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Comparative Histological, Phytochemical, Microbiological, and
Pharmacological Characterization of Some Lythrum salicaria L.
Populations
Ph.D. dissertation
Tímea Bencsik, Pharm.D.
Supervisors:
Györgyi Horváth PhD
Nóra Papp PhD
Program leader:
József Deli PhD, DSc
Head of the Doctoral School:
Erika Pintér MD, PhD, DSc
Department of Pharmacognosy,
Medical School
University of Pécs, Hungary
Pécs, 2014
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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
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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,
9
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.
10
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
extracts effectively inhibited Ca2+
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
11
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).
12
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).
13
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)
SSSOOO KKKCCC
SSSIII
SSSLLL AAARRR
CCCBBB LLLSSS
LLLMMM
CCCRRR
KKKPPP
LLLDDD
LLLAAA
14
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).
15
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.
16
Table 2. Features of the microclimates according to Dövényi, 2010.
Wat
er P
rop
erti
es
of
calc
ium
and
mag
nes
ium
carb
on
ate
char
acte
r,
har
dn
ess:
15
-25
nk
°; i
n t
he
Kap
os
Val
ley
, th
e n
itra
te
con
ten
t: 6
0 m
g/L
of
calc
ium
and
mag
nes
ium
carb
on
ate
char
acte
r,
har
dn
ess:
15
-25
nk
°; t
he
sulf
ate
con
ten
t: <
60 m
g/L
alk
alin
e-h
ydro
gen
carb
on
ate
and
ch
lori
de
ther
mal
wat
er
of
calc
ium
and
mag
nes
ium
carb
on
ate
char
acte
r,
har
dn
ess:
25
-35
nk
°, s
ulf
ate
con
ten
t: <
60 m
g/L
of
calc
ium
and
mag
nes
ium
hy
dro
gen
carb
onat
e
char
acte
r, h
ard
nes
s: i
n
Szi
get
vár
<2
5 n
k°,
else
wh
ere
25
-35
nk
°;
sulf
ate
con
ten
t: ~
60
mg
/L
Ari
dit
y
ind
ex
1.0
0
0.9
6-1
.00
0.9
0-0
.94
1.0
0-1
.10
0.9
8-1
.00
An
nu
al
pre
cip
itat
ion
(mm
) /
Pre
cip
itat
ion
in
the
veg
etat
ion
per
iod
(m
m)
68
0-7
00
/
39
0-4
10
68
0-7
30
/
39
0-4
20
75
0 /
40
0-4
50
64
0-6
70
/
37
0-4
00
60
0-7
20
/
39
0-4
30
Mea
n a
nn
ual
tem
per
atu
re
(°C
) /
Mea
n
tem
per
atu
re o
f
the
gro
win
g
seas
on
(°C
)
10
.0-1
0.3
(su
mm
er:
16
.6-1
7.0
)
10
.0 (
sum
mer
:
16
.5)
9.0
-9.5
(su
mm
er:
15
.8-1
6.5
)
10
.6-1
0.8
(su
mm
er:
17
.0)
10
.0 (
sum
mer
:
16
.5)
Su
nsh
ine
du
rati
on
(ho
urs
/
yea
r)
20
00-2
02
0
(su
mm
er:
81
0)
20
00-2
02
0
(su
mm
er:
81
0)
20
30-2
05
0
(su
mm
er:
81
0-8
20
)
20
60
(su
mm
er:
82
0)
20
00-2
02
0
(su
mm
er:
81
0)
Cli
mat
e
mo
der
atel
y
war
m,
mo
der
atel
y
wet
mo
der
atel
y
war
m,
mo
der
atel
y
wet
mo
der
atel
y
war
m,
mo
der
atel
y
wet
mo
der
atel
y
war
m,
mo
der
atel
y
wet
mo
der
atel
y
war
m,
mo
der
atel
y
wet
Nam
e o
f th
e
hab
itat
Lak
e D
esed
a
(LD
)
Kap
osv
ár (
KP
)
Cse
rén
fa (
CR
)
Sik
on
da
(LS
)
Lak
e M
alo
m-
vö
lgy
(L
M)
Sze
ntl
őri
nc
(SO
)
Kac
sóta
(K
C)
Szi
get
vár
(S
I)
Sze
ntl
ászl
ó
(SL
) A
lmam
ellé
k
(AR
)
Lak
e A
lma-
mel
lék
(L
A)
Cse
bén
y (
CB
)
Mic
rore
gio
n
Dél
-Kü
lső
-
So
mo
gy
Ész
ak-Z
seli
c
Bar
any
ai-
Heg
yh
át
Dél
-
Bar
any
ai-
Do
mb
ság
Dél
-Zse
lic
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
17
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).
