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molecules
Article
Molecular Mechanisms of Leonurus Cardiaca L.
Extract Activity in Prevention of Staphylococcal
Endocarditis—Study on in Vitro and ex Vivo Models
Beata Sadowska 1, * , Dariusz Laskowski 2, Przemysław Bernat 3, Bartłomiej Micota 1,
Marzena Wi˛eckowska-Szakiel 1, Anna Pods˛edek 4and Barbara Ró˙zalska 1
1Department of Immunology and Infectious Biology, Institute of Microbiology, Biotechnology and
Immunology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16,
90-237 Lodz, Poland; bartlomiejmicota@o2.pl (B.M.); marzena.wieckowska@biol.uni.lodz.pl (M.W.-S.);
barbara.rozalska@biol.uni.lodz.pl (B.R.)
2Department of Microbiology, Faculty of Biology and Environmental Protection, Nicolaus Copernicus
University in Torun, Lwowska 1, 87-100 Torun, Poland; laskosd@umk.pl
3Department of Industrial Microbiology and Biotechnology, Institute of Microbiology, Biotechnology and
Immunology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16,
90-237 Lodz, Poland; przemyslaw.bernat@biol.uni.lodz.pl
4Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences,
Lodz University of Technology, Stefanowskiego 4/10, 90-924 Lodz, Poland; anna.podsedek@p.lodz.pl
*Correspondence: beata.sadowska@biol.uni.lodz.pl; Tel.: +48-42-635-45-25
Academic Editor: Vincenzo De Feo
Received: 22 August 2019; Accepted: 10 September 2019; Published: 12 September 2019
Abstract:
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.
Keywords:
Leonurus cardiaca L.; infective endocarditis; blood platelets; Staphylococcus aureus; microbial
adhesion; cell–pathogen interaction
1. Introduction
Infective endocarditis (IE) is an acute, life-threatening disease, in which microbial colonization
leads to progressive damage of heart tissue (usually valves) via the formation of complex aggregates
called vegetations containing microbes, fibrin, activated platelets, and phagocytes. The most common
etiological agents of IE include Staphylococcus aureus, oral streptococci from Streptococcus Viridans group,
Enterococcus spp., and coagulase-negative staphylococci (CNS). Although many bacteria and even fungi,
Molecules 2019,24, 3318; doi:10.3390/molecules24183318 www.mdpi.com/journal/molecules
Molecules 2019,24, 3318 2 of 15
if present in the bloodstream, may colonize heart tissue or the biomaterials placed in it (e.g., artificial
valves, stents, pacemakers), S. aureus seems to be best adapted to settle both normal and damage
endocardium thus it is responsible for 27–32% of IE cases [
1
–
3
]. The deposition of the microorganisms
into the vegetations (usually as biofilm form) depends on their affinity for adhesion. Many surface
molecules called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules),
including clumping factor A (ClfA), fibronectin binding proteins (FnBPs), staphylococcal protein
A (SpA), and secretory proteins such as coagulase (Coa) or von Willebrand factor binding protein
(vWbp) participate in S. aureus adherence, biofilm formation, and myocardial/endovascular diseases
development [
2
,
4
,
5
]. Due to their complex structure and physiology, biofilms enhance microbial
cell survival under environmental stress conditions, including exposure to antimicrobials and the
components of host immune system. Therefore, biofilm-associated infections (BAI) are hard to treat
and frequently require the removal of tissues or biomaterials colonized by the microorganisms, which
prolongs hospitalization and elevates the costs of treatment [
6
–
8
]. There is no doubt that it is much better
to prevent biofilm formation than to cure a BAI. However, because of antibiotic therapy shortage (the
21st century is even called the “post-antibiotic era”), very high drug-resistance among microorganisms
and the ability to induce resistance mechanisms, the usage of antibiotics for preventive purposes
should be limited. Thus, looking for alternative methods to inhibit microbial adhesion and biofilm
formation has recently attracted much attention. Some plant-origin products containing biologically
active secondary metabolites such as phenolic compounds possess the properties interesting in this
respect. According to Quave et al. [
8
], for instance, the fraction of butanol extract from the root of Rubus
ulmifolius Schott rich in ellagic acid and its derivatives inhibited S. aureus biofilm formation. Methanolic
extract from Opuntia ficus-indica (L.) Mill. reduced biofilm formation by Escherichia coli and S. aureus in a
dose-dependent manner [
9
]. Kaiser et al. [
10
] demonstrated that the isothiocyanates mixture consisted of
38% (v/v) allylisothiocyanate, 50% benzylisothiocyanate, and 12% phenylethyl-isothiocyanate according
to the proportion of nasturtium (Tropaeolum majus L.) and horseradish (Armoracia rusticana P. Gaertn., B.
Mey. & Scherb.) in the phytomedical preparation Angocin, used at sub-MIC significantly reduced
Pseudomonas aeruginosa biofilm biomass, bacterial metabolic activity, and proliferation. The extract from
the leaves of Eugenia uniflora L. was able to reduce the adhesion of Candida albicans and non-C. albicans
clinical isolates to human buccal epithelial cells, biofilm formation, and cell surface hydrophobicity [
11
].
Alternatively, originally obtained polyphenol-rich extract from Leonurus cardiaca L. (LCE) is the subject
of our interest, since motherwort herb possesses known beneficial effects on the heart and circulatory
system and is used to strengthen the heart muscle, regulate blood pressure and heart rhythm in such
herbal products as Cardiosan, Cardionervit, or Cardiogran. In our previous work we demonstrated that
S. aureus aggregate formation in plasma, microbial adherence to the deposit of fibrin network, plasma
clotting by staphylocoagulase, as well as expression of virulence factors participating in S. aureus
adhesion and biofilm formation (SpA, α-toxin) were negatively affected by LCE in a dose-dependent
manner. On the other hand, staphylococci exposed to LCE also showed higher tolerance to exogenous
hydrogen superoxide, which may help to avoid host immune response [12].
To confirm the legitimacy of LCE use in prevention or supporting the treatment of IE and
other systemic infections caused by staphylococci, the molecular mechanisms of the effect of LCE on
staphylococci–blood platelet interactions occurring in the growing vegetations were investigated in the
present study. The direct impact of LCE on S. aureus adhesive properties using atomic force microscopy,
lipid composition of staphylococcal cell membranes by HPLC, blood platelet adhesion to fibrinogen
using colorimetric assay, and the expression of their important receptors with FACS were assessed.
