatogr. A 1603 (2019) 355-360.
[2] Á.M. Móricz, V. Lapat, G.E. Morlock, P.G. Ott, Talanta 219 (2020) 121306.
[3] Á.M. Móricz, E. Mincsovics, P.G. Ott, J. Liq. Chromatogr. Relat. Technol. 43 (2020) 300-304.

Mario Aranda1, Karem Henriquez-Aedo2, Jonathan Carrasco-Sandoval2, Pedro Aqueveque2
1Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, 7820436, Santiago, Chile
2Universidad de Concepción, Barrio Universitario s/n, 4070139, Concepción, Chile
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Chronic non-communicable diseases (CNCD) are responsible for 41 million of deaths per year, which represents 71% of all deaths worldwide. The most relevant ones are cardiovascular, cancer, diabetes, and chronic respiratory diseases accounting for around 80% of all CNCD deaths. These figures have increased the interest in searching bioactive molecules in natural sources with therapeutic potential over CNCD and neurodegenerative diseases. Several analytical techniques have been applied to detect this kind of molecules but each one has critical drawbacks. In this scenario, high performance thin layer chromatography (HPTLC) emerges as the technique of choice for bioactive molecules detection and identification because is unique features like the capacity of performing effect-directed analysis (bioassay) straight on the HPTLC plate and the direct coupling with mass spectrometry (MS). This presentation describes some examples of HPTLC/bioassay/MS approach to detect and identify bioactive molecules with therapeutic potential against CNCD and neurodegenerative diseases, in particular, those with inhibitory activity over key enzymes such as acetylcholinesterase, alpha-glucosidase and cyclooxygenase. In this respect, a new HPTLC-bioassay to detect cyclooxygenase inhibitors is presented, which is based on the oxidation of N,N,N,N-tetramethyl-p-phenyl diamine (TMPD) during the reduction of prostaglandin G2(PGG2) to prostaglandin H2(PGH2), reaction monitored at 590 nm. Microplate bioassay conditions, i.e., arachidonic acid and TMPD concentration and incubation time, were optimized via design of experiments and then transferred to HPTLC showing promising results such as inhibitors detection at ug level.

Acknowledgments
Authors want to thank to Chilean National Agency of Research and Development (ANID) for doctoral scholarship granted. This work was financially supported by projects FONDECYT Nº1211803 and REDES Nº180178.

Eike Reich
HPTLC Association, Dianastrasse 10, 4310 Rheinfelden, Switzerland
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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.

Lin-Nan Li, Li-Hua Gu, Zi-Jia Zhang, Li Yang, Fan Li, Yi-Lin Chen, Zheng-Tao Wang
Institute of Chinese Materia Medica/Shanghai University of Traditional Chinese Medicine, 1200 Cailun Rd, 201203, Zhangjiang Hi-Tech Park, Shanghai, China
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Thin-layer chromatography (TLC) is a most economic and conventional technique in authentication of herbal drugs and has been adopted as an official method in Chinese Pharmacopoeia (ChP) since 1977 edition. In the latest edition of ChP 2020, TLC/HPTLC methods are applied in more than 80% of the monographs of Chinese crude drugs. In order to provide intuitive TLC images with more informative characters which will be very helpful for the better understanding and application of the standards, the TLC atlas of Chinese crude drugs in ChP (Volumes 1, 2 and 3) were conducted and published including 117, 111, and 109 crude drugs respectively. The present report briefly introduces the advances in TLC atlas of Chinese crude drugs (Vol 3) completed by our laboratory. Among the 109 species conducted, the TLC/HPTLC authentications of 70 species were carried out by following the methods as described in ChP (2015 edition). Yet the other ones were revised to get more diagnostic and informative TLC images, including the use of new chemical markers, modified samples preparation, chromatographic and detection conditions. In addition, new TLC identification items were established for two crude drugs (Eucommiae Cortex and Nelumbinis Folium). In total, the revision of 24 species have been adopted in ChP (2020 edition). As a continuation, a new volume of the TLC atlas of Chinese crude drugs in ChP (Volumes 4) is in progress which will also include 100 species of Chinese crude drugs. New technologies, such as Characterization of bioactive / chemical markers in herbal medicines aided by HPTLC analyses combined with bioautography-mass spectrometry，or aided with laser ablation-assisted direct analysis in real-time mass spectrometry，were developed.