18
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).
19
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.
20
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).
Habitat "free" water content
(m/m%)
Mean
pH
NO3- (mg/
100 g)
NO2- (mg/
100 g)
SO42- (mg/
100 g)
PO43- (mg/
100 g)
K+ (mg/
100 g)
NH4+ (mg/
100 g)
Lake
Almamellék 46.47 7.91 5.00 0 <50 1.25 25.00 0.00
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
Malomvölgy 4.49* 7.97 13.75 0 <50 0.92 33.33 0.00
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
21
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.
22
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
23
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
24
(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).
25
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).
26
Fig. 6. Stem and leaves of L. salicaria
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).
epidermal cells
xylem
phloem
primary cortex
27
Table 5. Mean ±SD of the measured histological parameters of the stem.
Stem Thick stem Thin 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).
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
epidermal cells
epidermal cells
vascular bundle
trichome
Ca(COO)2 crystal
mesomorphic stoma
spongy cells
palisade cells
28
(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.
29
Table 6. Mean ±SD of the measured histological parameters of the leaf of L. salicaria. (continued overleaf)
Kap
osv
ár
(Mea
n ±
SD
)
±10
.89
±13
.25
±3.5
2
±10
.78
±7.6
5
±3.2
1
±7.7
7
±8.5
5
±6.4
4
±4.7
4
±0.5
6
±69
.03
±23
.73
21
.49
37
.58
12
.20
22
.50
70
.65
14
.58
19
.10
21
.22
24
.96
26
.16
1.7
6
21
1.1
9
18
1.9
1
Kac
sóta
(Mea
n ±
SD
)
±6.4
1
±14
.73
±4.6
7
±7.9
8
±10
.64
±2.6
9
±6.2
1
±8.3
2
±8.2
3
±7.5
5
- -
±25
.32
19
.03
34
.99
11
.94
17
.55
63
.61
13
.10
19
.10
20
.87
29
.92
30
.66
no
t
fou
nd
no
t
fou
nd
19
5.1
0
Lak
e D
esed
a
(Mea
n ±
SD
)
±9.3
2
±21
.90
±3.7
7
±8.5
1
±8.3
8
±5.0
5
±8.3
2
±8.9
2
±10
.25
±8.5
8
±0.5
3
±59
.14
±17
.68
25
.94
46
.97
12
.61
21
.20
57
.71
15
.43
20
.90
23
.55
32
.15
35
.75
1.3
6
13
5.9
1
16
6.6
0
Cse
rén
fa
(Mea
n ±
SD
)
±8.1
1
±18
.75
±3.1
3
±5.7
6
±8.6
7
±2.8
7
±6.1
8
±7.5
7
±5.7
9
±5.0
2
±0.3
8
±28
.48
±26
.83
20
.36
36
.50
11
.59
16
.96
48
.88
13
.70
16
.66
18
.31
29
.59
30
.12
1.0
7
80
.97
13
6.9
4
Cse
bén
y
Mea
n ±
SD
)
±5.5
5
±7.4
7
±3.5
3
±7.1
6
±7.1
1
±4.0
5
±7.6
1
±8.3
0
±5.8
4
±7.0
9
±0.5
2
±74
.98
±31
.68
23
.70
37
.82
13
.51
19
.18
57
.93
15
.97
20
.79
24
.30
30
.16
30
.07
1.4
8
17
7.6
3
18
3.0
8
Lak
e
Alm
amel
lék
(Mea
n ±
SD
)
±10
.23
±23
.73
±4.8
6
±10
.74
±9.3
7
±3.5
5
±7.0
1
±7.6
2
±5.4
0
±6.5
4
±0.5
1
±53
.50
±20
.69
19
.95
40
.08
14
.05
21
.07
56
.39
14
.93
18
.05
19
.37
25
.38
28
.07
1.2
5
15
8.6
2
15
7.1
4
Alm
amel
lék
(Mea
n ±
SD
)
±7.4
5
±16
.53
±5.3
3
±9.9
7
±6.8
7
±2.3
5
±7.7
2
±5.4
6
±8.3
9
±7.1
2
±0.5
1
±81
.22
±26
.68
22
.98
38
.19
13
.99
17
.73
54
.50
15
.65
20
.46
20
.47
34
.21
35
.26
1.6
0
16
7.3
9
16
0.1
9
Lea
f
Hei
gh
t o
f th
e ep
