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Leonurus cardiaca L. herb--a derived extract and an ursolic acid as the factors affecting the adhesion capacity of Staphylococcus aureus in the context of infective endocarditis


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The objective was an assessment of the impact of Leonurus cardiaca L. extract (LCE) and ursolic acid (UA) on the adhesive properties of Staphylococus aureus NCTC 8325 strain, expressing virulence factors important in the pathogenesis of infective endocarditis. The adhesion and biofilm formation of bacteria cultured in the presence of subinhibitory concentrations of LCE or UA on the abiotic surface or covered with fibrinogen, fibronectin or collagen, were evaluated. Inhibitory effects of LCE and UA on staphylococcal adherence to both types of surface were demonstrated. This, in the case of UA, resulted in a significant reduction of biofilm formation.
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Leonurus cardiaca L. herb — a derived extract and an
ursolic acid as the factors aecting the adhesion capacity of
Staphylococcus aureus in the context of infective endocarditis*
Bartłomiej Micota1, Beata Sadowska1, Anna Podsędek2, Małgorzata Redzynia2 and
Barbara Różalska1*
1Department of Immunology and Infectious Biology, Institute of Microbiology, Biotechnology and Immunology, University of Lodz, Łódź, Po-
land, 2Institute of Technical Biochemistry, Department of Biotechnology and Food Sciences Lodz Technical University, Poland, Łódź, Poland
The objective was an assessment of the impact of
Leonurus cardiaca L. extract (LCE) and ursolic acid (UA)
on the adhesive properties of Staphylococus aureus NCTC
8325 strain, expressing virulence factors important in the
pathogenesis of infective endocarditis. The adhesion and
biolm formation of bacteria cultured in the presence of
subinhibitory concentrations of LCE or UA on the abiotic
surface or covered with brinogen, bronectin or colla-
gen, were evaluated. Inhibitory eects of LCE and UA on
staphylococcal adherence to both types of surface were
demonstrated. This, in the case of UA, resulted in a sig-
nicant reduction of biolm formation.
Key words: ECM, Leonurus cardiaca, ursolic acid, Staphylococcus au-
Infective endocarditis (IE) is often a fatal disease, which
mostly develops as a complication of heart surgery or
anatomical disorders, mainly in the valves. Occasionally,
but in last few years with signicantly growing frequency,
it may occur in individuals without clear risk factors. The
native valve staphylococcal IE is usually caused by Staphy-
lococcus aureus. In contrast, the prosthetic valve infection
is more often caused by a coagulase-negative staphylo-
cocci (S. epidermidis). Both bacteria species possess nu-
merous surface-bound and extracellular proteins recog-
nizing extracellular matrix components (ECM), which are
exposed on the inamed heart tissue. The resulting “veg-
etations” are composed of the bacterial cells forming
biolm, brin, platelet clots, leukocytes and erythrocytes.
It has been found that plasma-derived protein “bridges”,
between staphylococcal receptors and thrombocytes, are
formed from brinogen, brin, thrombospondin and
von Willebrand factor (Hauck & Ohlsen, 2006). Staph-
ylococci bind also avidly to endothelial cells through
adhesin-receptor interactions (SpA, von Willebrand-fac-
tor binding protein, FbpA and ClfA, ClfB). Two other
S. aureus products (secretable) interact with hemostasis
system, but at two different stages. Coagulase- prothrom-
bin binding results in the formation of active ‘’staphylo-
thrombin’’ and brin generation, whereas staphylokinase,
which binds plasminogen and facilitates plasmin activity
is a thrombolytic agent. On the other hand, targets of
α-toxin include lymphocytes, macrophages, epithelium,
endothelium, and erythrocytes. This toxin has also been
proposed to play a role in biolm formation by increas-
ing the bacterial cell-to-cell interactions (Anderson et al.,
2012; McAdow et al., 2012; Zhang et al., 2011).
Because of the risk of serious sequelae, patients with
IE undergo compulsory treatment with antibiotics, un-
fortunately, due to the drug-resistance and the tendency
of bacteria to grow in a biolm, such treatment is often
ineffective (Widmer et al., 2006). Since the most com-
mon cause of death of IE patients is an appearance of
clots derived from vegetations, they are routinely admin-
istered antiplatelet/anticoagulation drugs. Taking into ac-
count that resistance to some of these drugs is also an
emerging clinical entity, it would be interesting to deter-
mine similar properties of naturally occurring substances
(e.g. plant extracts or their components), which in future
could be used as the alternatives to the currently known
chemotherapeutics (Fraga et al., 2010). The results of our
unpublished preliminary studies on the biological activity
of several plant extracts rich in polyphenols drew atten-
tion to the Leonurus cardiaca herb extract and one of its
compounds — an ursolic acid. Both products demon-
strated interesting antimicrobial, antiplatelet and immu-
nomodulatory activities. Another reason for an interest
in L. cardiaca herb extract is its known benecial effect
on heart and cardiocirculatory system. It is a part of the
preparations, such as Cardiosan, Cardionervit, Cardiogran
and many others used, for example, to strengthen the
heart muscle, possessing biological features which are
compared in many respects with those derived from Va-
lerianae radix and Melissae folium (Wojtyniak et al., 2013).
However, due to the simultaneous antimicrobial activity
of L. cardiaca extract / ursolic acid, the main objective
of the present study is an assessment of their impact on
the expression of S. aureus attributes, taking part in the
initial steps of pathogenesis of infective endocarditis. The
study includes evaluation of: i) adhesion of pathogens
to an inert plastic surface or to a surface covered with
ECM components (brinogen, bronectin, collagen),
resembling for example a heart valve prosthesis, ii) bio-
lm formation in the above circumstances, reproducing
roughly the bacterial behavior involved in the formation
of vegetations.