2. Results and Discussion
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
–
14
], showed that such secondary
Molecules 2019,24, 3318 3 of 15
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).
The force spectroscopy analysis revealed significant differences in the adhesive properties of S. aureus
after exposure to LCE (p<0.001) (Figure 2). Adhesion force were markedly reduced following LCE
treatment at both concentrations, with a substantial increase (ca. 16–32%) in the number of curves
exhibiting no adhesion (Figure 3). Interestingly, the highest decrease in adhesion force by 58% was
observed for the cells exposed on LCE at lower concentration (0.5
×
MIC). Thus, the adhesion properties
of S. aureus were substantially affected by the LCE. Moreover, the effect of LCE treatment on S. aureus
cell morphology was demonstrated (Figure 1). AFM is seen as a proper tool for the observation the
impact of antimicrobial preparations on single microbial cell morphology to better understand the
mechanisms of their activity [
17
,
18
]. In our study bacteria in native conditions existed mostly in clusters
and showed the typical near-spherical shape of the cells. The untreated cells had a relatively smooth
surface without pores or any ruptures in comparison with treated cells. Exposure to LCE caused the
deformation of the cell wall, with a visible increase of in roughness. Moreover, some cells collapsed,
indicating a loss of cellular content. The effect was tightly dependent on the LCE concentration, being
stronger for higher concentration. 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.
Molecules 2019, 24, x FOR PEER REVIEW 3 of 15
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 [12–14],
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). The force spectroscopy analysis
revealed significant differences in the adhesive properties of S. aureus after exposure to LCE (p < 0.001)
(Figure 2). Adhesion force were markedly reduced following LCE treatment at both concentrations,
with a substantial increase (ca. 16–32%) in the number of curves exhibiting no adhesion (Figure 3).
Interestingly, the highest decrease in adhesion force by 58% was observed for the cells exposed on
LCE at lower concentration (0.5 × MIC). Thus, the adhesion properties of S. aureus were substantially
affected by the LCE. Moreover, the effect of LCE treatment on S. aureus cell morphology was
demonstrated (Figure 1). AFM is seen as a proper tool for the observation the impact of antimicrobial
preparations on single microbial cell morphology to better understand the mechanisms of their
activity [17,18]. In our study bacteria in native conditions existed mostly in clusters and showed the
typical near-spherical shape of the cells. The untreated cells had a relatively smooth surface without
pores or any ruptures in comparison with treated cells. Exposure to LCE caused the deformation of
the cell wall, with a visible increase of in roughness. Moreover, some cells collapsed, indicating a loss
of cellular content. The effect was tightly dependent on the LCE concentration, being stronger for
higher concentration. 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.
Figure 1. Atomic force microscopy (AFM) images of S. aureus 8325-4 control cells and the cells exposed
on L. cardiaca L. extract (LCE) at 0.5 × MIC or 0.75 × MIC for 24 h.
Figure 1.
Atomic force microscopy (AFM) images of S. aureus 8325-4 control cells and the cells exposed
on L. cardiaca L. extract (LCE) at 0.5 ×MIC or 0.75 ×MIC for 24 h.
Cell-to-cell interactions, also including those between pathogens and host cells, as well as microbial
adhesion to abiotic surfaces depend on many factors, such as physical forces, surface hydrophobicity,
charge, diversity of microbial cell envelopes, expression of specific adhesins, and others [
4
,
19
–
21
].
Modifications in microbial cell wall and membrane composition may play an important role in those
processes, too. To verify this hypothesis the effect of LCE on the profile of the lipids in S. aureus cell
membrane using qualitative and quantitative tests were assessed. The cytoplasmic membranes of
Gram-positive bacteria, including Staphylococcus spp., very commonly contain phosphatidylglycerols
esterified with lysine, alanine, or ornithine [
22
,
23
]. The content of both phosphatidylglycerol (PG) and
lysyl-phosphatidylglycerol (LPG), and also cardiolipin (CL), was assessed in our study. In S. aureus cells
exposed to LCE the general level of LPG was growing at the expense of PG and CL as compared to
control (untreated) cells (Figure 4), however these differences seem very small. Therefore, it is hard to say
Molecules 2019,24, 3318 4 of 15
if observed modifications are sufficient to affect membrane function. On the other hand, it was reported
that in staphylococci exposed to antimicrobial cationic peptides, the charge of membrane was modulated
and an anionic PG was converted to cationic LPG. Such reaction was mediated by a membrane protein
called the multiple peptide resistance factor F (MprF), which transfers an aminoacyl group from
lysine-tRNA to PG [
22
,
23
]. The described modification is therefore the answer of staphylococci to stress
conditions, so exposure to LCE seems to be treated by S. aureus as a stress factor. Moreover, analyzing
the composition of the main phospholipid species (with content above 2%) in the cell membrane of
control and LCE-treated S. aureus (Figure 5), an almost 0.5-fold increase in the level of LPG 14:0/14:0
was demonstrated in LCE-exposed samples. Although, for other phospholipids different directions
of change were noticed. Small increase in the level of branched fatty acids (BCFAs) with isomerism
anteiso (aiC15:0) as compared with control cells was also observed (Figure 6). A mixture of BCFAs
and straight-chain fatty acids (SCFAs) additionally complicated by the presence of staphyloxanthin, a
triterpenoid carotenoid with a C30 chain being one of important S. aureus virulence factors, are the main
components of these bacteria membrane. BCFAs are produced de novo from the branched-chain amino
acids, such as isoleucine (anteiso odd-numbered fatty acids), leucine (iso odd-numbered fatty acids),
valine (iso even-numbered fatty acids), and the position of branching cannot be later modified. Thanks
to this, BCFAs disrupt the close packing of fatty acyl chains, thus their presence increases the fluidity of
cell membrane and prevents forming of crystal structures [
24
]. Therefore, it can be assumed that the
observed changes in the composition of phospholipids and fatty acids in the presence of LCE, followed
by membrane charge, fluidity, and hydrophobicity, at least partially determine the adhesive properties
of S. aureus cells, leading to the desired effects such as reduction of adhesion forces measured using
single-bacterial contact probe atomic force microscopy, and inhibition both of staphylococcal adhesion
and biofilm formation, which we demonstrated previously [
12
,
14
]. On the other hand, the same
modifications can cause the increased resistance of staphylococci to antimicrobial cationic peptides being
important component of innate immunity or cationic antibiotics (e.g., daptomycin), by changing the
charge of the bacterial surface [
22
,
23
]. Moreover, environmental factors, mainly chemical stress stimuli
targeting the cell wall or membrane, but also mechanical stress as this triggered by cell adhesion and
thus deformation of bacterial cell wall/membrane, can change staphylococcal genes expression by one-
or two-component regulatory systems, finally limiting the effects of stress conditions and promoting the
survival of microbial cells. It was shown, for instance, that the presence of nisin upregulates the NsaAB
efflux pump by NsaRS two-component regulatory system for more effective efflux of the antimicrobial
peptide from S. aureus cytoplasm [
25
]. We can assume that LCE may have a similar effect on genes
expression of staphylococci, modifying diverse cell properties.
