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
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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
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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
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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 acyltransferase in vitro (due to their triterpene components) suggesting that they might be effective in the prevention and treatment of hypercholesterolemia or atherosclerosis (KIM et al. 2011). Additionally, its polysaccharide- polyphenolic conjugates showed antitussive and bronchodilatory effects on guinea-pig
(ŠUTOVSKÁ et al. 2012).
SHAMSI and WHITEHEAD (1974a, 1974b, 1976, 1977a, 1977b) studied several adaptation mechanisms of L. salicaria caused by environmental changes at different ecological habitats focusing on the modified morphological characters and plant responses to various water supply, temperature, light, and mineral nutrition of the areas. It is evident that these changes in the environment may influence the production of the active compounds, and therefore the quality of the drug. L. salicaria is a traditionally used medicinal plant, and many of its effects have been confirmed by recent investigations. To find and investigate new medicinal plants and to prove the efficacy of ancient medicinal plant taxa are important tasks recently. Most of the active substances of drugs are derived from natural sources. The former and new challenges, for example the antibiotic resistance, have necessitated a continuous search for new active
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compounds. Medicinal plants may be veritable sources of potential antimicrobial compounds for drug industry because of their great chemical diversity.
According to the previously mentioned directions of recent researches on medicinal plants, the specific aims of this study were: to collect and investigate some Hungarian populations of L. salicaria living at different areas (drainage ditches: Szentlőrinc, Szigetvár, Kaposvár, Cserénfa; mesotrophic wet meadows: Csebény, Kacsóta, Almamellék, Szentlászló; and lake edges: Lake of Almamellék, Deseda, Malomvölgy and Sikonda). to measure some histological parameters of leaves, flowers and stems of L. salicaria and to investigate the histological variations among populations with various ecological conditions. to investigate the phytochemical variations of the above mentioned L. salicaria populations. to compare the polyphenol contents of different plant parts (leaf, flower, stem) of L. salicaria populations. to identify and quantify some polyphenolic compounds of L. salicaria extracts by UHPLC-MS. to prove antimicrobial effects of L. salicaria with different microbiological tests and microbes. to evaluate the pharmacological effects of L. salicaria extracts on guinea pig ileum and to try to find whether the identified phenolic compounds could be responsible for these effects or not.
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II. Characterization of Lythrum salicaria L.
II. 1. Taxonomy (according to Borhidi, 2008) Division Magnoliophyta Subdivision Rosophytina Class Rosopsida Subclass Rosidae Superorder Myrtanae Order Myrtales Suborder Lythrineae Family Lythraceae Species Lythrum salicaria L. Purple loosestrife (Lythrum salicaria L.) is one of the representatives of Lythraceae family. The Lythraceae family consists of about 25 genus and 550 species including herbs, shrubs and trees not only in the tropics but also in the temperate climate regions (BORHIDI
2008). The majority of the species in the Lythrum genus are wetland plants (BORHIDI 2008) growing in mesotrophic wet meadows, fen communities, edges of streams, rivers and lakes. About 10 Lythrum species can be found in Europe and altogether 6 species in Hungary: L. thesioides M. B., L. linifolium KARELIN et KIRILOW, L. hyssopifolia L., L. tribracteatum
SALZM., L. virgatum L. and L. salicaria L (SIMON 2004). L. hyssopifolia L. is common in Hungary. L. virgatum L. is rare in the Transdanubian Mountains (Dunántúli-középhegység), the North Hungarian Mountains (Északi-középhegység) and the Transdanubian Hills (Dunántúl), sporadic in the Little Hungarian Plain (Kisalföld), and common in the Great
Hungarian Plain (Alföld). L. thesioides M. BIEB. can be found in the Great Hungarian Plain and the Danube-Tisza Interfluve (Duna-Tisza-köze). L. linifolium KAR. et KIR. lives in the Great Hungarian Plain and the Trans-Tisza Region (Tiszántúl), while L. tribracteatum Salzm. can be found in the North Hungarian Mountains and the Great Hungarian Plain (KIRÁLY
2009). The latter two species are protected taxa in Hungary (SIMON 2004).
II. 2. Nomenclature
According to TUNALIER et al. (2007) Lythrum salicaria L. is also called as L. tomentosum DC. and L. cinereum GRIS. Its Hungarian name is "réti füzény", in English it is called "purple loosestrife", "blooming sally", "purple willow-herb", "rainbow weed", "spiked lythrum", "salicaire" and "bouquet violet", in German "Blutweiderich", in French "Salicaire", in Rumanian "rǎchitan", in Swedish "fackelblomster", in Finnish "rantakukka", in Turkish
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"Tibbi hevhulma", in Chinese "Qian Qu Cai", in Japan "ezo-misohagi", and in Bosnia- Hercegovina "Potočnjak", etc. The generic name of L. salicaria originates from the Greek word "Lytron", meaning blood and indicating the styptic properties of the plant or referring to the red-purple colour of the flower. The specific epithet comes from the fact that the shape of its leaves resembles to those of willow species (Salix spp.) (TEALE 1982; BALOGH 1986).
II. 3. Geographic distribution It is a cosmopolitan and circumpolar plant widely distributed in the northern hemisphere (Europe, Asia, North Africa, and North America), but it can also be found in
Australia and New Zealand (HULTÉN 1971). It is native to Europe and Asia, its pollens were found in eastern Macedonia in Pleistocene deposits (MAL et al. 1992). In Europe, it occurs from Great Britain to central Russia, from the southern coast of Norway, southern Sweden, and Finland to Italy. It is absent from north-west Finland, Denmark, and Iceland (TUTIN et al. 1968). In Asia it is native to Japan, Manchuria, China, and Korea, from south-east Asia to the
Himalayan region, North India, and Pakistan (HULTÉN 1971; BOKHARI 1982). Its North
American distribution is well known (THOMPSON et al. 1987; KEDDY et al. 1994); due to the advantageous circumstances, it is widespread in the whole territory of the USA and Canada
(STUCKEY 1980; ANDERSON 1995). Purple loosestrife was introduced into North America in the early 1910’s maybe via seeds contained in sailing ships’ ballast that was dumped into the east coast harbors (STUCKEY 1980); or by beekeepers, who cultivated it as a honey source plant in summertime (HAYES 1979); or with the wool industry while imported wool contaminated with the seeds of purple loosestrife was washed in various river systems around
New England (THOMPSON et al. 1987). It has been introduced not only into North America, but also into Australia, Tasmania and New Zealand (HULTÉN 1971; HOLM et al. 1979;
HULTEN and FRIES 1986).
II. 4. Interactions of L. salicaria with other plants and animals It is a widespread opinion in the literature that purple loosestrife has a negative impact on the North American flora and fauna. Many researchers suggested that this plant outcompetes all other plants and create monocultures (RAWINSKI and MALECKI, 1984;
THOMPSON et al., 1987). However many articles contradict with this idea. For example, according to HAGER and VINEBROOKE (2004) the plant diversity was higher in patches recently invaded by L. salicaria than in uninvaded patches. RAWINSKI (1982) reported that seedlings of this plant cannot successfully outcompete with those of Phalaris arundinacea,
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however this is also an invasive species. According to LEVIN (1979) as well as LAZRI and
BARROWS (1984), the insects, which pollinate the flowers of L. salicaria include leaf cutter bees (Megachilinae), honey bees (Apinae), carpenter bees (Xylopinae), common sulphur
(Colias philodice), and wood nymph (Cercyonis pegala). KIVIAT (1989) found several species of butterflies (Pieris raphae, Colias philodice, Papilio glaucus) feeding on the nectar of L. salicaria. Besides the pollinators, several insects, e.g., Galerucella calmariensis, G. pusilla (Chrysomelidae), Dasineura salicariae (Cecidomyiidae); Hylobius transversovittatus, Nanophyes marmoratus, N. brevis (Curculionidae); Pyrrhalta calmariensis, P. pusilla, Aphthona lutescens, Altica lythri (Chrysomelidae), Acleris lorquiniana (Tortricidae), and some fungi species were described as herbivores and main natural enemies of purple loosestrife (BATRA et al. 1986; NYVALL, 1995). Some of these species were tried to use as biological control of the dispersion of purple loosestrife (MAL et al. 1992). The plant can also be consumed by herbivorous mammals, as well, e.g. by white-tailed deer (RAWINSKI 1982), muskrat (THOMPSON et al. 1987), rabbits (ANDERSON, 1995), and possibly meadow voles
(KIVIAT 1989). The germinated seeds can provide food for herbivorous fishes (ANDERSON
1995). The following waterfowl and songbirds have been observed nesting in stands of purple loosestrife: red-winged blackbirds (Agelaius phoeniceus), American coots (Fulica americana), pied-billed grebes (Podilymbus podiceps), black-crowened night-herons (Nycticorax nycticorax), American goldfinches (Caeduelis tristis), and gray catbirds
(Dumetella carolinenseis) (RAWINSKI 1982; KIVIAT 1989; ANDERSON 1995). According to
RIDLEY (1930), birds living in wet habitats play a significant role in the dispersion of the seeds in mud.
II. 5. Ethnobotany Purple loosestrife is traditionally used internally as a decoction, or as extracts for diarrhoea, chronic intestinal catarrhs, haemorrhoids, and eczema, or externally for varicose veins, venous insufficiency, and bleeding of the gums (MANTLE et al 2000; RAUHA et al.
2000; SZENDREI and CSUPOR 2009). In some ancient herbal medicinal books, we found several monographs about this species, e.g., in Historia Plantarum (TEOPHRASTUS 1644),
Historia Plantarum (CLUSIUS 1601), Materia Medica (DIOSCORIDES 1557), Stirpium Historiae
(DODONEUS 1583), Herbarium and Kreuterbuch (HIEROMYMUS TRAGUS BOCK), Kräuterbuch
(LONICERUS 1593), and Commentarii in pedacii libros Dioscoridis Anazarbei de Materia
Medica (MATTHIOLUS 1554) mentioned as Lysimachia or Pseudolysimachia. The aims of this study do not include the investigation of ancient herbal medicinal books.
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II. 6. Pharmacological aspects of L. salicaria Lythri herba, the whole flowering aerial parts, is official in the Hungarian Pharmacopoeia 8th ed (Ph. Hg. VIII.), which corresponds to the European Pharmacopoeia 4- 7th edition. It contains not less than 5.0% of tannins, expressed as pyrogallol and calculated with reference to the dried drug. The pharmacopoeias order the macroscopic and microscopic identification of the drug, examination of its flavonoids by thin-layer chromatography, tests for foreign matter, loss on drying and total ash, as well as the determination of its total tannin content. Several ancient and medieval use of the plant has been proved recently. The use against diarrhoea is based on ethnopharmacological observations but no human clinical trials have been carried out in connection with this effect. Salicarine®, a natural product elaborated in France is made from the flowering shoots of purple loosestrife. The antidiarrhoeal activity of this product was compared to loperamide in the case of castor oil-induced diarrhoea in mice and bisacodyl-induced increase in large intestine transit of rats. Salicarine® partially reduced the contractions of isolated rat duodenum induced by barium chloride and acetylcholine, but it did not change the normal gastrointestinal transit in mice (BRUN et al. 1998). Significant antioxidant properties of purple loosestrife have been reported in several articles (RAUHA 2001; TUNALIER et al. 2007; LÓPEZ et al. 2008; BORCHARDT et al. 2009).
RAUHA (2001) investigated the in vitro calcium antagonistic activity of L. salicaria, and its 2+ extracts effectively inhibited Ca fluxes into rat pituitary GH4C1 cells. KIM et al. (2011) suggested that L. salicaria might be effective in the prevention and treatment of hypercholesterolemia or atherosclerosis, because its extracts showed in vitro inhibitory effects on human acyl-CoA-cholesterol acyltransferase due to its triterpene components, i.e. betulinic acid and its derivatives. PAWLACZYK et al. (2010, 2011) investigated the glycoconjugates of L. salicaria and their effects on haemostasis. In vitro and ex vivo experiments revealed inhibitory effects on plasma clot formation, but in vivo a pro-coagulant activity was detected.
According to ŠUTOVSKÁ et al. (2012), polysaccharide-polyphenolic conjugates of purple loosestrife showed antitussive and bronchodilatory effects in guinea-pigs and reduced the number of cough efforts even 5 hours after administration; however, their antitussive effect was lower compared to codeine. Many investigations have been carried out on the therapeutic use of flavonoids against inflammation (HARBORNE and WILLIAMS 2000; HAVSTEEN 2002). TUNALIER et al. (2007) investigated the anti-nociceptive activity (with p-benzoquinone-induced abdominal constriction test) and anti-inflammatory activity (with carrageenan-induced hind paw edema
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model) of L. salicaria in vivo in mice. Only the methanol extract showed inhibitory activity against carrageenan-induced hind paw edema and p-benzoquinone-induced writhings. In their study, none of the extracts caused any gastric damage. PIWOWARSKI et al. (2011) reported notable anti-hyaluronidase and anti-elastase activity of Lythri herba extracts supporting its anti-inflammatory effect. The extracts of the stem and flowers showed significant hypoglycemic effect both on hyper- and normo-glycemic rats by increasing the level of insulin in the blood (LAMELA et al.
1985, 1986). YOSHIDA et al. (2008) searched for effective α-glucosidase inhibitors by screening of 218 plants, to contribute to the development of physiological functional foods or lead compounds against diabetes. Leaves of L. salicaria showed significant inhibitory activity against rat intestinal maltase (90%) and sucrase (93%).
II. 7. Other possible uses of L. salicaria Besides the therapeutical use, purple loosestrife can also be utilized for other purposes, for example previously its purple flowers were used as dyeing plant for foods (SZENDREI and
CSUPOR 2009). Its extracts have been reported to have molluscicidal acitivity
(SCHAUFELBERGER and HOSTETTMANN 1983). Cultivars of purple loosestrife have been developed and widely cultivated due to its conspicuous flowers (MAL et al. 1992). Its honey has a characteristic colour and flavour, in some areas it is quite good, but in some cases it can be dark with rather objectionable flavour maybe due to the different soil conditions of the areas (BUNCH 1977). Pollen grains of the medium and short stamens are yellow, those of long stamens are green (MAL et al. 1992), and presumably for this reason, the honey has also a greenish color (HAYES 1979). Its superior competitive activity was found to be beneficial in the control of the spread of Alopecurus pratensis L. in the floodplain of the Tisza
(BODROGKÖZY and HORVÁTH 1979). BUSH et al. (1986) suggest that L. salicaria can accumulate greater amounts of polychlorobiphenyl (PCB) congeners and could be used as an environmental biomonitor agent. MUDROCH and CAPOBIANCO (1978) observed that L. salicaria can accumulate metals, as well. Metal concentrations (Zn, Pb, Cu, Cr, Ni, Co, and Cd) were found to be higher in the roots than in the above-ground plant parts. Recent studies aim at using L. salicaria for the removal of pollutants from household, urban, winery, and industrial wastewaters (ZHAO et al. 2009, MENA et al. 2009).
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III. Investigation of the selected habitats of L. salicaria populations
In this chapter, the habitats, as well as the weather and soil conditions of the selected habitats are summarized. Purple loosestrife is a wetland plant growing in wet habitats with variable water supply (SOÓ 1966): in marshes, mesotrophic wet meadows, fen communities, lake shores, riversides, ditches, etc. (SOÓ 1966; SHAMSI and WHITEHEAD 1974a). But its elevational range rarely extends 600 m (POST 1932). The social behaviour type of purple loosestrife is a generalist of wide ecological range and tolerance, living in various communities (BORHIDI 1993).
III. 1. Materials and methods
III. 1. 1. Selection and description of habitats Whole herbs of 12 L. salicaria populations were collected from different ecological habitats in south-west Hungary (Baranya and Somogy counties) in summer of 2010 and 2011: drainage ditches (Szentlőrinc, Szigetvár, Kaposvár, Cserénfa), mesotrophic wet meadows (Csebény, Kacsóta, Almamellék, Szentlászló), and lake edges (Lake of Almamellék, Deseda, Malomvölgy, and Sikonda) (Fig. 1). Specimens were identified by the botanist Dr. Nóra Papp, and voucher specimens have been deposited in the Department of Pharmacognosy at University of Pécs (Nos.: L. s. 10SO, L. s. 10KC, L. s. 10SI, L. s. 10SA, L. s. 10AR, L. s. 10LA, L. s. 10CB, L. s. 10KP, L. s. 10LD, L. s. 10CR, L. s. 10LS, L. s. 10LM, L. s. 11SO, L. s. 11KC, L. s. 11SI, L. s. 11SA, L. s. 11AR, L. s. 11LA, L. s. 11CB, L. s. 11KP, L. s. 11LD, L. s. 11CR, L. s. 11LS, L. s. 11LM). Herbs were dried at room temperature for 3 weeks, ground, and stored in dark. The geographical coordinates and altitudes were measured by a GPS Navigation System (VayteQ N720BT).
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LLDD
KKPP
CCRR
LLAA LLSS CCBB SSLL AARR KKCC SSOO SSIII
LLMM
Fig. 1. Study areas of the surveyed region (the selected habitats are marked with red points). (Abbreviations: SO – Szentlőrinc, KC – Kacsóta, SI – Szigetvár, SL – Szentlászló, AR – Almamellék, LA – Lake Almamellék, CB – Csebény, KP – Kaposvár, LD – Lake Deseda, CR – Cserénfa, LS – Lake Sikonda, LM – Lake Malomvölgy)
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III. 1. 2. Ecological and phytosociological character of L. salicaria Phytosociological relevés were conducted on 12 selected areas in September, 2011, and the species co-occurring with L. salicaria were determined by the coenologist Dr. Róbert Pál. The size of individual plots was 1 m2, in a square shape.
III. 1. 3. Weather conditions of the habitats The monthly precipitation was measured in Szentlászló, as well as data of monthly precipitation, average temperature, and sunshine could be asked from the Hungarian Meteorological Service (Országos Meteorológiai Szolgálat - OMSZ) in the case of the habitats of Szigetvár, Kaposvár, and Pécs. Because of their request, we cannot give exact numbers of these values. Unfortunately, these data could not be gained in the case of the other habitats.
III. 1. 4. Soil investigation The fieldwork soil samples (0.5 kg/habitat) were collected in the selected areas near the plant samples from the above 10 cm layer of the ground soil in July 2010, and July and August 2011. The foreign elements (coming either of vegetable, animal, or mineral origin) were removed from the collected soil samples. "Free" water content was determined by gravimetric method and expressed in m/m%. The collected samples were measured, dried for 3 weeks at room temperature and then measured again. Physisorbed water content was not investigated. After determination of “free” water content, the soil samples were ground. For determination of the pH, 50.0 mL distilled water was added to 20.0 g soil samples, homogenized, allowed to stand for 1 hour, then filtered. The pH values were measured with DZS-708 Multi-parameter Analyzer and WTW SenTix 60 combination electrode. The content of nitrate, nitrite, sulphate, phosphate, potassium, and ammonium ions were detected by Quantofix test strips (Macherey Nagel, Germany).
III. 2. Results and discussion III. 2. 1. Selection and description of habitats The altitude of the habitats varied from 120 m (Szigetvár) to 185 m (Lake Sikonda, in Mecsek Hills) (Table 1).
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Table 1. Abbreviations, latitude, longitude, and altitude of the selected habitats.
Habitats Latitude Longitude Altitude
Szentlőrinc N 46º 02'43.3” E 17º 59'26.2” 131 Kacsóta N 46º 02'16.5” E 17º 56'51.8” 121 Szigetvár N 46º 03'15.6” E 17º 48'39.9” 120 Szentlászló N 46º 08'32.6” E 17º 49'47.5” 135 Almamellék N 46º 10'03.3” E 17º 53'25.5” 147 Lake Almamellék N 46º 10'06.9” E 17º 53'57.4” 141 Csebény N 46º 11'01.5” E 17º 55'15.1” 149 Kaposvár N 46º 20'54.9” E 17º 46'46.0” 140 Lake Deseda N 46º 26'26.1” E 17º 47'25.4” 139 Cserénfa N 46º 18'07.2” E 17º 52'54.4” 146 Lake Sikonda N 46º 10'53.3” E 18º 12'59.6” 185 Lake Malomvölgy N 46º 01'06.8” E 18º 12'01.8” 142
"Szentlőrinc" is a ruderal habitat, samples were collected in a drainage ditch near to the road. Near "Kacsóta", samples were collected from a mesotropic wet meadow. Plant and soil samples could only be collected in 2010, while in 2011 L. salicaria extincted from this area. Near "Szigetvár" the samples were collected from a drainage ditch, in a tall-sedge vegetation. There is a mesotropic wet meadow near a fishpond besides "Szentlászló", where our samples were collected. Near Almamellék, we collected samples from two areas. One of them was a waterside woody vegetation of a drainage ditch of a lake along the road ("Lake Almamellék"). On the other hand samples were collected from a tall-sedge vegetation of a mesotropic wet meadow, besides the rails of an operating narrow-gauge railway ("Almamellék"). Near "Csebény" the samples were collected from a grazed tall-sedge vegetation in a mesotropic wet meadow, this place can be considered as the habitat least affected by humans. Samples were collected from a drainage ditch in the downtown of "Kaposvár", in a bulrush sedge vegetation on the edge of "Lake Deseda", from a drainage ditch near "Cserénfa", and from the shore of "Lake Sikonda". "Lake Malomvölgy" is a popular place of excursion near to Pécs (the county town of Baranya county); and therefore an anthropogenically disturbed habitat. Samples were collected from the shore of this lake. For snapshots of the habitats, see Appendix 1; for features of the microclimates of the habitats see Table 2.
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Table 2. Features of the microclimates according to Dövényi, 2010.
the
mg/L
mg/L
nk°;
nk°; innk°; the nk°, sulfate
<60
the nitrate
:
nk°,
35
,
25 25 nk°; 35
mg/L
-
- - -
mg/L
tent
hydrogen
60 <60
re 25
-
:
Water Properties of calcium and magnesium carbonate character, hardness:15 Kapos Valley content of calcium and magnesium carbonate character, hardness:15 sulfate con alkaline carbonate and chloride thermal water of calcium and magnesium carbonate character, hardness:25 content: of calcium and magnesium hydrogencarbonate character, hardness:in Szigetvár <25 elsewhe sulfate content: ~ 60
1.00 0.94 1.10 1.00
- - - -
Aridity index 1.00 0.96 0.90 1.00 0.98
430
700 / 410 730 / 420 450 670 / 400 720 /
-
------
0
90
3
Annual precipitation (mm) / Precipitation in the vegetation period(mm) 680 390 680 390 750 / 400 640 370 60
0.3
1 17.0) 16.5) 10.8
- - - -
9.5
-
Mean annual temperature (°C) Mean / temperatureof the growing season (°C) 10.0 (summer: 16.6 10.0(summer: 16.5) 9.0 (summer: 15.8 10.6 (summer: 17.0) 10.0(summer: 16.5)
r:
2020 2020 2050 2020
- - - -
820)
-
Sunshine duration (hours / year) 2000 (summe 810) 2000 (summer: 810) 2030 (summer: 810 2060 (summer: 820) 2000 (summer: 810)
, ,
Climate moderately warm, moderately wet moderately warm, moderately wet moderately warm, moderately wet moderately warm, moderately wet moderately warm moderately wet
-
-
(KP)
(SI)
(CR)
(CB)
(LS)
(KC)
alom
(LA)
(LM)
Name of the habitat Lake Deseda (LD) Kaposvár Cserénfa Sikonda Lake M völgy Szentlőrinc (SO) Kacsóta Szigetvár Szentlászló (SL) Almamellék (AR) Lake Alma mellék Csebény
-
- -
Zselic
-
Külső Zselic
- - -
mogy
Microregion Dél So Észak Baranyai Hegyhát Dél Baranyai Dombság Dél
III. 2. 2. Ecological and phytosociological character of L. salicaria The number of the species varied between 9 (Szigetvár) and 28 (Cserénfa) within the plots. The most frequently occurring species were Calystegia sepium (L.) R. BR. (in 8 habitats), Ranunculus repens L. (in 6 habitats), Carex acutiformis EHRH., Elymus repens (L.)