ider
mal
cel
ls a
t
the
adax
ial
surf
ace
of
the
leaf
(μm
):
Wid
th o
f th
e ep
ider
mal
cel
ls a
t
the
adax
ial
surf
ace
of
the
leaf
(μm
):
Hei
gh
t o
f th
e ep
ider
mal
cel
ls a
t
the
abax
ial
surf
ace
of
the
leaf
(μm
):
Wid
th o
f th
e ep
ider
mal
cel
ls a
t
the
abax
ial
surf
ace
of
the
leaf
(μm
):
Hei
gh
t o
f th
e p
alis
ade
cell
s
(μm
):
Wid
th o
f th
e p
alis
ade
cell
s
(μm
):
Hei
gh
t o
f th
e sp
on
gy
cel
ls (
μm
):
Wid
th o
f th
e sp
on
gy
cel
ls (
μm
):
Hei
gh
t o
f th
e C
a(C
OO
) 2 c
ryst
als
(μm
):
Wid
th o
f th
e C
a(C
OO
) 2 c
ryst
als
(μm
):
Nu
mb
er o
f th
e ce
lls
wit
hin
th
e
tric
ho
mes
:
Hei
gh
t o
f th
e tr
ich
om
es (
μm
):
Av
erag
e th
ickn
ess
of
the
leaf
(μm
):
30
Table 6. (Continued) Mean ±SD of the measured histological parameters of the leaf. (continued overleaf)
Szi
get
vár
(Mea
n ±
SD
)
±5.5
2
±13
.48
±4.7
7
±9.4
0
±7.6
8
±2.8
2
±5.0
0
±5.4
4
±6.4
1
±6.3
0
±0.3
5
±46
.01
±10
.63
24
.20
34
.03
15
.14
20
.83
49
.94
14
.06
15
.94
16
.02
22
.17
24
.04
1.4
6
16
3.7
1
17
1.3
6
Sze
ntl
őri
nc
(Mea
n ±
SD
)
±6.4
8
±15
.79
±5.3
8
±7.4
4
±8.2
1
±3.6
0
±6.9
9
±7.3
0
±4.7
7
±4.9
4
±0.3
5
±62
.64
±32
.25
22
.33
41
.96
12
.28
19
.66
46
.53
16
.59
19
.10
22
.21
29
.18
33
.69
1.3
0
18
2.9
8
13
1.2
5
Sze
ntl
ászl
ó
(Mea
n ±
SD
)
±7.1
6
±15
.13
±3.0
8
±8.3
4
±6.6
4
±3.0
4
±9.7
3
±9.0
5
±6.9
4
±5.5
0
±1.0
1
±67
.30
±24
.94
21
.91
41
.91
11
.84
20
.77
54
.56
17
.22
22
.71
24
.18
27
.65
29
.32
1.8
5
16
1.6
8
17
3.2
7
Lak
e S
iko
nd
a
(Mea
n ±
SD
)
±8.0
2
±17
.71
±4.1
2
±10
.44
±6.0
7
±5.6
8
±4.5
5
±7.9
2
±3.3
0
±5.3
0
±0.4
7
±57
.39
±18
.61
23
.06
43
.43
11
.05
21
.54
37
.09
15
.95
16
.35
20
.14
24
.53
31
.20
1.6
3
17
5.0
5
11
1.7
4
Lak
e M
alo
mvö
lgy
(Mea
n ±
SD
)
±9.8
3
±19
.12
±4.2
7
±7.4
0
±7.0
1
±3.9
5
±7.4
9
±11
.88
±7.1
8
±7.5
1
±0.7
0
±99
.31
±21
.40
22
.44
40
.80
12
.52
21
.65
48
.64
16
.20
18
.96
25
.30
26
.35
29
.57
1.5
0
21
5.4
8
13
9.5
5
Lea
f
Hei
gh
t o
f th
e ep
ider
mal
cel
ls a
t
the
adax
ial
surf
ace
of
the
leaf
(μm
):
Wid
th o
f th
e ep
ider
mal
cel
ls a
t
the
adax
ial
surf
ace
of
the
leaf
(μm
):
Hei
gh
t o
f th
e ep
ider
mal
cel
ls a
t
the
abax
ial
surf
ace
of
the
leaf
(μm
):
Wid
th o
f th
e ep
ider
mal
cel
ls a
t
the
abax
ial
surf
ace
of
the
leaf
(μm
):
Hei
gh
t o
f th
e p
alis
ade
cell
s
(μm
):
Wid
th o
f th
e p
alis
ade
cell
s
(μm
):
Hei
gh
t o
f th
e sp
on
gy
cel
ls (
μm
):
Wid
th o
f th
e sp
on
gy
cel
ls (
μm
):
Hei
gh
t o
f th
e C
a(C
OO
) 2 c
ryst
als
(μm
):
Wid
th o
f th
e C
a(C
OO
) 2 c
ryst
als
(μm
):
Nu
mb
er o
f th
e ce
lls
wit
hin
th
e
tric
ho
mes
:
Hei
gh
t o
f th
e tr
ich
om
es (
μm
):
Av
erag
e th
ickn
ess
of
the
leaf
(μm
):
31
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)
Fig. 11. Bract structure of L. salicaria (cross section)
epidermal cells
epidermal cells
trichome
Ca(COO)2 crystal
spongy cells
palisade cells
intercellular space
abaxial surface
adaxial surface
32
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
33
(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.
