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Communication
Leonurus cardiaca L. herb — a derived extract and an
ursolic acid as the factors aecting 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
biolm 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 eects of LCE and UA on
staphylococcal adherence to both types of surface were
demonstrated. This, in the case of UA, resulted in a sig-
nicant reduction of biolm formation.
Key words: ECM, Leonurus cardiaca, ursolic acid, Staphylococcus au-
reus
INTRODUCTION
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 signicantly 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 inamed heart tissue. The resulting “veg-
etations” are composed of the bacterial cells forming
biolm, 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 biolm 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 biolm, 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 benecial 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.
*e-mail: rozab@biol.uni.lodz.pl
*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
385–388
on-line at: www.actabp.pl
386 2014B. Micota and others
MATERIALS AND METHODS
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. Briey, 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-
tication and maximum of UV-Vis absorption, phenolics
were qualied 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 specied 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-biolm properties of LCE
and UA. The suspension of S. aureus strain (1–5 × 107
CFU/ml) was prepared in adhesion/biolm 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-biolm 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/biolm
formation of bacteria, Alamar Blue (AB, Trek Diag-
nostic Systems, Inc. USA) staining protocol was used
as recommended by the manufacturer. Briey, 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 signicance 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 signicant.
RESULTS AND DISCUSSION
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/biolm
formation, ½ and ¾ MIC of these products were used.
Why were the subinhibitory concentrations exmined?
Because it is known that the benecial 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 specic pathogenesis course of
infective endocarditis, it can be assumed that achieving the
MIC concentration of a product at the inammation site
is unlikely. Moreover, the products tested by us possess
Vol. 61 387Adhesive properties of S. aureus inuenced 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 specic 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%),
signicant only in the case of a collagen coated surface.
When LCE and UA were used at a lower concentration
(½ 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 inammation, 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 inammatory 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
coagulation.
Thus, the weakened S. aureus adherence caused by the
presence of LCE and UA in the culture medium is a
benecial effect, especially because it resulted in the sta-
tistically signicant reduction in the metabolic potency of
bacterial mass (biolm) 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-biolm 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 biolm 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 biolm,
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 biolm
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 biolm
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 biolm development is at the moment unknown, it
can be assumed that it is based on modication of the
surface adhesins molecular architecture, their expression
and/or inuence 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-inammatory, 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-
search.
Conict of Interest
The authors have declared that there is no conict of
interest.
Acknowledgements
The work was supported by University of Lodz
(2013) for B.M. and by grant No.: PRO-2013/09/N/
NZ6/00826.
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