GOULD, Solidago gigantea AITON, Symphytum officinale L. (in 5 habitats), Carex riparia
16
CURTIS, Epilobium hirsutum L., Equisetum arvense L., Erigeron annuus (L.) PERS., Galium mollugo L., Glechoma hederacea L., Lycopus europeus L., Potentilla reptans L., Taraxacum officinale WEBER ex WIGGERS, and Urtica dioica L. (in 4 habitats). The list of the species in the investigated fields is given in Appendix 2.
Compared to our results, THOMPSON et al. (1987) found that in Northeast, Midwest and Northwest United States and southern Canada the following taxa were associated with L. salicaria: Typha latifolia, Phalaris arundinacea, Carex spp., Scirpus spp., Salix spp., Equisetum fluviatile, Typha angustifolia, Phragmites australis, Cyperus sp., Alisma plantago- aquatica, Urtica dioica, Sparganium eurycarpum, Agrostis gigantea (alba), Acorus calamus, Populus deltoides, Chenopodium album, Rubus spp., Euphorbia spp., Impatiens capensis, Cornus stolonifera, Convolvulus arvensis, Verbena hastata, Solanum dulcamara, Eupatorium perfoliatum, Solidago spp., Aster sp., Cirsium arvense, and Lactuca scariola.
If we compare our list (Appendix 2) to the results of THOMPSON et al. (1987), the following species are common: Carex spp., Solidago spp., Urtica dioica, Typha latifolia, Phalaris arundinacea, Salix spp., Alisma plantago-aquatica, Rubus spp., Convolvulus arvensis, Solanum dulcamara, and Cirsium arvense.
III. 2. 3. Weather conditions of the habitats The climate, sunshine duration, mean annual temperature, mean temperature of the growing season, annual precipitation, precipitation in the vegetation period, and water properties of the habitats are detailed in Table 2. The warmest month is July (when purple loosestrife starts to flower), its mean temperature is 19-21ºC. The main genetic soil types are brown forest soils and meadow soils, and the soil water management is moderate (KOCSIS and
SCHWEITZER 2009). In both 2010 and 2011, the least precipitation was measured in Pécs. The highest precipitation could be observed July 2011, the lowest ones were measured in July 2010 and August 2011 (Fig. 2).
17
Fig. 2. Mean average precipitation (mm) of Szentlászló, Pécs, Kaposvár, and Szigetvár in 2010 and 2011.
The monthly average temperature values were available only in Pécs, Kaposvár, and Szigetvár. The highest average temperature could be measured in Pécs in all examined months, while in Szigetvár and Kaposvár the values were very similar to each other within one month (Fig. 3).
Fig. 3. Mean average temperature (°C) of Pécs, Kaposvár and Szigetvár in 2010 and 2011.
Values of montly sunshine duration were available only from Pécs. The sum of these values was higher in 2011 than in 2010 (Fig. 4).
18
Fig. 4. Mean average sunshine duration (hours) of Pécs in 2010 and 2011.
WHITEHEAD (1962) showed that plants are capable of advantageous modifications of their phenotype in response to environmental changes. Several studies have been carried out related to various species which may react differently and although such compensating mechanism may aid survival, the adverse effects mostly affect the growth, development, or reproduction (MYERSCOUGH and WHITEHEAD 1967; PAPP et al. 2005; PAPP et al. 2012).
SHAMSI and WHITEHEAD (1974b) studied the effects of light on growth and development of L. salicaria, and they found a comparable morphological variability. It seems to be tolerant to moderate shade, but there was a progressive increase in leaf size with reduction of light intensity. In full light, the leaves were thick and had many trichomes, but with decreasing light intensity, there was a reduction in leaf thickness and a decrease in the amount of tomentum. With decrease in light intensity, there was a marked delay and reduction in dry weight production, number of flowers, and subsequently in the number of fruits and seeds. The root was only little affected by light intensity. They showed that the 13-hour daylength period is critical and required for the changes in morphogenesis and flowering. According to
SHAMSI and WHITEHEAD (1977b), the temperature is a crucial factor in growth and development of purple loosestrife. Only a little effective germination occurred below 20°C and none below 14°C, but rapid growth could be seen at high temperature required for germination (SHAMSI and WHITEHEAD 1974a). The temperature and the amount of precipitation are considered to be the most important factors from the point of view of the development and reproduction (SHAMSI and WHITEHEAD 1974a; THOMPSON et al. 1987), however, well-established plants can persist in dry sites for many years (POWELL et al. 1994).
LEMPE et al. (2001) investigated whether the tolerance to flooding contributes to the invasive nature of purple loosestrife in North America. But while other members of the Lythraceae family responded similarly to flooding as L. salicaria, this feature is considered not to be responsible for the invasiveness of this plant.
19
Unfortunately, we could not collect enough meteorological data to be able to draw firm conclusions regarding how the weather conditions influence the morphological and phytochemical features of L. salicaria.
III. 2. 4. Soil investigation Table 3 summarizes the average results of the investigation of the soil samples. The pH of the soil was slightly alkaline in all of the habitats except for Cserénfa (having neutral soil). The highest soil pH value was measured in Szigetvár (8.03), the lowest one in Cserénfa (6.95). The sulphate concentration of the soil samples was under the detection limit of the test strips in all samples, except for Cserénfa (56 mg/100 g soil). Nitrite could not be detected with this method. The average "free" water content was high in all of the soil samples (Table 3 and Appendix 3). In Lake Sikonda and Lake Malomvölgy, soil samples could not be collected directly around the plant, because it was too deep to reach the bottom of the lake. Accordingly, samples were obtained from the shore, and for this reason, the "free" water content is low in these samples. Table 3. Average results of the investigation of soil samples. (For detailed results, see Appendix 3).
- - 2- 3- + + "free" water content Mean NO3 (mg/ NO2 (mg/ SO4 (mg/ PO4 (mg/ K (mg/ NH4 (mg/ Habitat (m/m%) pH 100 g) 100 g) 100 g) 100 g) 100 g) 100 g)
Lake 46.47 7.91 5.00 0 <50 1.25 25.00 0.00 Almamellék Almamellék 39.45 7.74 9.58 0 <50 1.08 50.00 0.25 Csebény 44.72 7.75 3.33 0 <50 2.00 41.67 0.17 Cserénfa 29.66 6.95 31.25 0 56.25 0.92 41.67 0.42 Lake Deseda 27.78 7.81 4.58 0 <50 1.25 33.33 0.17 Kacsóta 28.40 7.61 5 0 <50 1.75 50.00 0.75 Kaposvár 25.59 7.98 3.33 0 <50 0.92 33.33 0.17 Lake 4.49* 7.97 13.75 0 <50 0.92 33.33 0.00 Malomvölgy Lake Sikonda 2.47* 7.79 2.08 0 <50 1.25 25.00 0.00 Szentlászló 65.97 7.25 2.50 0 <50 1.25 41.67 1.08 Szentlőrinc 17.69 7.79 7.08 0 <50 1.08 33.33 0.17 Szigetvár 31.31 8.03 5.42 0 <50 0.67 25.00 0.25 * The plants grew in the bottom of the lake and soil samples could not be collected directly around the plant; therefore they were obtained from the shore, and for this reason, the free water content is low in these samples.
L. salicaira can be found on humid, both calcareous and acid soils (BORHIDI 2003).
SHAMI and WHITEHEAD (1977a) investigated the tolerance of this plant to nutrient deficiency, and it seems to be more affected by nitrogen (N) than phosphorus (P) and potassium (K) deficiency. It showed a progressive reduction of flowering and fruiting by an increase in the deficiency of N, P and K, and responded to each reduction of nutrient concentration by an
20
increase of the root/shoot ratio. Therefore they concluded that potassium deficiency may play a crucial and differentiating role in the establishment and growth of this species. SHAMI and
WHITEHEAD (1977b) published that germination and seedling growth of L. salicaria can be successful both in basic and acidic (pH 4 and above), nutrient-rich and nutrient-poor soil, and these properties may contribute to its occurrence in a wide variety of the habitats. But according to HASLAM (1965), low pH suppresses the germination of L. salicaria. According to the relative "temperature figures", it can be found in the montane mesophilous broad-leaved forest belt (TB=5). Based on the relative "moisture figures", the plant prefers wet and not very well aerated soils (WB=9). It is a basifrequent plant occurring mostly on basic soils (its soil reaction values, RB = 7). Concerning the ammonia and nitrate supply, it prefers the submesotrophic areas (NB=4). It is a salt tolerant species living mainly at non saline soils (its salt tolerance, SB = 1), and occasionally at mildly saline soils (0–0,1% Cl-). It is a halflight plant mostly occurring in full light (light intensity, LB = 7), and occasionally as a shadow tolerant species. Purple loosestrife represents an intermediate type with slight suboceanic-subcontinental character (degree of continentality, CB = 5) (BORHIDI 1993). In agree with literature data, our L. salicaria samples were found mostly in basic soils and wet habitats, and it seems, that there was no nutrient deficiency in either habitats. Most of the plants lived in full light, there was found only a partial shade of trees in Lakes Malomvölgy and Sikonda, Cserénfa, and Kaposvár.
21
IV. Morphology and histology
Lythrum salicaria L. can vary in some morphological and phytochemical features according to its habitat. The great diversity of the plant may arise from genetic reasons. In the Lythrum genus the basic chromosome number is 5 with haploid numbers of 5, 10, 15, 25 and
30. In L. salicaria, n can be 15, 20 or 30 (TOBE et al. 1986; GRAHAM et al. 1987). In our study genetic variations has not been studied. L. salicaria is tolerant to a wide range of various ecological conditions, for example, it is able to make direct developmental responses in submerged stems, increased production of aerenchyma, or changes in leaf morphology (THOMPSON et al. 1987). Previously, some histological studies of the plant organs of purple loosestrife have been reported (SCHOCH-
BODMER 1938-1939; STEVENS et al. 1997; KEMPERS et al. 1998; LEMPE et al. 2001; STEVENS et al. 2002; RUDALL 2010). In North America, where L. salicaria is an invasive species, several studies were carried out associated with the histological structure of the stem and root. In the plants living in humid areas, aerenchyma functions as an adaptive tissue type in the case of changed environmental conditions. Adaptive features of parts of L. salicaria were studied by STEVENS et al. (1997, 2002). Plants have been germinated in standard conditions, transferred to pots filled with sand, and grown in wet conditions. The aerenchymatous phellem, which can be found in the stem and plays a significant role in oxygen transport and storage, was removed from the root, and the gaseous exchange was measured with gas chromatography after seven weeks. The oxygen level decreased and influenced the morphology of the root, which consequently became smaller in size (STEVENS et al. 2002). In another study, plants grown in wet treatment had more adventitious roots and thicker phellem in the submerged tissues, which refer to the responsive ability of the species through the change of the gas exchange in modified environmental conditions (STEVENS et al. 1997).
LEMPE et al. (2001) have also studied 6 Lythrum species in flooding treatment. Among the histological characters of L. salicaria investigated with scanning electron microscope (SEM), they observed multiplied layers of phellem, bigger diameter, numerous lacunes, and Casparian bands in the cell walls of the submerged stem. In addition, the height of the plant has decreased together with the reduced availability of water. Vascular elements and vessels of the family Lythraceae and L. salicaria were summarized by SCHWEINGRUBER et al. (2011). Ring boundaries of xylem are diffuse-porous to semi-ring-porous in all species. Vessel diameter varies from 20-40 μm in herbaceous taxa and 50-100 μm in shrubs, with simple perforations arranged opposite to each other. Fibres are thin or thick-walled with small pits. Axial parenchyma is sometimes paratracheal. Vessels of L. salicaria contain various phenolic
22
substances in the heartwood. Sieve-tubes and parenchyma are difficult to distinguish on transverse sections in the phloem, having several crystal druses in the parenchyma cells.
IV. 1. Materials and methods
Plant materials. Whole herbs of twelve selected populations of L. salicaria were collected at different ecological habitats in South-West Hungary in summer 2010 (for details see Chapter III. 1. 1.) Chemicals and solvents. Ethanol and glycerine were purchased from Molar Chemicals Ltd., Hungary, Technovit 7100 from Realtrade Ltd., Hungary, and Canada balsam from Merck Ltd., Hungary. Histological preparation. Studied plant materials (stem, leaf, bract, and flower) in each population (2 pieces/2 specimens/population) were fixed in the mixture of 96% ethanol : glycerine : distilled water (1:1:1). It was followed by dehydration in ethanol series (in 30, 50, 70, and 96% for 12, 12, 24, and 3 hours, respectively), then samples were embedded in synthetic resin (Technovit 7100). Longitudinal and transverse sections (10-15 μm thick) were prepared by a rotation microtome (Anglia Scientific 0325), stained in toluidine blue (0,02%) for 5 minutes, rinsed in distilled water, then transferred to 96% ethanol for 3 minutes twice, to isopropanol for 2 minutes, to xylene for 3 minutes then for 10 minutes, and finally covered with Canada balsam (SÁRKÁNY and SZALAI 1957). Cleared preparations. The structure of the trichomes (shape, cell number) was investigated in cleared preparations. Firstly the fixed samples (2 pieces/2 specimens/population) were cut into 2 x 2 cm pieces and stored in 5% potassium hydroxide for 72 hours, then in 5% sodium hypochlorite for 10 minutes, until each sample lost its colour. Then they were rinsed with distilled water three times (for 10 minutes, each), and dehydrated in ethanol series (in 25, 50, 75, and 100% for 10 minutes, respectively). Finally, the cleared leaf pieces were placed in the mixture of 96% ethanol : xylene (1:1) for 10 minutes and fixed in Canada balsam. Programs used for evaluation. Slides were studied by Nikon Eclipse 80i and Opton Anxiovert 35 microscopes, Olympus 12 MP digital camera, SPOT BASIC 4.0, and Olympus Cell D programs. Histological data were measured by Image Tool 3.00 program. At least 30 measurements per 2 specimens of each population were performed for all parameters. The studied parameters were the following: thickness of the leaves and bracts (µm), height and width of the epidermal cells (µm), height and width of Ca(COO)2 crystals in all plant parts
23
(µm), height and width of palisade and spongy cells in mesophyll of leaves and bracts (µm), and finally the ratio (%) of the thickness of phloem and xylem in the stem. Statistical analysis. Data were presented as mean values. For the analysis of the primary data Microsoft Excel 2010 Software was used in all experiments. Statistical evaluation of the data was performed with the SPSS 13.0 for Windows (SPSS Inc., Chicago, Illinois, USA). Histological data were evaluated by ANOVA with Bonferoni, Tukey, Tahmane and Dunnet post hoc tests. The correlation between the plant height in 2010 and 2011 was tested by One-way-ANOVA.
IV. 2. Results and discussion
Root
Purple loosestrife is a hemicryptophyte plant (SOÓ 1966), its overwintering buds are at the surface of the soil (DARÓK 2011). It can be characterised by climbing root-stock (KIRÁLY
2009), but adventitious roots have not been described (SHAMSI és WHITEHEAD 1974a). Up to 30-50 herbaceous stems rise from a persistent perennial tap root (Fig. 5) and a spreading rootstock (up to 0.5 m diameter) (THOMPSON et al. 1987; MALECKI et al. 1993). A well developed plant may have a rootstock weighing up to 1 kg (online source 1). This species may spread vegetatively with the parts of the rootstock (BENDER 1987; ROYER and
DICKINSON 1999), but this phenomenon is less frequent compared to the propagation via seeds (SHAMSI and WHITEHEAD 1974a). In our study, the rootstock was not investigated.
Fig. 5. Tap root system of L. salicaria
Stem Purple loosestrife can be characterised by about 1-1.5 m height in average, however, in some articles, specimens with 2.7 m height were also reported (MAL et al. 1992). According to the literature, a lower plant height may result from nutrient deficiency (e.g., nitrogen, phosphorous, potassium), less sunlight, or reduced availability of water (SHAMSI and
WHITEHEAD 1977a, b; LEMPE et al. 2001).
24
Table 4. Average height of the plants at the selected habitats (in cm). The values are averages of 6-10 parallel measurements. Habitat 2010 2011 Szentlőrinc 125 70 Kacsóta 120 -* Szigetvár 130 120 Szentlászló 140 115 Lake Almamellék 160 100 Almamellék 160 90 Csebény 140 105 Kaposvár 145 120 Lake Deseda 130 90 Cserénfa 110 80 Lake Sikonda 80 60 Lake Malomvölgy 185 155 Average 135 100 *At this place, we could not collect samples, therefore this data point is missing.
In our study (Table 4), the average height of the plants was between 80 and 185 cm in 2010 (the average of all populations: 135 cm), and between 60 and 155 cm in 2011 (the average of all populations: 100 cm). It can be seen clearly, that the plants grew higher in 2010 than in 2011 in all habitats probably due to the different weather conditions (p< 0.005), resulting in smaller biomass production. The angular stem (Fig. 6 and 7) is usually covered by trichomes in whole length
(KIRÁLY 2009), but sometimes it can be hairless. It is brownish-green, rigid, four-angled, longitudinally wrinkled, and branching at the top (Ph. Hg. VIII.). Under the single cell layer epidermis, the subepidermal region consists of the elements of angular collenchyma in two cell rows. The primary cortex found between the epidermis and vascular tissues is composed of isodiametric cells. The compressed cell layers of the phloem are followed by the ring of the secondary cambium and xylem elements comprising several concentric circles in the stem.
The xylem has intraxylary phloem elements (CUTLER 1978) and tracheas forming groups with 1 to 4 elements. The pith consists of several isodiametric parenchyma cells and intercellular spaces in the central part of the stem. In the phloem, symplastic connection with plasmodesmata was described between the sieve elements, companion cells, and the phloem parenchyma cells. The cytoplasm is not dense, and the cell walls have no protrusions in the species (KEMPERS et al. 1998).
25
Fig. 6. Stem and leaves of L. salicaria
primary cortex
xylem
phloem
epidermal cells
Fig. 7. Stem of L. salicaria (cross section)
The ratio of the xylem and phloem was similar in all samples collected from various habitats (xylem: 91.0-95.0% in thick stems and 88.0-93.0% in thin stems), except for the sample collected in Kacsóta, where the ratio of the xylem was only 74.0% maybe due to the lack of optimal conditions. This theory may be supported by the fact, that L. salicaria extincted from this habitat in 2011. The epidermal cells of the thick stem were elongated and flat, while those of the thin stem were nearly isodiametric (Table 5). There was no difference in the size of Ca(COO)2 crystal druses between the thicker and thinner stems (Table 5).
26
Table 5. Mean ±SD of the measured histological parameters of the stem. Thick stem Thin stem Stem Mean ±SD Mean ±SD Thickness of the primary cortex (μm): 93.27 ±17.83 109.92 ±22.60 Height of the epidermal cells (μm): 16.32 ±4.35 19.15 ±6.57 Width of the epidermal cells (μm): 35.98 ±9.13 23.17 ±8.64
Height of the Ca(COO)2 crystals (μm): 22.73 ±5.63 25.93 ±5.84 Width of the Ca(COO)2 crystals (μm): 30.96 ±7.25 30.31 ±5.51
Leaf The cauline leaves (Fig. 6) are opposite, decussate, rarely in whorls of three. They are 3-15 cm long and 1-2.5 cm wide, sessile, lanceolate to ovate, with entire leaf margin and hairs on the abaxial surface. The subsidiary veins form arcs that anastomose near the leaf margin
(Ph. Hg. VIII.; CLAPHAM et al., 1962; MAL et al. 1992; KIRÁLY 2009).
epidermal cells
palisade cells
trichome spongy cells
epidermal cells
mesomorphic stoma
Ca(COO)2 crystal
vascular bundle
Fig. 8. Leaf structure of L. salicaria (cross section)
Both the adaxial and abaxial surface of the leaf (Fig. 8) is covered by 1-, 2- or 3-celled trichomes (Fig. 9). The abaxial surface is characterised by significantly protruding veins 27
(corresponding to vascular bundles) and mesomorphic stomata (hypostomatic leaf). These stomata are anomocytic (Ph. Hg. VIII.) without subsidiary cells and with cavities under the guard cells. Epidermal cells are flattened, composed of a single cell layer, and they are higher on the adaxial than the abaxial surface in all samples. The heterogenous mesophyll of the dorsiventral leaf is composed of palisade cells at the adaxial and isodiametric spongy cells in 3-4 rows at the abaxial surface. Several intercellular spaces and special idioblasts with
Ca(COO)2 crystals can be observed among the spongy cells. Collateral bundles are closed without cambium among the vascular tissue elements. The central bundle and the isodiametric cells of the angular collenchyma perform a supporting function in the leaf.
Fig. 9. Trichomes and Ca-oxalate crystal druses in the leaf of L. salicaria (cleared leaf)
Table 6 summarizes the measured histological parameters of the leaf of L. salicaria, but these results are detailed and compared with the corresponding results of the bracts after the descriptive characterization of the bract.