Molecules 2019, 24, x FOR PEER REVIEW 4 of 15
Figure 2. Changes of S. aureus 8325-4 adhesive properties after exposure to L. cardiaca L. extract (LCE)
at 0.5 × MIC or 0.75 × MIC for 24 h, measured using atomic force microscopy (AFM). Statistical analysis
was estimated with nonparametric Kruskal
–
Wallis one-way ANOVA test. The differences were
significant in comparison to the control (p < 0.001).
Figure 3. Adhesion force histogram obtained for S. aureus 8325-4 control cells and the cells exposed
on L. cardiaca L. extract (LCE) at 0.5 × MIC or 0.75 × MIC for 24 h. Lines denote fitted function
describing normal distribution of experimental points. Mean ± standard deviation. N—number of
force
–
distance curve.
Cell-to-cell interactions, also including those between pathogens and host cells, as well as
microbial adhesion to abiotic surfaces depend on many factors, such as physical forces, surface
hydrophobicity, charge, diversity of microbial cell envelopes, expression of specific adhesins, and
others [4,19–21]. Modifications in microbial cell wall and membrane composition may play an
important role in those processes, too. To verify this hypothesis the effect of LCE on the profile of the
Figure 2.
Changes of S. aureus 8325-4 adhesive properties after exposure to L. cardiaca L. extract (LCE)
at 0.5
×
MIC or 0.75
×
MIC for 24 h, measured using atomic force microscopy (AFM). Statistical
analysis was estimated with nonparametric Kruskal–Wallis one-way ANOVA test. The differences
were significant in comparison to the control (p<0.001).
Molecules 2019,24, 3318 5 of 15
Molecules 2019, 24, x FOR PEER REVIEW 4 of 15
Figure 2. Changes of S. aureus 8325-4 adhesive properties after exposure to L. cardiaca L. extract (LCE)
at 0.5 × MIC or 0.75 × MIC for 24 h, measured using atomic force microscopy (AFM). Statistical analysis
was estimated with nonparametric Kruskal
–
Wallis one-way ANOVA test. The differences were
significant in comparison to the control (p < 0.001).
Figure 3. Adhesion force histogram obtained for S. aureus 8325-4 control cells and the cells exposed
on L. cardiaca L. extract (LCE) at 0.5 × MIC or 0.75 × MIC for 24 h. Lines denote fitted function
describing normal distribution of experimental points. Mean ± standard deviation. N—number of
force
–
distance curve.
Cell-to-cell interactions, also including those between pathogens and host cells, as well as
microbial adhesion to abiotic surfaces depend on many factors, such as physical forces, surface
hydrophobicity, charge, diversity of microbial cell envelopes, expression of specific adhesins, and
others [4,19–21]. Modifications in microbial cell wall and membrane composition may play an
important role in those processes, too. To verify this hypothesis the effect of LCE on the profile of the
Figure 3.
Adhesion force histogram obtained for S. aureus 8325-4 control cells and the cells exposed
on L. cardiaca L. extract (LCE) at 0.5
×
MIC or 0.75
×
MIC for 24 h. Lines denote fitted function
describing normal distribution of experimental points. Mean
±
standard deviation. N—number of
force–distance curve.
Molecules 2019, 24, x FOR PEER REVIEW 6 of 15
Figure 4. Composition of cell membrane phospholipid classes of control S. aureus 8325-4 cells or the
cells treated with L. cardiaca L. extract (LCE) at 0.5 × MIC or 0.75 × MIC for 24 h, measured by HPLC
–
MS. PG—phosphatidylglycerol, LPG—lysyl-phosphatidylglycerol, CL—cardiolipin.
Figure 5. Content of the main phospholipid species (achieving above 2% in control) in cell membrane
of control S. aureus 8325-4 cells or the cells treated with L. cardiaca L. extract (LCE) at 0.5 × MIC or 0.75
× MIC for 24 h, measured by HPLC
–
MS. PG—phosphatidylglycerol, LPG—lysyl-
phosphatidylglycerol, CL—cardiolipin.
Figure 4.
Composition of cell membrane phospholipid classes of control S. aureus 8325-4 cells or the
cells treated with L. cardiaca L. extract (LCE) at 0.5
×
MIC or 0.75
×
MIC for 24 h, measured by HPLC–MS.
PG—phosphatidylglycerol, LPG—lysyl-phosphatidylglycerol, CL—cardiolipin.
Molecules 2019,24, 3318 6 of 15
Molecules 2019, 24, x FOR PEER REVIEW 6 of 15
Figure 4. Composition of cell membrane phospholipid classes of control S. aureus 8325-4 cells or the
cells treated with L. cardiaca L. extract (LCE) at 0.5 × MIC or 0.75 × MIC for 24 h, measured by HPLC
–
MS. PG—phosphatidylglycerol, LPG—lysyl-phosphatidylglycerol, CL—cardiolipin.
Figure 5. Content of the main phospholipid species (achieving above 2% in control) in cell membrane
of control S. aureus 8325-4 cells or the cells treated with L. cardiaca L. extract (LCE) at 0.5 × MIC or 0.75
× MIC for 24 h, measured by HPLC
–
MS. PG—phosphatidylglycerol, LPG—lysyl-
phosphatidylglycerol, CL—cardiolipin.
Figure 5.
Content of the main phospholipid species (achieving above 2% in control) in cell
membrane of control S. aureus 8325-4 cells or the cells treated with L. cardiaca L. extract (LCE)
at 0.5
×
MIC or 0.75
×
MIC for 24 h, measured by HPLC–MS. PG—phosphatidylglycerol,
LPG—lysyl-phosphatidylglycerol, CL—cardiolipin.