34
Table 7. Mean ±SD of the measured histological parameters of the bract of L. salicaria. (continued overleaf)
Kap
osv
ár
(Mea
n ±
SD
)
±4.4
9
±12
.71
±2.8
9
±4.9
3
±6.2
8
±3.6
9
±4.2
4
±6.7
2
±4.3
3
±7.5
3
±0.5
2
±73
.11
±16
.21
14
.91
23
.08
10
.60
13
.43
32
.72
13
.53
14
.40
17
.59
21
.26
23
.75
1.5
5
17
8.9
9
10
1.8
3
Kac
sóta
(Mea
n ±
SD
)
±4.7
2
±11
.54
±5.0
3
±7.0
1
±5.1
8
±2.9
5
±6.7
6
±7.7
8
±3.6
9
±4.1
3
- - ±31
.56
17
.93
26
.47
16
.09
19
.74
35
.30
14
.07
17
.68
19
.73
24
.98
27
.02
no
t
fou
nd
no
t
fou
nd
15
8.5
9
Lak
e D
esed
a
(Mea
n ±
SD
)
±6.9
3
±10
.47
±4.0
4
±8.0
1
±5.8
9
±3.5
4
±6.9
7
±7.0
0
±4.2
0
±5.2
5
±0.5
2
±81
.96
±39
.94
19
.65
28
.59
16
.78
22
.93
34
.71
14
.13
17
.42
20
.30
21
.14
21
.18
1.5
3
21
0.8
8
12
5.4
4
Cse
rén
fa
(Mea
n ±
SD
)
±4.6
4
±9.4
5
±3.9
4
±7.3
5
±6.4
0
±2.9
0
±6.5
9
±10
.01
±6.2
1
±8.7
0
±0.5
1
±37
.06
±26
.88
16
.17
24
.04
16
.58
18
.33
35
.50
14
.87
18
.71
21
.98
23
.35
27
.62
1.4
2
15
6.9
1
10
9.7
1
Cse
bén
y
(Mea
n ±
SD
)
±4.6
8
±10
.83
±4.1
1
±5.0
3
±6.1
1
±3.1
5
±5.0
7
±7.7
9
±6.1
7
±6.4
6
±0.6
2
±64
.27
±25
.36
19
.43
25
.06
14
.24
17
.14
37
.45
15
.17
17
.58
21
.37
28
.76
30
.03
1.6
7
19
5.0
4
12
0.5
7
Lak
e A
lmam
ellé
k
(Mea
n ±
SD
) ±4.5
1
±10
.75
±3.5
5
±7.4
2
±5.3
8
±1.5
7
±4.3
4
±6.2
3
±4.8
4
±4.2
0
±0.5
1
±78
.77
±23
.65
16
.25
24
.21
14±.