28
Table 6. Mean ±SD of the measured histological parameters of the leaf of L. salicaria. (continued overleaf)
3.52 7.65 3.21 7.77 8.55 6.44 4.74 0.56
10.89 13.25 10.78 69.03 23.73
± ± ± ± ± ± ± ±
± ± ± ± ±
Kaposvár
(Mean ±SD)
1.76
21.49 37.58 12.20 22.50 70.65 14.58 19.10 21.22 24.96 26.16
211.19 181.91
- -
6.41 4.67 7.98 2.69 6.21 8.32 8.23 7.55
14.73 10.64 25.32
± ± ± ± ± ± ± ±
± ± ±
Kacsóta
(Mean ±SD)
not not
19.03 34.99 11.94 17.55 63.61 13.10 19.10 20.87 29.92 30.66
found found
195.10
9.32 3.77 8.51 8.38 5.05 8.32 8.92 8.58 0.53
21.90 10.25 59.14 17.68
± ± ± ± ± ± ± ± ±
± ± ± ±
(Mean ±SD)
Lake Deseda
1.36
25.94 46.97 12.61 21.20 57.71 15.43 20.90 23.55 32.15 35.75
135.91 166.60
8.11 3.13 5.76 8.67 2.87 6.18 7.57 5.79 5.02 0.38
18.75 28.48 26.83
± ± ± ± ± ± ± ± ± ±
± ± ±
Cserénfa
(Mean ±SD)
1.07
20.36 36.50 11.59 16.96 48.88 13.70 16.66 18.31 29.59 30.12 80.97
136.94
5.55 7.47 3.53 7.16 7.11 4.05 7.61 8.30 5.84 7.09 0.52
74.98 31.68
± ± ± ± ± ± ± ± ± ± ±
± ±
Csebény
Mean ±SD)
1.48
23.70 37.82 13.51 19.18 57.93 15.97 20.79 24.30 30.16 30.07
177.63 183.08
0.69
4.86 9.37 3.55 7.01 7.62 5.40 6.54 0.51
10.23 23.73 10.74 53.50 2
± ± ± ± ± ± ± ±
± ± ± ± ±
Lake
Almamellék
(Mean ±SD)
1.25
19.95 40.08 14.05 21.07 56.39 14.93 18.05 19.37 25.38 28.07
158.62 157.14
7.45 5.33 9.97 6.87 2.35 7.72 5.46 8.39 7.12 0.51
16.53 81.22 26.68
± ± ± ± ± ± ± ± ± ±
± ± ±
Almamellék
(Mean ±SD)
1.60
22.98 38.19 13.99 17.73 54.50 15.65 20.46 20.47 34.21 35.26
167.39 160.19
:
(μm):
(μm)
crystals
crystals
cells at cells
2
2
)
)
of the leaf
COO
COO
cells withincells the
Leaf
epidermal cells at at epidermal cells
hickness hickness
Heightof the at epidermal cells the adaxial surfaceof leaf the (μm): Width of the epidermal at cells the adaxial surfaceof leaf the (μm): Heightof the the abaxial surfaceof leaf the (μm): Width of the epidermal the abaxial surfaceof leaf the (μm): Heightof palisade the cells (μm): Width of the palisade cells (μm): Heightof the spongy cells Width of the spongy cells Heightof the Ca( (μm): Width of the Ca( (μm): Number theof trichomes: Heightof the trichomes(μm): Average t (μm):
29
Table 6. (Continued) Mean ±SD of the measured histological parameters of the leaf. (continued overleaf)
5.52 4.77 9.40 7.68 2.82 5.00 5.44 6.41 6.30 0.35
13.48 46.01 10.63
± ± ± ± ± ± ± ± ± ±
± ± ±
Szigetvár
(Mean ±SD)
1.46
24.20 34.03 15.14 20.83 49.94 14.06 15.94 16.02 22.17 24.04
163.71 171.36
6.48 5.38 7.44 8.21 3.60 6.99 7.30 4.77 4.94 0.35
15.79 62.64 32.25
± ± ± ± ± ± ± ± ± ±
± ± ±
Szentlőrinc
(Mean ±SD)
1.30
22.33 41.96 12.28 19.66 46.53 16.59 19.10 22.21 29.18 33.69
182.98 131.25
5
7.16 3.08 8.34 6.64 3.04 9.73 9.0 6.94 5.50 1.01
15.13 67.30 24.94
± ± ± ± ± ± ± ± ± ±
± ± ±
Szentlászló
(Mean ±SD)
1.85
21.91 41.91 11.84 20.77 54.56 17.22 22.71 24.18 27.65 29.32
161.68 173.27
8.02 4.12 6.07 5.68 4.55 7.92 3.30 5.30 0.47
17.71 10.44 57.39 18.61
± ± ± ± ± ± ± ± ±
± ± ± ±
.95
(Mean ±SD)
Lake Sikonda
1.63
23.06 43.43 11.05 21.54 37.09 15 16.35 20.14 24.53 31.20
175.05 111.74
9.83 4.27 7.40 7.01 3.95 7.49 7.18 7.51 0.70
19.12 11.88 99.31 21.40
± ± ± ± ± ± ± ± ±
± ± ± ±
(Mean ±SD)
1.50
22.44 40.80 12.52 21.65 48.64 16.20 18.96 25.30 26.35 29.57
215.48 139.55
Lake Malomvölgy
at at
(μm):
(μm):
crystals
crystals
2
2
he leafhe
of the leaf
Leaf
e e at epidermal cells e spongy cells
hickness
ichomes:
Heightof th the adaxial surfaceof leaf the (μm): Width of the epidermal cells the adaxial surfaceof leaf the (μm): Heightof the at epidermal cells the abaxial surfaceof leaf the (μm): Width of the epidermal at cells the abaxial surfaceof t (μm): Heightof palisade the cells (μm): Width of the palisade cells (μm): Heightof th Width of the spongy cells Heightof the Ca(COO) (μm): Width of the Ca(COO) (μm): Number theof withincells the tr Heightof the trichomes(μm): Average t (μm):
30
Table 6. (Continued) Mean ±SD of measured histological parameters of the selected habitats of L. salicaria. Leaf Mean of all investigated populations ±SD Height of the epidermal cells at the adaxial surface of the leaf (μm): 22.28 ±7.91 Width of the epidermal cells at the adaxial surface of the leaf (μm): 39.52 ±16.47 Height of the epidermal cells at the abaxial surface of the leaf (μm): 12.73 ±4.20 Width of the epidermal cells at the abaxial surface of the leaf (μm): 20.05 ±8.66 Height of the palisade cells (μm): 53.87 ±7.86 Width of the palisade cells (μm): 15.28 ±3.57 Height of the spongy cells (μm): 19.01 ±7.05 Width of the spongy cells (μm): 21.33 ±8.03 Height of the Ca(COO)2 crystals (μm): 28.02 ±6.58 Width of the Ca(COO)2 crystals (μm): 30.33 ±6.35 Number of the cells within the trichomes: 1.48 ±0.54 Height of the trichomes (μm): 166.42 ±63.55 Average thickness of the leaf (μm): 159.01 ±23.37
Bract The single flowers in the inflorescence are surrounded by several bracts (Fig. 10).
Fig. 10. Bracts of L. salicaria (indicated by the arrow)
intercellular space epidermal cells
adaxial surface
trichome epidermal cells
palisade cells Ca(COO)2 crystal
spongy cells abaxial surface
Fig. 11. Bract structure of L. salicaria (cross section)
31
These bracts are covered by several trichomes consisting of 1-3, rarely 4-5 cells (Fig. 11 and 12). Among the flattened epidermal cells numerous mesomorphic stomata can be observed on both surfaces (amphistomatic bract). The heterogenous mesophyll includes palisade and isodiametric cells with intercellular spaces. The leaf is dorsiventral. The collateral closed bundle corresponding to the central vein is surrounded by parenchimatic, sometimes collenchimatic bundle sheath.
Fig. 12. Trichomes in the bract of L. salicaria (cleared leaf)
Thickness of the leaves measured in the top, middle, and base of the leaf blade varies between 111.74 μm (Lake Sikonda) and 195.10 μm (Kacsóta), and that of the bracts varies from 101.83 μm (Kaposvár) to 158.59 μm (Kacsóta) in all plant samples. The length of trichomes changes from 156.91 μm (Cserénfa) to 383.81 μm (Szentlőrinc) in the bracts, and from 67.14 μm (Cserénfa) to 224.13 μm (Lake Malomvölgy) in the leaves. The longer trichomes of the bracts play important role in the protection of the flowers. The cell number of the trichomes is regularly one to two in the leaves (Fig. 9) and one to three in the bracts (Fig. 12) except for the samples collected from Szentlőrinc, Lake Malomvölgy, and Szigetvár having three to four cells in the leaves, and samples collected from Szentlőrinc having four to five cells in the bracts. The size of the epidermal cells was highly variable in all plant parts and in all populations (Table 6 and 7). Leaf epidermal cells were higher on the adaxial than the abaxial surface in all samples. In bracts, the height and width of the cells were the lowest at Kacsóta and highest at Lake Deseda on the adaxial side, the smallest at Lake Sikonda and highest at Szigetvár on the abaxial surface. The lowest cell height and width values of the abaxial epidermal cells of the bracts could be measured in the plants living at Kaposvár. Several differences were measured in connection with the palisade and spongy cells of the leaves and bracts, as well (Table 6 and 7). Palisade cells of the leaves were variable in their height (37.09-70.65 μm, Lake Sikonda – Kaposvár) and width (13.10-17.22 μm, Kacsóta – Szentlászló), similarly to the measured extreme data of spongy cells in the cell height 32
(15.94-22.71 μm, Szigetvár – Szentlászló) and width (16.02-25.30 μm, Szigetvár – Lake Malomvölgy). Data of palisade cells in bracts were similar in all populations except for the extremely high value of the plants living at Lake Sikonda (53.28 μm). Spongy cells were the highest at Cserénfa (18.71 μm) and the lowest at Szigetvár (13.83 μm) in the bracts.
The measured parameters of Ca(COO)2 crystals (Fig. 9) were similar to each other, except for the fact that these crystals were wider than high in the stems and the leaves, while they were rather isodiametric in the bracts. In the stem, the average height of crystals for all populations was 22.73 μm and the average width for all populations was 30.96 μm. The size of crystals in the leaves was 28.02 × 30.33 μm, and that of the bracts was 23.44 × 24.85 μm. The crystals of the leaves were the highest at Almamellék, the widest at Lake Deseda, and lowest at Szigetvár. These characters of the crystals present small variance in the bracts of the studied populations.
33
Table 7. Mean ±SD of the measured histological parameters of the bract of L. salicaria. (continued overleaf)
28
4.49 12.71 2.89 4.93 6. 3.69 4.24 6.72 4.33 7.53 0.52 73.11 16.21
± ± ± ± ± ± ± ± ± ± ± ± ±
Kaposvár (Mean ±SD) 14.91 23.08 10.60 13.43 32.72 13.53 14.40 17.59 21.26 23.75 1.55 178.99 101.83
4.72 11.54 5.03 7.01 5.18 2.95 6.76 7.78 3.69 4.13 31.56
± ± ± ± ± ± ± ± ± ± ± - -
30
Kacsóta (Mean ±SD) 17.93 26.47 16.09 19.74 35. 14.07 17.68 19.73 24.98 27.02 not found not found 158.59
6.93 10.47 4.04 8.01 5.89 3.54 6.97 7.00 4.20 5.25 0.52 81.96 39.94
± ± ± ± ± ± ± ± ± ± ± ± ±
Lake (Mean ±SD) Deseda 19.65 28.59 16.78 22.93 34.71 14.13 17.42 20.30 21.14 21.18 1.53 210.88 125.44
4.64 9.45 3.94 7.35 6.40 2.90 6.59 10.01 6.21 8.70 0.51 37.06 26.88
± ± ± ± ± ± ± ± ± ± ± ± ±
Cserénfa (Mean ±SD) 16.17 24.04 16.58 18.33 35.50 14.87 18.71 21.98 23.35 27.62 1.42 156.91 109.71
4.68 10.83 4.11 5.03 6.11 3.15 5.07 7.79 6.17 6.46 0.62 64.27 25.36
± ± ± ± ± ± ± ± ± ± ± ± ±
Csebény (Mean ±SD) 19.43 25.06 14.24 17.14 37.45 15.17 17.58 21.37 28.76 30.03 1.67 195.04 120.57
4.51 10.75 3.55 7.42 5.38 1.57 4.34 6.23 4.84 4.20 0.51 78.77 23.65
± ± ± ± ± ± ± ± ± ± ± ± ±
.30
±
7.31
Lake Almamellék (Mean ±SD) 16.25 24.21 14 21.46 36.36 14.47 15.01 1 20.62 21.76 1.43 212.33 114.40
8.39 9.98 3.77 7.94 4.86 2.85 5.31 6.01 3.18 4.91 0.52 89.03 25.19
± ± ± ± ± ± ± ± ± ± ± ± ±
Almamellék (Mean ±SD) 19.29 23.12 14.23 18.13 33.28 14.61 15.70 17.97 23.27 25.86 1.55 233.62 119.90
at at
bract bract bract bract bract
(μm):
(μm):
crystals crystals
crystals
2
2
)
)
of the
mes(μm):
COO
COO
hickness
htof the tricho
Bract Height of the epidermal cells the adaxial surface of the (μm): Width of the epidermal cells at the adaxial surface of the (μm): Height of the epidermal cells at the abaxial surface of the (μm): Width of the epidermal cells at the abaxial surface of the (μm): Height of the (μm): palisade cells Width of (μm): the palisade cells Heightof the spongy cells Width of the spongy cells Height of the Ca( (μm): Width of the Ca( (μm): Number of the cells within the trichomes: Heig Average t (μm):
34
Table 7. (Continued) Mean ±SD of the measured histological parameters of the bract of L. salicaria. (continued
overleaf)
7.17 11.32 5.86 8.52 5.46 2.56 4.71 6.53 3.78 4.05 0.64 99.86 18.55
± ± ± ± ± ± ± ± ± ± ± ± ±
Szigetvár (Mean ±SD) 17.93 23.65 17.09 19.90 32.39 12.40 13.83 16.08 20.44 21.37 1.47 205.03 138.06
3.13 8.75 3.71 8.03 5.87 3.07 4.75 7.91 3.22 3.56 1.58 192.21 29.39
± ± ± ± ± ± ± ± ± ± ± ± ±
entlőrinc (Mean
Sz ±SD) 15.69 25.81 13.21 20.18 32.85 13.75 16.59 21.30 21.57 22.36 3.00 383.81 106.65
5.57 10.34 3.80 6.19 6.20 4.15 4.69 6.95 4.01 4.66 0.80 106.81 21.56
± ± ± ± ± ± ± ± ± ± ± ± ±
Szentlászló (Mean ±SD) 16.83 25.56 13.30 16.97 39.68 16.25 17.04 20.20 27.79 28.83 2.27 330.09 129.76
4.29 11.57 4.18 7.71 12.51 2.54 6.18 7.37 5.00 4.70 0.63 114.49 28.02
± ± ± ± ± ± ± ± ± ± ± ± ±
.10
Lake (Mean ±SD) Sikonda 16.67 31.39 14.85 19.09 53.28 16 17.77 20.04 25.55 24.22 1.64 237.18 150.36
5.71 8.10 2.27 7.11 6.68 3.11 4.90 7.94 4.61 6.66 0.50 63.93 27.10
± ± ± ± ± ± ± ± ± ± ± ± ±
Lake Malomvölgy (Mean ±SD) 17.94 21.75 11.40 16.14 34.05 13.12 14.21 18.44 22.55 24.18 1.36 169.38 109.15
bract bract bract bract bract
(μm):
(μm):
crystals crystals
crystals
the
2
2
)
)
of the
COO
COO
epidermal cells at
epidermal cells at
hickness
the
of
Bract Height of the the adaxial surface of the (μm): Width of the epidermal cells at the adaxial surface of the (μm): Height of the epidermal cells at the abaxial surface of the (μm): Width the abaxial surface of (μm): Height of the (μm): palisade cells Width of (μm): the palisade cells Heightof the spongy cells Width of the spongy cells Height of the Ca( (μm): Width of the Ca( (μm): Number of the cells within the trichomes: Heightof the trichomes(μm): Average t (μm):
35
Table 7. (Continued) Mean ±SD of measured histological parameters of the selected habitats of L. salicaria. Bract Mean of all investigated populations ±SD Height of the epidermal cells at the adaxial surface of the bract (μm): 17.39 ±5.35 Width of the epidermal cells at the adaxial surface of the bract (μm): 25.23 ±10.49 Height of the epidermal cells at the abaxial surface of the bract (μm): 14.39 ±3.93 Width of the epidermal cells at the abaxial surface of the bract (μm): 18.62 ±7.10 Height of the palisade cells (μm): 36.46 ±6.40 Width of the palisade cells (μm): 14.37 ±3.01 Height of the spongy cells (μm): 16.33 ±5.38 Width of the spongy cells (μm): 19.36 ±7.35 Height of the Ca(COO)2 crystals (μm): 23.44 ±4.44 Width of the Ca(COO)2 crystals (μm): 24.85 ±5.40 Number of the cells within the trichomes: 1.72 ±0.67 Height of the trichomes (μm): 228.48 ±91.04 Average thickness of the bract (μm): 123.70 ±26.12
Flowers L. salicaria can be mentioned as a classic representative example of the phenomenon of tristyly, which means that it has stamens and styles in three different lengths: namely long-, medium-, and short-styled forms (HARASZTY 2004). This is a rare phenomenon in the plant world, which has already been studied by DARWIN in 1877, and can also be observed in the Oxalidaceae and Pontederiaceae families. We found all three morphs in most of the habitats, except for Lake Malomvölgy, where only the short-styled form could be found; Lake Sikonda, where only long- and short-styled forms could be found; and Lake Almamellék, Cserénfa, and Szentlászló, where only long- and mid-styled forms could be observed.
short stamen medium style long stamen
Fig. 13. Flower of L. salicaria (with long stamens, short stamens, and medium style).
The length of the spike can vary from a few cm up to 1 m (BALOGH 1986). The polypetalous corolla consists of 6 petals, each expanded at the top with a wavy outline narrowing at the base (Ph. Hg. VIII.). Although the petals are typically magenta, scattered pink, or purple, nearly white flowers have also been observed (BALOGH 1986). The petals are 5-12.5 mm long, obovate, inserted near the top of the calyx tube, and they alternate with 36
sepals (MAL et al. 1992). The flowers have a pubescent, tubular, persistent gamosepalous calyx, 6 sepals with 4-8 mm length bearing 6 small, triangular teeth alternating with 6 large acute teeth at least half as the length of the tube (Ph. Hg. VIII.). The development of the floral parts of the species namely centrifugal interzonal development can be characterized by the following steps observed in SEM photos (RUDALL 2010): after inner sepals the outer sepals develop forming the epicalyx. The next one is the gynoecium, and then the inner and outer stamens appear in the flower of the plant. The androecium consists of 2 verticils of 6 stamens (one verticil with short, barely emerging stamens, the other with long stamens extending well out of the corolla, Fig. 13) (Ph. Hg. VIII.). Anthers are 1.25 × 1 mm, introrse, two-celled, dorsifixed, and in a longitudinally dehiscent position (MAL et al. 1992). The filaments of the longest stamens are generally bright pink with purple anthers. Those of mid-length stamens are colourless, while the short stamens have greenish filaments with greenish anthers (MAL et al. 1992). The anthers of the mid-length set are larger than the others (EAST 1927; DARWIN 1865). Pollen grains produced in the long stamens have significantly larger size than the others in the medium and short stamens (MAL and HERMANN 2000). The size and the colour of the pollen grains varies depending on whether they come from long, medium or short stamens: pollen grains from medium and short stamens are yellow and those from long stamens are green (MAL et al. 1992). The larger pollen contains greater amount of starch
(GANDERS 1979). The pollen is uniform and can be distinguished by three prominent granular pseudocolpi alternating with three apertures, in which the colpi are also granular (GRAHAM et al. 1987). The ovary position is superior, comprising of two fused carpels, simple style, capitate stigma, and two loculi containing numerous anatropous ovules. The placentation type is axile. Long-styled flowers have larger stigma with globular surface, but that of the other two morphs can be characterised by papillae on the surface (MAL et al. 1992). The many-seeded fruit is a small capsule included in the persistent calyx (Ph. Hg. VIII.) and surrounded by fused sepals. This oblong-ovoid capsule (Fig. 14) (3-4 mm long and 2 mm broad) with two valves dehisces septicidally; it has chambers inside and breaks apart along the septums of the wall during the fruit ripening period. It bears an average of 93, 130, and 83.5 seeds per capsule in long-, mid- and short-styled forms, respectively (DARWIN 1865). The seeds are 0.5- 0.6 mg in mass, 400 μm long and 200 μm wide (Fig. 14). They are nonendospermic, light, flat and thin-walled with two cotyledons (THOMPSON et al. 1987).
37
Fig. 14. Seeds and capsules of L. salicaria
The flowering period lasts from June to September (KIRÁLY, 2009). During sample collection, it was found that flowering started earlier in 2010 than in 2011. We were able to collect whole flowering herbs in all habitats in July 2010, but there were some habitats, where the plants have not started to flower in July 2011 yet (Csebény, Lakes Almamellék and Deseda), in spite of the fact that we collected the samples in the same calendar week (27th and 34th weeks) in both years. This phenomenon may be due to different weather conditions.
Pollination is performed by insects, mostly by honey bees (SOÓ 1966; THOMPSON et al. 1987).
Table 8. Measurements of floral parts in each of the three sex morphs. Lengths expressed in mm (±SE) and range
is stated in square brackets (from MAL et al.1992). Floral morphs Floral part Long Mid Short Ovary 2.07 (0.019) 2.12 (0.022) 2.15 (0.023) [1.4-2.7] [1.4-2.8] [1.3-2.9] Pistil 10.71 (0.082) 6.95 (0.060) 3.28 (0.031) [7.7-13.4] [5.6-8.8] [2.4-4.4] Long stamen 9.76 (0.091) 9.76 (0.082) - [5.7-12.6] [6.8-13.6] Medium stamen 6.15 (0.060) 6.12 (0.062) - [3.8-8.2] [4.3-8.6] Short stamen 2.97 (0.046) 3.12 (0.061) - [1.1-4.6] [1.9-5.7] Perianth 14.66 (0.153) 14.76 (0.177) 14.80 (0.176) Index of heterostyly* 0.422 (0.005) 0.283 (0.006) 0.288 (0.005) [0.262-0.58] [0.050-0.435] [0.062-0.446] *The index of heterotristyly, shown in the last row is calculated as
distance between stigma and nearest set of anthers, I = according to Eiten (1963). height of longest sporophyll
MAL et al.’s (1992) measurements of various floral parts can be seen in Table 8, which is complemented by our data in Table 9. Our data contribute to the full understanding of the 38
histological structure of the inflorescence and the flowers. Besides providing a detailed anatomical description of each floral part, the most important quantitative features were also measured.
Table 9. Histological data of some flower parts Histological characters of some flower parts Mean ±SD Thickness of the calyx (μm): 96.39 ±30.89 Height of the epidermal cells in the calyx (μm): 24.78 ±6.74 Width of the epidermal cells in the calyx (μm): 41.32 ±14.67 Thickness of the petal (μm): 36.93 ±17.94 Thickness of the ovary wall (μm): 74.91 ±18.77
A central vascular bundle was observed in the inflorescence axis branching at the base of each flower and running along the whole length of the flowers (Fig. 15).
sepal central vascular bundle
ovary wall
annular nectary
epidermal cells
ovule Fig. 15. Central vascular bundle in the receptacle indicated by the arrow (longitudinal section)
Sepals are composed of 4 or 6 cell layers with flattened epidermal cells and unicellular trichomes on the abaxial surface (Fig. 16). The base and the top of the sepals consist of spongy cells exclusively, whereas the middle part can be characterised by both spongy and palisade cells together with numerous intercellular spaces.
39
pollen grains
ovary
trichome anther
sepal
annular nectary
Fig. 16. Histological structure of the sepal, ovary, and an anther with pollen grains (longitudinal section)
In our samples, the petals were composed of only 1-2 cell layers (Fig. 17).
petal sepal
Fig. 17. Histological structure and origin of the petal (cross section)
The filament of the stamen has a collateral closed bundle in central position. The anther is covered by the exothecium, followed by a fibrous layer with characteristic cell wall thickening (Fig. 16 and Fig 18).
40
pollen grains
Fig. 18. Histological structure of the anther and the pollen grains (cross section)
The ovary is divided by the septum bearing ovules on both sides (axile placentation) (Fig. 15, 16, and 19). The vascular bundle of the septum is surrounded exclusively by spongy cells at the top and the base. The annular nectary is located in the valley between the receptacle and the ovary. Trichomes and intercellular cavities could not be observed in the structure of the ovary. The average thickness of the wall of the ovary is composed of 4-5 cell layers (Table 9).
stigma
papillae
Fig. 19. Short-styled stigma (longitudinal section)
41
V. Phytochemical evaluation of L. salicaria
In this chapter, the polyphenol composition of twelve populations of L. salicaria collected from different habitats in south-west Hungary was investigated with official methods of the Ph. Hg. VIII. (measurement of the total polyphenol, tannin, and flavonoid content, as well as TLC analysis) and UHPLC-MS. Polyphenols constitute the largest group of plant secondary metabolites, this group includes compounds with highly diverse structures (Fig. 20).
Fig. 20. Classification of the analyzed compounds within polyphenols
(transmitted from BOROS et al. 2010, with some modifications).