Acknowledgements
This work was supported by Chinese Pharmacopoeia Commission.

References
S.M. Shi, Z.T. Wang, Y.Q. Jiang. TLC Atlas of Chinese Crude Drugs in Pharmacopoeia of the People’s Republic of China, China Medical Science and Technology Press, Beijing, 2019
F. Yang, L.H. Gu, Z.Z. Han, Z.T. Wang. J Chromatogr. B 1170(2021):122599
Y.L. Chen, L.N. Li, R. Xu, F. Li, L.H. Gu, H.W. Liu, Z.T. Wang, L. Yang. J Chromatogr. A 1654(2021):462461

Colin F. Poole
Wayne State University, Department of Chemistry, Detroit, MI 48202, USA
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The limited capability of models to describe retention for silica gel layers arises from difficulties in separating out the simultaneous contributions of solute and solvent adsorption interactions as well as limitations of the descriptors employed by current models to represent interactions at the adsorbent surface. The heterogeneous energy distribution of adsorption sites results in site-specific interactions and the steric requirements associated with immobile sites for solute adsorption at the adsorbent surface are not easily handled for varied compounds. The separate solute and solvent adsorption parameters of the competition model can be modeled with reasonable success but are difficult to reconcile with their stated properties. The reduction in the contribution of site-specific and steric interactions for RPTLC separations and the formation of a more extensive solvated interphase region provides a more favorable fit to the solvation parameter model. These models emphasize the importance of water in the mobile phase and the general contributions of mobile phase interactions on the retention mechanism. This allows system maps and correlation diagrams based on contribution of individual intermolecular interactions to the retention mechanism to be used for the comparison of selectivity for different layers with the same mobile phase composition or different mobile phase compositions for the same layer.

Vicente Cebolla1, Carmen Jarne1, María Sancho-Albero2, Luis Membrado1, Jesús Vela3, María Savirón4, José-Manuel Escuín1, Javier Galbán5, Pilar Martín-Duque6, Jesús Santamaría2
1Instituto de Carboquímica/CSIC, Miguel Luesma Castán 4, 50018, Zaragoza, Spain
2Aragon Institute of Nanoscience/Universidad de Zaragoza, Campus Rio Ebro, 50018, Zaragoza, Spain
3Analytical Chemistry Department/Universidad de Zaragoza, Campus Río Ebro, 50018, Zaragoza, Spain
4CEQMA/CSIC-Universidad de Zaragoza, Campus San Francisco, 50009, Zaragoza, Spain
5Analytical Chemistry Department/Universidad de Zaragoza, Campus San Francisco, 50009, Zaragoza, Spain
6Instituto Aragonés de Ciencias de la Salud/Fundación ARAID, Campus San Francisco, 50009, Zaragoza, Spain
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The current scientific paradigm to understand the role of lipids in human diseases is based on the hypothesis that some diseases have distinctive lipid profiles (biomarkers) with respect to the respective healthy status.
HPTLC separation of sample, followed by densitometry and MS coupling provides a simple but powerful approach for determining lipid classes and obtaining a detailed structural elucidation of lipids present in biological extracts, allowing their exact recognition by their m/z, and confirmation by their collision-induced dissociation MS/MS data [1].
The application of HPTLC to identification in human plasma of 19 molecular species related to globotriaosylceramides (Gb3) which are considered biomarkers of Fabry disease, a lysosomal storage disorder, will be first discussed [2].
Another example is the open question of whether exosome lipids can be considered as potential cancer biomarkers. This faces our current limited knowledge of their composition. A procedure to isolate exosomes and obtain a phospholipid (PL)-class determination and identification of their molecular species will be also discussed, from embryonic murine fibroblasts (NIH-3T3 cell line), and metastatic murine skin melanoma cells with different degrees of malignancy [3].
For both examples, a detailed structural characterization of molecular species of each concerned lipid class was performed directly from the chromatographic plate, thanks to an elution-based interface, using ion-trap technology by different approaches that involve simultaneous positive and negative ESI-MS, MS/MS, or APCI.