30
21
.46
36
.36
14
.47
15
.01
17
.31
20
.62
21
.76
1.4
3
21
2.3
3
11
4.4
0
Alm
amel
lék
(Mea
n ±
SD
)
±8.3
9
±9.9
8
±3.7
7
±7.9
4
±4.8
6
±2.8
5
±5.3
1
±6.0
1
±3.1
8
±4.9
1
±0.5
2
±89
.03
±25
.19
19
.29
23
.12
14
.23
18
.13
33
.28
14
.61
15
.70
17
.97
23
.27
25
.86
1.5
5
23
3.6
2
11
9.9
0
Bra
ct
Hei
gh
t o
f th
e ep
ider
mal
cel
ls a
t
the
adax
ial
surf
ace
of
the
bra
ct
(μm
):
Wid
th o
f th
e ep
ider
mal
cel
ls a
t
the
adax
ial
surf
ace
of
the
bra
ct
(μm
):
Hei
gh
t o
f th
e ep
ider
mal
cel
ls a
t
the
abax
ial
surf
ace
of
the
bra
ct
(μm
):
Wid
th o
f th
e ep
ider
mal
cel
ls a
t
the
abax
ial
surf
ace
of
the
bra
ct
(μm
):
Hei
gh
t o
f th
e p
alis
ade
cell
s
(μm
):
Wid
th
of
the
pal
isad
e ce
lls
(μm
):
Hei
gh
t o
f th
e sp
on
gy
cel
ls (
μm
):
Wid
th o
f th
e sp
on
gy
cel
ls (
μm
):
Hei
gh
t o
f th
e C
a(C
OO
) 2 c
ryst
als
(μm
):
Wid
th o
f th
e C
a(C
OO
) 2 c
ryst
als
(μm
):
Nu
mb
er o
f th
e ce
lls
wit
hin
th
e
tric
ho
mes
:
Hei
gh
t o
f th
e tr
ich
om
es (
μm
):
Av
erag
e th
ick
nes
s o
f th
e b
ract
(μm
):
35
Table 7. (Continued) Mean ±SD of the measured histological parameters of the bract of L. salicaria. (continued
overleaf)
Szi
get
vár
(Mea
n ±
SD
) ±7.1
7
±11
.32
±5.8
6
±8.5
2
±5.4
6
±2.5
6
±4.7
1
±6.5
3
±3.7
8
±4.0
5
±0.6
4
±99
.86
±18
.55
17
.93
23
.65
17
.09
19
.90
32
.39
12
.40
13
.83
16
.08
20
.44
21
.37
1.4
7
20
5.0
3
13
8.0
6
Sze
ntl
őri
nc
(Mea
n
±S
D)
±3.1
3
±8.7
5
±3.7
1
±8.0
3
±5.8
7
±3.0
7
±4.7
5
±7.9
1
±3.2
2
±3.5
6
±1.5
8
±19
2.2
1
±29
.39
15
.69
25
.81
13
.21
20
.18
32
.85
13
.75
16
.59
21
.30
21
.57
22
.36
3.0
0
38
3.8
1
10
6.6
5
Sze
ntl
ászl
ó
(Mea
n
±S
D)
±5.5
7
±10
.34
±3.8
0
±6.1
9
±6.2
0
±4.1
5
±4.6
9
±6.9
5
±4.0
1
±4.6
6
±0.8
0
±10
6.8
1
±21
.56
16
.83
25
.56
13
.30
16
.97
39
.68
16
.25
17
.04
20
.20
27
.79
28
.83
2.2
7
33
0.0
9
12
9.7
6
Lak
e S
iko
nd
a
(Mea
n ±
SD
) ±4.2
9
±11
.57
±4.1
8
±7.7
1
±12
.51
±2.5
4
±6.1
8
±7.3
7
±5.0
0
±4.7
0
±0.6
3
±11
4.4
9
±28
.02
16
.67
31
.39
14
.85
19
.09
53
.28
16
.10
17
.77
20
.04
25
.55
24
.22
1.6
4
23
7.1
8
15
0.3
6
Lak
e M
alo
mvö
lgy
(Mea
n ±
SD
) ±5.7
1
±8.1
0
±2.2
7
±7.1
1
±6.6
8
±3.1
1
±4.9
0
±7.9
4
±4.6
1
±6.6
6
±0.5
0
±63
.93
±27
.10
17
.94
21
.75
11
.40
16
.14
34
.05
13
.12
14
.21
18
.44
22
.55
24
.18
1.3
6
16
9.3
8
10
9.1
5
Bra
ct
Hei
gh
t o
f th
e ep
ider
mal
cel
ls a
t
the
adax
ial
surf
ace
of
the
bra
ct
(μm
):
Wid
th o
f th
e ep
ider
mal
cel
ls a
t
the
adax
ial
surf
ace
of
the
bra
ct
(μm
):
Hei
gh
t o
f th
e ep
ider
mal
cel
ls a
t
the
abax
ial
surf
ace
of
the
bra
ct
(μm
):
Wid
th o
f th
e ep
ider
mal
cel
ls a
t
the
abax
ial
surf
ace
of
the
bra
ct
(μm
):
Hei
gh
t o
f th
e p
alis
ade
cell
s
(μm
):
Wid
th
of
the
pal
isad
e ce
lls
(μm
):
Hei
gh
t o
f th
e sp
on
gy
cel
ls (
μm
):
Wid
th o
f th
e sp
on
gy
cel
ls (
μm
):
Hei
gh
t o
f th
e C
a(C
OO
) 2 c
ryst
als
(μm
):
Wid
th o
f th
e C
a(C
OO
) 2 c
ryst
als
(μm
):
Nu
mb
er o
f th
e ce
lls
wit
hin
th
e
tric
ho
mes
:
Hei
gh
t o
f th
e tr
ich
om
es (
μm
):
Av
erag
e th
ick
nes
s o
f th
e b
ract
(μm
):
36
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.