*The preliminary report on the same subject was presented at
MIKROBIOT 2013 Workshop, Łódź, Poland
Abbreviations: ECM, extracellular matrix proteins; IE, infective en-
docarditis; LC E, Leonurus cardiaca extract; UA, ursolic acid
Received: 28 October, 2013; revised: 11 March, 2014; accepted: 27
March, 2014; available on-line: 11 June, 2014
Vol. 61, No 2/2014
on-line at:
386 2014B. Micota and others
Bacterial strains to be tested. The study was per-
formed on a model Staphylococcus aureus 8325-4 (NCTC
8325) strain expressing virulence factors of the
MSCRAMMs/SERAMMs group α-toxin+, SpA+,
Coa+, ClfA+ ClfB-, SAK+. Their expression was earlier
proved in our Lab. Briey, SpA (staphylococcal protein
A) was detected using goat anti-SpA FITC-labeled IgG;
SAK (staphylokinase) was detected on the basis of the
reduction of the chromogenic substrate; α-hemolysin
secretion was evaluated as an intensity of hemolysis of
the sheep red blood cells and with the Western blot
using anti-α-tox mAb; expression of a brinogen re-
ceptor (ClfA, clumping factor A) was assessed using a
semi-quantitative Latex S. aureus test; coagulase (Coa)
production was evaluated in the rabbit plasma coagula-
tion test (Sadowska, 2010).
Preparation and chemical characterization of the
Leonurus cardiaca extract (LCE). Motherwort (Leonurus
cardiaca L.) was a commercial sample supplied by
KAWON-HURT Nowak sp.j. (Gostyń, Poland). Leaves
were extracted with acetone-water (70:30, v/v) at a solid
to liquid ratio 1:10 (w/v), at room temperature for 30
min and then centrifuged for 15 min (4000 rpm). The
pellets were re-extracted twice with 70% aqueous ace-
tone for 15 min and the supernatants were combined.
After the removal of acetone with a vacuum rotary evap-
orator (Rotavapor RII, Büchi, Switzerland) at <40°C,
the extracts were subjected to liquid-liquid partition with
chloroform (1:1 v/v) nine times. The water fractions
were concentrated in vacuum, and lyophilized (Alpha
1–2 LD plus, Christ) with the yield of 14.47%. Dried
extracts were reconstituted at 5 mg/ml of water before
analysis. Qualitative and quantitative composition of
LCE was determined using spectrophotometric methods
and HPLC-PDA analysis. On the basis of spectral iden-
tication and maximum of UV-Vis absorption, phenolics
were qualied in four subclasses: avanols and hydroxy-
benzoic acids (expressed as gallic acid equivalents; detec-
tion at 280 nm), hydroxycinnamic acids (as chlorogenic
acid equivalents; detection at 320 nm), avonols (as rutin
equivalents; detection at 360 nm), and anthocyanins (as
cyanidin 3-glucoside equivalents; detection at 520 nm).
The results were expressed as mg/g of extract.
Ursolic acid (UA) 90% purity was purchased from
Sigma, USA.
Antimicrobial activity of LCE and UA. MIC (min-
imal inhibitory concentration) values were specied by
a standard microdilution broth assay. Stock solution of
LCE was prepared in 50% DMSO, while UA in 96.0%
EtOH (PoCh, Poland). The concentration ranges of the
compounds (in the two-fold dilutions system in 96-well
microplates) were of: 0.0625–2.0 mg/ml (UA) and 0.5–
6.0 mg/ml (LCE). Subsequently, the bacterial suspension
(100 μl) was added (1:1) to the wells. Phytocompounds
dilutions without bacteria were prepared as the negative
control, while for a positive control bacterial suspensions
in Mueller-Hinton broth were added to the wells. The -
nal highest DMSO concentration was 1.25% and EtOH
was 4.25%, which did not affect bacterial growth. Plates
were incubated at 37°C for 18 h and the lowest con-
centration showing no turbidity was recorded as MIC. In
each case, experiments were carried out in quadruplicate
in two separate occasions.
Anti-adhesive and anti-biolm properties of LCE
and UA. The suspension of S. aureus strain (1–5 × 107
CFU/ml) was prepared in adhesion/biolm promoting
medium — tryptic soya broth (TSB) supplemented with
0.25% glucose (TSB/Glu). The experiment scheme was
as follows: (A) — 96-well microplates with immobi-
lized brinogen 2 µg/well (Sigma, USA), collagen I, rat
tail (Life Technologies, USA) or bronectin, 1 µg/well
(R&D Systems, Inc.) were used; (B) 96-well polystyrene
microplates (Nunc, Roskilde, Denmark) with uncoated
wells were used. In all cases 100 µl of S. aureus sus-
pension and 100 µl of LCE or UA at nal ½ or ¾
MIC were added and incubated for 2 h at 37°C; (C)
anti-biolm activity of LCE and UA was tested when
the above described experiments (A) and (B) were pro-
longed to 24 h. In this case only ¾ MIC of LCE and
UA was used. Negative controls in each experiment
version (A, B, C) were wells containing only dilutions
of phytocompounds in DMSO (½ or ¾ MIC), while
positive controls were wells containing bacterial sus-
pensions and TSB/Glu. To evaluate adhesion/biolm
formation of bacteria, Alamar Blue (AB, Trek Diag-
nostic Systems, Inc. USA) staining protocol was used
as recommended by the manufacturer. Briey, 5 µl of
Alamar Blue (AB, BioSource, USA) was added to the
wells of 96-well tissue culture polystyrene plate (Nun-
clon Surface, Nunc, Denmark), containing bacterial cul-
tures after an appropriate time of their exposition on
phytochemicals or medium (control). Then, the plates
were incubated for 1 h at 37°C (with shaking). Finally,
the absorbance was determined at 550 nm and 600 nm
using a multifunctional counter (Victor2, Wallac, Fin-
land). The percentage of AB reduction was calculated
according to the manufacturer formula. Relative chang-
es in bacterial adhesion were expressed as a percentage
of attached cells incubated in the presence of subMIC
of phytocompounds ± S.D., compared to the positive
control. In each case, experiments were carried out in
quadruplicate in two separate occasions.