The situation
in vivo
is much more complicated because of cell–cell, cell–protein, and cell–pathogen
interactions. In the development of IE and some other cardiovascular diseases (CVD), such components as
endothelium, platelets, immunocompetent cells, fibrinogen (Fg)/fibrin (Fb), physiological platelet agonists
(e.g., adenosine diphosphate (ADP), arachidonic acid (AA), thrombin, collagen), and microorganisms
are involved [
1
,
3
,
26
]. To define comprehensively an activity of L. cardiaca L. extract after potential oral
administration the effect of LCE on blood platelets—the cells pivotal in IE pathogenesis—was assessed ex
vivo. Both platelet adhesion to Fg and selected receptors expression (P-selectin, GP IIb/IIIa) on activated
platelets were evaluated. Taking into consideration the limited intestinal absorption and availability in
other tissues of plant-origin preparations after oral administration, as well as their bioconversion by
intestinal microbiota [
27
,
28
], such high concentrations as previously used for bacteria, 4.5 mg/mL, which
correspond with 0.75
×
MIC and 3 mg/mL (0.5
×
MIC), do not seem possible to achieve in tissue. Therefore,
we decided to use much lower concentrations (50–350
µ
g/mL) totest the LCE impact on eukaryotic cells. It
was shown that the adhesion of freshly isolated blood platelets to fibrinogen in the presence of LCE used
at the whole range of tested concentrations decreased by 9.98
±
4.51% to 31.50
±
5.09%. The inhibitory
effect of LCE was concentration-dependent and the observed differences were statistically significant
(Figure 7). Because of platelet adhesion to Fg/Fb mediates their aggregation and thus activation [
29
–
31
],
we demonstrated anti-aggregative and inactivating properties of LCE relative to blood platelets, pointing
at the same time on the disruption of Fg–platelet interactions as the most probable mechanism of such
motherwort extract activity. This hypothesis has also been supported by the results of testing the LCE effect
on the expression of platelet surface receptors as another relevant determinant of these cells activation.
The expression of CD62P (P-selectin) and CD41 (GP IIb/IIIa) on ADP-activated platelets was assessed
using flow cytometry (representative dot plots for cytometry analysis are presented on Figure 8). There
was no impact of LCE on the level of P-selectin expression on ADP-activated cells, but in response to
the growing concentration of extract the expression of GP IIb/IIIa on platelet surface was reduced (max.
Molecules 2019,24, 3318 7 of 15
by 35% at 350
µ
g/mL of LCE; Figure 9). The percentage of platelets expressing GP IIb/IIIa was also
significantly reduced (by 11–29%) in the population of these cells after LCE treatment in comparison to
the control cells. Moreover, LCE used at the highest concentration (350
µ
g/mL) significantly reduced also
a percentage of platelets with P-selectin expression (Figure 10). Since CD41 is an integrin complex acting
as a receptor for fibrinogen [
29
,
31
], these results correlate very well with observed decrease in platelet
adhesion to fibrinogen in the presence of LCE. Thus, it can be assumed that molecular mechanism of
antiplatelet effect of L. cardiaca L. extract is based on the disruption of platelets-fibrinogen interaction
by LCE ability to reduce GP IIb/IIIa expression and the number of the cells among whole population
expressing this fibrinogen receptor.
Molecules 2019, 24, x FOR PEER REVIEW 7 of 15
Figure 6. Composition of cell membrane fatty acids of control S. aureus 8325-4 cells or the cells treated
with L. cardiaca L. extract (LCE) at 0.5 × MIC or 0.75 × MIC for 24 h, measured by HPLC
–
MS. iC/aiC—
branched fatty acids with isomerism, respectively, iso/anteiso.
The situation in vivo is much more complicated because of cell–cell, cell–protein, and cell–
pathogen interactions. In the development of IE and some other cardiovascular diseases (CVD), such
components as endothelium, platelets, immunocompetent cells, fibrinogen (Fg)/fibrin (Fb),
physiological platelet agonists (e.g., adenosine diphosphate (ADP), arachidonic acid (AA), thrombin,
collagen), and microorganisms are involved [1,3,26]. To define comprehensively an activity of L.
cardiaca L. extract after potential oral administration the effect of LCE on blood platelets—the cells
pivotal in IE pathogenesis—was assessed ex vivo. Both platelet adhesion to Fg and selected receptors
expression (P-selectin, GP IIb/IIIa) on activated platelets were evaluated. Taking into consideration
the limited intestinal absorption and availability in other tissues of plant-origin preparations after
oral administration, as well as their bioconversion by intestinal microbiota [27,28], such high
concentrations as previously used for bacteria, 4.5 mg/mL, which correspond with 0.75 × MIC and 3
mg/mL (0.5 × MIC), do not seem possible to achieve in tissue. Therefore, we decided to use much
lower concentrations (50–350 µg/mL) to test the LCE impact on eukaryotic cells. It was shown that
the adhesion of freshly isolated blood platelets to fibrinogen in the presence of LCE used at the whole
range of tested concentrations decreased by 9.98 ± 4.51% to 31.50 ± 5.09%. The inhibitory effect of LCE
was concentration-dependent and the observed differences were statistically significant (Figure 7).
Because of platelet adhesion to Fg/Fb mediates their aggregation and thus activation [29–31], we
demonstrated anti-aggregative and inactivating properties of LCE relative to blood platelets,
pointing at the same time on the disruption of Fg–platelet interactions as the most probable
mechanism of such motherwort extract activity. This hypothesis has also been supported by the
results of testing the LCE effect on the expression of platelet surface receptors as another relevant
determinant of these cells activation. The expression of CD62P (P-selectin) and CD41 (GP IIb/IIIa) on
ADP-activated platelets was assessed using flow cytometry (representative dot plots for cytometry
analysis are presented on Figure 8). There was no impact of LCE on the level of P-selectin expression
on ADP-activated cells, but in response to the growing concentration of extract the expression of GP
IIb/IIIa on platelet surface was reduced (max. by 35% at 350 µg/mL of LCE; Figure 9). The percentage
Figure 6.
Composition of cell membrane fatty acids of control S. aureus 8325-4 cells or the cells
treated with L. cardiaca L. extract (LCE) at 0.5
×
MIC or 0.75
×
MIC for 24 h, measured by HPLC–MS.
iC/aiC—branched fatty acids with isomerism, respectively, iso/anteiso.
