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Comparative Histological, Phytochemical, Microbiological, and Pharmacological Characterization of Some Lythrum salicaria L. Populations Ph.D. dissertation Tímea Bencsik, Pharm.D. Supervisors: Györgyi Horváth PhD Nóra Papp PhD Program leader: József Deli PhD, DSc Head of the Doctoral School: Erika Pintér MD, PhD, DSc Department of Pharmacognosy, Medical School University of Pécs, Hungary Pécs, 2014 1 Table of contents Table of contents ......................................................................................................................... 2 Abbreviations .............................................................................................................................. 4 I. General introduction and aims ................................................................................................. 5 II. Characterization of Lythrum salicaria L. ............................................................................... 7 II. 1. Taxonomy ....................................................................................................................... 7 II. 2. Nomenclature ................................................................................................................. 7 II. 3. Geographic distribution .................................................................................................. 8 II. 4. Interactions of L. salicaria with other plants and animals ............................................. 8 II. 5. Ethnobotany .................................................................................................................... 9 II. 6. Pharmacological aspects of L. salicaria ....................................................................... 10 II. 7. Other possible uses of L. salicaria ............................................................................... 11 III. Investigation of the selected habitats of L. salicaria populations ...................................... 12 III. 1. Materials and methods ................................................................................................ 12 III. 1. 1. Selection and description of habitats ................................................................... 12 III. 1. 2. Ecological and phytosociological character of L. salicaria ................................ 14 III. 1. 3. Weather conditions of the habitats ...................................................................... 14 III. 1. 4. Soil investigation ................................................................................................. 14 III. 2. Results and discussion................................................................................................. 14 III. 2. 1. Selection and description of habitats ................................................................... 14 III. 2. 2. Ecological and phytosociological character of L. salicaria ................................ 16 III. 2. 3. Weather conditions of the habitats ...................................................................... 17 III. 2. 4. Soil investigation ................................................................................................. 20 IV. Morphology and histology .................................................................................................. 22 IV. 1. Materials and methods ................................................................................................ 23 IV. 2. Results and discussion ................................................................................................ 24 V. Phytochemical evaluation of L. salicaria ............................................................................ 42 V. 1. Materials and methods ................................................................................................. 45 2 V. 1. 1. Total polyphenol and tannin content (according to the Ph. Hg. VIII.) ................. 45 V. 1. 2. Total flavonoid content (according to the Ph. Hg. VIII.) ..................................... 46 V. 1. 3. TLC ....................................................................................................................... 48 V. 1. 4. Qualitative-quantitative UHPLC-MS analysis of L. salicaria extracts ................ 49 V. 2. Results and discussion .................................................................................................. 51 V. 2. 1. Total polyphenol and tannin content (according to the Ph. Hg. VIII.) ................. 51 V. 2. 2. Total flavonoid content (according to the Ph. Hg. VIII.) ..................................... 56 V. 2. 3. TLC ....................................................................................................................... 60 V. 2. 4. Qualitative-quantitative UHPLC-MS analysis of L. salicaria extracts ................ 61 VI. Antimicrobial activities of L. salicaria extracts ................................................................. 63 VI. 1. Materials and methods ................................................................................................ 66 VI. 2. Results and discussion ................................................................................................ 67 VII. Pharmacological study of L. salicaria extracts on guinea pig ileum preparation ............. 70 VII. 1. Materials and methods ............................................................................................... 70 VII. 2. Results and discussion ............................................................................................... 72 VIII. Summary and novel findings ........................................................................................... 77 IX. Acknowledgements ............................................................................................................ 82 X. References ............................................................................................................................ 84 XI. Appendix ............................................................................................................................ 95 XI. 1. Snapshots of the habitats ............................................................................................. 95 XI. 2. Plant community composition of the selected habitats and the percentage of plant species in the records (Floristic composition and attributes of the species of the surveyed weed community) .................................................................................................................. 99 XI. 3. Results of the investigation of soil samples .............................................................. 108 XI. 4. Chemical structure of the standard compounds detected in Lythrum extracts by LC- MS method .......................................................................................................................... 110 XI. 5. Total ion chromatograms .......................................................................................... 114 XI. 6. Tube dilution method ................................................................................................ 116 XII. Publications, posters and oral presentations .................................................................... 117 3 Abbreviations 50% ethanol Pharm. 50% ethanol in water extract used for pharmacological experiments 50% ethanol Microb. 50% ethanol in water extract used for microbiological experiments ADM Agar Diffusion Method CB Continentality Figures COX Cyclooxygenase DDM Disk Diffusion Method LB Light Figures MBC Minimum Bactericidal Concentration MIC Minimum Inhibitory Concentration MRSA Methicillin-resistant Staphylococcus aureus MS Mass Spectrometry NB Nitrogen Figures Ph. Hg. VIII. 8th Edition of the Hungarian Pharmacopoeia RB Reaction Figures SB Salt Figures TB Temperature Figures TLC Thin Layer Chromatography UHPLC Ultra High Pressure/Performance Liquid Chromatography WB Moisture Figures 4 I. General introduction and aims Lythrum salicaria L. (purple loosestrife) is native to Europe and Asia, but nowadays it is also widely distributed in North America, Northwest Africa, and Northeastern Australia. It was a well-known and frequently used medicinal plant already in the ancient times. Extracts of the dried aerial parts of this plant were traditionally used in the treatment of diarrhoea, chronic intestinal catarrh, stomach aches, haemorrhoids, eczema, varicose veins, and bleeding gums. Nowadays, Lythri herba, the flowering aerial part of this plant, is an official drug in the Ph. Hg. VIII. Today, its application is less important but even more and more in vitro and in vivo pharmacological investigations are carried out to prove the ethnopharmacological observations (e.g., antidiarrhoeal or haemostyptic effects) and discover new therapeutic purposes (e.g., against hypercholesterolemia or atherosclerosis). Recently, Lythrum salicaria L. has been reported to have astringent, antidiarrhoeal, hypoglycemic, antioxidant, anti- inflammatory, antinociceptive, antifungal, antibacterial, and calcium antagonistic properties (TORRES and SUAREZ 1980; LAMELA et al. 1985, 1986; BRUN et al. 1998, RAUHA et al. 1999; RAUHA et al. 2000; BECKER et al. 2005; TUNALIER et al. 2007; PAWLACZYK et al. 2010), which are attributed to the major groups of its compounds, i.e. tannins and flavonoids (KAHKÖNEN 1999; RAUHA et al. 2001; TOKAR 2007; HUMADI and ISTUDOR 2009; MOLLER et al. 2009; PIWOWARSKI and KISS 2013). L. salicaria extracts also showed inhibitory effect on human acyl-CoA-cholesterol