Statistic analysis of data. Differences in parameters
were tested for signicance using the Mann-Whitney U
test and the program Statistica 10.0 (Stat Soft Inc.). The
differences with P<0.05 were considered to be statisti-
cally signicant.
The phytochemical analysis showed that the total con-
tent of polyphenols in L. cardiaca L. extract (LCE), ex-
pressed as a gallic acid equivalent averaged from three
measurements, was 115.12±11.5 mg/g. The HPLC
analysis demonstrated their amount as 182.75±14.57
mg/g. Hydroxycinnamic acids (caffeic acid, ferulic acid
and p-coumaric acid) content calculated as chlorogenic
acids was 172.01±14.34 mg/g. The minimal inhibito-
ry concentration (MIC) of this extract against S. aureus
8325-4 (NCTC 8325) was 6 mg/ml, whereas MIC of ur-
solic acid (UA) was 0.25 mg/ml. In subsequent experi-
ments dedicated to the assessment of adhesion/biolm
formation, ½ and ¾ MIC of these products were used.
Why were the subinhibitory concentrations exmined?
Because it is known that the benecial effect of antibi-
otic or other drug is not only achieved when the con-
centration is above the minimal inhibitory concentration
(MIC) between consecutive doses. It has been shown in
vitro that, depending on the pharmacokinetic and phar-
macodynamic properties, the subMIC of a given product
is able to affect important bacterial characteristics (Sad-
owska, 2010). Due to the specic pathogenesis course of
infective endocarditis, it can be assumed that achieving the
MIC concentration of a product at the inammation site
is unlikely. Moreover, the products tested by us possess
Vol. 61 387Adhesive properties of S. aureus inuenced by plant extract
(at MIC) unfavorable biocompatibility index, which ex-
cludes their use at a higher concentration (not shown).
The microplate Alamar Blue assay (MABA) used in
the study showed the inhibitory effects of LCE and UA
on staphylococcal adherence to an inert surface. The
percentage of the inhibition of adherence to uncoated
wells of polystyrene plates ranged from 14.2% to 72.4%,
dependent on the type (LCE, UA) and concentration of
each phytocompound (½ or ¾ MIC). This limitation of
bacterial settlement was not caused by the decrease in
their viability per se, since the growth rate of S. aureus sus-
pension culture (tested in parallel after 2 h co-incubation
by MABA) was not affected. The specic adherence of
bacteria to the microplate wells containing immobilized
extracellular matrix proteins (ECM) was also diminished.
For example, ursolic acid (UA) used in a concentration
of ¾ MIC limited bacterial adhesion to collagen-coated
wells by 73.2%, to bronectin-coated wells by 58.8%,
and to brinogen-coated surface by 65.9%. The L. car-
diaca extract used at ¾ MIC showed the activity twice
as weak as ursolic acid (in the range of 23.2–32.4%),
signicant only in the case of a collagen coated surface.
When LCE and UA were used at a lower concentration
MIC), the obtained inhibitory effect was weaker. The
data in Fig. 1A represents the results obtained in experi-
ments where ¾ MICs of the compounds were used.
The exposure of ECM neoepitopes, deposition of
platelets and brin at the site of inammation, as well
as the expression of the various molecules on capillary
endothelial cells, occur in the heart area. These condi-
tions lead to a local inammatory process and coagula-
tion, and created a perfect surface for the attachment of
a pathogen (Chavakis et al., 2005; Edwards & Massey,
2011; McAdow et al., 2011; Kim et al., 2012). Therefore,
a limitation of the adhesion by the L. cardiaca extract
and ursolic acid indicate a new potential application of
these compounds. In another study, conducted by us at
the same time in a different direction than the one pre-
sented here, it was found that both phytocompounds (at
concentrations of 0.5 to 50 μg/ml) inhibit the adhesion
of platelets to immobilized collagen. Moreover, these
products, when co-incubated with human plasma, inhibit
(in a dose dependent manner) brinogen polymerization
(data not yet published). Thus, the L. cardiaca extract as
well as ursolic acid, in addition to the very interesting
anti-staphylococcal activity detected in the present study,
not only modulate platelet functions but may also change
properties of brinogen, which is a key protein in blood
Thus, the weakened S. aureus adherence caused by the
presence of LCE and UA in the culture medium is a
benecial effect, especially because it resulted in the sta-
tistically signicant reduction in the metabolic potency of
bacterial mass (biolm) formed during subsequent 24 h
incubation (Fig. 1B). If we relate these observations to
the real situation in vivo, we may suppose and anticipate
that vegetations would be smaller/weaker and less prone
to detachment, if such a type of products is present in
Figure 1. Anti-adherent and anti-biolm activity of Leonurus cardiaca-derived extract and ursolic acid against S. aureus NCTC 8325.
Bacteria were cultured for 2 or 24 h on microplates without or with immobilized brinogen, bronectin or collagen, in absence or con-
stant presence of the phytocompounds used at their ¾ MIC. Adhesion (A) and biolm formation (B) were evaluated by staining with
Alamar Blue. Results are presented as the percentage of the biomass viability, compared to the control, accepted as 100%. All presented
results are mean from 2 independent experiments performed in quadruplicate ± S.D. *p<0.05
388 2014B. Micota and others
a given microenvironment. The comparison of the de-
gree of the Alamar Blue reduction by cells in biolm,
which developed in the presence of phytochemicals,
showed a decrease in their formation. LCE was a weak
inhibitor, since it caused only 5% inhibition of biolm
formation on the inert polystyrene surface and approxi-
mately 1-10% reduction on an surface coated with ECM
proteins, whereas ursolic acid proved to be very effec-
tive. At a concentration of ¾ MIC UA limited biolm
formation on the surface, both on inert (by 85%) and
on coated with ECM proteins (approximately 70-86%).