Molecules 2019, 24, x FOR PEER REVIEW 8 of 15
of platelets expressing GP IIb/IIIa was also significantly reduced (by 11–29%) in the population of
these cells after LCE treatment in comparison to the control cells. Moreover, LCE used at the highest
concentration (350 µg/mL) significantly reduced also a percentage of platelets with P-selectin
expression (Figure 10). Since CD41 is an integrin complex acting as a receptor for fibrinogen [29,31],
these results correlate very well with observed decrease in platelet adhesion to fibrinogen in the
presence of LCE. Thus, it can be assumed that molecular mechanism of antiplatelet effect of L. cardiaca
L. extract is based on the disruption of platelets-fibrinogen interaction by LCE ability to reduce GP
IIb/IIIa expression and the number of the cells among whole population expressing this fibrinogen
receptor.
Figure 7. Effect of L. cardiaca L. extract (LCE) on human blood platelet adhesion to fibrinogen. The
results are presented as a percentage of control for single individuals and expressed also as a median
% ± SD. Statistical analysis was estimated with nonparametric Kruskal
–
Wallis one-way ANOVA test
with Bonfferoni's correction (significant differences: *p < 0.05; **p < 0.01, ***p < 0.001).
Figure 7.
Effect of L. cardiaca L. extract (LCE) on human blood platelet adhesion to fibrinogen. The results
are presented as a percentage of control for single individuals and expressed also as a median
%±SD.
Statistical analysis was estimated with nonparametric Kruskal–Wallis one-way ANOVA test with
Bonfferoni’s correction (significant differences: * p<0.05; ** p<0.01, *** p<0.001).
Molecules 2019,24, 3318 8 of 15
Molecules 2019, 24, x FOR PEER REVIEW 8 of 15
of platelets expressing GP IIb/IIIa was also significantly reduced (by 11–29%) in the population of
these cells after LCE treatment in comparison to the control cells. Moreover, LCE used at the highest
concentration (350 µg/mL) significantly reduced also a percentage of platelets with P-selectin
expression (Figure 10). Since CD41 is an integrin complex acting as a receptor for fibrinogen [29,31],
these results correlate very well with observed decrease in platelet adhesion to fibrinogen in the
presence of LCE. Thus, it can be assumed that molecular mechanism of antiplatelet effect of L. cardiaca
L. extract is based on the disruption of platelets-fibrinogen interaction by LCE ability to reduce GP
IIb/IIIa expression and the number of the cells among whole population expressing this fibrinogen
receptor.
Figure 7. Effect of L. cardiaca L. extract (LCE) on human blood platelet adhesion to fibrinogen. The
results are presented as a percentage of control for single individuals and expressed also as a median
% ± SD. Statistical analysis was estimated with nonparametric Kruskal
–
Wallis one-way ANOVA test
with Bonfferoni's correction (significant differences: *p < 0.05; **p < 0.01, ***p < 0.001).
Figure 8.
Representative dot plots for flow cytometry analysis. (
a
,
c
) Control: P-selectin and GP IIb/IIIa
expression on platelet surface after ADP activation. (
b
,
d
) P-selectin and GP IIb/IIIa expression on
platelet surface after ADP activation preceded by platelets co-incubation with L. cardiaca L. extract
(LCE) used at 350 µg/mL.
Molecules 2019, 24, x FOR PEER REVIEW 9 of 15
Figure 8. Representative dot plots for flow cytometry analysis. (a), (c) Control: P-selectin and GP
IIb/IIIa expression on platelet surface after ADP activation. (b), (d) P-selectin and GP IIb/IIIa
expression on platelet surface after ADP activation preceded by platelets co-incubation with L.
cardiaca L. extract (LCE) used at 350 µg/mL.
Figure 9. Effect of L. cardiaca L. extract (LCE) on human blood platelet receptor expression. The results
are presented as a percentage of relative fluorescence unit (RFU) in comparison to control (RFU
considered as 100%) and expressed as a mean % ± SEM. Statistical analysis was estimated with
nonparametric Kruskal
–
Wallis one-way ANOVA test with Bonfferoni's correction (significant
differences: *p< 0.05).
Figure 10. Effect of L. cardiaca L. extract (LCE) on human blood platelet activation. The results are
presented as a percentage of activated cells (with P-selectin or GP IIb/IIIa exposure) in comparison to
total amount of analyzed cells and expressed as the mean ± SEM. Statistical analysis was estimated
with nonparametric Kruskal
–
Wallis one-way ANOVA test with Bonfferoni's correction (significant
differences: *p < 0.05; **p < 0.01, ***p < 0.001).
Blood platelet activation is a basic process of hemostasis but is also involved in some
pathological changes, such as cardiovascular disorders (e.g., atherosclerosis, thrombosis, IE).
Intensified blood platelet aggregation is noted in obese people and diabetics, which increases the risk
of CVD. In such cases a therapy with antiplatelet drugs (e.g., aspirin) is usually applied. Dietary
recommendations to enrich our food with flavonoids, being biologically active components of many
plant products (including tested LCE), are other unconventional but quite popular strategies to
reduce CVD risk [15,32,33]. Wright et al. [34] showed that such flavonoids as quercetin, apigenin,
tamarixetin, and galangin were able to inhibit the aggregation of platelets in plasma (PRP).
Polyphenol-rich extracts from berries of Aronia melanocarpa (Michx.) Elliott (black chokeberry) and
from grape seeds reduced blood platelet adhesion to collagen and fibrinogen, the platelet aggregation
Figure 9.
Effect of L. cardiaca L. extract (LCE) on human blood platelet receptor expression. The results are
presented as a percentage of relative fluorescence unit (RFU) in comparison to control (RFU considered
as 100%) and expressed as a mean %
±
SEM. Statistical analysis was estimated with nonparametric
Kruskal–Wallis one-way ANOVA test with Bonfferoni’s correction (significant differences: * p<0.05).
Molecules 2019,24, 3318 9 of 15
Molecules 2019, 24, x FOR PEER REVIEW 9 of 15
Figure 8. Representative dot plots for flow cytometry analysis. (a), (c) Control: P-selectin and GP
IIb/IIIa expression on platelet surface after ADP activation. (b), (d) P-selectin and GP IIb/IIIa
expression on platelet surface after ADP activation preceded by platelets co-incubation with L.
cardiaca L. extract (LCE) used at 350 µg/mL.
Figure 9. Effect of L. cardiaca L. extract (LCE) on human blood platelet receptor expression. The results
are presented as a percentage of relative fluorescence unit (RFU) in comparison to control (RFU
considered as 100%) and expressed as a mean % ± SEM. Statistical analysis was estimated with
nonparametric Kruskal
–
Wallis one-way ANOVA test with Bonfferoni's correction (significant
differences: *p< 0.05).