Tannins can be distinguished from other phenolic compounds based on their chemical reactions and biological activities. Tannins were traditionally used for "tanning", converting animal hides to leather. This refers to one of their leading properties, the ability to interact with proteins and precipitate them. Tannins can be classified into two main groups: condensed and hydrolysable tannins (HAGERMAN 2002). L. salicaria contains hydrolysable tannins, such as gallotannins and ellagitannins. Tannin-containing drugs have been used traditionally as styptics. They can be used internally for the protection of inflamed surfaces of the mouth and throat. They have antidiarrhoeal effect; and they may be applied as antidotes in heavy metal and alkaloid poisoning. Recently, it was found that tannin derivatives (especially tannic acid) may lead to severe liver necrosis; and therefore their use has been declined (EVANS 2008). Flavonoids also belong to the large group of polyphenols. Flavonoid compounds of L. salicaria, namely orientin, isoorientin, vitexin and isovitexin, are flavone-C-glycosides (for chemical structures see Appendix 4). Flavonoids are important constituents of plants; they are responsible for UV-B protection, and along with other polyphenols, they have undisputed role 42
in protection of plants against microbial invasion as well as both insect and mammalian herbivory. Flavonoids are recognized by pollinators, which contribute to the dispersion of seeds. They play part in growth in association with the actions of auxin. Flavonoids also have medicinal importance; they have been found to inhibit oxidation of low-density lipoprotein (LDL), reduce the risk of coronary heart disease, have antitumour, antispasmolytic, antibacterial, antifungal, anti-inflammatory, hepatoprotective, oestrogenic, or analgesic activities. Since flavonoids are ubiquitous in plant foods, a large quantity is consumed in our daily diet (HARBONE and WILLIAMS 2000; HAVSTEEN 2002). Recently, L. salicaria has been the subject of several phytochemical studies. Reported chemical constituents of L. salicaria are given in Table 10. with appropriate references.
43
Table 10. Phenolic compounds detected in Lythrum salicaria L. (from Rauha et al. 2001, with some supplements).
Class Compound Reference Tannins HHDP-glucose RAUHA et al. (2001) 1-O-galloylglucose RAUHA et al. (2001) 6-O- galloylglucose RAUHA et al. (2001) 1,6-di-O- galloylglucose RAUHA et al. (2001) galloyl-HHDP-glucose RAUHA et al. (2001) trigalloylglucose RAUHA et al. (2001) galloyl-bis-HHDP-glucose RAUHA et al. (2001) trigalloyl-HHDP-glucose RAUHA et al. (2001) vescalagin RAUHA et al. (2001) pedunculagin RAUHA et al. (2001) castalagin MA et al. (1996) lythrine A MA et al. (1996) lythrine B MA et al. (1996) lythrine C MA et al. (1996) lythrine D MA et al. (1996) Flavonoids isoorientin PARIS (1967) orientin PARIS (1967) isovitexin PARIS (1967) vitexin PARIS (1967) Anthocyanins cyanidin-3-galactoside PARIS and PARIS (1964) malvidin-3,5-diglucoside PARIS and PARIS (1964) Phenolics gallic acid PARIS (1967) methyl-gallate PARIS (1967) chlorogenic acid PARIS (1967) ellagic acid PARIS (1967) vanoleic acid dilactone RAUHA et al. (2001) isochlorogenic acid TORRENT MARTI (1975) caffeic acid TORRENT MARTI (1975) p-coumaric acid TORRENT MARTI (1975) Phtalates dibutyl phthalate FUJITA et al. (1972) diisobutyl phthalate FUJITA et al. (1972) diisoheptyl phthalate FUJITA et al. (1972) diisooctyl phthalate FUJITA et al. (1972) butyl-2-metylpropyl phthalate FUJITA et al. (1972) phtalic acid FUJITA et al. (1972) Sterols β-sitosterol FUJITA et al. (1972) Terpenes loliolide FUJITA et al. (1972) betulinic acid KIM et al. (2011)
Its main components are tannins and flavonoids. In lower amounts sterols (β- sitosterol), terpenes (e.g. loliolid), and phtalates were also reported from the plant. The attractive colour of the flowers is due to the anthocyanins (PARIS and PARIS 1964; PARIS
1967; TORRENT MARTI 1975; FUJITA et al. 1972; MA et al. 1996; RAUHA et al. 2001). In the
Lythraceae family, many species are rich in alkaloids, e.g., Decodon verticillatus (L.) ELL.,
Heimia salicifolia (KUNTH) LINK, or Lythrum anceps MAKINO (FERRIS et al. 1966; FUJITA et al. 1967), but in L. salicaria no remarkable amounts of them could be detected and none of them would identified (STEINFELD 1969; FUJITA et al. 1972).
44
V. 1. Materials and methods
V. 1. 1. Total polyphenol and tannin content (according to the Ph. Hg. VIII.) The Ph. Hg. VIII. prescribes the determination of total tannin content of Lythri herba which could not be less than 5.0% of tannins expressed as pyrogallol and calculated with reference to the dried drug. Chemicals and solvents. Hide powder and phosphomolybdotungstic reagent were purchased from Sigma Aldrich, Hungary, sodium carbonate from VWR International Ltd., Hungary. Total polyphenols. 0.750 g of the powdered drug was added to 150 mL of distilled water and heated for 30 min, diluted to 250.0 mL with distilled water, then filtered. 5.0 mL of the filtrate were diluted to 25.0 mL with distilled water, 2.0 mL of this solution were mixed with 1.0 mL of phosphomolybdotungstic reagent and 10.0 mL of distilled water and diluted to 25.0 mL with a 290 g/L solution of sodium carbonate. After 30 min the absorbance was measured at 760 nm (A1), using distilled water as a compensation liquid. Polyphenols not adsorbed by hide powder. 0.10 g of hide powder was added to 10.0 mL of the previously described filtrate, shaken vigorously for 60 min, then filtered. 5.0 mL of the filtrate were diluted to 25.0 mL with distilled water. 2.0 mL of this solution were mixed with 1.0 mL of phosphomolybdotungstic reagent and 10.0 mL of distilled water, and diluted to 25.0 mL with a 290 g/L solution of sodium carbonate. After 30 min the absorbance was measured at 760 nm (A2) using distilled water as compensation liquid. Standard. 50.0 mg of pyrogallol were dissolved immediately before use in distilled water and diluted to 100.0 mL with the same solvent. 5.0 mL of the solution were diluted to 100.0 mL with distilled water. 2.0 mL of this solution were mixed with 1.0 mL of phosphomolybdotungstic reagent and 10.0 mL of distilled water, and diluted to 25.0 mL with a 290 g/L solution of sodium carbonate. After 30 min the absorbance was measured at 760 nm
(A3), using distilled water as compensation liquid. The percentage content of tannins were calculated and expressed as pyrogallol with the help of the following expression:
62.5 × (A1-A2) × m2
A3 × m1 where m1 = mass of the sample to be examined (in grams), and m2 = mass of pyrogallol (in grams). A Metertech SP-8001 UVVIS spectrophotometer (Spektrum 3D Ltd., Debrecen, Hungary) was used for all absorbance measurements.
45
Statistical analysis. The correlation between the polyphenol content of the populations in 2010 and 2011 was tested by Past 2.17 Program with One-way-ANOVA method.
V. 1. 2. Total flavonoid content (according to the Ph. Hg. VIII.)
Method 1 (for drugs containing O-glycosides)
Chemicals and solvents. Hexamethylenetetramine, aluminium chloride, and anhydrous sodium sulphate were purchased from VWR International Ltd., Hungary; hydrochloric acid, glacial acetic acid, acetone, ethyl acetate, and methanol from Molar Chemicals Ltd., Hungary. Stock solution. 0.500 g of the powdered drug was boiled with 1.0 mL of a solution of hexamethylenetetramine (5 g/L), 20 mL of acetone, and 2.0 mL of 25% hydrochloric acid under a reflux condenser for 30 min. The liquid was filtered through a plug of absorbent cotton into a flask, and the residue was extracted two times with 15 mL of acetone, each time boiling under a reflux condenser for 10 min. After boiling, the extract was cooled and filtered through a plug of absorbent cotton into the same flask as previously. The combined acetone extracts were filtered through a paper-filter into a 50.0 mL volumetric flask and diluted to 50.0 mL with acetone by rinsing the flask and the filter-paper. 10.0 mL of the solution were introduced into a separating funnel, 20 mL of distilled water were added, and the mixture was shaken with 15 mL of ethyl acetate and then 10-10 mL of ethyl acetate three times. The ethyl acetate extracts were combined in a separating funnel, washed with two quantities, each of 50 mL of distilled water, filtered over 10 g of anhydrous sodium sulphate into a volumetric flask, and diluted to 50.0 mL with ethyl acetate. Test solution. To 10.0 mL of the stock solution, 1.0 mL of aluminium chloride reagent (2.0 g aluminium chloride in 100.0 mL of 5% solution of glacial acetic acid in methanol) was added and diluted to 25.0 mL with a 5% solution of glacial acetic acid in methanol. Compensation solution. 10.0 mL stock solution was diluted to 25.0 mL with a 5% solution of glacial acetic acid in methanol. The absorbance of the test solution was measured after 30 min by comparison with the compensation solution at 425 nm. The percentage contents of flavonoids were calculated related to hyperoside, from the following expression: A × 1.25 m where A = absorbance at 425 nm; m = mass of the drug to be examined, in grams. 46
Statistical analysis. The correlation between the flavonoid content of the populations in 2010 and 2011 was tested by Past 2.17 Program with One-way-ANOVA method.
Method 2 (for drugs containing C-glycosides) Chemicals and solvents. Ethanol, methanol, and glacial acetic acid were purchased from Molar Chemicals Ltd., Hungary, anhydrous formic acid from VWR International Ltd., Hungary, boric acid and oxalic acid from Sigma Aldrich, Hungary. Stock solution. 0.150 g of the powdered drug was heated with 20 mL of 60% ethanol in water under a reflux condenser at 60°C for 10 min. The liquid was filtered through a plug of absorbent cotton into a flask and the residue was extracted again with 20 mL 60% ethanol in water under a reflux condenser at 60°C for 10 min. After boiling, the extract was cooled and filtered through a plug of absorbent cotton into the same flask. The combined ethanol extracts were filtered through a paper-filter into a 50.0 mL volumetric flask and diluted to 50.0 mL with 60% ethanol in water by rinsing the flask and the filter-paper. Test solution. 5.0 mL of the stock solution were introduced into a round bottomed flask and evaporated to dryness. The residue was taken up with 8 mL of a mixture of 10 volumes of methanol and 100 volumes of glacial acetic acid and transferred into a 25 mL volumetric flask. The round-bottomed flask was rinsed with 3 mL of a mixture of 10 volumes of methanol and 100 volumes of glacial acetic acid and transfered into the same 25 mL volumetric flask as previously. 10.0 mL of a solution containing 25.0 g/L of boric acid and 20.0 g/L of oxalic acid in anhydrous formic acid were added, and diluted to 25.0 mL with anhydrous acetic acid. Compensation solution. 5.0 mL of the stock solution were introduced into a round bottomed flask and evaporated to dryness. The residue was taken up with 8 mL of a mixture of 10 volumes of methanol and 100 volumes of glacial acetic acid and transferred into a 25 ml volumetric flask. The round-bottomed flask was rinsed with 3 mL of a mixture of 10 volumes of methanol and 100 volumes of glacial acetic acid and transferred into the same 25 mL volumetric flask as previously. 10.0 mL of anhydrous formic acid were added and diluted to 25.0 mL with anhydrous acetic acid. The absorbance of the test solution was measured after 30 min, by comparison with the compensation solution at 410 nm. The percentage contents of flavonoids were calculated related to hyperoside, with the help of the following expression: A × 1.235 m where A = absorbance of the test solution at 410 nm; m = mass of the drug to be examined, in grams.
47
A Metertech SP-8001 UV/VIS Spectrophotometer (Spektrum 3D Ltd., Hungary) was used for all absorbance measurements.
V. 1. 3. TLC
In recent years, there is an increasing need for quick and cheap quality assurance tools to ensure the identity, purity, and quality of medicinal plants and drugs. Thin-layer chromatography (TLC) is a quick, cheap, reliable, and therefore frequently used technique for rutine, screening, and preliminary measurements. It can also be used for chemotaxonomic investigations (HERNANDEZ et al. 1987; JANICSÁK et al. 1999; REICH and SCHIBLI 2007). The Ph. Hg. VIII. prescribes TLC for identification and quality assurance of most of the herbal drugs, as also in the case of Lythri herba. The Ph. Hg. VIII. recommends the application of only chlorogenic acid, hyperoside, rutin, and vitexin as reference compounds, but we also used orientin. Thin layer chromatographic examination of leaf, flower, and stem parts of L. salicaria were performed in the case of all habitats. Our aim was to check whether there are any qualitative differences among the flavonoid pattern of the drug parts and the habitats. Chemicals and solvents. Methanol was purchased from Molar Chemicals Ltd., Hungary, diphenylboric acid aminoethyl ester, vitexin, orientin, caffeic acid, hyperoside, chlorogenic acid, rutin, and PEG 4000 from Sigma Aldrich, Hungary. TLC. 2.0 mL of methanol were added to 0.200 g of the powdered drug. It was heated at 40°C in ultrasonic waterbath (Elmasonic S 30H, Elma GmbH & Co, Germany) for 5 min, filtered, and diluted to 2.0 mL with methanol. Reference solutions included 0.2 mg/mL vitexin, 0.5 mg/mL orientin, 2.0 mg/mL caffeic acid, 1.0 mg/mL hyperoside, 1.0 mg/mL chlorogenic acid and 1.0 mg/mL rutin, in methanol. Chromatography was performed on
10 cm × 10 cm silica gel 60 F254 aluminium sheet TLC plates (Merck, Darmstadt, Germany). 10, 20, and 30 μL of plant extracts were applied as bands, and 3 μl of the reference solutions were applied as spots with Minicaps capillary pipettes (Ringcaps, Germany). The position of the starting line was 1.0 cm from the bottom and 1.5 cm from the sides. After sample application, the plates were developed (over a path of 8 cm) with the mobile phase optimized for flavonoids (ethyl acetate : formic acid : glacial acetic acid : distilled water = 100:11:11:26). Ascendant development chromatography was used in a saturated twin trough chamber (Camag, Switzerland). All TLC separations were performed at room temperature (20°C). After chromatographic separation, the plates were dried at room temperature, dipped into Naturstoff/PEG reagent (mixture of 10 g/L solution of diphenylboric acid aminoethyl
48
ester in methanol and 50 g/L solution of PEG 4000 in methanol), dried at 105°C and examined at 365 nm.
V. 1. 4. Qualitative-quantitative UHPLC-MS analysis of L. salicaria extracts
Preparation of extracts. The flowering branches used in the microbiological experiments were collected from Kaposvár in August 2010 (50% ethanol Microb.), and the flowering branches used in the pharmacological experiments were collected from Szigetvár in August 2011 (50% ethanol Pharm.). We selected these samples because these habitats were mostly invaded by L. salicaria within these terms and we could collect bigger amounts of samples there. The extracts used in the microbiological experiments were prepared as follows: 3.00 g drug (flowering tops collected from Kaposvár in August 2010) was extracted three times with 30 mL 50% ethanol in water with ultrasonic water bath at 40ºC, the solution was filtered, evaporated by vacuum-distillation, and the concentration was set to be 10 mg dried extract/1.0 mL 50% ethanol in water (50% ethanol Microb.). The extracts used in the pharmacological experiments were prepared as follows: 0.50 g drug (flowering tops collected from Szigetvár in August 2011) was extracted with 10.0 mL solvent (hexane, chloroform, ethyl acetate, or 50% ethanol in water) using ultrasonic water bath at 40ºC for 3 min. The solution was filtered, evaporated by vacuum-distillation, and diluted to 10.0 mL with DMSO in the case of the hexane, chloroform, and ethyl acetate extracts, and with 50% ethanol in the case of the ethanol extract. DMSO was used to ensure the blending with the organ bath in the case of the hexane, chloroform, and ethyl acetate extracts and qualitative-quantitative analyses were carried out with these extracts. Chemicals and solvents. All of the standards (gallic acid, ellagic acid, caffeic acid, catechin, vitexin, orientin, hyperoside, chlorogenic acid, rutin, apigenin, luteolin) were purchased from Sigma Aldrich Ltd., Hungary, and formic acid, methanol, and water (all of them were HPLC grade) from VWR International Ltd., Hungary. Hexane, chloroform, ethyl- acetate, and absolute ethanol were purchased from Molar Chemicals Ltd., Hungary. UHPLC-MS system and method. The qualitative and quantitative analyses of polyphenolic compounds were performed by LC–MS. The hyphenated LC system was an Agilent 1290 Infinity UHPLC device consisted of a 1290 Infinity binary pump (G4220A), Infinity TCC (LC column compartment temperature control device, G1316C), Infinity autosampler (G4226A), Infinity DAD (UV diode-array detector, G4212A). The Q-TOF MS instrument (Agilent 6530 Accurate Mass Q-TOF LC/MS system; Agilent Technologies,
49
USA) was operated with an Agilent Jet Stream ESI source in negative ionization mode with a mass accuracy <1 ppm, a mass resolution of 10,000–20,000 (150–966 m/z), a measuring frequency of 10,000 transients/s and a detection frequency of 2 GHz (200,000 points/transient). The Jet Stream ion source was operated using the following conditions: pressure of nebulising gas (N2) was 35 psi, the temperature and flow rate of drying gas (N2) were 325°C and 7 L/min, the temperature and flow rate of sheath gas were 200°C and 10 L/min. The capillary voltage was 4000 V, fragmentor and skimmer potential was set to 100 V and 65 V, respectively. Reference ions used for mass calibration were as follows: purine 119.036319 [M−H]−, HP-921 = hexakis (1H,1H,3H-tetrafluoropropoxy)phosphazine − 966.000725 [M+HCO2] . The measurements and post-run analyses were controlled by the MassHunter Acquisition software. Chromatographic separations were performed on Zorbax Extend C18 column (50 × 2.1 mm, 1.8 μm particle diameter, Agilent, USA). Two mobile phases, solvent A containing water : formic acid (99.5:0.5 v/v) and solvent B containing methanol : formic acid (99.5:0.5 v/v) were used. The gradient profile was set as follows: 0.00 min 3% B eluent, 7.00 min 20% B eluent, 7.10 min 30% B eluent, 17.00 min 40% B eluent, 25.00 min 100% B eluent, 25.10 min 3% B eluent and 30.00 min 3% B eluent. The flow rate was 0.5 mL/min, the column temperature was 50°C. The injection volume was 0.5 μL for Lythrum extracts and for standard mixtures. UV detection wavelengths were 280 nm and 360 nm. Components were identified by comparison of their retention times, UV spectra (Fig. 21) and mainly by the mass of their deprotonated molecules ([M–H]-) with those of the standards and in the published literatures (RAUHA et al., 2001; BOROS et al., 2010; BEELDERS et al., 2012). Standard solutions. A standard stock solution (0.5 mg/mL) was prepared, which contained the standard compouns (gallic acid, ellagic acid, caffeic acid, catechin, vitexin, orientin, isovitexin, isoorientin, hyperoside, chlorogenic acid, rutin, apigenin, luteolin) dissolved in pure methanol with ultrasonication for 10 min. Samples used for LC–MS analyses were diluted in water : methanol = 4:6 (v/v) with 0.5% (v/v) formic acid to a series of appropriate concentrations (18.5–100.000 ng/mL) for the construction of calibration curves. All the stock solutions were stored in amber flask at 4°C.
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Fig. 21. Chromatogram of the standards’ stock solution. Retention factors of the standards are indicated in parentheses. Chemical structure of these compounds can be seen in Appendix 4. (Isoorientin and isovitexin were identified based on their molecular mass and their quantities were calculated based on the calibration curve of orientin and vitexin standards.)
V. 2. Results and discussion
V. 2. 1. Total polyphenol and tannin content (according to the Ph. Hg. VIII.)
The polyphenol and tannin contents showed difference among the studied plant organs and populations. They were higher in the flowering top than in the other organs. In 2010, the measured total polyphenol values ranged from 1.2 to 27.3% (8.3-27.3% in the flowering top, 5.3-23.3% in the leaves, and 1.2-9.9% in the stems; Table 11). Total tannin values varied between 1.0 and 21.9% (6.6-21.9% in the flowering top, 4.0-20.9% in the leaves, and 1.0- 8.4% in the stems). The highest tannin content was detected in the population of Szentlőrinc in July and Szentlászló in August (in flowering top; Table 12). In 2011, the measured total polyphenol values ranged from 4.7 to 36.8% (13.5-36.8% in the flowering top, 8.4-28.7% in the leaves, and 4.7-9.6% in the stems; Table 11). Total tannin values varied between 3.2 and 25.7% (9.0-25.7% in the flowering top, 6.1-22.0% in the leaves, and 3.2-7.2% in the stems). The highest tannin content was detected in the population of Szentlőrinc in July and Lake Almamellék in August (in the flowering top; Table 12). According to the Ph. Hg. VIII., Lythri herba contains not less than 5.0% of tannins, expressed as pyrogallol, and calculated with reference to the dried drug. This criterion was met in herbs of all selected populations. Our 51
results are also in agreement of those of MIHAJLOVIC (1988), who detected tannins in 6% per dry weight in petioles and 24% in leaves. The statistical analysis showed that the total polyphenol content of the populations was significantly higher (p = 0.012) in 2011 than in 2010, which might be caused by the weather conditions. There was no significant difference between July and August 2010 (p = 0.822), July and August 2011 (p = 0.971), July 2010 and 2011 (p = 0.063), as well as August 2010 and 2011 (p = 0.091), respectively.