Although the exact mechanism by which the tested phy-
tocompounds affects S. aureus cells during the adhesion
and biolm development is at the moment unknown, it
can be assumed that it is based on modication of the
surface adhesins molecular architecture, their expression
and/or inuence on the cell-surface hydrophobicity. This
remains to be determined in further studies just under-
taken in the our Lab. The original plant extract used in
the study — L. cardiaca is known as a herb used in tra-
ditional medicine, being applied in the cases for a va-
riety of illnesses. It contains alkaloid leonurine, which
is a mild vasodilator, and therefore, has long been used
as a sedative in heart neurosis. Among other biochemi-
cal constituents, it also contains bitter iridoid glycosides,
di- and triterpenoids, avonoids, tannins and volatile oils
(Wojtyniak et al., 2013). The L. cardiaca extract tested in
the present study was characterized as presented above
and was shown to contain high amounts of known bio-
logically active components. Another product tested in
this study — ursolic acid, belongs to the main group
of triterpenoids represented by pentacyclic derivatives,
which are ubiquitous in the plant kingdom, including
L. cardiaca. Several biochemical and pharmacological
effects of UA such as anti-inammatory, antioxidant,
anti-proliferative, anti-cancer, anti-mutagenic, anti-ather-
osclerotic, anti-hypertensive, anti-leukemic and antiviral
properties are reported in a number of experimental sys-
tems (Checker & Sainis, 2012). Through the presented
results we provide additional valuable information on the
biological properties of these phytochemicals, which had
not been tested before in the range covered by our re-
Conict of Interest
The authors have declared that there is no conict of
The work was supported by University of Lodz
(2013) for B.M. and by grant No.: PRO-2013/09/N/
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... Phytochemical analysis of originally prepared Leonurus cardiaca L. extract (LCE) used in the present study was presented previously by Sadowska et al. [13] and demonstrated a high content of polyphenols (137.0 ± 0.8 mg/g) with hydroxycinnamic acid derivatives (81.3 ± 5.7 mg/g) as predominant phenolic compounds. Several researches, inclusive of our previous studies [12][13][14], showed that such secondary metabolites of plants exhibit a wide range of biological activity and may influence both physiological processes in human body (e.g., hemostasis, immune response, maintenance of physiological barriers of skin, mucosa, and endothelium) and host-pathogen interactions important for the course of infections. Motherwort herb is known to possess antihypertensive, heart-strengthening, antioxidant, analgesic, anti-inflammatory, neuroprotective, and antimicrobial effects [15,16]. ...
... However, observed morphological changes of staphylococcal cell surfaces can only partly explain antiadhesive properties of L. cardiaca L. extract, since LCE used at 0.5 × MIC was more potent in the reduction of S. aureus adhesion simultaneously exhibiting moderate effect on cell morphology compare to 0.75 × MIC. [12][13][14], showed that such secondary metabolites of plants exhibit a wide range of biological activity and may influence both physiological processes in human body (e.g., hemostasis, immune response, maintenance of physiological barriers of skin, mucosa, and endothelium) and host-pathogen interactions important for the course of infections. Motherwort herb is known to possess antihypertensive, heart-strengthening, antioxidant, analgesic, anti-inflammatory, neuroprotective, and antimicrobial effects [15,16]. ...
... In our previous studies we demonstrated unknown antiadhesive and anti-biofilm activity of LCE on staphylococcal infective endocarditis in vitro model. LCE used at sub-inhibitory concentrations (0.75 × MIC and 0.5 × MIC) negatively affected S. aureus adhesion to both native and conditioned with extracellular matrix proteins (ECM) surfaces, as well as to the deposit of fibrin network [12,14]. In the present study we confirmed these results testing adhesive properties of S. aureus by atomic force microscopy (AFM) (Figure 1). ...
Full-text available
Better understanding the mechanisms of Leonurus cardiaca L. extract (LCE) activity is necessary to prepare recommendations for the use of LCE-based herbal products for preventive/supportive purposes in case of infective endocarditis (IE) and other staphylococcal invasive infections. The aim of the study was to analyze molecular mechanisms of LCE effect on Staphylococcus aureus and blood platelets in the context of their interactions playing a pivotal role in such disorders. Using atomic force microscopy, we demonstrated that adhesion forces of S. aureus were markedly reduced after exposure to LCE at subinhibitory concentrations. The effect resulted from the impact of LCE on S. aureus cell morphology and the composition of phospholipids and fatty acids in bacterial membranes (assessed by HPLC), which modulated their stabilization, hydrophobicity, and charge. Moreover, using FACS we showed also that LCE significantly reduced GP IIb/IIIa expression on blood platelets, thus the disruption of platelet-fibrinogen interactions seems to explain antiplatelet effect of LCE. The obtained results prove the usefulness of LCE in the prevention of S. aureus adhesion, platelet activation, and vegetations development, however, also pointed out the necessity of excluding the cationic antibiotics from the treatment of S. aureus-associated IE and other invasive diseases, when motherwort herb is used simultaneously as an addition to the daily diet.
... UA showed moderate activity (10 < MIC < 100 mg/L) against 13 species of Gram-positive bacteria [46,54,63,69,70,72,73,75,[78][79][80][81][82][83][84][85][86][87] and 6 species of Gram-negative bacteria [75,79,82]. On the other hand, the low activity of UA (MIC > 100 mg/L) was described for 10 species of Gram-positive bacteria [13,46,63,72,80,[88][89][90][91] and 8 species of Gram-negative bacteria [10,47,63,69,70,72,82,84,88,92,93]. The lowest sensitivity to UA (MIC ≥ 1000 mg/L) showed 5 species of Gram-positive bacteria: B. cereus [82], L. monocytogenes [82], S. aureus [82], S. mutans [94], S. pneumoniae [80], and 5 species of Gramnegative bacteria: A. caveae [82], E. coli [10,47,82,95], K. pneumoniae [80], S. choleraesuis [80], V. cholerae [80,82]. ...