Figure 10. Effect of L. cardiaca L. extract (LCE) on human blood platelet activation. The results are
presented as a percentage of activated cells (with P-selectin or GP IIb/IIIa exposure) in comparison to
total amount of analyzed cells and expressed as the mean ± SEM. Statistical analysis was estimated
with nonparametric Kruskal
–
Wallis one-way ANOVA test with Bonfferoni's correction (significant
differences: *p < 0.05; **p < 0.01, ***p < 0.001).
Blood platelet activation is a basic process of hemostasis but is also involved in some
pathological changes, such as cardiovascular disorders (e.g., atherosclerosis, thrombosis, IE).
Intensified blood platelet aggregation is noted in obese people and diabetics, which increases the risk
of CVD. In such cases a therapy with antiplatelet drugs (e.g., aspirin) is usually applied. Dietary
recommendations to enrich our food with flavonoids, being biologically active components of many
plant products (including tested LCE), are other unconventional but quite popular strategies to
reduce CVD risk [15,32,33]. Wright et al. [34] showed that such flavonoids as quercetin, apigenin,
tamarixetin, and galangin were able to inhibit the aggregation of platelets in plasma (PRP).
Polyphenol-rich extracts from berries of Aronia melanocarpa (Michx.) Elliott (black chokeberry) and
from grape seeds reduced blood platelet adhesion to collagen and fibrinogen, the platelet aggregation
Figure 10.
Effect of L. cardiaca L. extract (LCE) on human blood platelet activation. The results are
presented as a percentage of activated cells (with P-selectin or GP IIb/IIIa exposure) in comparison to
total amount of analyzed cells and expressed as the mean
±
SEM. Statistical analysis was estimated
with nonparametric Kruskal–Wallis one-way ANOVA test with Bonfferoni’s correction (significant
differences: * p<0.05; ** p<0.01, *** p<0.001).
Blood platelet activation is a basic process of hemostasis but is also involved in some pathological
changes, such as cardiovascular disorders (e.g., atherosclerosis, thrombosis, IE). Intensified blood
platelet aggregation is noted in obese people and diabetics, which increases the risk of CVD. In such cases
a therapy with antiplatelet drugs (e.g., aspirin) is usually applied. Dietary recommendations to enrich
our food with flavonoids, being biologically active components of many plant products (including
tested LCE), are other unconventional but quite popular strategies to reduce CVD risk [
15
,
32
,
33
].
Wright et al. [34]
showed that such flavonoids as quercetin, apigenin, tamarixetin, and galangin
were able to inhibit the aggregation of platelets in plasma (PRP). Polyphenol-rich extracts from
berries of Aronia melanocarpa (Michx.) Elliott (black chokeberry) and from grape seeds reduced blood
platelet adhesion to collagen and fibrinogen, the platelet aggregation and superoxide anion radicals
production at
in vitro
model of hyperhomocysteinemia, which suggests their protective potential [
35
].
Rahman et al. [36]
showed antiplatelet activity of aged garlic extract, which inhibited the binding of
activated platelets to fibrinogen and thus preventing their shape changes by increasing in the amounts
of cyclic nucleotides (cGMP, cAMP) as intracellular signaling molecules. Epidemiological
in vivo
studies on the diet effect on CVD demonstrated that consumption of fruit and berries by elderly men
impacted on the intima–media thickness (IMT) of the carotid artery and may be protective against
carotid atherosclerosis [
37
]. Since recently, Fruitflow containing water-soluble tomato extract as a source
of lycopene able to inhibit platelet aggregation stimulated by ADP or collagen has been commercially
available in Europe as the first natural product with cardioprotective action [
38
]. The results of
our
in vitro
and ex vivo studies indicate relevant L. cardiaca L. extract ability to change adhesive
properties of staphylococci and to prevent of platelet activation, which may be beneficial
in vivo
to
protect the host against the development of serious infection-associated cardiovascular disorders such
as vegetations growing or intravascular coagulation. Moreover, in previously published works on
health-promoting effect of plant-origin preparations mainly attention has been paid to their antioxidant
properties [
35
,
37
,
38
], while we demonstrated other mechanisms of motherwort extract favorable
activity (the modifications of eukaryotic cell surface receptors expression and microbial membranes
composition). However, the concentrations of herbal products in practice achieved
in vivo
are probably
the most difficult problem to solve to determine their pro-health doses. After our
in vitro
and ex vivo
studies we can only point to certain range of concentrations, which should be exceeded to exert positive
effects, e.g., above 50
µ
g/mL LCE will be health-promoting because of the antiplatelet effect. We still do
not know how to achieve such concentrations in target tissues. Moreover, since the activity of even a
Molecules 2019,24, 3318 10 of 15
single compound depends on many factors (e.g., the structure, time of platelets exposure, presence of
plasma proteins) [
34
],
in vivo
testing such complex preparations as LCE to assess their bioavailability,
pharmacodynamics and pharmacokinetics seem to be necessary.
3. Materials and Methods
3.1. Bacterial Strain
Staphylococcus aureus 8325-4 derived from NCTC 8325, ATCC 35556 parent strain, was
used. The strain has been described as expressing of surface-associated adhesive molecules
such as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), SpA
(staphylococcal protein A), and ClfA (clumping factor A). Bacteria from frozen stock were grown for
24 h at 37
◦
C on Tryptic Soy Agar (TSA; BTL, Poland) as a primary culture. Then ready-to-use culture
at a required density was freshly prepared in Tryptic Soy Broth (BTL, Poland) with 0.25% glucose
(TSB/Glu) using spectrophotometer (Densi-La-Meter II, Erba Lachema, Czech Republic).
3.2. Platelets
The study involved seven healthy volunteers and was conducted according to the approval of the
University Commission for Bioethics Research No. KBBN-UŁ/II/26/2012. The interview before the
study concerned the lack of taking any anticoagulants for 30 days and the exclusion of thrombotic
disorders. Blood taken from the ulnar vein was collected into tubes with sodium citrate and centrifuged
for 15 min at 190×gat 37 ◦C to obtain platelet rich plasma (PRP).
3.3. Preparation of L. cardiaca L. Extract (LCE) Solutions
Commercially available L. cardiaca L. (Motherwort) basic plant material (KAWON-HURT Nowak
Sp.j., Gosty ´n, Poland) was extracted and chemically characterized as we previously described [
14
].