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Table 11. Total polyphenol content of the drug parts. Yellow colour indicates the stems, green colour the leaves, and purple colour the flowers. (Mean of 2 parallel measurements.) ID July 2010 % ID August 2010 % 1 Lake Deseda stem 2.6 1 Csebény stem 1.2 2 Csebény stem 3.3 2 Szigetvár stem 1.6 3 Lake Sikonda stem 3.4 3 Lake Sikonda stem 1.7 4 Cserénfa stem 3.9 4 Lake Deseda stem 1.8 5 Lake Almamellék stem 4.3 5 Szentlőrinc stem 2.6 6 Lake Malomvölgy stem 4.6 6 Kaposvár stem 3.1 7 Szigetvár leaf 5.3 7 Kacsóta stem 3.1 8 Almamellék stem 5.8 8 Almamellék stem 3.7 9 Kacsóta stem 5.9 9 Lake Almamellék stem 4.1 10 Szigetvár stem 6.5 10 Lake Malomvölgy stem 4.3 11 Szentlőrinc stem 6.7 11 Szentlászló stem 5.6 12 Kaposvár stem 7.3 12 Cserénfa stem 5.8 13 Lake Almamellék leaf 7.5 13 Lake Deseda leaf 6.5 14 Lake Deseda leaf 7.9 14 Szigetvár leaf 6.5 15 Lake Deseda flowering top 8.3 15 Lake Sikonda leaf 8.8 16 Szentlászló stem 9.9 16 Kaposvár flowering top 10.0 17 Almamellék leaf 10.9 17 Szentlászló leaf 10.2 18 Lake Sikonda leaf 11.1 18 Szentlőrinc leaf 10.7 19 Cserénfa leaf 11.7 19 Kacsóta flowering top 10.7 20 Lake Almamellék flowering top 12.1 20 Almamellék leaf 12.2 21 Lake Malomvölgy leaf 13.3 21 Kacsóta leaf 12.5 22 Szigetvár flowering top 13.3 22 Kaposvár leaf 13.7 23 Szentlászló flowering top 14.1 23 Lake Malomvölgy leaf 15.0 24 Szentlászló leaf 16.3 24 Cserénfa leaf 16.7 25 Kacsóta leaf 17.0 25 Lake Almamellék leaf 16.7 26 Cserénfa flowering top 17.2 26 Lake Almamellék flowering top 18.1 27 Lake Malomvölgy flowering top 17.4 27 Csebény flowering top 19.7 28 Kaposvár flowering top 17.4 28 Lake Deseda flowering top 20.6 29 Szentlőrinc leaf 18.0 29 Lake Sikonda flowering top 22.3 30 Lake Sikonda flowering top 19.3 30 Almamellék flowering top 23.2 31 Almamellék flowering top 21.2 31 Csebény leaf 23.3 32 Kaposvár leaf 22.0 32 Lake Malomvölgy flowering top 23.8 33 Csebény leaf 22.2 33 Szentlőrinc flowering top 25.0 34 Csebény flowering top 22.9 34 Cserénfa flowering top 25.0 35 Kacsóta flowering top 23.1 35 Szigetvár flowering top 25.4 36 Szentlőrinc flowering top 25.5 36 Szentlászló flowering top 27.3
(continued overleaf)
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Table 11. (continued) Total polyphenol content of the drug parts. Yellow colour indicates the stems, green colour the leaves, and purple colour the flowers. (Mean of 2 parallel measurements.) ID July 2011 % ID August 2011 % 1 Lake Sikonda stem 4.7 1 Szentlőrinc stem 5.2 2 Szentlőrinc stem 5.4 2 Csebény stem 5.5 3 Cserénfa stem 6.6 3 Kaposvár stem 5.7 4 Lake Almamellék stem 7.4 4 Cserénfa stem 6.5 5 Szentlászló stem 7.7 5 Almamellék stem 7.1 6 Lake Deseda stem 7.8 6 Lake Almamellék stem 7.9 7 Lake Malomvölgy stem 7.8 7 Szentlászló stem 8.0 8 Almamellék stem 8.3 8 Lake Deseda stem 8.4 9 Cserénfa leaf 8.4 9 Lake Malomvölgy stem 8.7 10 Kaposvár stem 8.6 10 Szigetvár stem 9.5 11 Szigetvár stem 8.8 11 Lake Sikonda stem 9.6 12 Lake Deseda leaf 10.8 12 Cserénfa leaf 11.1 13 Lake Sikonda leaf 11.2 13 Lake Deseda leaf 11.5 14 Szentlőrinc leaf 14.2 14 Csebény leaf 12.1 15 Lake Malomvölgy leaf 16.4 15 Lake Sikonda leaf 12.6 16 Cserénfa flowering top 17.1 16 Szentlászló flowering top 13.5 17 Szentlászló leaf 17.7 17 Szentlőrinc leaf 13.9 18 Szigetvár leaf 19.2 18 Kaposvár leaf 14.2 19 Lake Sikonda flowering top 19.6 19 Kaposvár flowering top 16.3 20 Lake Almamellék leaf 22.2 20 Szentlászló leaf 16.5 21 Kaposvár leaf 22.3 21 Almamellék leaf 17.6 22 Szentlászló flowering top 23.9 22 Lake Malomvölgy leaf 18.0 23 Szigetvár flowering top 24.7 23 Almamellék flowering top 19.0 24 Almamellék flowering top 25.7 24 Lake Deseda flowering top 19.5 25 Almamellék leaf 29.2 25 Lake Sikonda flowering top 20.8 26 Kaposvár flowering top 29.2 26 Cserénfa flowering top 22.8 27 Lake Malomvölgy flowering top 30.9 27 Szigetvár leaf 22.9 28 Szentlőrinc flowering top 31.2 28 Csebény flowering top 25.5 29 Csebény flowering top - 29 Szentlőrinc flowering top 25.6 30 Csebény leaf* - 30 Szigetvár flowering top 27.8 31 Csebény stem* - 31 Lake Malomvölgy flowering top 28.5 32 Kacsóta flowering top* - 32 Lake Almamellék leaf 28.7 33 Kacsóta leaf* - 33 Lake Almamellék flowering top 36.8 34 Kacsóta stem* - 34 Kacsóta flowering top* - 35 Lake Almamellék flowering top* - 35 Kacsóta leaf* - 36 Lake Deseda flowering top* - 36 Kacsóta stem* - *At some places we could not collect samples, or plants had no flowers in July yet, therefore some data are missing.
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Table 12. Total tannin content of the drug parts. Yellow colour indicates the stems, green colour the leaves, and purple colour the flowers. (Mean of 2 parallel measurements.) ID July 2010 % ID August 2010 % 1 Lake Deseda stem 2.0 1 Lake Sikonda stem 1.0 2 Csebény stem 2.6 2 Csebény stem 1.0 3 Lake Sikonda stem 2.8 3 Szigetvár stem 1.2 4 Cserénfa stem 2.9 4 Lake Deseda stem 1.6 5 Lake Malomvölgy stem 3.7 5 Szentlőrinc stem 2.0 6 Szigetvár leaf 3.9 6 Kaposvár stem 2.3 7 Lake Almamellék stem 4.1 7 Kacsóta stem 2.4 8 Almamellék stem 4.7 8 Almamellék stem 3.2 9 Kacsóta stem 5.0 9 Lake Almamellék stem 3.2 10 Szigetvár stem 5.4 10 Lake Malomvölgy stem 3.8 11 Szentlőrinc stem 5.4 11 Cserénfa stem 4.4 12 Lake Almamellék leaf 6.2 12 Szentlászló stem 4.7 13 Kaposvár stem 6.3 13 Szigetvár leaf 5.1 14 Lake Deseda leaf 6.3 14 Lake Deseda leaf 6.0 15 Lake Deseda flowering top 6.6 15 Lake Sikonda leaf 7.1 16 Lake Almamellék flowering top 7.8 16 Szentlászló leaf 8.2 17 Szentlászló stem 8.4 17 Szentlőrinc leaf 8.7 18 Almamellék leaf 8.6 18 Kacsóta flowering top 8.8 19 Lake Sikonda leaf 9.1 19 Kaposvár flowering top 9.2 20 Cserénfa leaf 10.1 20 Almamellék leaf 9.5 21 Szigetvár flowering top 11.3 21 Kacsóta leaf 10.5 22 Lake Malomvölgy leaf 11.5 22 Kaposvár leaf 10.5 23 Szentlászló flowering top 12.2 23 Lake Malomvölgy leaf 11.7 24 Kaposvár flowering top 13.3 24 Cserénfa leaf 14.2 25 Lake Malomvölgy flowering top 14.4 25 Lake Almamellék flowering top 15.0 26 Cserénfa flowering top 14.8 26 Lake Almamellék leaf 15.2 27 Szentlászló leaf 15.5 27 Csebény flowering top 16.1 28 Kacsóta leaf 16.1 28 Lake Deseda flowering top 16.9 29 Lake Sikonda flowering top 16.4 29 Csebény leaf 18.4 30 Szentlőrinc leaf 16.7 30 Lake Sikonda flowering top 18.9 31 Kacsóta flowering top 18.2 31 Almamellék flowering top 19.0 32 Almamellék flowering top 18.4 32 Cserénfa flowering top 19.6 33 Csebény flowering top 19.6 33 Szentlőrinc flowering top 20.2 34 Kaposvár leaf 20.8 34 Lake Malomvölgy flowering top 20.3 35 Csebény leaf 20.9 35 Szigetvár flowering top 21.0 36 Szentlőrinc flowering top 21.5 36 Szentlászló flowering top 21.9
(continued overleaf)
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Table 12. (Continued) The total tannin content of the drug parts. Yellow colour indicates the stems, green colour the leaves, and purple colour the flowers. (Mean of 2 parallel measurements.) ID July 2011 % ID August 2011 % 1 Lake Sikonda stem 3.2 1 Szentlőrinc stem 3.5 2 Szentlőrinc stem 4.3 2 Csebény stem 4.0 3 Cserénfa stem 4.9 3 Kaposvár stem 4.1 4 Szentlászló stem 5.8 4 Cserénfa stem 4.4 5 Lake Almamellék stem 5.9 5 Almamellék stem 4.6 6 Cserénfa leaf 6.1 6 Szentlászló stem 5.4 7 Lake Malomvölgy stem 6.3 7 Lake Almamellék stem 5.6 8 Kaposvár stem 6.4 8 Lake Deseda stem 6.5 9 Almamellék stem 6.6 9 Lake Malomvölgy stem 6.6 10 Szigetvár stem 7.1 10 Lake Sikonda stem 6.6 11 Lake Sikonda leaf 8.1 11 Szigetvár stem 7.2 12 Szentlőrinc leaf 11.1 12 Cserénfa leaf 8.2 13 Lake Malomvölgy leaf 12.5 13 Lake Sikonda leaf 8.3 14 Cserénfa flowering top 12.8 14 Csebény leaf 8.6 15 Szentlászló leaf 13.9 15 Lake Deseda leaf 8.9 16 Lake Sikonda flowering top 14.7 16 Szentlászló flowering top 9.0 17 Szigetvár leaf 15.2 17 Kaposvár leaf 10.4 18 Kaposvár leaf 15.4 18 Szentlőrinc leaf 10.5 19 Szentlőrinc flowering top 18.4 19 Lake Malomvölgy leaf 11.4 20 Szentlászló flowering top 18.7 20 Kaposvár flowering top 11.5 21 Szigetvár flowering top 18.8 21 Szentlászló leaf 11.5 22 Almamellék leaf 19.5 22 Almamellék leaf 13.4 23 Almamellék flowering top 19.5 23 Almamellék flowering top 13.7 24 Kaposvár flowering top 21.0 24 Lake Deseda flowering top 13.9 25 Lake Malomvölgy flowering top 22.6 25 Lake Sikonda flowering top 14.5 26 Csebény flowering top* - 26 Cserénfa flowering top 16.2 27 Csebény leaf* - 27 Szigetvár leaf 18.1 28 Csebény stem* - 28 Csebény flowering top 18.3 29 Kacsóta flowering top* - 29 Szentlőrinc flowering top 18.4 30 Kacsóta leaf* - 30 Szigetvár flowering top 19.2 31 Kacsóta stem* - 31 Lake Malomvölgy flowering top 20.7 32 Lake Almamellék flowering top* - 32 Lake Almamellék leaf 22.0 33 Lake Almamellék leaf* - 33 Lake Almamellék flowering top 25.7 34 Lake Deseda flowering top* - 34 Kacsóta flowering top* - 35 Lake Deseda leaf* - 35 Kacsóta leaf* - 36 Lake Deseda stem* - 36 Kacsóta stem* - *At some places we could not collect samples, or plants had no flowers in July yet, therefore some data are missing.
V. 2. 2. Total flavonoid content (according to the Ph. Hg. VIII.)
The Ph. Hg. VIII. prescribes different spectrophotometric methods for identification of total flavonoid content of drugs containing O-glycosides (Method 1) and C-glycosides (Method 2). Although L. salicaria contains mainly C-glycosides, at first, we carried out some test measurements (with three different samples); because we were intended to compare 56
which method gives better results, and then we used this method for measurement of all samples. Despite the C-glycoside content of Lythri herba, greater values of total flavonoids were measured with Method 1 except for one occasion (Table 13); therefore this method was used in our study. Previously, the two methods were also compared by BÖSZÖRMÉNYI and
LEMBERKOVICS (2005) as well as VUKICS and KÉRY (2005). They investigated the total flavonoid content of Betulae folium (containing mostly O-glycosides), Crataegi folium cum flore (containing mostly C-glycosides) and Violae tricoloris herba (containing both O- and C- glycosides). They found that the results of Method 1 and 2 were nearly the same in the case of Betulae folium, but in contrast with our observations Method 2 resulted in higher total flavonoid amounts in the case of the C-glycoside containing drugs.
Table 13. Comparison of the results of Method 1 and Method 2. (Mean of 2 parallel measurements.) Plant sample Method 1 Method 2 Lythrum leaf - Lake Almamellék, 2010 August 1.7 1.2 Lythrum flowering branches - Kaposvár, 2010 August 0.4 0.3 Lythrum leaf – Szigetvár, 2011 August 1.4 1.1 Lythrum flowering branches – Szigetvár, 2011 August 0.6 0.4 Lythrum leaf – Kaposvár, 2011 August 1.7 2.5 Lythrum flowering branches – Kaposvár, 2011 August 0.6 0.3
Generally, total flavonoid content was higher in the populations collected during the main blooming period in August than at the beginning of flowering in July. The highest flavonoid content was measured in the leaves, followed by the flowering branches and stems. In 2010, the detected values ranged from 0.0 to 2.7% (0.3-2.7% in the leaves, 0.1-0.7% in the flowering tops, and 0.0-0.2% in the stems). The highest flavonoid content was recorded in the populations of Lake Almamellék in July and Lake Desesa in August (in the leaves). In 2011, the detected values ranged from 0.1 to 2.9% (0.2-2.9% in the leaves, 0.2-0.9% in the flowering tops, and 0.1-0.3% in the stems). The highest flavonoid content was measured in the populations of Cserénfa in July and Lake Deseda in August (Table 14). The statistical analysis showed that the total flavonoid content of the populations (p = 0.007) was significantly higher in July 2011 than in July 2010 in all plant parts (p = 0.027, 0.028, and 0.006 in the stem, leaves, and flowers, respectively), which might be caused by different weather conditions. Among the possible meteorological data, which could influence the amount of the active compounds, we could obtain only the mean average temperature and the mean average precipitation. According to these data, the monthly average precipitation was about three times higher, but the monthly average temperature was a little lower in July
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2011 compared to July 2010. The flavonoid content of the populations was higher in August 2010 than in July 2010 (p = 0.010; there was about two times more precipitation in August than in July, and the temperature was a little lower), but there was no significant difference between July and August 2011 (p = 0.936; the precipitiation in August was about the half than those in July, but there was not so big difference between the mean temperatures). According to the literature, not only the weather conditions may influence the flavonoid content of plants, but also the type of the soil and its capacity to hold water (RUSSEL 1961; DOWNEY et al. 2006).
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Table 14. Total flavonoid content of the drug parts. Yellow colour indicates the stems, green colour the leaves and purple colour the flowers. (Mean of 2 parallel measurements.) ID July 2010 % ID August 2010 % 1 Csebény stem 0.0 1 Lake Malomvölgy stem 0.1 2 Lake Sikonda stem 0.1 2 Lake Sikonda stem 0.1 3 Szentlőrinc stem 0.1 3 Lake Almamellék stem 0.1 4 Lake Deseda stem 0.1 4 Csebény stem 0.1 5 Kacsóta stem 0.1 5 Szentlászló stem 0.1 6 Cserénfa stem 0.1 6 Almamellék stem 0.1 7 Szentlászló stem 0.1 7 Szentlőrinc stem 0.1 8 Lake Malomvölgy stem 0.1 8 Kaposvár stem 0.2 9 Szentlászló flowering top 0.1 9 Szentlászló flowering top 0.2 10 Almamellék stem 0.1 10 Lake Deseda stem 0.2 11 Szigetvár stem 0.1 11 Kacsóta stem 0.2 12 Csebény flowering top 0.1 12 Szigetvár stem 0.2 13 Kaposvár stem 0.1 13 Szentlőrinc flowering top 0.2 14 Kacsóta flowering top 0.1 14 Cserénfa stem 0.2 15 Szentlőrinc flowering top 0.2 15 Almamellék flowering top 0.3 16 Cserénfa flowering top 0.2 16 Lake Malomvölgy flowering top 0.3 17 Lake Almamellék stem 0.2 17 Csebény flowering top 0.3 18 Lake Malomvölgy flowering top 0.2 18 Kaposvár flowering top 0.4 19 Lake Deseda flowering top 0.2 19 Kacsóta flowering top 0.4 20 Almamellék flowering top 0.2 20 Cserénfa flowering top 0.4 21 Kaposvár flowering top 0.2 21 Lake Almamellék flowering top 0.4 22 Cserénfa leaf 0.3 22 Szigetvár flowering top 0.5 23 Csebény leaf 0.3 23 Lake Sikonda flowering top 0.6 24 Lake Deseda leaf 0.3 24 Lake Deseda flowering top 0.7 25 Lake Sikonda flowering top 0.3 25 Csebény leaf 0.8 26 Kacsóta leaf 0.3 26 Szentlászló leaf 0.9 27 Szigetvár flowering top 0.4 27 Lake Malomvölgy leaf 0.9 28 Lake Malomvölgy leaf 0.4 28 Szentlőrinc leaf 1.1 29 Szentlászló leaf 0.4 29 Almamellék leaf 1.2 30 Almamellék leaf 0.5 30 Kacsóta leaf 1.3 31 Szentlőrinc leaf 0.5 31 Kaposvár leaf 1.5 32 Lake Almamellék flowering top 0.6 32 Szigetvár leaf 1.6 33 Kaposvár leaf 0.7 33 Lake Almamellék leaf 1.7 34 Lake Sikonda leaf 0.7 34 Lake Sikonda leaf 1.7 35 Szigetvár leaf 1.0 35 Cserénfa leaf 1.8 36 Lake Almamellék leaf 1.3 36 Lake Deseda leaf 2.7
(continued overleaf)
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Table 14. (Continued) Total flavonoid content of the drug parts. Yellow colour indicates the stems, green colour the leaves and purple colour the flowers. (Mean of 2 parallel measurements.) ID July 2011 % ID August 2011 % 1 Lake Sikonda stem 0.1 1 Lake Malomvölgy stem 0.1 2 Szentlőrinc stem 0.1 2 Szentlászló stem 0.1 3 Lake Almamellék stem 0.1 3 Lake Sikonda stem 0.1 4 Szentlászló stem 0.1 4 Kaposvár stem 0.1 5 Szigetvár stem 0.1 5 Szentlászló flowering top 0.2 6 Kaposvár stem 0.2 6 Szentlőrinc stem 0.2 7 Cserénfa stem 0.2 7 Csebény stem 0.2 8 Szentlászló flowering top 0.2 8 Szigetvár stem 0.2 9 Lake Malomvölgy stem 0.2 9 Cserénfa stem 0.2 10 Almamellék stem 0.2 10 Lake Almamellék stem 0.2 11 Szentlászló leaf 0.3 11 Almamellék stem 0.2 12 Szigetvár flowering top 0.3 12 Almamellék flowering top 0.2 13 Lake Deseda stem 0.3 13 Lake Deseda stem 0.3 14 Kaposvár flowering top 0.4 14 Csebény flowering top 0.3 15 Szentlőrinc flowering top 0.5 15 Lake Malomvölgy flowering top 0.3 16 Lake Malomvölgy flowering top 0.5 16 Szentlőrinc flowering top 0.4 17 Lake Sikonda flowering top 0.6 17 Cserénfa flowering top 0.5 18 Almamellék flowering top 0.6 18 Lake Almamellék flowering top 0.5 19 Szigetvár leaf 0.9 19 Szigetvár flowering top 0.6 20 Cserénfa flowering top 0.9 20 Lake Deseda flowering top 0.6 21 Lake Almamellék leaf 1.0 21 Kaposvár flowering top 0.6 22 Szentlőrinc leaf 1.2 22 Lake Sikonda flowering top 0.6 23 Almamellék leaf 1.5 23 Szentlászló leaf 0.7 24 Lake Sikonda leaf 1.6 24 Almamellék leaf 0.9 25 Lake Malomvölgy leaf 1.7 25 Csebény leaf 1.2 26 Kaposvár leaf 1.9 26 Lake Malomvölgy leaf 1.3 27 Lake Deseda leaf 2.2 27 Szigetvár leaf 1.4 28 Cserénfa leaf 2.9 28 Lake Almamellék leaf 1.5 29 Csebény flowering top* - 29 Szentlőrinc leaf 1.7 30 Csebény leaf* - 30 Kaposvár leaf 1.7 31 Csebény stem* - 31 Lake Sikonda leaf 1.7 32 Kacsóta flowering top* - 32 Cserénfa leaf 2.2 33 Kacsóta leaf* - 33 Lake Deseda leaf 2.5 34 Kacsóta stem* - 34 Kacsóta flowering top* - 35 Lake Almamellék flowering top* - 35 Kacsóta leaf* - 36 Lake Deseda flowering top* - 36 Kacsóta stem* - *At some places we could not collect samples, or plants had no flowers in July yet, therefore some data are missing.
V. 2. 3. TLC
The chromatogram (Fig. 22) showed two yellow fluorescent zones in the upper half of the plate, one of them corresponds to the zone of the orientin reference solution (Rf ~ 0.62), and presumably the other zone is isoorientin (Rf ~ 0.5) based on WAGNER and BLADT (2001).
At Rf ~ 0.72 it could be found a light green fluorescent zone corresponding to the zone of the 60
vitexin reference solution and the other light green zone (Rf ~ 0.44) under the two yellow zones might be isovitexin based on WAGNER and BLADT (2001). The Ph. Hg. VIII. does not identify these compounds, it says only that the bright green and yellow fluorescent zones are in similar positions to the reference solutions (hyperoside, chlorogenic acid, and rutine). All parts of the plant contained all of these flavonoid components but in different amounts. In agreement with the results of the total flavonoid measurements, our observation was that the greatest amount of flavonoids could be found in the leaves followed by flowering branches, and the smallest amounts of them are in the stems. There was no qualitative difference between the habitats. Rutin, hyperoside, chlorogenic acid, and caffeic acid could not be detected in the samples by TLC.
Fig. 22. TLC fingerprint of the studied parts of L. salicaria. Reference compounds: vitexin, orientin, caffeic acid, hyperoside, chlorogenic acid, rutin. Solvent system: ethyl acetate - formic acid - glacial acetic acid - distilled water 100:11:11:26. Detection: Naturstoff/PEG reagent under 365 nm.
V. 2. 4. Qualitative-quantitative UHPLC-MS analysis of L. salicaria extracts
We were interested in the evaluation of some polyphenolic compounds, whether they could be responsible for spasmogenic effects of L. salicaria extracts, so different solvents (hexane, chloroform, ethyl-acetate, 50% ethanol in water) were used for the extraction, and solvents were analysed with LC-MS system. In the case of microbiological studies, the most potent extract was the 50% ethanol in water, and therefore this extract was also analysed with LC-MS system.
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The yield of dried extracts of hexane, chloroform, ethyl acetate and 50% ethanol in water were 11.1 mg, 13.5 mg, 28.2 mg, and 90.5 mg, respectively. Only the 50% ethanol extract contained all of the selected standards. Ellagic acid could be detected in the largest amount followed by isoorientin and orientin. Catechin, caffeic acid, hyperoside, and rutin could not be detected in the hexane, chloroform, and ethyl acetate extracts (Table 15). (Total ion chromatograms can be seen in Appendix 5.) The considerable difference between the amount of compounds in the 50% ethanol extract and the other extracts can be explained by the fact that polyphenols can be dissolved in polar solvents rather than in nonpolar solvents. Differences between 50% ethanol Pharm. extract and 50% ethanol Microb. extract arise from the fact that their plant samples were collected from different habitats.
Table 15. Compounds identified in the extracts used in pharmacological and microbiological procedures. Mean of 3 experiments. Extract used in the Extracts used in the pharmacological experiments microbiological Compounds (μg/g) experiments 50% ethanol 50% ethanol hexane chloroform ethyl acetate Pharm. Microb. gallic acid 4.78 3.58 39.40 166.26 189.12 catechin - - - 29.56 10.10 caffeic acid - - 3.86 4.60 1.22 chlorogenic acid 1.77 1.96 1.33 171.10 97.92 orientin 2.34 5.22 7.70 471.42 456.74 isoorientin 8.92 23.78 25.38 1319.96 1678.96 vitexin 1.18 1.74 3.52 88.26 143.38 ellagic acid 16.44 27.70 49.48 1718.56 1173.00 hyperoside - - - 25.06 17.34 isovitexin 2.84 4.10 7.38 130.62 344.12 rutin - - - 1.14 1.00 luteolin 1.84 13.30 44.38 98.36 39.14 apigenin 2.60 6.18 3.40 3.06 3.70
Recently, some quantitative data were reported on the flavonoid content of the whole aerial parts of L. salicaria. The ethyl acetate extract contained 1.3 mg/g isoorientin and 0.67 mg/g isovitexin (compared to our 25.38 μg/g and 7.38 μg/g, respectively), and 50% methanol in water extract contained 6.18 mg/g isoorientin and 1.94 mg/g isovitexin (compared to our 50% ethanol in water extract 1.32 mg/g and 0.13 mg/g, respectively)
(TUNALIER et al. 2007), so our extracts contained lower quantities of isoorientin and isovitexin.