... The aim of a study by Micota et al. [90] was to determine the effect of UA on adhesion and biofilm formation by coagulase-positive S. aureus strains that are a common cause of infective endocarditis. Titre plates with wells coated with fibrinogen, fibronectin, and collagen were used in that study. ...
Full-text available
Due to the ever-increasing number of multidrug-resistant bacteria, research concerning plant-derived compounds with antimicrobial mechanisms of action has been conducted. Pentacyclic triterpenes, which have a broad spectrum of medicinal properties, are one of such groups. Asiatic acid (AA) and ursolic acid (UA), which belong to this group, exhibit diverse biological activities that include antioxidant, anti-inflammatory, diuretic, and immunostimulatory. Some of these articles usually contain only a short section describing the antibacterial effects of AA or UA. Therefore, our review article aims to provide the reader with a broader understanding of the activity of these acids against pathogenic bacteria. The bacteria in the human body can live in the planktonic form and create a biofilm structure. Therefore, we found it valuable to present the action of AA and UA on both planktonic and biofilm cultures. The article also presents mechanisms of the biological activity of these substances against microorganisms.
... The BuOH extract of L. cardiac, which contains lavandulifolioside, also exhibited the heart ratereducing activity. However, the mechanism of action for lavandulifolioside and the BuOH extract were different, indicating a high chance that other principles in the extracts might also contribute to the cardio modulation effect (Miłkowska-Leyck et al., 2002). ...
... Extract of L. cardiaca and ursolic acid (144), a triterpenoid with anti-inflammatory activity (Ali et al., 2007), were active to decrease different pathogenic characteristics of Staphylococus aureus, including aggregation, adherence, plasma clotting as well as survival in whole human blood. However, L. cardiaca extract also enhanced the staphylococcal tolerance to exogenous hydrogen peroxide, possibly due to the increased anti-oxidant activity of bacterial (Micota et al., 2014(Micota et al., , 2016. The CHCl 3 extract of L. cardiaca has notable antiplasmodial activity against the multidrug resistant K1 Plasmodium falciparum strain with an IC 50 value of 3.1 mg/ml (best among three L. cardiaca extracts). ...
Plants belonging to the genus Leonurus, also named motherwort, are traditionally used for anti-gynecological disorder in East Asia, and for sedative in Europe. Chemical investigation of the genus Leonurus not only enriched the natural products library, but also enlarged the pharmacological application of this traditional herb. In this review, we systematically summarized the structures of 259 compounds isolated from the genus Leonurus, featured with 147 labdane diterpenoids. The reported bioactivity studies up to 2017 are presented in the second part, with the main focus on the isolated compounds and also concerning the extracts. In addition to the traditional uterine contraction and sedative activity, recently the cardiovascular protection effect of leonurine has drawn most attention. Other than that, neuroprotection, anti-inflammation, anti-cancer, anti-platelet aggregation and many other activities have been assigned to various compounds from the genus Leonurus. Among 70 bioactivity references cited in this review, 57% of them were concentrated on two alkaloids (leonurine and stachydrine), whereas only 20% are about the 147 diterpenoids. Anti-inflammation is the major bioactivity discovered so far for the labdane diterpenoids from the genus Leonurus, whose further therapeutic potential still remains for exploration.
... UA kills bacteria by altering the structure of the bacterial cell, particularly by interfering with the cell membrane and adhesion proteins (93). UA also affects cell morphology and controls the expression of genes related to virulence factors, such as pili and fritillary (94). UA inhibits bacterial growth by reducing the ability of bacteria to adhere to host cells, as well as disrupting biofilm formation (95). ...
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Ursolic acid (UA) is a plant-derived pentacyclic triterpenoid with 30 carbon atoms. UA has anti-inflammatory, antioxidative, antimicrobial, hepato-protective, anticancer, and other biological activities. Most studies on the biological functions of UA have been performed in mammalian cell ( in vitro ) and rodent ( in vivo ) models. UA is used in animal husbandry as an anti-inflammatory and antiviral agent, as well as for enhancing the integrity of the intestinal barrier. Although UA has been shown to have significant in vitro bacteriostatic effects, it is rarely used in animal nutrition. The use of UA as a substitute for oral antibiotics or as a novel feed additive in animal husbandry should be considered. This review summarizes the available data on the biological functions of UA and its applications in animal husbandry.
... The above studies included the determination of the minimal inhibitory concentration (MIC) value of UA and its influence on the survival of bacteria, on the morphology of cells and bacterial membranes as well as on the impact on bacterial virulence factors such as hydrophobicity of the surface of bacterial cells, ability to move, and synthesis of the curli fimbria and P-type fimbria. Few research centers have investigated the effect of UA on the ability of bacteria to form biofilms, and these studies have focused on singlespecies consortia [30,41,[43][44][45][46][47][48][49][50][51][52][53][54][55][56][57]. ...
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Multispecies bacterial biofilms are the often cause of chronic recurrent urinary tract infections within the human population. Eradicating such a complex bacterial consortium with standard pharmacotherapy is often unsuccessful. Therefore, plant-derived compounds are currently being researched as an alternative strategy to antibiotic therapy for preventing bacterial biofilm formation and facilitating its eradication. Therefore, our research aimed to determine the effect of secondary plant metabolite ursolic acid (UA) on the growth and survival, the quantity of exopolysaccharides formed, metabolic activity, and morphology of uropathogenic Gram-negative rods living in single- and mixed-species biofilms at various stages of their development. Spectrophotometric methods were used for biofilm mass formation and metabolic activity determination. The survival of bacteria was established using the serial dilution assay. The decrease in survival and inhibition of biofilm creation, both single- and multispecies, as well as changes in the morphology of bacterial cells were noticed. As UA exhibited better activity against young biofilms, the use of UA-containing formulations, especially during the initial steps of urinary tract infection, seems to be reasonable. However, the future direction should be a thorough understanding of the mechanisms of UA activity as a bioactive substance.