References
[1] V.L. Cebolla, C. Jarne, J. Vela, R. Garriga, L. Membrado, J. Galbán, J. Liq. Chromatogr. & Rel. Technol. 44 (2021) 148-170.
[2] C. Jarne, L. Membrado, M. Savirón, J. Vela, J. Orduna, R. Garriga, J. Galbán, V.L. Cebolla, J. Chromatogr. A 1638 (2021), 461895.
[3] M. Sancho-Albero, C. Jarne, M. Savirón, P. Martín-Duque, L. Membrado, V.L. Cebolla, J. Santamaría, Int. J. Mol. Sci. 2021, in press.

Acknowledgements
This work was supported by DGA-FEDER (E25_20R, N&SB), AES PI21/00036, CSIC 202180E076, ISCIII PI19/01007, ERC Advanced Grant CADENCE (grant no. ERC-2016-ADG-742684)

Gertrud Morlock
Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany
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Is it a dozen or even more? The more complex the samples, the more information is helpful in understanding about their composition. Chromatographic systems and detectors are coupled in hyphenated techniques to extract the maximum amount of information from a chromatographic run. Combining the best features of planar chromatography, effect-directed assays, column chromatography, and spectral detections results in powerful hyphenations [1]. It reveals how efficiently and effectively our natural plant-based food contributes to our homeostasis, and straightforwardly gives important answers [1-4]. Miniaturization and method greenness (eco-friendliness) are also important keys. To achieve this, the simulation of the coordinated processes of all digestion segments was miniaturized and combined with separation and effect-detection. [5] The potential of new technologies was used to create a miniaturized all-in-one open-source 2LabsToGo system [6].

References
[1] G.E. Morlock, Anal. Chim. Acta 1180 (2021) 338644.
[2] T. Schreiner, A. Ronzheimer, M. Friz, G.E. Morlock, Multiplex planar bioassay with reduced diffusion on normal phase: androgens and verified antiandrogens in botanicals, in submission.
[3] I. Klingelhoefer, N. Hockamp, G.E. Morlock, Anal. Chim. Acta 1125 (2020) 288-298.
[4] T. Schreiner, D. Sauter, M. Friz, J. Heil, G.E. Morlock, Front. Pharmacol. 12 (2021) 755941.
[5] G.E. Morlock, L. Drotleff, S. Brinkmann, Anal. Chim. Acta 1154 (2021) 338307.
[6] L. Sing, W. Schwack, R. Göttsche, G.E. Morlock, 2LabsToGo − Recipe for building your own chromatography equipment including biological effect-detection in complex samples, in submission.

Danica Agbaba1, Darija Obradović1, Marija Čarapić2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy-University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia
2Medicines and Medical Devices Agency of Serbia, Vojvode Stepe 450, 11000 Belgrade, Serbia
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Thin Layer Chromatography (TLC) is one of the oldest chromatographic techniques which has widely been used in research on small molecules of natural and synthetic origin with physiological or pharamacological importance. Regulatory requirements for monitoring of quality and safety of substances which remain in pharmaceutical use throughout their whole life cycle (i.e., not only in human body, but also in the environment) become increasingly more strict. This trend is accompanied by development of instrumentation to establish novel and better conditions for drug analysis. The Quality by Design (QbD) methodology is going to be implemented by pharmaceutical industry in production and quality control of active pharmaceutical substances (APIs) and dosage formulations, although it still is not an obligatory regulatory demand. Nowdays, classical yet DoE-supported, or eco-friendly TLC/HPTLC methods are used for qualitative and quantitative pharmaceutical analysis, which includes identification, purity and chiral purity testing, dissolution, and content and uniformity of content with APIs as raw materials and in the dosage form.