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VI. Antimicrobial activities of L. salicaria extracts
The development of antibiotic resistance is an urgent problem necessitating the search for new antimicrobial substances. A wide range of medicinal plants are used traditionally to treat different infectious diseases (SIBANDA and OKOH 2007). Numerous secondary metabolites (e.g., tannins, terpenoids, alkaloids, and flavonoids) have been found to have antimicrobial properties in vitro (COWAN 1999; PAPP 2004; PAPP 2005; LEWIS and AUSUBEL 2006). Unfortunately, despite the great number of publications on the antimicrobial activities of plant extracts, none of the plant derived chemicals have successfully been exploited yet for clinical use as antibiotics (GIBBONS 2004). TEGOS et al. (2002) suggested that a number of plant compounds are not antimicrobial, but might have antibiotic resistance modifying effect (e.g., efflux pump inhibitors), and they can promote the effectiveness of antimicrobial compounds. Diffusion methods are widely used to investigate antibacterial activity of plant extracts. We selected disk and agar diffusion methods as well as tube dilution method to examine the antibacterial activity of L. salicaria extracts. The activity of the sample solution can be estimated from the diameter of the inhibition zones in the disk and agar diffusion methods. The following bacterial and fungal strains were used in our microbiological experiments. Staphylococcus aureus and S. epidermidis are Gram-positive cocci, they can be found on the normal human skin flora and mucous membranes. S. epidermidis is an opportunistic pathogen, only patients with compromised immune system are at risk for developing infections. But S. aureus can infect healthy people as well, in the case of skin or mucous membrane injuries. Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for 25-60% of the severe nosocomial infections. MRSA has developed resistance to β-lactam antibiotics, e.g., penicillins, carbapenems, and cephalosporins. This resistance can be accompanied by other resistances (e.g., aminoglycoside resistance) resulting in multidrug resistance. Bacillus subtilis is a Gram-positive, rod-shaped bacterium. It can be found not only in the soil but also in the human intestines. Escherichia coli is a Gram-negative, rod-shaped bacterium. It is an important part of the normal flora of the gut, but sometimes E. coli strains can be found on the skin, pharyngeal mucous membrane, or female external genitalia, as well
(GERGELY 2003). E. coli and S. aureus are common causes of bacterial food-poisoning
(MADIGAN et al. 2000). Micrococcus luteus is a Gram-positive, spherical, saprotrophic bacterium. It can be found in soil, dust, water, and air, and also colonizes the human mouth, mucosae, oropharynx, and upper respiratory tract. Pseudomonas aeruginosa is a Gram- negative, rod-shaped, opportunistic pathogen and it can be the causative agent for example of
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nosocomial infections or corneal ulcer. Its multidrug-resistant form is one of the most dangerous nosocomial pathogens (GERGELY 2003). Candida albicans is an opportunistic pathogen fungus. It commonly causes oral and genital infections in humans with suppressed immune system. It is a part of the normal human flora and can be found in the airways, gastrointestinal system, and mucous membrane of the female genitalia (GERGELY 2003). C. albicans is quite frequently responsible for clinical infections (BODEY et al. 1992). The aim of our microbiological experiments was to confirm the antimicrobial effects of L. salicaria extracts published previously in the literature (Table 16), and to compare the results of the most frequently used microbiological techniques.
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Table 16. Summary of the results of previously published papers in connection with antimicrobial activity of L. salicaria
CITOGLU DULGER ALTANLAR RAUHA Barteria and and BECKER et BORCHARDT BORCHARDT and et al., et al., our results fungi ALTANLAR, al., 2005 et al., 2008 et al., 2009 GONUZ, 2006 2000 2003 2004 Gram-positive cocci Staphylococcus n.t. + + + + + + + aureus MRSA n.t. n.t. n.t. n.t. n.t. n.t. n.t. + S. epidermidis n.t. n.t. n.t. n.t. n.t. n.t. n.t. + Micrococcus n.t. n.t. + n.t. n.t. + n.t. + luteus Gram-positive bacilli Bacillus cereus n.t. n.t. n.t. n.t. n.t. + n.t. n.t. B. subtilis n.t. + n.t. n.t. n.t. n.t. n.t. + Listeria + n.t. n.t. n.t. n.t. - n.t. n.t. monocytogenes Mycobacterium n.t. n.t. n.t. n.t. n.t. + n.t. n.t. smegmatis Gram- negative rods Escherichia n.t. + - + + - + + coli Klebsiella n.t. n.t. n.t. n.t. n.t. - n.t. n.t. pneumonia Proteus n.t. n.t. n.t. n.t. n.t. - n.t. n.t. vulgaris P. mirabilis n.t. n.t. + n.t. n.t. n.t. n.t. n.t. Pseudomonas n.t. + - + + - n.t. + aeruginosa MDR P. n.t. n.t. n.t. n.t. n.t. n.t. n.t. + aeruginosa Citrobacter n.t. n.t. - n.t. n.t. n.t. n.t. n.t. freundii Fungi Aspergillus n.t. n.t. n.t. n.t. n.t. n.t. - n.t. niger Candida n.t. + + + + - + + albicans C. glabrata n.t. + n.t. n.t. n.t. n.t. n.t. n.t. C. krusei n.t. + n.t. n.t. n.t. n.t. n.t. n.t. Kluyveromyces n.t. n.t. n.t. n.t. n.t. - n.t. n.t. fragilis Rhodotorula n.t. n.t. n.t. n.t. n.t. - n.t. n.t. rubra Saccaromyces n.t. n.t. + n.t. n.t. n.t. n.t. n.t. cerevisiae tube dilution, agar disk disk disk disk cylinder disk and Applied tests diffusion diffusion bioautography diffusion diffusion diffusion diffusion agar method method method method method method diffusion methods Legend: + active; - inactive; n.t. not tested RAUHA et al. (2000) investigated not only the antibacterial effects of plant extracts but also those of their isolated phenolic compounds. S. aureus was sensitive to quercetin, naringenin, morin, and kaempferol compounds. S. epidermidis showed sensitivity to quercetin, naringenin, and morin. B. subtilis was sensitive to quercetin, and naringenin. Micrococcus luteus was sensitive against flavone, quercetin, naringenin, methyl gallate, and 65
morin. Flavone, quercetin, naringenin, methyl gallate, and morin inhibited the growth of M. luteus. E. coli was sensitive to flavone, quercetin, naringenin, and methyl gallate. P. aeruginosa was sensitive to gallic acid. AFIFI et. al. (1997) investigated the antimicrobial activity of isoorientin and vitexin, and they found that it exerts certain antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa. BECKER et al. (2005) identified oleanolic acid, ursolic acid and vescalagin in purple loosestrife extracts as active principles of the antifungal and antibacterial activity by bioautography.
VI. 1. Materials and methods Preparation of extracts. 3.00-3.00 g drug (flowering tops, leaves, and stems collected from Kaposvár in August 2010) was extracted three times (with 30 mL 50% ethanol in water, hexane, chloroform, and water, respectively) with ultrasonic water bath at 40ºC, the solution was filtered, evaporated by vacuum-distillation, and the concentration was set to be 10 mg dried extract/1.0 mL solvent). Antibiotics and solvents. The used antibiotics were as follows: Amphotericin B: Fungizone 50mg powder for sterile concentrate – Bristol-Myers Squibb; Gentamicin: Gentamicin 40mg/ml injection – Sandoz; Cefuroxime: powder for injection – GlaxoSmithKline; Ciprofloxacin: Ciprinol 2 mg/ml solution for infusion – KRKA d.d.; Polymyxin B: Polymyxin B sulfate salt powder – Sigma Aldrich Ltd.; Vancomycin – Vancomycin Human 50 mg/ml powder for infusion, TEVA. Absolute ethanol, hexane, chloroform, ethyl-acetate, and absolute ethanol were purchased from Molar Chemicals Ltd., Hungary. Disk (DDM) and agar diffusion method (ADM). Mueller-Hinton agar (Oxoid, UK) was used in DDM and ADM. The tested microorganisms were Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Candida albicans ATCC 90028, and bacteria isolated from patients were MRSA 4262, Staphylococcus epidermidis 122228, Bacillus subtilis, Micrococcus luteus, and a multi-drug resistant P. aeruginosa. These test microorganisms were maintained on Mueller-Hinton agar in the Institute of Medical Microbiology and Immunology at University of Pécs. From each microorganism a solution was prepared, which corresponds to >105 colony-forming units (cfu)/mL. In DDM 20 μL of plant extracts were applied to the sterile paper disks (diameter: 5 mm), dried and arranged equidistant on the agar surface in sterile Petri dishes (diameter: 8 cm); in ADM 50 and 100 μL of leaf and flower extracts prepared with 50% ethanol in water were measured into the holes made in the agar using sterile metal borers. The inhibition zones
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were measured after 48 hours incubation at 37ºC (except for C. albicans which was incubated at 30ºC), and the effect was calculated as a mean of triplicate tests. Evaluation of minimum inhibitory (MIC) and bactericidal concentrations (MBC) by tube dilution method (according to Lóránd et al. 1999). 1.0 mL broth (in the case of C. albicans, the broth contained 3% glucose) was measured into 10 tubes, then 1.0 mL plant extract (10 mg/mL) or antibiotic solution (200 μg/mL) was added to the first tube and a twofold dilution was performed through the series of tubes. 10 μL of bacterial culture (A600 5 nm≈0.1, which corresponds to >10 colony-forming units (cfu)/mL) were added to each tube. After a 24 hours incubation at 37ºC (except for C. albicans which was incubated at 30ºC), the bacterial cultures were spread on the surface of Mueller-Hinton agar. The lowest inhibitory and bactericidal concentrations were evaluated after a 48 hours incubation period at 37ºC
(except for C. albicans which was incubated at 30ºC). The number of bacterial colonies was compared to the untreated control. Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial substance that could inhibit the visible growth of bacteria. The minimum bactericidal concentration (MBC) is the lowest concentration of an antimicrobial substance required to kill all germs in a tube (Appendix 6).
VI. 2. Results and discussion Disk diffusion method. S. aureus, MRSA, S. epidermidis, and M. luteus were sensitive to the 50% ethanol in water and distilled water extracts. The greatest inhibition zones were produced by the flowering branches then the leaves and finally the stems. S. epidermidis was the most sensitive bacterium in this experiment. The solvents caused no inhibition by themselves (Table 17). The hexane and chloroform extracts of L. salicaria failed to show any activity against the investigated microbial strains; therefore they are not listed in Table 17, and we used only the ethanolic extracts in the subsequent experiments. The reason of this phenomenon is probably, that polyphenols, which are thought to be responsible for the antimicrobial effect, are less soluble in the nonpolar solvents compared to the polar ones
(DAGLIA 2012).
Table 17. Inhibition zones (in mm) of 50% ethanol in water and distilled water extracts of L. salicaria with disk diffusion method. Mean of 3 parallel measurements. 50% Ethanol in water extracts Distilled water extracts Bacteria Solvent Leaf Flowering branches Stem Solvent Leaf Flowering branches Stem Staphylococcus 0.0 11.0 13.5 8.0 0.0 14.5 17.0 13.5 aureus Micrococcus luteus 0.0 12.5 14.0 8.0 0.0 9.5 15.0 7.0 MRSA 0.0 13.0 14.0 12.0 0.0 11.5 16.0 12.5 Staphylococcus 0.0 16.0 18.5 14.0 0.0 13.5 18.5 14.5 epidermidis 67
Agar diffusion method. All of the selected strains except for B. subtilis were sensitive to 50% ethanol in water extract. The Gram positive Staphylococcus strains were the most sensitive (Table 18). The inhibition zones could be evaluated even 2 weeks after the application.
Table 18. Inhibition zones of 50% ethanol in water extract on different bacteria and fungus with agar diffusion method. Mean of 3 parallel measurements. Inhibition zone (mm) 50% Ethanol in water extracts Bacteria flower flower solvent leaf (50 μL) leaf (100 μL) (50 μL) (100 μL) Bacillus subtilis 0.0 0.0 0.0 0.0 0.0 Escherichia coli 0.0 0.0 0.0 14.0 19.0 Micrococcus luteus 0.0 15.5 17.5 16.5 19.0 MRSA 0.0 16.0 17.5 18.0 22.0 Pseudomonas aeruginosa 0.0 15.0 19.0 18.5 22.5 Candida albicans 0.0 18.0 20.0 19.0 23.0 Staphylococcus aureus 0.0 18.0 22.5 22.5 25.5 Staphylococcus epidermidis 0.0 19.0 21.0 23.5 25.5
Minimum inhibitory and bactericidal concentration. All of the selected strains were sensitive to 50% ethanol in water extract of L. salicaria in appropriate concentrations (Table 19). The antibiotic resistant S. aureus and P. aeruginosa were similarly sensitive than the S. aureus and P. aeruginosa having no antibiotic resistance. Compared to the antibiotics, L. salicaria approximates mostly the effectiveness of vancomycin against MRSA. The other antibiotics can be more than 1000 times potent than our extracts. (For quantitative evaluation of the 50% ethanol extract used in this experiment, see Chapter V. 2. 4.)
Table 19. MBC and MIC values of the 50% ethanol in water extract on different bacteria and fungus with tube dilution method compared to the antibiotic standards. Mean of 3 parallel measurements. L. salicaria extract Antibacterial, antifungal drugs Microorganisms MBC MIC MBC (mg/mL) MIC (mg/mL) (μg/mL) (μg/mL) Candida albicans 2.50 5.00 Amphotericin B 0.78 1.56 Micrococcus luteus 2.50 5.00 Gentamicin 0.78 1.56 Bacillus subtilis 1.25 2.50 Cefuroxime 1.56 3.12 Escherichia coli 1.25 2.50 Cefuroxime 6.25 12.5 Pseudomonas aeruginosa 1.25 2.50 Ciprofloxacin 0.39 0.78 MDR Pseudomonas 1.25 2.50 Polymyxin B 1.56 3.12 aeruginosa Staphylococcus aureus 0.63 1.25 Gentamicin 0.20 0.39 MRSA 0.63 1.25 Vancomycin >100* n.t.* Staphylococcus epidermidis 0.63 1.25 Gentamicin 3.12 6.25 *The MRSA strains were not sensitive enough to the used standard concentrations of vancomycin.
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We can conclude that the antimicrobial effect of L. salicaria is dose-dependent. The higher doses were given (in the different methods) the more microbes were sensitive to the extract. The antibacterial effects of L. salicaria extracts have been investigated in the case of many bacteria and fungi. Table 16 shows the results of these publications including our results. Most of the publications listed in this table concluded that L. salicaria extracts are effective against S. aureus. Our experiments also confirmed this result, moreover, methicillin- resistant S. aureus also showed sensitivity to L. salicaria extracts. We also confirmed that M. luteus and B. subtilis are sensitive to Lythrum extracts. To the best of our knowledge, this is the first time, when antimicrobial effects of Lythrum extracts have been proven on MRSA, S. epidermidis, and a multidrug-resistent P. aeruginosa. In the literature, we found only one article stating that L. salicaria is ineffective against C. albicans, our result confirms the opinion of most of the authors, i.e. it is effective. Literature results do not agree, wheather L. salicaria has antimicrobial effects on Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans or not. These differences may arise from the differences in the used techniques and dosage.
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VII. Pharmacological study of L. salicaria extracts on guinea pig ileum preparation
The purpose of our work was to investigate the effect and the mechanism of action of hexane, chloroform, ethyl acetate, and 50% ethanol in water extracts of L. salicaria on isolated guinea pig ileum. The extracts of flowering tops were used in the experiments, because they were more active in the preliminary tests than the leaf. The 50% ethanol in water extract was tested both in the absence and presence of various drugs having specific mechanisms of action. The guinea pig small intestine was chosen as an isolated organ capable of detecting a whole range of neuronal and smooth muscle inhibitory and excitatory effects (Table 20). In order to attempt to explain the background of the obtained results, phytochemical analyses of the extracts were also carried out (see Chapter V. 2. 4.).
Table 20. Neuronal and smooth muscle inhibitory and excitatory effects on guinea pig small intestine. • Inhibitors of nerve conduction: tetrodotoxin, local anaesthetics (Na-channel Neuronal inhibitory effects: blockers) inhibition of the cholinergic • N-type Ca-channel blockers "twitch" response without • Inhibitors of acetylcholine release: adrenergic a-receptor stimulants*, opioid influencing direct smooth receptor agonists*, agonists acting at some histamine or serotonin receptors*, muscle stimulation nociceptin receptor agonists*, cannabinoid receptor agonists*, adenosine receptor agonists* • Cholinesterase inhibitors Neuronal excitatory effects: • Ganglionic stimulants (e.g., nicotine)* enhancement of the cholinergic • TRPV1 receptor agonists (e.g., capsaicin)* "twitch" response or of • Tachykinin NK-3 receptor agonists* spontaneous tone, without • Cholecystokinin receptor agonists* influencing direct smooth • P2X purinoceptor agonists (e.g., α,β-methyleneATP)* muscle stimulation • Muscimol and other GABA-A receptor agonists* • Agonists acting at some serotonin receptors (5-HT-3, 5-HT-4)* • Enhancers of intracellular cAMP or cGMP, e.g., β-adrenoceptor* or Inhibition of both neuronally- VIP/PACAP receptor* agonist, phosphodiesterase inhibitor, NO donor# evoked contraction and direct • Smooth muscle relaxants (e.g., papaverin-like) smooth muscle stimulation • L-type Ca-channel blockers Acetylcholine-like*, histamine-like*, substance P-like*, prostaglandin-like*, Smooth muscle excitatory leukotriene-like* effects • P2X purinoceptor agonists (e.g., ATP)* * The effect can be inhibited by the respective antagonist, hence, antagonist effects can also be tested. # The effect can be blocked by inhibitors of the soluble guanylate cyclase.
VII. 1. Materials and methods All pharmacological experimental procedures were carried out according to the 1998/XXVIII Act of the Hungarian Parliament on Animal Protection and Consideration Decree of Scientific Procedures of Animal Experiments (243/1988) and complied with the recommendations of the International Association for the Study of Pain and the Helsinki 70
Declaration. The studies were approved by the Ethics Committee on Animal Research of University of Pécs according to the Ethical Codex of Animal Experiments, and the licence was given (licence no.: BA 02/2000-7/2006). Chemicals and solvents. Sources of drugs were as follows, alpha,beta-methyleneATP (α,β-meATP), atropine sulfate, capsaicin, tetrodotoxin, pyridoxal-5'-phosphate-6-azophenyl- 2',4'-disulfonate (PPADS), suramin sodium salt, indomethacin, and DMSO (Sigma, Hungary); chloropyramine hydrochloride (Suprastin® inj., Egis, Hungary). Hexane, chloroform, ethyl- acetate, and absolute ethanol were purchased from Molar Chemicals Ltd., Hungary. Preparation of extracts. 0.50 g drug (flowering tops collected from Szigetvár in August 2011) was extracted with 10.0 mL solvent (hexane, chloroform, ethyl acetate, or 50% ethanol in water) using ultrasonic water bath at 40ºC for 3 min. The solution was filtered, evaporated by vacuum-distillation, and diluted to 10.0 mL with DMSO in the case of the hexane, chloroform, and ethyl acetate extracts, and with 50% ethanol in the case of the ethanol extract. DMSO was used to ensure the blending of hexane, chloroform, and ethyl acetate extracts with the organ bath and qualitative-quantitative analyses were carried out with these solutions. Animals and preparation. Short-haired, coloured guinea pigs of both sexes, weighing 400-600 g were killed by stunning and bled out. The pre-terminal ileum was excised and put in Krebs’ solution. Whole segments of the ileum (approx. 3 cm in length) were made up as longitudinally-oriented preparations that were put into organ baths containing 5 mL of
Krebs’ solution (composition [mM]: NaCl 119, NaHCO3 25, KH2PO4 1.2, CaCl2 2.5, MgSO4 1.5, KCl 2.5, and glucose 11) and kept at 37°C with the help of a circulating thermostat
(Experimetria, Budapest, Hungary). The bathing fluid was bubbled with a mixture of 5% CO2 and 95% O2. The preparations were connected to isotonic lever transducers (Hugo Sachs Elektronik/Harvard Instruments, March-Hugstetten, FRG). Movements of the tissues were recorded on compensographic ink writers. The load on the tissues was 7 mN. Experiments were commenced after 40 min equilibration of the preparations. Contractions were compared to and expressed as percentage of the maximal longitudinal spasm evoked by histamine (0.5 μM) administered at the beginning of the experiment and washed out carefully. L. salicaria extracts were administered twice, each for 15 min, with a washout period of 30 min. Drugs and contact times. The following concentrations and contact times were used for pre-treatments: α,β-meATP, 15 + 15 μM, 10 min each; PPADS (50 μM) plus suramin (100 μM), 20 min; chloropyramine (0.2 μM), 15 min; indomethacin (2 μM), 15 min; atropine (0.5 μM), 15 min; tetrodotoxin (0.5 μM), 15 min; atropine plus tetrodotoxin, 15 min. capsaicin (10 μM) was administered for 10 min for producing tachyphylaxis (desensitization),
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but it was removed from the organ bath and a 40-min washout period was allowed to the preparations to recover from its smooth muscle-depressant effect (BARTHÓ et al. 1987). Electrical field stimulation. Electrical field stimulation was delivered by a high- performance stimulator (Experimetria, Budapest, Hungary) through platinum wire electrodes placed in the bathing solution above and below the preparation, at a distance of 4 cm from each other. Parameters of electrical field stimulation were as follows: 60 V amplitude, 0.1 ms pulse width, single shocks at a frequency of 0.05 Hz. Statistical analysis. For the analysis of the primary data Microsoft Excel 2010 Software was used. Statistical evaluation of the data was performed with Microsoft Excel WinSTAT. Ileum contractions were expressed in mean ± SEM as percentage of the maximal longitudinal spasm triggered by histamine (0.5 μM). Mann-Whitney test was used for two independent samples, Kruskal-Wallis test was used for more than two independent samples and Wilcoxon’s signed rank test was used for two related samples. Data were considered to be significant if p<0.05.
VII. 2. Results and discussion The hexane, chloroform, ethyl acetate and 50% ethanol in water extracts (10 μL/5 mL organ bath) produced contractile responses (n = 5-7) on guinea-pig ileum preparations (Fig. 23 and 24). The largest contractions were elicited by the 50% ethanol in water extract. The effect was concentration-dependent (Fig. 25). Higher concentrations could not be investigated because of the effect of the solvent on the preparations. The spasmogenic effect partially faded away without washing the tissue with fresh bathing fluid (Fig. 23). The magnitude of contractions induced by electrical stimulation was not significantly influenced by the presence of extracts (Fig. 26). The solvents by themselves caused no response.
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Fig. 23. Contraction of the guinea-pig ileum preparation in response to Lythrum extract (10 μL/5 mL bath fluid of 50% ethanol in water extract). Administration of the extract at the circle. Vertical calibration (left) shows the amplitude of the maximal longitudinal spasm evoked by 0.5 μM histamine. Upward deflections on the left (ES) are cholinergic ‘twitch’ responses evoked by single pulses of electrical field stimulation of intramural nerves. Stimuli were stopped 120 s before administration of the extract.
Fig. 24. Contractions of the guinea-pig ileum elicited by different extracts of L. salicaria (10 μL/ 5 mL organ bath for each extract), expressed as percentage of the maximal longitudinal spasm evoked by histamine. Mean ± SEM of 5-7 experiments.
Fig. 25. Concentration-dependent contractions of the guinea pig ileum elicited by 50% ethanol extract of L. salicaria. Mean ± SEM of 5-7 experiments.
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Fig. 26. The presence of 50% ethanol in water extract at different concentrations failed to significantly influence the magnitude of contractions induced by electrical stimulation. Present: the presence of 50% ethanol in water extract, before: before the administration of the extract. Mean ± SEM of 5-7 experiments.