... Micota et al. [37] studied the antimicrobial potential of L. cardiaca acetone-water extract and its component ursolic acid by determining the minimal inhibitory concentration, as well as the antiadhesive and antibiofilm properties against Staphylococcus aureus strain (potential etiological agent of infective endocarditis). Their results (MIC = 6 mg/ml for extract and 0.25 mg/ml for ursolic acid) showed weak biostatic activity of L. cardiaca extract in comparison to ursolic acid, but both preparations possessed antiadhesive potential. ...
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Leonurus cardiaca L. (motherwort) is a perennial herb, native to Asia and southeastern Europe, with widespread global occurrence in present days. The plant was historically used as cardiotonic and for treating gynaecological afflictions (such as amenorrhea, dysmenorrhea, menopausal anxiety, or postpartum depression). Although its use in oriental and occidental medicine is relatively well documented, the recent progress registered raises the need for an update of the Medicines Agency assessment report on Leonurus cardiaca L., herba (2010). The current study presents the progress made within the 2010-2018 timeframe regarding the potential applications and scientific evidences supporting the traditional use of motherwort, in the same time suggesting future research opportunities.
... NO and platelet-activating factor (PAF)] are released. [1][2][3][4][5] These activities are intended to restore homeostasis in the body, which depends on the relevant repair processes in damaged tissues. Therefore, the reasons for the development of many chronic lesions, such as infective endocarditis (IE), diabetic foot ulcerations (DFU) or chronic wound infections (CWI), include both microbial infection and pathological changes within the host tissue, which disturb these physiological repair processes. ...
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The immunomodulatory activity of Leonurus cardiaca L. polyphenol-rich extract (LCE) was tested in vitro on HUVECs to explore its potential therapeutic usefulness in the treatment of inflammatory lesions. The phytochemical composition of LCE, its antioxidant and cytotoxic activity, and the influence of LCE on NO and platelet-activating factor (PAF) secretion by HUVECs and platelet aggregation were all assessed. Total polyphenol contents in LCE reached 137.0 ± 0.8 mg/g, with hydroxycinnamic acid derivatives as the predominant phenolic compounds. LCE expressed antioxidant capacity, which was, however, 13- to 16-fold lower than the antioxidant activity of ascorbic acid. The plant extract was not cytotoxic up to a concentration 4500 µg/ml and did not exhibit proapoptotic activity. LCE significantly increased NO production in HUVECs in a concentration-dependent manner and led to the inhibition of PAF secretion induced by staphylococcal peptidoglycan. The extract used at the concentration of 100 µg/ml significantly reduced platelet aggregation in the presence of arachidonic acid. We provide in vitro data demonstrating the immunomodulatory potential of LCE, which may be beneficial in preventing the development of difficult-to-treat inflammatory lesions within chronically infected tissues.
... Micota et al. investigated L. cardiaca L. extract (LCE) and ursolic acid on the adhesive properties of Staphylococus aureus NCTC 8325 strain, expressing virulence factors important in the pathogenesis of infective endocarditis [127]. The adhesion and biofilm formation of bacteria cultured in the presence of subinhibitory concentrations of L. cardiaca extract or ursolic acid (triterpenoid included in L. cardiaca) on the abiotic surface or covered with fibrinogen, fibronectin or collagen, were evaluated. ...
Plants have been always a fruitful source of active metabolites against many human disorders, where cardiovascular (CV) diseases have been one of the major health problems all over the world. There are some clinically proved medicinal plants with cardioprotective effects such as Crataegus monogyna and C. oxyacantha (hawthorn) from Rosaceae. On the other hand, cardiac glycosides, present in a number of plant species, are well-known for their cardiotonic activity. However, we encountered such a fact that very less number of studies are available on the source plants; e.g. Adonis vernalis, Convallaria majalis, Strophanthus kombe, Thevetia peruviana, Leonurus cardiaca, etc. Consequently, we herein aimed to cover all available data consisting of in vitro, in vivo, and human studies (if any) on cardiotonic effects of the aforementioned species through a wide literature search using Scopus, Web of Science as well as Pubmed.
Little or no information is available concerning online high‐performance liquid chromatography (HPLC) antioxidants and the antibiofilm effect of Leonurus cardiaca . Five distinct extractions of methanolic, ethyl acetate, dichloromethane, hexane, and water were obtained from L. cardiaca . In the online‐HPLC‐antioxidant analysis of all examined samples, rosmarinic acid emerged as the primary antioxidant, registering concentrations ranging from 6 to 15 ppm at wavelengths of 517 and 734 nm. Notably, the water extract exhibited robust antioxidant activity In vitro. Regarding acetylcholinesterase and butrylcholinesterase inhibition, the n‐hexane extract exhibited superior inhibition with values of 3.08 and 5.83 galanthamine equivalent, respectively. Except for the water extract, all tested extracts (at a concentration of 20 μg/mL) exhibited substantial inhibitory activity against biofilm formation, in many cases superior to 80%, and reached even 94.52% against Escherichia coli . Although less vigorous, the extracts also acted against the mature biofilm (inhibition up 76.50% against Staphylococcus aureus ). They could work against the metabolism inside an immature and mature biofilm, with inhibition percentages up to 93.18% (vs. Pseudomonas aeruginosa ) and 76.50% (vs. Acinetobacter baumannii ), respectively. Considering its significant antioxidants, enzyme inhibition, and antimicrobial activity, L. cardiaca emerges as a promising candidate for therapeutic potential.