Radosław Gwarda, Rafał Gajos, Michał Żmuda Trzebiatowski, Adam Chomicki, Tadeusz Dzido
Department of Physical Chemistry, Medical University of Lublin, Chodźki Street 4a, 20-093 Lublin, Poland
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For the first time, planar electrochromatography in an open system was introduced to the scientific literature in 1974 [1], and in a closed system in 2004 [2]. The latter technique was called pressurized planar electrochromatography (PPEC). Inspired by the results of the papers mentioned, our team also conducted research in this area. In this presentation, we provide a retrospective review of developments in pressurized planar electrochromatography and present the latest research that documents further progress in this separation technique, and two other techniques, which have been evolving because of investigations based on the former:
- pressurized planar electrochromatography, in which the flow of the mobile phase is generated by the action of an electric field (electroosmotic effect);
- orthogonal pressurized planar electrochromatography, in which the flow of the mobile phase is hydrodynamic, and an electric field is applied perpendicular to the direction of this flow;
- high-pressure layer electrochromatography, in which the flow of the mobile phase is hydrodynamic, and the electric field is applied parallel to the direction of this flow.
We also discuss the advantages and problems that should be the subject of further research on the techniques presented.

References
[1] V. Pretorius, B. J. Hopkins, J. D. Schieke, J. Chromatogr. A 99 (1974) 23-30.
[2] D. Nurok, J. M. Koers, A. L. Novotny, M. A. Carmichael, J. J. Kosiba, R. E. Santini, G. L. Hawkins, R. W. Replogle, Anal. Chem. 76 (2004) 1690-1695.

Acknowledgements
This work was supported by National Science Centre, Poland, Agreement No. UMO 2016/23/B/ST4/02877

Snezana Agatonovic-Kustrin1,2, David Morton1,2
1Department of Pharmaceutical and Toxicological Chemistry named after Arzamastsev of the Institute of Pharmacy, I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow 119991, Russia
2Department of Rural Clinical Sciences, La Trobe University, Edwards Rd, Bendigo 3550, Australia
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Olea europaea L. or European olive, is one of the oldest known agricultural fruit trees cultivated to produce olives and olive oil. Cultivation of olive trees for extraction of olive oil generates large quantities of olive leaves as a by-product. Due to their high polyphenolic content, olive leaves present an important raw material. It has been reported by many researchers that olive leaf extract has antimicrobial activity due to its high phenolic content. The aim of this study was to analyse the effect of spontaneous microbial maceration on the release and extraction of the flavonoids and phenolics from olive leaves, and to identify compounds responsible for the antimicrobial activity of olive leaf extracts via thin layer chromatography - direct bioautography (TLC-DB). Results show that fermentation significantly enhances extraction of phenolic compounds and flavonoids. Polyphenolic content was increased from 6.7 µg GAE (gallic acid equivalents) to 25.5 µg GAE, antioxidants from 10.3 µg GAE to 25.3 µg GAE, and flavonoid content from 42 µg RE (rutin equivalents) to 238 µg RE per 20 µL of extract. Increased antioxidant activity of fermented ethyl acetate extracts was attributed to the increased extraction of flavonoids and phenolic terpenoids, while increased antioxidant activity in fermented ethanol extract was due to increased extraction of flavonoids. This increase in extraction may be due, in part, to in situ generated lactic acid based natural deep eutectic solvents (NADES) from the plant sample. Lactic acid that is released during fermentation and glycine present in the olive leaves form a Natural Deep Eutectic Solvent (NADES) with significantly increased solubility for flavonoids. TLC screening and effect-directed analysis of olive leaf extracts revealed two zones that inhibit the growth of different Enterococcus and Staphylococcus bacterial strains. The active compounds were further identified as maslinic and oleanolic acid by comparison of the R_F values of the bands with the standard reference materials and confirmed with ATR-FTIR and NMR spectra after isolation with flash chromatography.