Stock solution of polyphenol-enriched LCE was prepared in 50% DMSO and then diluted in appropriate
liquid media up to the concentrations used in each experiment.
3.4. LCE Impact on S. Aureus Adhesive Properties Tested Using Atomic Force Microscopy (AFM)
S. aureus 8325-4 cells obtained by centrifugation (2500 rpm/10 min) from 1 mL of microbial
suspension at OD
535
=1.8 were exposed to LCE (0.5 mL) at a concentration of 4.5 mg/mL, which
correspond with 0.75
×
MIC (minimum inhibitory concentration) and 3 mg/mL (0.5
×
MIC) for 24 h at
37
◦
C. Stock solution of LCE (90 mg/mL in 50% DMSO) was diluted 20 or 30 times in TSB/Glu to obtain
the final LCE concentrations (4.5 or 3 mg/mL, respectively). The highest concentration of DMSO as
LCE primary solvent reached then 2.5%. Therefore, the control bacteria were prepared in TSB/Glu
containing 2.5% DMSO to exclude any solvent effect on microbial viability. After exposure, bacteria
were centrifuged (in conditions as above), suspended in 0.5 mL fresh TSB/Glu and supplied to the
Center of Quantum Optics at the Faculty of Physics, Astronomy and Informatics, Nicolaus Copernicus
University in Toru´n (Poland) to perform AFM measurements.
S. aureus treated with LCE and untreated (control) were immobilized on poly-l-lysine (PLL,
Sigma-Aldrich, Saint Louis, MO, USA) coated glass surfaces. Cell suspensions in phosphate-buffered
saline (PBS) were deposited for 45 min. on PLL-coated slides. Loosely bound cells were removed by
thorough rinsing with fresh Milli-Q water. Samples were used immediately for force spectroscopy
experiments. AFM measurements were performed in PBS with a Bioscope II AFM equipped with a
NanoScope V controller (Veeco) and using an MLCT-D silicon nitride cantilever with a nominal tip
apex radius of 20 nm. The cantilevers were calibrated prior to the experiments by a thermal noise
method, an average spring constant was 0.03 N/m. To localize individual cells, images were obtained
in an intermittent contact mode. Force spectroscopy measurements were then performed in the contact
mode. At least 5 force-distance curves (each curve in a different location) were collected for each within
a batch of 30–50 different bacterial cells, using a maximum applied force of 1 nN, a constant retraction
Molecules 2019,24, 3318 11 of 15
speed of 2
µ
m/s and retract delay 1 s. Adhesion forces were calculated as described before [
39
]. Control
force–distance curves were registered on bacteria-free area before and after proper measurements on
bacterial cells to confirm that the AFM tip had not been contaminated by bacterial biopolymers. Force
curves were analyzed in NanoScope Analysis 1.7 software (Bruker, Billerica, MA, USA). The effect of
the LCE on bacteria cell were obtained in contact mode in air. The topography and deflection images
were obtained simultaneously at a can rate of 0.5 Hz at a resolution of 512 pixel per line on 5
×
5
µ
m
2
area. The data were analyzed with Gwyddion 2.47 software [40].
3.5. The Effect of LCE on Staphylococcal Cell Membrane Lipid Profile
S. aureus 8325-4 cells obtained by centrifugation (2500 rpm/10 min) from 8 mL of microbial
suspension at OD
535
=1.8 were exposed to LCE (8 mL) at a concentration of 4.5 mg/mL (0.75
×
MIC)
and 3 mg/mL (0.5
×
MIC) for 24 h at 37
◦
C with shaking. Stock solution of LCE (90 mg/mL in 50%
DMSO) was diluted as described in Section 3.4. The highest concentration of DMSO as primary solvent
reached then 2.5%. Therefore, the control bacteria were prepared in TSB/Glu containing 2.5% DMSO to
exclude any solvent effect on microbial viability. Then, each sample was divided into 4 tubes of 2 mL
and analyzed by chromatography and mass spectrometry as described below.
3.5.1. Chemicals
The following reagents were used: 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol)
(sodium salt) (14:0/14:0 PG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (12:0/12:0 PE) and
1’,3’-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (sodium salt) (14:0 Cl) purchased from
Avanti Polar Lipids (Alabaster, AL, USA). The other chemicals were acquired from Sigma-Aldrich
(Saint Louis, MO, USA), and POCh (Gliwice, Poland). All chemicals were high purity grade reagents.
3.5.2. Phosholipid Extraction and HPLC Analysis
Phospholipids (PLs) from S. aureus suspensions were extracted according to the method proposed
by Folch et al. [
41
], with some modifications. The bacterial biomass was transferred into Eppendorf
tubes containing glass beads, 0.66 mL of chloroform and 0.33 mL of methanol. The homogenization
process using a ball mill (FastPrep) was carried out for 1 min. The mixture was extracted for 2 min.
In order to facilitate the separation of two layers, 0.2 mL of 0.9% saline was added. The lower layer
was collected and evaporated. The phospholipid extracts were dissolved in 750
µ
L of chloroform:
methanol (1:9, v/v) solution.
The polar lipids were measured using an Agilent 1200 HPLC system (Santa Clara, CA, USA)
and a 4500 QTRAP mass spectrometer (Sciex, Torrance, CA, USA) with an ESI source. For the
reversed-phase chromatographic analysis, 10
µ
L of the lipid extract was injected onto a Kinetex C18
column
(50 mm ×2.1 mm,
particle size: 5
µ
m; Phenomenex, Torrance, CA, USA). The mobile phase
consisted of 5-mM ammonium formate in water (A) and 5-mM ammonium formate in methanol (B).
The solvent gradient was initiated at 70% B, increased to 95% B over 1.25 min, and maintained at 95%
B for 16 min before returning to the initial solvent composition over 3 min. The column temperature
was maintained at 40
◦
C, and the flow rate was 500
µ
L/min. The instrumental settings were as follows:
spray voltage
−
4.500 V, curtain gas (CUR) 25, nebulizer gas (GS1) 50, turbo gas (GS2) 60, and ion source
temperature of 600 ◦C. The data analysis was performed with the Analyst™v1.6.2 software (Sciex).