The aim of the study was to determine whether Leonurus cardiaca L. herb extract (LCE) used at subinhibitory concentration modifies the characteristics of S. aureus, important in the pathogenesis of invasive infections originating from the bloodstream, in a way favorable for the human host. Polyphenol rich LCE, a common ingredient in the pharmaceutical products used for various cardiovascular and nervous system disorders, had shown interesting antibacterial and anti-biofilm properties in our previous studies. Our current findings indicate that the following S. aureus characteristics were decreased, depending on the LCE concentration: i) formation of aggregates in plasma, ii) adherence to a fibrin coated surface, iii) staphylocoagulase-dependent plasma clotting, iv) bacterial survival in whole human blood in an ex vivo model, v) expression of cell surface protein A (SpA), vi) synthesis of -toxin. However, staphylococcal tolerance to exogenous hydrogen peroxide was enhanced after pre-incubation with LCE, possibly due to the increased activity of bacterial antioxidant enzymes: a possibility confirmed by the higher production of superoxide dismutase and slightly higher production of catalase. Summarizing, the use of LCE at sub-MIC in in vitro and an ex vivo models resulted in the weakening of some important staphylococcal immunoprotective attributes but the strengthening of such virulence factors as those responsible for oxidative stress tolerance. Some of these results, and the fact that LCE has direct anticoagulant properties, reflected in a reduced thrombin-dependent fibrinogen polymerization rate, suggest a risk of adverse effects, which could be important in the context of S. aureus survival in the host.
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Staphylococcus aureus causes many diseases in humans, ranging from mild skin infections to serious, life-threatening, superantigen-mediated Toxic Shock Syndrome (TSS). S. aureus may be asymptomatically carried in the anterior nares or vagina or on the skin, serving as a reservoir for infection. Pulsed-field gel electrophoresis clonal type USA200 is the most widely disseminated colonizer and the leading cause of TSS. The cytolysin α-toxin (also known as α-hemolysin or Hla) is the major epithelial proinflammatory exotoxin produced by TSS S. aureus USA200 isolates. The current study aims to characterize the differences between TSS USA200 strains [high (hla(+)) and low (hla(-)) α-toxin producers] in their ability to disrupt vaginal mucosal tissue and to characterize the subsequent infection. Tissue viability post-infection and biofilm formation of TSS USA200 isolates CDC587 and MN8, which contain the α-toxin pseudogene (hla(-)), MNPE (hla(+)), and MNPE isogenic hla knockout (hlaKO), were observed via LIVE/DEAD® staining and confocal microscopy. All TSS strains grew to similar bacterial densities (1-5 × 10(8) CFU) on the mucosa and were proinflammatory over 3 days. However, MNPE formed biofilms with significant reductions in the mucosal viability whereas neither CDC587 (hla(-)), MN8 (hla(-)), nor MNPE hlaKO formed biofilms. The latter strains were also less cytotoxic than wild-type MNPE. The addition of exogenous, purified α-toxin to MNPE hlaKO restored the biofilm phenotype. We speculate that α-toxin affects S. aureus phenotypic growth on vaginal mucosa by promoting tissue disruption and biofilm formation. Further, α-toxin mutants (hla(-)) are not benign colonizers, but rather form a different type of infection, which we have termed high density pathogenic variants (HDPV).
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Ursolic acid (UA), a pentacyclic triterpenoid carboxylic acid, is the major component of many plants including apples, basil, cranberries, peppermint, rosemary, oregano and prunes and has been reported to possess antioxidant and anti-tumor properties. These properties of UA have been attributed to its ability to suppress NF-κB (nuclear factor kappa B) activation. Since NF-κB, in co-ordination with NF-AT (nuclear factor of activated T cells) and AP-1(activator protein-1), is known to regulate inflammatory genes, we hypothesized that UA might exhibit potent anti-inflammatory effects. The anti-inflammatory effects of UA were assessed in activated T cells, B cells and macrophages. Effects of UA on ERK, JNK, NF-κB, AP-1 and NF-AT were studied to elucidate its mechanism of action. In vivo efficacy of UA was studied using mouse model of graft-versus-host disease. UA inhibited activation, proliferation and cytokine secretion in T cells, B cells and macrophages. UA inhibited mitogen-induced up-regulation of activation markers and co-stimulatory molecules in T and B cells. It inhibited mitogen-induced phosphorylation of ERK and JNK and suppressed the activation of immunoregulatory transcription factors NF-κB, NF-AT and AP-1 in lymphocytes. Treatment of cells with UA prior to allogenic transplantation significantly delayed induction of acute graft-versus-host disease in mice and also significantly reduced the serum levels of pro-inflammatory cytokines IL-6 and IFN-γ. UA treatment inhibited T cell activation even when added post-mitogenic stimulation demonstrating its therapeutic utility as an anti-inflammatory agent. The present study describes the detailed mechanism of anti-inflammatory activity of UA. Further, UA may find application in the treatment of inflammatory disorders.
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Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci--coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA)--were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans.
Leonurus cardiaca is a perennial plant indigenous to central Europe and Scandinavia, but it is also found in the area spanning temperate Russia to central Asia. It has been introduced to North America and has become established locally in the wild. Motherwort (Leonuri cardiacae herba) consists of aerial parts of Leonurus cardiaca gathered during the flowering period, dried at 35 °C and, according to European Pharmacopoeia 7th edition, should contain a minimum of 0.2% flavonoids, expressed as hyperoside. Compounds belonging to the group of monoterpenes, diterpenes, triterpenes, nitrogen- containing compounds, phenylpropanoids, flavonoids and phenolic acids, as well as volatile oils, sterols and tannins, have been identified in motherwort. Traditionally, extracts of the herb have been used internally, mainly for nervous heart conditions and digestive disorders. However, they have also been used for bronchial asthma, climacteric symptoms and amenorrhoea, as well as externally in wounds and skin inflammations. Mild negative chronotropic, hypotonic and sedative effects can be attributed to the herb and preparations thereof. Pharmacological studies have confirmed its antibacterial, antioxidant, anti-inflammatory and analgesic activity, as well as its effects on the heart and the circulatory system. Sedative and hypotensive activity has been demonstrated in clinical trials. Copyright © 2012 John Wiley & Sons, Ltd.