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
long stamen
medium style
short stamen
37
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).
38
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
I = distance between stigma and nearest set of anthers,
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
39
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).
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.
central vascular bundle
sepal
ovary wall
epidermal cells
annular nectary
ovule
40
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).
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).
ovary
sepal
trichome anther
pollen grains
sepal
annular nectary
petal
41
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).
Fig. 19. Short-styled stigma (longitudinal section)
pollen grains
stigma
papillae
42
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
43
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.
44
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
betulinic acid
FUJITA et al. (1972)
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).
45
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.
46
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.
47
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.
48
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
49
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,
50
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.
51
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
52
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.
53
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)
54
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.
55
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)
56
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
57
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
58
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).
59
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)
60
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
61
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.
62
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.
Compounds (μg/g)
Extracts used in the pharmacological experiments
Extract used in the
microbiological
experiments
hexane chloroform ethyl acetate 50% ethanol
Pharm.
50% ethanol
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.
63
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
64
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.
65
Table 16. Summary of the results of previously published papers in connection with antimicrobial activity of L.
salicaria
Barteria and
fungi
ALTANLAR
et al.,
2006
CITOGLU
and
ALTANLAR,
2003
BECKER et
al., 2005
BORCHARDT
et al., 2008
BORCHARDT
et al., 2009
DULGER
and
GONUZ,
2004
RAUHA
et al.,
2000
our results
Gram-positive
cocci
Staphylococcus
aureus n.t. + + + + + + +
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
luteus n.t. n.t. + n.t. n.t. + n.t. +
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
monocytogenes + n.t. n.t. n.t. n.t. - n.t. n.t.
Mycobacterium
smegmatis n.t. n.t. n.t. n.t. n.t. + n.t. n.t.
Gram-
negative rods
Escherichia
coli n.t. + - + + - + +
Klebsiella
pneumonia n.t. n.t. n.t. n.t. n.t. - n.t. n.t.
Proteus
vulgaris n.t. n.t. n.t. n.t. n.t. - n.t. n.t.
P. mirabilis n.t. n.t. + n.t. n.t. n.t. n.t. n.t.
Pseudomonas
aeruginosa n.t. + - + + - n.t. +
MDR P.
aeruginosa n.t. n.t. n.t. n.t. n.t. n.t. n.t. +
Citrobacter
freundii n.t. n.t. - n.t. n.t. n.t. n.t. n.t.
Fungi
Aspergillus
niger n.t. n.t. n.t. n.t. n.t. n.t. - n.t.
Candida
albicans n.t. + + + + - + +
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
fragilis n.t. n.t. n.t. n.t. n.t. - n.t. n.t.
Rhodotorula
rubra n.t. n.t. n.t. n.t. n.t. - n.t. n.t.
Saccaromyces
cerevisiae n.t. n.t. + n.t. n.t. n.t. n.t. n.t.
Applied tests
agar
diffusion
method
disk
diffusion
method
bioautography
disk
diffusion
method
disk
diffusion
method
disk
diffusion
method
cylinder
diffusion
method
tube
dilution,
disk and
agar
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
66
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
67
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
nm≈0.1, which corresponds to >105 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.
Bacteria 50% Ethanol in water extracts Distilled water extracts
Solvent Leaf Flowering branches Stem Solvent Leaf Flowering branches Stem
Staphylococcus
aureus 0.0 11.0 13.5 8.0 0.0 14.5 17.0 13.5
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
epidermidis 0.0 16.0 18.5 14.0 0.0 13.5 18.5 14.5
68
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.