Two approaches were applied to identify PLs: targeted and untargeted. The untargeted approach
was performed with the precursor ion scanning (precursor for m/z153) survey scan, triggering the EPI
experiments. On the basis of the untargeted analysis, a comprehensive list of the multiple reaction
monitoring (MRM) transitions was generated (parent fatty acyl fragment) for the following PL classes:
phosphatidylglycerol (PG), lysyl-phosphatidylglycerol (LPG), and cardiolipin (CL).
The LC-MS/MS method was validated with the standards of (14:0/14:0 PG), (12:0/12:0 PE) and
(14:0 CL). The LOD range of phospholipid reference compound varied between 3 ng/mL for PG,
5 ng/mL for PE, and 35 ng/mL for CL. CL was the most difficult lipid to quantify, since the shape of
Molecules 2019,24, 3318 12 of 15
the peaks. The lipids belonging to each class in examined samples were quantified upon comparison
with the standard of the relevant class. The percent coefficient of variation was less than 22% for
all phospholipids.
3.5.3. Fatty Acids Extraction and GC-MS Analysis
S. aureus cell pellet in Eppendorf tube was diluted in mixture of methanol (0.75 mL), toluene
(0.1 mL), and 8.0% HCl solution in methanol (0.15 mL) [
42
]. The tube was vortexed and then incubated
overnight at 45
◦
C. After cooling to room temperature, 1 mL of hexane and 1 mL of water were added
for the extraction of fatty acid methyl esters (FAMEs). The tube was vortexed, and then 0.3 mL of
hexane layer was moved to the chromatographic vial. 1
µ
L of the extract samples were analyzed using
gas chromatography.
FAMEs analysis was performed with a Agilent Model 7890 gas chromatograph, equipped with
a 5975C mass detector. The separation was carried out in the capillary column HP 5 MS methyl
polysiloxane (30 m
×
0.25 mm i.d.
×
0.25 mm ft). The column temperature was maintained at 60
◦
C for
3 min, then increased to 212
◦
C at the rate of 6
◦
C/min, followed by an increase to 245
◦
C at the rate
of 2
◦
C/min, and finally to 280
◦
C at the rate of 20
◦
C/min. The column temperature was maintained
at 280
◦
C for 10 min. Helium was used as the carrier gas at the flow rate of 1 mL/min. The injection
port temperature was 250
◦
C. Split injection was employed. Bacterial fatty acids were identified by
comparison with the retention times of the authentic standards (Sigma, Supelco, Sigma-Aldrich) or
by their mass spectra, and finally, the results were expressed as a percentage of the total amount of
fatty acids.
3.6. LCE Influence on Blood Platelet Adhesion to Fibrinogen
The suspension of blood platelets (1
×
10
8
cells/mL) treated with LCE at concentration of 50,
100, and 350
µ
g/mL or untreated (control) were applied to the wells of culture 96-well plate (Nunc,
Denmark) previously coated with fibrinogen as described by Micota et al. [
14
]. The samples were
incubated for 1 hour at 37
◦
C, 5% CO
2
. Then the wells were washed with 200
µ
L of TBS followed
by addition of 50 mL lysis buffer for 10 min (stirring constantly). The supernatants (25
µ
L) were
transferred to a new 96-well plate to assess the protein concentration using colorimetric Bicinchoninic
Acid Protein Assay Kit (Sigma).
3.7. Flow Cytometric Analysis of Blood Platelets Activation after Exposure to LCE
PRP was exposed on LCE at a concentration of 50, 100, and 350
µ
g/mL with the highest DMSO
concentration of 0.2%, which has no effect of cell viability. To the control PRP Tris-buffered saline (TBS;
50 mM Tris-HCl, 150 mM NaCl, pH 7.6) was added. All samples were stimulated with 20 mM ADP
(Sigma) at 37
◦
C for 5 min. Then the samples were transferred to cytometric tubes and 10
µ
L mouse
monoclonal Ab anti human CD62P (P-selectin) labeled with AlexaFluor 488 (Bio-Rad, Hercules, CA,
USA) and 10
µ
L mouse monoclonal Ab anti human CD41 (GP IIb/IIIa) labeled with PE (Bio-Rad) were
added. The required isotypic controls were included. The samples were incubated for 30 min on ice
and then analyzed using LSR II Digital Analytical Flow Cytometer (Becton Dickinson, Franklin Lakes,
NJ, USA).
3.8. Statistics
The results were analyzed for significance using nonparametric Kruskal–Wallis one-way ANOVA
test and the program Statistica 12.0 (Stat Soft Inc., Tulsa Shock, OK, USA). The differences with p<0.05
were considered to be statistically significant.
Molecules 2019,24, 3318 13 of 15
4. Conclusions
The results of our
in vitro
and ex vivo studies demonstrated essential L. cardiaca L. extract
biological activity against both staphylococcal adhesion and blood platelet activation, confirming its
pro-health potential. Molecular mechanism of LCE antiplatelet effect was based on the disruption of
platelet–fibrinogen interactions by altering GP IIb/IIIa expression. We also showed the effect of LCE on
the morphology of S. aureus cells, as well as the composition of phospholipids and fatty acids in their
membranes, which may both positive and negative consequences. Thus, by an application of LCE (e.g.,
as an addition to the daily diet) staphylococcal adhesion, aggregation, and biofilm formation could be
inhibited. However, the resistance of these bacteria to some antibiotics may be simultaneously enhanced,
which indicates the necessity of exclusion the cationic antibiotics (e.g., daptomycin) from infection
treatment, when motherwort herb is used at the same time. Therefore, improved understanding the
mechanisms of LCE activity may serve to develop proper recommendations for the use of herbal
products based on L. cardiaca L. extracts as preventive preparations or supportive products for classic
antibiotics in cases of infective endocarditis and other invasive infections.
Author Contributions:
Conceptualization, B.S.; methodology, D.L., P.B., B.M., M.W.-S. and A.P.; formal analysis,
B.S., D.L., P.B.; investigation, B.S., D.L., P.B., B.M. and M.W.-S.; resources, A.P.; data curation, B.S., D.L., P.B. and
B.M.; writing—original draft preparation, B.S.; writing—review and editing, B.S.; visualization, B.S., D.L. and
B.M.; supervision, B.R.; project administration, B.S.; funding acquisition, B.S., B.M. and B.R.
Funding:
The research was partially funded by The National Science Center, Poland, grant number
2013/09/N/NZ6/00826.
Conflicts of Interest:
The authors declare no conflict of interest. The funder had no role in the design of the study;
in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish
the results.
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Sample Availability: Samples of LCE are not available.
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