Fig. 27. Responses of the preparations to 50% ethanol extract of L. salicaria (10 μL/ 5 mL organ bath) after different pre-treatments. Significant differences are shown above the columns; * p< 0.05 vs. control (open column), ** p< 0.01 vs. control (open column), # p<0.05 according the mark. Mean ± SEM of 5-8 experiments except for chloropyramine (mean ± SEM of 3 experiments) showing minimal inhibitory effect.
BRUN et al. (1998) reported that Salicarine®, an antidiarrhoeal preparation from flowering tops of L. salicaria elaborated in France, significantly inhibited the barium- chloride- or acetylcholine induced contractions of rat duodenum in a concentration-dependent manner, but its effect was limited in magnitude (about 60% inhibition), in spite of increasing concentrations. Spasmolytic effects of many flavonoids have been proven, e.g. vitexin produced a non-competitive antagonism on the acetylcholine dose-response curve of rat duodenum while isovitexin lacked this inhibitory effect (RAGONE et al. 2007). Isoorientin caused concentration-dependent inhibition of the amplitude and the frequency of the phasic contractions of the rat and guinea-pig uterus, but did not affect the isolated aorta, ileum or trachea (AFIFI et al. 1999). By contrast, our results clearly demonstrate that the extracts of L. salicaria flowering tops produce contractile responses on guinea-pig ileum. A similar 74
phenomenon has already been reported (VINCENT and SEGONZAC 1954; TORRENT MARTI 1975). The following pre-treatments significantly reduced the responses of the ileum preparations due to 10 μL of 50% ethanol in water extract in a 5 mL organ bath: atropine, atropine combined with tetrodotoxin, indomethacin, and PPADS combined with suramin. The strongest inhibition was caused by atropine, i.e., tetrodotoxin did not add to the inhibitory effect of atropine. The effect of the extract was insensitive (no or small, non-significant difference) to pre-treatment with tetrodotoxin, capsaicin, chloropyramine, and α,β-meATP (Fig. 27). None of the drugs applied could completely abolish the contractile responses due to the plant extract showing that more mechanisms are involved in its spasmogenic effect. We found that the contractile response of the ethanol extract was reduced strongly, but not completely, in the presence of the nonselective muscarinic antagonist atropine, indicating that muscarinic receptor activation could partially explain the contractile response. Since the response was not significantly inhibited by tetrodotoxin (a Na+ channel blocker that fully suppresses the cholinergic ‘twitch’ response), it seems probable that cholinergic stimulation of the L. salicaria extract is a post-junctional phenomenon, i.e. takes place at the level of the smooth muscle. According to literature data bioactive constituents of L. salicaria show inhibition of acetylcholine esterase (10-19%) (THE LOCAL FOOD-NUTRACEUTICALS
CONSORTIUM 2005). We doubt, however, that such an effect can account for the contractile response; if our extracts had acetylcholine esterase inhibitory activity, they should have increased the cholinergic contractions elicited by electrical stimuli. Responses due to the 50% ethanol in water extract were also significantly reduced by the cyclooxygenase inhibitor indomethacin and the purinergic P2 receptor inhibitor PPADS plus suramin, the former suggesting that the contractile responses are also mediated through prostanoids. A weak inhibitory effect of the P2 purinoceptor antagonists might indicate some involvement of a purinergic mechanism, although endogenous purinoceptor agonists may positively modulate cholinergic excitation (BARTHÓ et al. 1997). Overall, the inhibitory effects of fairly highly- dosed indomethacin or the purinoceptor antagonists were only moderate. This could indicate some release of prostanoids and an ATP-like substance by the extract (in addition to a contractile effect of unknown origin) and/or, alternatively, a positive modulation of the contractile effect of the extract by prostanoids or ATP-like purinergic agonists. Pre-treatment of the tissues with chloropyramine, a histamine (H1) receptor blocker, did not alter the response to the plant extract, demonstrating that no histamine receptor activation has a role in the contractile responses. Pre-treatments with α,β-meATP, a purinergic P2X receptor agonist and desensitizer (BENKÓ et al. 2005), and capsaicin, that damages the afferent nerve endings
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(BARTHÓ et al. 2004), elicit no changes in the responses demonstrating that these pathways are also not involved in the contractile mechanism. The polar fraction (50% ethanol in water extract) was found to be rich in polyphenols. Ellagic acid could be detected in the highest amount, followed by isoorientin and orientin
(Table 15). Recently, TUNALIER et al. (2007) reported some quantitative data on the flavonoid content of the whole aerial parts of L. salicaria. The ethyl acetate extract contained 1.3 mg/g isoorientin and 0.67 mg/g isovitexin, compared to our 25.4 μg/g and 7.4 μg/g, respectively. Their 50% methanol in water extract contained 6.18 mg/g isoorientin and 1.94 mg/g isovitexin, compared to 1.32 mg/g and 0.13 mg/g, respectively, in our 50% ethanol in water extract. In the present study, the extracts contained lower quantity of isoorientin and isovitexin. Hexane, chloroform and ethyl acetate extracts also showed spasmogenic effects while catechin, caffeic acid, hyperoside and rutin could not be detected in these extracts. For this reason we do not think that these compounds play a part in the mechanism of the spasmogenic effect. Further isolation, purification and pharmacological analysis of the different components are necessary to identify the components which can be responsible for the antispasmodic and spasmogenic effects. Cholinergic receptor stimulants are used against gastrointestinal atony. The present results may suggest that a diluted extract of L. salicaria p.o. could be used as a mild stimulant of gastrointestinal motility.
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VIII. Summary and novel findings
Plants usually vary in some morphological and phytochemical features according to the different ecological parameters of the habitats. Lythrum salicaria is a species investigated widely throughout the world, but rarely in Hungary. Therefore we examined some morphological and phytochemical parameters of twelve populations of L. salicaria collected from different habitats in south-west Hungary in 2010 and 2011. The great diversity of this plant may also arise from genetic reasons, but genetic investigations were out of the scope of this dissertation. The altitude of the habitats varied from 120 m (Szigetvár) to 185 m (Lake Sikonda, in Mecsek Hills), but we do not think that these small differences could influence either the amount of the active compounds or the morphological parameters. We also investigated the plant community in the selected areas. The number of the species living together with L. salicaria varied between 9 (Szigetvár) and 28 (Cserénfa). The most frequently occurring species were Calystegia sepium (L.) R. BR. (in 8 habitats),
Ranunculus repens L. (in 6 habitats), Carex acutiformis EHRH., Elymus repens (L.) GOULD,
Solidago gigantea AITON, Symphytum officinale L. (in 5 habitats), Carex riparia CURTIS,
Epilobium hirsutum L., Equisetum arvense L., Erigeron annuus (L.) PERS., Galium mollugo L., Glechoma hederacea L., Lycopus europeus L., Potentilla reptans L., Taraxacum officinale
WEBER ex WIGGERS, and Urtica dioica L. (in 4 habitats). In both 2010 and 2011, the least precipitation was measured in Pécs. The highest precipitation could be observed in July 2011, the lowest ones were measured in July 2010 and August 2011. The monthly average temperature values were available only in Pécs, Kaposvár, and Szigetvár. The highest average temperature could be measured in Pécs in all examined months, while in Szigetvár and Kaposvár the values were very similar to each other within one month. Values of montly sunshine duration were available only from Pécs. The sum of these values was higher in 2011 than in 2010. Unfortunately, we were unable to collect enough meteorological data and to draw firm conclusions regarding the influence of weather conditions on the morphological and phytochemical features of L. salicaria. In our study, the selected populations of L. salicaria started to flower earlier in 2010 than in 2011, and the average height of the plants was larger in 2010 than in 2011 in all habitats, probably due to the different weather conditions, however we could not prove it with our observations. Our L. salicaria samples were found mostly in basic soils and wet habitats, and the nutrient supply was probably enough for the plants in each habitats. A partial shade of trees
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was found at Lakes Malomvölgy and Sikonda, Cserénfa, and Kaposvár, the other plants lived in full light. Considering these environmental circumstances, our results agree with literature. All of the habitats contained abundant amount of water, which is essential for the growth of L. salicaria. The pH values, nitrite, potassium, and ammonium ion contents of the soil samples were similar to each other at the different habitats, while we could measure prominent amounts of nitrate and sodium ions in soil samples collected from Cserénfa; and the most phosphate was measured in Csebény. We could not find unambiguous tendencies between the amounts of different ions in the soil samples and either the morphological parameters or the amount of the active compounds. Both the leaves and the bracts of L. salicaria are covered by trichomes, but trichomes of the bracts are longer than those of the leaves. The longer trichomes of the bracts may play an important role in the protection of the flowers. It can be observed in both the bracts and leaves that the epidermal cells are flattened, composed of a single cell layer; which is higher on the adaxial than the abaxial surface; the heterogenous mesophyll of the dorsiventral leaves and bracts are composed of palisade cells at the adaxial and isodiametric spongy cells at the abaxial surface; there are several intercellular spaces and special idioblasts with Ca(COO)2 crystals among the spongy cells, and these crystals were wider than high in the stems and the leaves, while they were rather isodiametric in the bracts. The central vascular bundle of the inflorescence axis branches at the base of each flower and runs along the whole length of the flowers. Sepals are composed of 4 or 6 cell layers with flattened epidermal cells and unicellular trichomes on the abaxial surface. Exclusively spongy cells can be observed at the base and the top of the sepals, whereas in the middle part, both spongy and palisade cells can be observed together with numerous intercellular spaces. In our samples, the petals were composed of only 1-2 cell layers. The filament of the stamen has a collateral closed bundle in central position. The anther is covered by the exothecium, and a fibrous layer with characteristic cell wall thickening. The ovary is divided by the septum bearing ovules on both sides (axile placentation). The vascular bundle of the septum is surrounded exclusively by spongy cells at the top and the base. The annular nectary is located in the valley between the receptacle and the ovary. In the structure of the ovary, trichomes and intercellular cavities could not be observed. The average thickness of the wall of the ovary is composed of 4-5 cell layers. The polyphenol and tannin contents showed difference among the studied plant organs and populations. The statistical analysis showed that the total polyphenol content of the populations was significantly higher (p = 0.012) in 2011 than in 2010, which might be caused by the weather conditions. Polyphenol and tannin contents were higher in the flowering tops
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than in the other organs (leaves or stems). According to the Ph. Hg. VIII., Lythri herba contains not less than 5.0% of tannins, expressed as pyrogallol, and calculated with reference to the dried drug. This criterion was met in the specimens of all selected populations. Generally, total flavonoid content was higher in the populations collected during the main blooming period in August than at the beginning of flowering in July. The highest flavonoid content was measured in the leaves, followed by the flowering branches and stems. The flavonoid compounds were investigated by TLC, as well. We found four zones at the plates as follows: vitexin (light green fluorescent zone, Rf ~ 0.72), orientin (yellow fluorescent zone, Rf ~ 0.62), isoorientin (yellow fluorescent zone, Rf ~ 0.5), and isovitexin (light green fluorescent zone, Rf ~ 0.45). All parts of the plants contained all of the identified flavonoid components but in different amounts. In agreement with the results of the total flavonoid measurements, our observation is that the greatest amount of flavonoids could be found in the leaves followed by flowering branches, and the smallest amounts of them are in the stems. There was no qualitative difference between the habitats. Rutin, hyperoside, chlorogenic acid, and caffeic acid could not be detected in our samples by TLC. UHPLC-MS measurements confirmed the presence of the above mentioned flavonoid aglycones (isoorientin, orientin, isovitexin, and vitexin). In the samples, ellagic acid could be detected in the largest amount followed by isoorientin and orientin. As mentioned above, rutin, hyperoside, chlorogenic acid, and caffeic acid could not be detected in the samples by TLC, but their presence could be proved by LC-MS. To the best of our knowledge, this is the first time, that catechin, hyperoside, rutin, luteolin, and apigenin has been detected in samples of L. salicaria, however in low amount. Based on our microbiological results, we can say that the antimicrobial effect of L. salicaria is dose-dependent. The greater doses were given (in the different methods) the more microbes were sensitive to the extract. All of the used bacteria and the fungus (Micrococcus luteus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, MDR Pseudomonas aeruginosa, Staphylococcus aureus, MRSA, Staphylococcus epidermidis, and Candida albicans) were sensitive to the 50% ethanol in water extract of L. salicaria; moreover, the drug resistant strains (MDR Pseudomonas aeruginosa and MRSA) were similarly sensitive to our extracts than the non-resistant ones. The most sensitive bacteria were the Staphylococcus strains. We prepared hexane, chloroform, ethyl acetate, and 50% ethanol in water extracts of L. salicaria, and all of them produced contractile responses on guinea-pig ileum preparations. The largest contractions were elicited by the 50% ethanol in water extract, and this effect was concentration-dependent. The spasmogenic effect partially faded away after some minutes
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without washing the tissues with fresh bathing fluid. The magnitude of the contractions induced by electrical stimulation was not significantly influenced by the presence of extracts. Several pre-treatments (atropine, atropine combined with tetrodotoxin, indomethacin, and PPADS combined with suramin) significantly reduced the contractile responses of the ileum preparations, but none of them was able to completely abolish the contractions showing that more mechanisms are involved in the mechanism of action. The effect of the extract was insensitive (showed no or small, non-significant difference) to pre-treatments with tetrodotoxin, capsaicin, chloropyramine, and α,β-meATP. Cholinergic receptor stimulants are used against gastrointestinal atony. The present results may suggest that a diluted extract of L. salicaria p.o. could be used as a mild stimulant of gastrointestinal motility. The polar fraction (50% ethanol in water extract) was found to be rich in polyphenols. Ellagic acid could be detected in the highest amount, followed by isoorientin and orientin. Hexane, chloroform and ethyl acetate extracts also showed spasmogenic effects while catechin, caffeic acid, hyperoside, and rutin could not be detected in these extracts. For this reason we do not think that these compounds would play a part in the mechanism of the spasmogenic effect. Further isolation, purification, and pharmacological analysis of the different components are necessary to identify which components are responsible for the antispasmodic and spasmogenic effects.
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Novel findings This study was the first which investigated populations of Lythrum salicaria in south- west Hungary. Our histological examinations showed that the leaf epidermal cells were higher on the adaxial than the abaxial surface in all samples, and the trichomes of the bracts were longer than the trichomes of the leaves. Based on the results of the phytochemical investigations, we can state that all selected specimens contained high amounts of tannins, and therefore the collection of all of them can be suggested for therapeutic purposes from this point of view. The flowering tops contained higher amounts of tannins than the leaves, while the leaves contained higher amounts of flavonoids than the flowers. TLC examination showed that all parts of the plant (flowers, leaves, stems) contain the same flavonoid compounds, but in different concentration. To the best of our knowledge, this is the first time, that catechin, hyperoside, rutin, luteolin, and apigenin has been detected in samples of L. salicaria, however in low amount. We applied first the tube dilution method for the investigation of the antimicrobial effects of L. salicaria. We confirmed that L. salicaria extracts are effective against S. aureus, moreover, methicillin-resistant S. aureus also showed sensitivity to L. salicaria extracts. Results of other authors are ambiguous in the case of Escherichia coli, Pseudomonas aeruginosa, and Candida albicans, but in our experiments they were also sensitive to Lythrum extracts. To the best of our knowledge, this is the first time, when antimicrobial effects of Lythrum extracts have been proven on MRSA, S. epidermidis, and a multidrug-resistent P. aeruginosa. In the pharmacological experiments, we found that the contractile response of the Lythrum extract was reduced strongly, but not completely, in the presence of the nonselective muscarinic antagonist atropine, indicating that muscarinic receptor activation could partially explain the contractile response. We suppose that cholinergic stimulation of the L. salicaria extract is a post-junctional phenomenon, i.e. takes place at the level of the smooth muscle.
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IX. Acknowledgements
This dissertation would not have been possible without the guidance and help of my supervisors, Dr. Györgyi Horváth and Dr. Nóra Papp. I am grateful to Prof. László Gy. Szabó for suggesting Lythrum salicaria L. as the topic of my PhD thesis and to Prof. Péter Molnár, previous head and to Prof. József Deli, present head of the Department of Pharmacognosy where my research could be completed. I am thankful to Prof. Loránd Barthó for helping and letting me carry out my pharmacological studies, to Veronika Szombati for her assistance in this field and to Dr. Rita Benkó for her advice in the literature of this field. Dr. Béla Kocsis and Erika Kocsis are thanked for their help in the microbiological experiments. I thank Dr. Róbert Pál for his help in the assessment of the habitats and vegetation. Dr. András Benkő and Dr. Pál Perjési are thanked for their help in the preliminary analytical measurements of my extracts. I am grateful to Viktor Sándor for performing the UHPLC-MS measurements as well as to Dr. Attila Felinger and Dr. Ferenc Kilár for enabling chemical analysis at the Department of Analytical and Environmental Chemistry. I am thankful to Dr. Attila Dévay for providing vacuum evaporator for preparation of the extracts and to Dr. Szilárd Pál, Dr. Tivadar Pernecker, and János Brunner for their technical assistance in this field. I acknowledge the help of Dr. Ágnes Farkas in teaching me the microscopic preparation techniques and evaluation of the histological samples. I thank Dr. Tamás Kőszegi for providing equipment for the preparation of histological microphotos and the evaluation of the TLC. Dr. Gábor Pauler, Dr. László Pótó, and Rita Filep are thanked for their advice and help in the statistical field. I would also like to thank Cathedral Library of Kalocsa for letting me search for L. salicaria in their old books. Attila Monostori is thanked for his informatics assistance and encouragement. I am grateful to Simex Ltd. for lending me equipment for the pH measurements. I must also thank for the technical contribution of Marika Fürdős in the phytochemical measurements. I am grateful to Dr. Gábor Rébék-Nagy and Dr. Zsófia Barcza for the language correction of some parts of my thesis. I am thankful to my dear mother and father, Ágnes and János Bencsik for collecting meteorological data in our home, Szentlászló, which is one of the selected habitats and I am indebted to my parents for their untiring effort to support me. I am grateful to my dear brother, Barnabás Bencsik, for his help in the collection of the plant and soil samples. I am also indebted to my partner, Dr. Miklós Poór, for his help, the valuable insights he has shared, his considerations, inspiration, patience and steadfast encouragement to complete my work.
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My research work was partially supported by TÁMOP / SROP-4.2.2/B-10/1-2010- 0029, ETT 03-372/2009 (Ministry of Welfare), grants No. OTKA T-81984, and NK-78059 of Hungarian Research Funds.