Clinical isolates of Staphylococcus aureus secrete coagulases, polypeptides that bind to and activate prothrombin, thereby converting fibrinogen to fibrin and promoting the clotting of plasma or blood. Two staphylococcal products, the canonical coagulase (Coa) as well as the recently identified von Willebrand factor binding protein (vWbp), promote similar modifications of the coagulation cascade during host infection. Staphylococcal binding to fibrinogen or fibrin is an important attribute of disease pathogenesis, which leads to the formation of abscesses and bacterial persistence in host tissues and also enables the pathogen to cause lethal sepsis. Circumstantial evidence suggests that the product of coagulase activity, staphylococci captured within a fibrin meshwork, enable this pathogen to disseminate as thromboembolic lesions and to resist opsonophagocytic clearance by host immune cells. In addition, the coagulation products of staphylococci appear to display discrete differences when compared to those of thrombin-mediated coagulation, the latter representing a key innate defense mechanism against many invading pathogens. Preclinical evidence suggests that inactivation or neutralization of coagulases may prevent the pathogenesis of staphylococcal infections, a strategy that could be used to combat the current epidemic of hospital-acquired infections with drug-resistant S. aureus isolates.
Staphylococcus aureus causes purulent skin and soft tissue infections (SSTIs) that frequently reoccur. Staphylococal SSTIs can lead to invasive disease and sepsis, which are among the most significant causes of infectious disease mortality in both developed and developing countries. Human or animal infections with S. aureus do not elicit protective immunity against staphylococcal diseases. Here we review what is known about the immune evasive strategies of S. aureus that enable the pathogen's escape from protective immune responses. Three secreted products are discussed in detail, staphylococcal protein A (SpA), staphylococcal binder of immunoglobulin (Sbi) and adenosine synthase A (AdsA). By forming a complex with V(H)3-type IgM on the surface of B cells, SpA functions as a superantigen to modulate antibody responses to staphylococcal infection. SpA also captures pathogen-specific antibodies by binding their Fcγ portion. The latter activity of SpA is shared by Sbi, which also associates with complement factors 3d and factor H to promote the depletion of complement. AdsA synthesizes the immune signaling molecule adenosine, thereby dampening innate and adaptive immune responses during infection. We discuss strategies how the three secreted products of staphylococci may be exploited for the development of vaccines and therapeutics.
Several fibrinogen binding proteins (Fibs) play important roles in the pathogenesis of Staphylococcus aureus (S. aureus). Most Fibs can promote the aggregation of platelets during infection, but the extracellular fibrinogen-binding protein (Efb) is an exception. It is reported that Efb can specifically bind fibrinogen and inhibit the aggregation of platelet with its N terminal. However, the biological significance of platelet aggregation inhibition in the infection caused by S. aureus is unclear until now. Here, we demonstrated that the persistence and aggregation of platelets were important for killing S. aureus in whole blood. It was found that the N terminal of Efb (EfbN) and platelets inhibitors could increase the survival of S. aureus in whole blood. The study in vivo also showed that EfbN and platelets inhibitors could reduce the killing of S. aureus and increase the lethality rate of S. aureus in the acute infection mouse model.
Staphylococcus aureus is a major cause of bacteraemia, which frequently leads to infective endocarditis, osteomyelitis, septic arthritis and metastatic abscess formation. The development of these secondary infections is due to bacterial dissemination from the blood into surrounding tissues and is associated with significantly increased morbidity and mortality. Despite the importance of S. aureus extravasation in disease progression, there is relatively little understanding of the molecular mechanisms by which this pathogen crosses the endothelial barrier and establishes new sites of infection. Recent work has identified a number of putative routes by which S. aureus can escape the bloodstream. In this article we review these new developments and set them in the context of strategies used by other established pathogens to traverse cellular barriers.
Polyphenols and consequently many flavonoids have several beneficial actions on human health. However, the actual molecular interactions of polyphenols with biological systems remain mostly speculative. This review addresses the potential mechanisms of action that have been so far identified, as well as the feasibility that they could occur in vivo. Those mechanisms include: i) non specific actions, based on chemical features common to most polyphenols, e.g. the presence of a phenol group to scavenge free radicals; and ii) specific mechanisms; based on particular structural and conformational characteristics of select polyphenols and the biological target, e.g. proteins, or defined membrane domains. A better knowledge about the nature and biological consequences of polyphenol interactions with cell components will certainly contribute to develop nutritional and pharmacological strategies oriented to prevent the onset and/or the consequences of human disease.
The intravascular manifestation of Staphylococcus aureus infection is often associated with a severe, and sometimes catastrophic disease. Many host factors contribute to endothelial tropism of S.aureus including subendothelial matrix proteins, endothelial cell receptors, and platelets that are engaged together with S. aureus cell wall adhesins such as the fibronectin binding proteins. Recently, the role of secreted staphylococcal factors that were initially identified by virtue of their binding function with host proteins and ligands, has been reappraised in this regard. Among these, bacterial proteins without significant homology among each other, coagulase (Coa), the extracellular fibrinogen binding protein (Efb), the extracellular matrix binding protein (Emp), or the extracellular adhesive protein (Eap), are the most prominent ones to be associated with endovascular disease. Newly discovered interactions with host components may account for profound effects on immunmodulation and wound healing which are summarized in this short review and which ascribe an important role of these molecules in acute and chronic endo- and extravascular staphylococcal disease. Further research in the complex functional role of these "secretable expanded repertoire adhesive molecules" (SERAM) may not only help to increase our understanding in the pathogenesis of S. aureus infection but can specify novel targets for preventive or therapeutic strategies.