Bacteria
Inhibition zone (mm)
50% Ethanol in water extracts
solvent leaf (50 μL) leaf (100 μL) flower
(50 μL)
flower
(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.
Microorganisms
L. salicaria extract Antibacterial, antifungal drugs
MBC (mg/mL) MIC (mg/mL) MBC
(μg/mL)
MIC
(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
aeruginosa 1.25 2.50 Polymyxin B 1.56 3.12
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.
69
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.
70
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.
Neuronal inhibitory effects:
inhibition of the cholinergic
"twitch" response without
influencing direct smooth
muscle stimulation
• Inhibitors of nerve conduction: tetrodotoxin, local anaesthetics (Na-channel
blockers)
• N-type Ca-channel blockers
• Inhibitors of acetylcholine release: adrenergic a-receptor stimulants*, opioid
receptor agonists*, agonists acting at some histamine or serotonin receptors*,
nociceptin receptor agonists*, cannabinoid receptor agonists*, adenosine
receptor agonists*
Neuronal excitatory effects:
enhancement of the cholinergic
"twitch" response or of
spontaneous tone, without
influencing direct smooth
muscle stimulation
• Cholinesterase inhibitors
• Ganglionic stimulants (e.g., nicotine)*
• TRPV1 receptor agonists (e.g., capsaicin)*
• Tachykinin NK-3 receptor agonists*
• Cholecystokinin receptor agonists*
• P2X purinoceptor agonists (e.g., α,β-methyleneATP)*
• Muscimol and other GABA-A receptor agonists*
• Agonists acting at some serotonin receptors (5-HT-3, 5-HT-4)*
Inhibition of both neuronally-
evoked contraction and direct
smooth muscle stimulation
• Enhancers of intracellular cAMP or cGMP, e.g., β-adrenoceptor* or
VIP/PACAP receptor* agonist, phosphodiesterase inhibitor, NO donor#
• Smooth muscle relaxants (e.g., papaverin-like)
• L-type Ca-channel blockers
Smooth muscle excitatory
effects
Acetylcholine-like*, histamine-like*, substance P-like*, prostaglandin-like*,
leukotriene-like*
• 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
71
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),
72
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.
73
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.
74
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
75
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
76
(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.
77
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
78
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
79
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
80
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.
81
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.
82
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.
83
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.
84
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wetlands planted with Acorus calamus and Lythrum salicaria. Journal of Health
Science 55: 757-766.
Online source 1:
http://wiki.bugwood.org/Archive:BCIPEUS/Purple_Loosestrife, 2012.01.26.
95
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
96
Fig. 31. Szentlászló
Fig. 32. Almamellék
Fig. 33. Lake Almamellék
97
Fig. 34. Csebény
Fig. 35. Kaposvár
Fig. 36. Lake Deseda
98
Fig. 37. Cserénfa
Fig. 38. Lake Sikonda
Fig. 39. Lake Malomvölgy
99
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
100
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
101
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
102
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
103
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
104
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
105
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
106
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
107
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