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XI. Appendix
XI. 1. Snapshots of the habitats Photos were prepared by Nóra Papp and Tímea Bencsik.
Fig. 28. Szentlőrinc
Fig. 29. Kacsóta
Fig. 30. Szigetvár
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Fig. 31. Szentlászló
Fig. 32. Almamellék
Fig. 33. Lake Almamellék
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Fig. 34. Csebény
Fig. 35. Kaposvár
Fig. 36. Lake Deseda
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Fig. 37. Cserénfa
Fig. 38. Lake Sikonda
Fig. 39. Lake Malomvölgy
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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) Szentlőrinc
ruderal plant community besides the road
46º02'43.3”; 17º59'26.2”; altitude:131 m Species % Festuca pratensis Huds. 40 Elymus repens (L.) Gould 15 Potentilla reptans L. 15 Ranunculus repens L. 15 Gallium mollugo L. 10 Pastinaca sativa L. 3 Achillea collina Becker ex Rchb. 2 Lathyrus tuberosus L. 2 Lythrum salicaria L. 2 Mentha longifolia (L.) Nath. 2 Taraxacum officinale Weber ex Wiggers 2 Convolvulus arvensis L. 1 Plantago lanceolata L. 1 Equisetum arvense L. 0,5 Glechoma hederacea L. 0,5 Rumex crispus L. 0,5 Sonchus oleraceus L. 0,5 Vicia cracca L. 0,5 Fallopia convolvulus (L.) A. Löve 0,1 Lotus corniculatus L. 0,1 Trifolium repens L. 0,1 Vicia angustifolia L. 0,1
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Kacsóta
mesic grassland
46º02'16.5”; 17º56'51.8”; altitude:121 m Species % Calamagrostis epigeios (L.) Roth 40 Carex acutiformis Ehrh. 15 Carex riparia Curtis 5 Solidago gigantea Aiton 5 Cirsium arvense (L.) Scop. 3 Sonchus arvensis L. 1 Epilobium hirsutum L. 0,5 Lythrum salicaria L. 0,5 Senecio erucifolius L. 0,5 Verbascum nigrum L. 0,5
Szigetvár
tall-sedge vegetation
46º03'15.6”; 17º48'39.9”; altitude:120 m Species % Carex riparia Curtis 70 Lythrum salicaria L. 20 Calystegia sepium (L.) R. Br. 5 Poa trivialis L. 5 Ranunculus repens L. 5 Symphytum officinale L. 2 Veronica beccabunga L. 1 Juncus articulatus L. 0,5 Stachys palustris L. 0,5
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Szentlászló
tall-sedge vegetation
46º08'32.6”; 17º49'47.5”; altitude:135 m Species % Carex riparia Curtis 60 Soligago gigantea Aiton 20 Epilobium hirsutum L. 10 Calystegia sepium (L.) R. Br. 5 Eupatorium cannabinum L. 5 Lycopus europaeus L. 5 Lysimachia vulgaris L. 5 Potentilla reptans L 5 Elymus repens (L.) Gould 2 Equisetum arvense L. 2 Vicia cracca L. 2 Galium mollugo L. 1 Lythrum salicaria L. 1 Ranunculus repens L. 1 Stellaria media (L.) Vill. 1 Atriplex patula L. 0,5 Glechoma hederacea L. 0,5 Myosoton aquaticum (L.) Moench 0,5 Sonchus arvensis L. 0,5 Lactuca serriola L. 0,1
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Almamellék tall-sedge vegetation
46º10'03.3”; 17º53'25.5”; altitude:147 m Species % Carex acutiformis Ehrh. 60 Rubus caesius L. 15 Solidago gigantea Aiton 15 Equisetum palustre L. 10 Equisetum arvense L. 5 Iris pseudacorus L. 5 Lythrum salicaria L. 5 Calystegia sepium (L.) R. Br. 3 Carex hirta L. 1 Erigeron annuus (L.) Pers. 1 Potentilla reptans L. 1 Sambucus ebulus L. 1 Acer negundo L. 0,5 Calamagrostis epigeios (L.) Roth 0,5 Geum urbanum L. 0,1
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Lake Almamellék stream sides
46º10'06.9”; 17º53'57.4”; altitude:141 m Species % Lycopus europeus L. 35 Solanum dulcamara L. 20 Urtica dioica L. 15 Alisma plantago-aquatica L. 5 Arrhenatherum elatius (L.) P. Beauv. Ex J. Presl et C. Presl 5 Elymus repens (L.) Gould 5 Calystegia sepium (L.) R. Br. 3 Lythrum salicaria L. 3 Symphytum officinale L. 3 Bidens tripartita L. 1 Erigeron annuus (L.) Pers. 1 Galium mollugo L. 1 Glechoma hederacea L. 1 Pastinaca sativa L. 1 Verbascum nigrum L. 1 Chenopodium polispermum L. 0,5 Scrophularia umbrosa Dumort. 0,5 Myosoton aquaticum (L.) Moench 0,1
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Csebény tall-sedge vegetation
46º11'01.5”; 17º55'15.1”; altitude:149 m Species % Carex acutiformis Ehrh. 60 Calamagrostis epigeios (L.) Roth 20 Soligago gigantea Aiton 15 Galega officinalis L. 10 Eupatorium cannabinum L. 5 Lythrum salicaria L. 5 Calystegia sepium (L.) R. Br. 2 Linaria vulgaris Mill. 2 Symphytum officinale L. 1 Hypericum perforatum L. 0,5 Scrophularia umbrosa Dumort. 0,1
Kaposvár ripparian vegetation
46º20'54.9”; 17º46'46.0”; altitude:140 m Species % Phalaris arundinacea L. 60 Equisetum palustre L. 20 Epilobium hirsutum L. 5 Lythrum salicaria L. 3 Lactuca serriola L. 2 Ranunculus repens L. 1 Taraxacum officinale Weber ex Wiggers 1 Urtica dioica L. 1 Calystegia sepium (L.) R. Br. 0,5 Galium aparine L. 0,5 Setaria pumila (Poir.) Schult. 0,5
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Lake Deseda reedbed vegetation
46º26'26.1”; 17º47'25.4”; altitude:139 m Species % Carex acutiformis Ehrh. 25 Epilobium hirsutum L. 25 Lythrum salicaria L. 10 Urtica dioica L. 5 Agrostis stolonifera L. 2 Glechoma hederacea L. 2 Poa compressa L. 2 Calystegia sepium (L.) R. Br. 1 Humulus lupulus L. 1 Solanum dulcamara L. 1 Solidago gigantea Aiton 1 Typha latifolia L. 1 Echinochloa crus-galli (L.) P. Beauv. 0,5
Lake Malomvölgy ruderal vegetation
46º01'06.8”; 18º12'01.8”; altitude:142 m Species % Cynodon dactylon (L.) Pers. 20 Elymus repens (L.) Gould 20 Carex riparia Curtis 10 Salix alba L. 10 Convolvulus arvensis L. 5 Humulus lupulus L. 5 Lycopus europeus L. 5 Rubus caesius L. 5 Plantago lanceolata L. 3 Achillea collina Becker ex Rchb. 2 Lythrum salicaria L. 1 Mentha arvensis L. 1 Symphytum officinale L. 1 Erigeron annuus (L.) Pers. 0,5 Reseda lutea L. 0,1
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Cserénfa ruderal vegetation
46º18'07.2”; 17º52'54.4”; altitude:146 m Species % Arrhenatherum elatius (L.) P. Beauv. Ex J. Presl et C. Presl 25 Elymus repens (L.) Gould 15 Galium mollugo L. 10 Lythrum salicaria L. 10 Carex acutiformis Ehrh. 5 Dactylis glomerata L. 5 Poa pratensis L. 5 Rubus caesius L. 5 Potentilla reptans L. 3 Torilis japonica (Houtt.) DC. 3 Calystegia sepium (L.) R. Br. 2 Carex hirta L. 2 Cruciata laevipes Opiz 2 Euonymus europaeus L. 2 Agrostis stolonifera L. 1 Daucus carota L. 1 Equisetum arvense L. 1 Quercus cerris L. 1 Ranunculus repens L. 1 Rumex acetosa L. 1 Urtica dioica L. 1 Veronica chamaedrys L. 1 Lathyrus pratensis L. 0,5 Medicago lupulina L. 0,5 Pastinaca sativa L. 0,5 Taraxacum officinale Weber ex Wiggers 0,5 Picris hieracioides L. 0,1 Silene alba (Mill.) E. H. L. Krause 0,1
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Lake Sikonda lakeshore vegetation
46º10'53.3”; 18º12'59.6”; altitude:185 m Species % Carex hirta L. 25 Cynodon dactylon (L.) Pers. 5 Ranunculus repens L. 5 Trifolium pratense L. 5 Tussilago farfara L. 5 Persicaria maculosa Gray 3 Lycopus europeus L. 2 Lythrum salicaria L. 2 Mentha arvensis L. 2 Erigeron annuus (L.) Pers. 1 Prunella vulgaris L. 1 Symphytum officinale L. 1 Verbena officinalis L. 1 Bidens tripartita L. 0,5 Daucus carota L. 0,5 Mentha aquatica L. 0,5 Plantago major L. 0,5 Rumex sp. 0,5 Taraxacum officinale Weber ex Wiggers 0,5 Achillea asplenifolia Vent. 0,2 Inula britannica L. 0,2 Agrimonia eupatoria L. 0,1 Conyza canadensis (L.) Cronquist 0,1 Picris hieracioides L. 0,1 Quercus cerris L. 0,1 Torilis japonica (Houtt.) DC. 0,1
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XI. 3. Results of the investigation of soil samples
"free" NO - SO 2- PO 3- K+ NH + NO - 3 NO - SO 2- 4 PO 3- 4 K+ NH + 4 Date of water Mean 3 (mg/ 2 4 (mg/ 4 (mg/ (mg/ 4 (mg/ Habitat (mg/ (mg/ (mg/ (mg/ (mg/ (mg/ collection content pH 100 100 100 100 100 mL) mL) mL) mL) mL) mL) (m/m%) g) g) g) g) g) Lake 2011 July 56,07 7,72 30,0 7,5 0 <200 <50 5 1,25 100 25,00 0 0,00 Almamellék Lake 2011 August 36,87 8,09 10,0 2,5 0 <200 <50 5 1,25 100 25,00 0 0,00 Almamellék Average 46,47 7,91 20,0 5,00 0 <200 <50 5 1,25 100 25,00 0 0,00
Almamellék 2011 July 33,42 7,82 75,0 18,75 0 <200 <50 5 1,25 200 50,00 0 0,00
Almamellék 2011 August 29,70 7,82 30,0 7,5 0 <200 <50 3 0,75 200 50,00 0 0,00
Almamellék 2010 July 49,20 7,58 10,0 2,5 0 <200 <50 5 1,25 200 50,00 3 0,75
Average 39,45 7,74 38,3 9,58 0 <200 <50 4 1,08 200 50,00 1 0,25
Csebény 2011 July 9,96 7,77 5,0 1,25 0 <200 <50 10 2,50 100 25,00 0 0,00 Csebény 2011 August 57,34 7,84 30,0 7,5 0 <200 <50 5 1,25 200 50,00 0 0,00 Csebény 2010 July 32,10 7,64 5,0 1,25 0 <200 <50 9 2,25 200 50,00 2 0,50 Average 44,72 7,75 13,3 3,33 0 <200 <50 8 2,00 167 41,67 1 0,17
Cserénfa 2011 July 27,96 6,48 200,0 50 0 <200 <50 3 0,75 200 50,00 3 0,75 Cserénfa 2011 August 26,52 6,86 170,0 42,5 0 250 62,5 5 1,25 100 25,00 0 0,00 Cserénfa 2010 July 32,80 7,53 5,0 1,25 0 <200 <50 3 0,75 200 50,00 2 0,50 Average 29,66 6,95 125,0 31,25 0 225,0 56,25 4 0,92 167 41,67 2 0,42
Lake 2011 August 23,77 8,10 30,0 7,5 0 <200 <50 5 1,25 100 25,00 0 0,00 Deseda Lake 2011 July 37,76 7,90 20,0 5 0 <200 <50 5 1,25 100 25,00 0 0,00 Deseda Lake 2010 July 17,80 7,43 5,0 1,25 0 <200 <50 5 1,25 200 50,00 2 0,50 Deseda Average 27,78 7,81 18,3 4,58 0 <200 <50 5 1,25 133 33,33 1 0,17
Kacsóta 2010 July 28,40 7,61 20,0 5 0 <200 <50 7 1,75 200 50,00 3 0,75
Kaposvár 2011 July 25,57 8,09 15,0 3,75 0 <200 <50 5 1,25 100 25,00 0 0,00 Kaposvár 2011 August 26,47 8,07 20,0 5 0 <200 <50 3 0,75 100 25,00 0 0,00 Kaposvár 2010 July 24,70 7,79 5,0 1,25 0 <200 <50 3 0,75 200 50,00 2 0,50 Average 25,59 7,98 13,3 3,33 0 <200 <50 4 0,92 133 33,33 1 0,17
(continued overleaf)
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"free" - - - 2- 2- 3- 3- + + + + Date of NO3 NO3 NO2 SO4 SO4 PO4 PO4 K K NH4 NH4 water Mean Habitat collec- (mg/ (mg/ (mg/ (mg/ (mg/ (mg/ (mg/ (mg/ (mg/ (mg/ (mg/ content pH tion mL) 100 g) mL) mL) 100 g) mL) 100 g) mL) 100 g) mL) 100 g) (m/m%) Lake 2011 3,57* 7,88 150,0 37,5 0 <200 <50 5 1,25 100 25,00 0 0,00 Malomvölgy July Lake 2011 4,67* 8,03 10,0 2,5 0 <200 <50 3 0,75 100 25,00 0 0,00 Malomvölgy August Lake 2010 4,30* 8,00 5,0 1,25 0 <200 <50 3 0,75 200 50,00 0 0,00 Malomvölgy July Average 4,49* 7,97 55,0 13,75 0 <200 <50 4 0,92 133 33,33 0 0,00
Lake 2011 2,35* 7,66 5,0 1,25 0 <200 <50 5 1,25 100 25,00 0 0,00 Sikonda July Lake 2011 0,83* 7,60 15,0 3,75 1 <200 <50 3 0,75 100 25,00 0 0,00 Sikonda August Lake 2010 4,10* 8,10 5,0 1,25 0 <200 <50 7 1,75 100 25,00 0 0,00 Sikonda July Average 2,47* 7,79 8,3 2,08 0 <200 <50 5 1,25 100 25,00 0 0,00
2011 Szentlászló 56,07 7,30 10,0 2,5 0 <200 <50 5 1,25 200 50,00 0 0,00 July 2011 Szentlászló 49,74 7,00 10,0 2,5 0 <200 <50 3 0,75 100 25,00 3 0,75 August 2010 Szentlászló 82,20 7,45 10,0 2,5 0 <200 <50 7 1,75 200 50,00 10 2,50 July Average 65,97 7,25 10,0 2,50 0 <200 <50 5 1,25 167 41,67 4 1,08
2011 Szentlőrinc 18,11 8,09 30,0 7,5 1 <200 <50 5 1,25 100 25,00 0 0,00 August 2011 Szentlőrinc 19,28 7,41 50,0 12,5 1 <200 <50 3 0,75 100 25,00 0 0,00 July 2010 Szentlőrinc 16,10 7,86 5,0 1,25 0 <200 <50 5 1,25 200 50,00 2 0,50 July Average 17,69 7,79 28,3 7,08 0 <200 <50 4 1,08 133 33,33 1 0,17
2011 Szigetvár 20,11 8,28 15,0 3,75 0 <200 <50 5 1,25 100 25,00 0 0,00 July 2011 Szigetvár 21,72 8,17 30,0 7,5 0 <200 <50 3 0,75 100 25,00 0 0,00 August 2010 Szigetvár 40,90 7,63 20,0 5 0 <200 <50 0 0,00 100 25,00 3 0,75 July Average 31,31 8,03 21,7 5,42 0 <200 <50 3 0,67 100 25,00 1 0,25 * The plants grew in the bottom of the lake and soil samples could not be collected directly around the plant; therefore they were obtained from the shore, and for this reason, the free water content is low in these samples. At some places we could not collect samples at all; therefore some data are missing.
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XI. 4. Chemical structure of the standard compounds detected in Lythrum extracts by LC-MS method O OH
HO OH OH
Fig. 40. Gallic acid
O HO O
HO OH
O OH O
Fig. 41. Ellagic acid
OH
HO O OH
OH OH
Fig. 42. Catechin
HO
O HO OH
Fig. 43. Caffeic acid
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HO COOH O
HO O OH OH OH
Fig. 44. Chlorogenic acid
OH OH
HO O OH O O OH O HO OH OH Fig. 45. Hyperoside
OH OH
HO O OH O OH OH O O OH OH HO O
O HO
CH3 Fig. 46. Rutin
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OH
HO O OH
OH O Fig. 47. Luteolin
OH
HO O
OH O Fig. 48. Apigenin
OH HO OH OH O OH HO HO O
OH O Fig. 49. Orientin
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OH OH
HO O OH O
OH O HO OH
OH Fig. 50. Isoorientin
OH HO OH O OH HO HO O
OH O Fig. 51. Vitexin
OH
HO O OH O
OH O HO OH
OH Fig. 52. Isovitexin
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XI. 5. Total ion chromatograms
Fig. 53. 50% ethanol in water extract of flowering branches used in microbiological experiments
Fig. 54. Hexane extract of flowering branches used in pharmacological experiments
Fig. 55. Chloroform extract of flowering branches used in pharmacological experiments
Fig. 56. Ethyl-acetate extract of flowering branches used in pharmacological experiments
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Fig. 57. 50% ethanol in water extract of flowering branches used in pharmacological experiments
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XI. 6. Tube dilution method
control 5.00 mg/mL 2.50 mg/mL
1.25 mg/mL 0.63 mg/mL 0.31 mg/mL
0.16 mg/mL 0.08 mg/mL 0.04 mg/mL
0.02 mg/mL 0.01 mg/ml
Fig. 58. Tube dilution method on MRSA. MBC means the lowest dilution showing total inhibition of bacterial growth, MIC means the lowest dilution that could inhibit the visible growth of bacteria.
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XII. Publications, posters and oral presentations
This doctoral thesis is based on the following publications and presentations: Original articles I. T. Bencsik, L. Barthó, V. Sándor, N. Papp, R. Benkó, A. Felinger, F. Kilár, Gy. Horváth. (2013): Phytochemical evaluation of Lythrum salicaria L. extracts and their effects on guinea pig ileum. Natural Product Communications 8: 1247-1250. (IF: 0.956) II. T. Bencsik, Gy. Horváth, N. Papp. (2012): A réti füzény (Lythrum salicaria L.) biológiai jellemzői és gyógyászati értéke. Botanikai Közlemények 99: 195– 210. III. T. Bencsik, Gy. Horváth, N. Papp. (2011): Variability of total flavonoid, polyphenol and tannin contents in some Lythrum salicaria L. populations. Natural Product Communications 6: 1417-1420. (IF: 1,242) Posters I. T. Bencsik, V. Sándor, L. Barthó, P. Molnár, F. Kilár, A. Felinger, Gy. Horváth. Lythrum salicaria L. kivonatok in vitro farmakológiai és fitoanalitikai vizsgálata. MET Elválasztástudományi Vándorgyűlés, Hungary, Hajdúszoboszló, 2012. November 7-9., Abstract: P-3, p. 72. II. T. Bencsik, N. Papp. Lythrum salicaria L. populációk polifenol- és cserzőanyag-tartalmának értékelése. XII. Magyar Gyógynövény Konferencia. Hungary, Szeged, 2011. May 5-7. Abstract: Gyógyszerészet Supplementum LV. S24. III. T. Bencsik, N. Papp. Variability of flavonoid content in some Lythrum salicaria L. populations. CIPAM 2011. Italy, Cagliari, 2011. April 13-15. Abstract: T1P8, p. 142. IV. T. Bencsik, Á. Farkas, N. Papp. A Lythrum salicaria L. hisztológiai értékelése. Greguss Pál Növényanatómiai Szimpózium. Hungary, Szeged, 2010. October 21. Oral presentations I. T. Bencsik, L. Barthó, B. Kocsis, Gy. Horváth. Lythrum salicaria kivonatok farmakológiai és mikrobiológiai jellemzése. Magyar Gyógyszerésztudományi Társaság Gyógynövény Szakosztályának Előadóülése. Hungary, Lajosmizse, 2012. June 8.
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II. T. Bencsik. Lythrum salicaria L. populációk fitokémiai és mikrobiológiai vizsgálata. X. Clauder Ottó Emlékverseny. Hungary, Budapest, 2011. October 13-14. Abstract: p. 19. III. T. Bencsik, Á. Farkas, N. Papp. Adatok a réti füzény (Lythrum salicaria L.) szövettanához. Magyar Biológiai Társaság Pécsi Csoportjának 236. szakülése. Hungary, Pécs, 2011. March 10.
Participation in the development of a herbal product AcneSolve® – containing extract of Lythrum salicaria (2012)
List of publications not related to the topic of this thesis Original articles I. N. Papp, K. Birkás-Frendl, T. Bencsik, Sz. Stranczinger, D. Czégényi (2014): Survey of traditional beliefs in the Hungarian Csángó and Székely ethnomedicine in Transylvania, Romania. Revista Brasileira de Farmácia. In press (IF: 0.676) II. P. Felső, Gy. Horváth, T. Bencsik, R. Godányi, É. Lemberkovics, A. Böszörményi, K. Böddi, A. Takátsy, P. Molnár, B. Kocsis (2013): Detection of antibacterial effect of essential oils on outer membrane proteins of Pseudomonas aeruginosa by Lab-on-a-chip and MALDI-TOF/MS. Flavour and Fragrance Journal 28: 367-372. (IF: 1.824) III. A. Végh, T. Bencsik, P. Molnár, A. Böszörményi, É. Lemberkovics, K. Kovács, B. Kocsis, Gy. Horváth (2012): Composition and antipseudomonal effect of essential oils isolated from different Lavender species. Natural Product Communication. 7(10): 1393-1396. (IF: 0.956) IV. M. Poór, S. Kunsági-Máté, T. Bencsik, J. Petrik, S. Vladimir-Knežević, T. Kőszegi (2012): Flavonoid aglycones can compete with ochratoxin A for human serum albumin: a new possible mode of action. International Journal of Biological Macromolecules, 51(3): 279-83. (IF: 2.502) V. N. Papp, T. Bencsik, K. Németh, K. Gyergyák, A. Sulc, Á. Farkas (2011): Histological study of some Echium vulgare L., Pulmonaria officinalis L. and Symphytum officinale L. populations. Natural Product Communications, 6 (10) 1475-1478. (IF: 1,242)
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Posters I. P. Felső, K. Böddi, Gy. Horváth, T. Bencsik, R. Godányi, É. Lemberkovics, A. Böszörményi, A. Takátsy, F. Kilár, P. Molnár, B. Kocsis. Néhány illóolaj bakteriális külső membránfehérjékre gyakorolt hatásának vizsgálata. MET Elválasztástudományi Vándorgyűlés, Hungary, Hajdúszoboszló, 2012. November 7-9., Abstract: P-11, p. 80. II. H. Békésiné Kallenberger, T. Bencsik, Á. Farkas, L. Balogh, N. Papp. A Fallopia sachalinensis és F. × bohemica fajok összehasonlító szövettani vizsgálata (Comparative histological study of Fallopia sachalinensis és F. × bohemica). XIVth Hungarian Plant Anatomy Symposium. Hungary, Pécs, 2012. September 28. Abstract: P3. pp. 45-46. III. Á. Farkas, R. Filep, T. Bencsik, E. Scheidné Nagy Tóth. Összehasonlító szövettani vizsgálatok Cotoneaster taxonok nektáriumstruktúrájára vonatkozóan (Comparative histological studies on the nectary structure of Cotoneaster taxa). XIVth Hungarian Plant Anatomy Symposium. Hungary, Pécs, 2012. September 28. Abstract: P12. pp. 63-64. IV. Gy. Horváth, T. Bencsik, R. Godányi, P. Felső, É. Lemberkovics, A. Böszörményi, A. Takátsy, P. Molnár, B. Kocsis. Lab-on-a-chip: a technique for detection of antibacterial effect of essential oils on outer membrane proteins. 43rd International Symposium on Essential Oils (ISEO2012). Lisbon, Portugal, 2012. September 5-8. Abstract: P.207. p. 251. V. A. Sulc, T. Bencsik, N. Papp. Symphytum officinale L. populációk összehasonlító szövettani jellemzése. XII. Magyar Gyógynövény Konferencia. Hungary, Szeged, 2011. May 5-7. Abstract: Gyógyszerészet Supplementum LV. S32. VI. N. Papp, Gy. Horváth, T. Bencsik, E. Turcsi, J. Deli, P. Molnár. Euphorbia taxonok karotinoid analízise. XII. Magyar Gyógynövény Konferencia. Hungary, Szeged, 2011. May 5-7. Abstract: Gyógyszerészet Supplementum LV. S31. VII. N. Papp, A. Sulc, T. Bencsik. Histological variability of Symphytum officinale L. populations. CIPAM 2011. Italy, Cagliari, 2011. April 13-15. Abstract: T1P34, p. 168. VIII. F. Budán, I. Szabó, K. Birkás-Frendl, Gy. Boris, T. Bencsik, N. Papp. A népgyógyászatban tumor kezelésére alkalmazott élelmiszer- és fűszernövények kemopreventív hatásainak irodalmi háttere. Fiatal Higénikusok Fóruma.
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Hungary, Debrecen, 2010. May 27-29. Abstract: Egészségtudomány LIV., p. 99. IX. N. Papp, K. Birkás-Frendl, Gy. Boris, V. Vántus, K. Csepregi, T. Bencsik, É. Vojkovics, D. Vincz, V. Bóna, I. Farkas, S. Bartha, L. Gajdos, I. Fancsali, T: Grynaeus, K. Csedő. Etnobotanikai kutatások a Pécsi Tudományegyetemen. XX. Tudományos Ülésszak Romanaia, Kézdivásárhely, 2010. April 22-24. Abstract: Orvostudományi Értesítő 83: 41. X. N. Papp, K. Birkás-Frendl, Gy. Boris, É. Vojkovics, T. Bencsik, T. Grynaeus. Gyógynövények a népi bőrgyógyászatban. Congressus Pharmaceuticus Hungaricus XIV., Hungary, Budapest, 2009. November 13-15. Abstract: Gyógyszerészet Supplementum I. 2009/11. p. 110. Oral presentations (the presenting author is underlined) I. Gy. Horváth, Á.M. Móricz, O. Péter, E. Kira, T. Bencsik, A. Böszörményi, É. Lemberkovics, B. Kocsis. Application of TLC-bioautography for detecting the antibacterial activity of essential oils. 44th ISEO, Budapest, Hungary. 2013. September 8-12. Abstract: O-11. II. F. Budán, T. Bencsik, Gy. Boris, K. Birkás-Frendl, I. Fancsali, Á. Farkas, Z. Gyöngyi, N. Papp. Synergies of anticarcinogenic and chemopreventive effects of edible herbs from populer medicine survey to secondary transmitter molecules (Kemopreventív élelmiszernövények hatásainak szinergiái - népgyógyászati felméréstől a másodlagos jelátvivő molekulákig). International Conference of Preventive Medicine and Public Health. Hungary, Pécs, 19-20 November 2010. Abstract: Egészségtudomány, LIV.(3):106. (2010) III. N. Papp, T. Bencsik, R. Molnár, R. Filep, Gy. Horváth, Á. Farkas. Gyógynövények hisztológiai jellemzői – Kutatásaink a pécsi Farmakognóziai Tanszéken. Greguss Pál Növényanatómiai Szimpózium. Hungary, Szeged, 2010. October 21. IV. Gy. Horváth, Á. Farkas, N. Papp, T. Bencsik, R. Molnár, P. Molnár, L. Gy. Szabó. Gyógynövénykutatás a Mecseken és környékén: múlt, jelen, jövő. MGYT Gyógynövény Szakosztályának előadóülése. Hungary, Lajosmizse, 2010. September 17. V. N. Papp, Á. Farkas, Gy. Horváth, T. Bencsik, R. Molnár, L. Gy . Szabó, P. Molnár. Bemutatkozik a Melius Gyógynövénykert. Magyar Biológiai Társaság Pécsi Csoportjának 231. szakülése. Hungary, Pécs, 2010. September 18.
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VI. T. Bencsik, N. Papp. A VIII. Magyar Gyógyszerkönyv néhány új gyógynövénye. Magyar Biológiai Társaság Pécsi Csoportjának 220. szakülése. Hungary, Pécs, 2009. March 18. VII. T. Bencsik. A VIII. Magyar Gyógyszerkönyv új gyógynövényei és gyógyászati felhasználásuk. PTE ÁOK Házi Tudományos Diákköri Konferencia. Hungary, Pécs, 2009. February 19-21. Abstract: p. 60.
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