108
XI. 3. Results of the investigation of soil samples
Habitat Date of collection
"free"
water content
(m/m%)
Mean pH
NO3-
(mg/
mL)
NO3-
(mg/ 100
g)
NO2-
(mg/
mL)
SO42-
(mg/
mL)
SO42-
(mg/ 100
g)
PO43-
(mg/
mL)
PO43-
(mg/ 100
g)
K+
(mg/
mL)
K+
(mg/ 100
g)
NH4+
(mg/
mL)
NH4+
(mg/ 100
g)
Lake
Almamellék 2011 July 56,07 7,72 30,0 7,5 0 <200 <50 5 1,25 100 25,00 0 0,00
Lake
Almamellék 2011 August 36,87 8,09 10,0 2,5 0 <200 <50 5 1,25 100 25,00 0 0,00
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 Deseda
2011 August 23,77 8,10 30,0 7,5 0 <200 <50 5 1,25 100 25,00 0 0,00
Lake
Deseda 2011 July 37,76 7,90 20,0 5 0 <200 <50 5 1,25 100 25,00 0 0,00
Lake Deseda
2010 July 17,80 7,43 5,0 1,25 0 <200 <50 5 1,25 200 50,00 2 0,50
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)
109
Habitat
Date of
collec-
tion
"free"
water
content
(m/m%)
Mean
pH
NO3-
(mg/
mL)
NO3-
(mg/
100 g)
NO2-
(mg/
mL)
SO42-
(mg/
mL)
SO42-
(mg/
100 g)
PO43-
(mg/
mL)
PO43-
(mg/
100 g)
K+
(mg/
mL)
K+
(mg/
100 g)
NH4+
(mg/
mL)
NH4+
(mg/
100 g)
Lake
Malomvölgy
2011
July 3,57* 7,88 150,0 37,5 0 <200 <50 5 1,25 100 25,00 0 0,00
Lake
Malomvölgy
2011
August 4,67* 8,03 10,0 2,5 0 <200 <50 3 0,75 100 25,00 0 0,00
Lake
Malomvölgy
2010
July 4,30* 8,00 5,0 1,25 0 <200 <50 3 0,75 200 50,00 0 0,00
Average 4,49* 7,97 55,0 13,75 0 <200 <50 4 0,92 133 33,33 0 0,00
Lake
Sikonda
2011
July 2,35* 7,66 5,0 1,25 0 <200 <50 5 1,25 100 25,00 0 0,00
Lake
Sikonda
2011
August 0,83* 7,60 15,0 3,75 1 <200 <50 3 0,75 100 25,00 0 0,00
Lake
Sikonda
2010
July 4,10* 8,10 5,0 1,25 0 <200 <50 7 1,75 100 25,00 0 0,00
Average 2,47* 7,79 8,3 2,08 0 <200 <50 5 1,25 100 25,00 0 0,00
Szentlászló 2011
July 56,07 7,30 10,0 2,5 0 <200 <50 5 1,25 200 50,00 0 0,00
Szentlászló 2011
August 49,74 7,00 10,0 2,5 0 <200 <50 3 0,75 100 25,00 3 0,75
Szentlászló 2010
July 82,20 7,45 10,0 2,5 0 <200 <50 7 1,75 200 50,00 10 2,50
Average 65,97 7,25 10,0 2,50 0 <200 <50 5 1,25 167 41,67 4 1,08
Szentlőrinc 2011
August 18,11 8,09 30,0 7,5 1 <200 <50 5 1,25 100 25,00 0 0,00
Szentlőrinc 2011
July 19,28 7,41 50,0 12,5 1 <200 <50 3 0,75 100 25,00 0 0,00
Szentlőrinc 2010
July 16,10 7,86 5,0 1,25 0 <200 <50 5 1,25 200 50,00 2 0,50
Average 17,69 7,79 28,3 7,08 0 <200 <50 4 1,08 133 33,33 1 0,17
Szigetvár 2011
July 20,11 8,28 15,0 3,75 0 <200 <50 5 1,25 100 25,00 0 0,00
Szigetvár 2011
August 21,72 8,17 30,0 7,5 0 <200 <50 3 0,75 100 25,00 0 0,00
Szigetvár 2010
July 40,90 7,63 20,0 5 0 <200 <50 0 0,00 100 25,00 3 0,75
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.
110
XI. 4. Chemical structure of the standard compounds detected in Lythrum extracts by
LC-MS method
HO
OH
OH
O OH
Fig. 40. Gallic acid
OH
OH
O
O
O
HO
HO
O
Fig. 41. Ellagic acid
O
OH
OH
OH
HO
OH
Fig. 42. Catechin
HO
HOO
OH
Fig. 43. Caffeic acid
111
HO
OH
COOH
O
O
OH
OH
HO
Fig. 44. Chlorogenic acid
O
OOH
HO
OH
OH
O O
OH
OH
HO
OH
Fig. 45. Hyperoside
O
OOH
HO
OH
OH
O
OOH
OH
OH
O
OHO
HO
OH
CH3
Fig. 46. Rutin
112
O
OOH
HO
OH
OH
Fig. 47. Luteolin
O
OOH
HO
OH
Fig. 48. Apigenin
O
O
OOH
HO
OH
OH
HO
HO
OH OH
Fig. 49. Orientin
113
O
OOH
HO
OH
OH
O
HO
OH
OH
OH
Fig. 50. Isoorientin
O
O
OOH
HO
OH
OH
HO
HO
OH
Fig. 51. Vitexin
O
OOH
HO
OH
O
HO
OH
OH
OH
Fig. 52. Isovitexin
114
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
115
Fig. 57. 50% ethanol in water extract of flowering branches used in pharmacological experiments
116
XI. 6. Tube dilution method
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.
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
117
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.
118
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)
119
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.
120
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.
121
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.
