Phytomedicine 22 (2015) 1172–1177

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Phytomedicine
journal homepage: www.elsevier.com/locate/phymed

Bronchipret® syrup containing thyme and ivy extracts suppresses

bronchoalveolar inflammation and goblet cell hyperplasia in
experimental bronchoalveolitis
Jan Seibel a,∗, Carlo Pergola b, Oliver Werz b, Kirill Kryshen c, Katja Wosikowski a,
Martin D. Lehner a, Jutta Haunschild a
a
Preclinical R&D, Bionorica SE, Kerschensteinerstr. 11-15, D-92318 Neumarkt, Germany
b
Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, D-07743 Jena, Germany
c
Saint-Petersburg Institute of Pharmacy, Leningrad Region, Vsevolozhsky District, 188663, Kuzmolovo P 245, Russia

a r t i c l e i n f o a b s t r a c t

Article history: Background/purpose: Acute bronchitis (AB) is a common lung condition characterized by inflammation of
Received 12 January 2015 the large bronchi in response to infection. Bronchipret® syrup (BRO), a fixed combination of thyme and ivy
Revised 12 August 2015
extracts has been effectively used for the treatment of AB. Combining in vivo and mechanistic in vitro studies
Accepted 2 September 2015
we aimed to provide a better understanding of the therapeutic potential of BRO on key aspects of AB and to
identify potential mechanisms of action.
Keywords: Methods: Bronchoalveolitis in rats was induced by intratracheal LPS instillation. BRO was administered p.o.
Acute bronchitis once daily at 1- to 10-fold equivalents of the human daily dose. Animals were sacrificed 24–72 h post LPS
Thyme challenge to analyze leukocyte numbers in lung tissue, bronchoalveolar lavage fluid (BALF) and blood as well
Ivy
as goblet cells in bronchial epithelium. Inhibitory effects of BRO analogue on leukotriene (LT) production were
Anti-inflammatory
determined in human neutrophils and monocytes as well as on isolated 5-lipoxygenase (5-LO).
Goblet cells
5-lipoxygenase Results: BRO significantly reversed the LPS-induced increase in leukocyte numbers in lung tissue, BALF and
blood as well as goblet cell numbers in bronchial epithelium. In vitro, BRO analogue suppressed cellular re-
lease of LTB4 (IC50 = 36 μgml−1 ) and cysLT (IC50 = 10 μgml−1 ) and inhibited the activity of isolated 5-LO
(IC50 = 19 μgml−1 ).
Conclusion: BRO exerts significant anti-inflammatory effects and attenuates goblet cell metaplasia in LPS-
induced bronchoalveolitis in vivo potentially via interference with 5-LO/LT signaling. These effects may con-
tribute to its observed clinical efficacy in AB.
© 2015 Elsevier GmbH. All rights reserved.

Introduction on the public health system (Holzinger et al. 2014). In addition to

Acute bronchitis (AB) is a predominantly self-limited respiratory
disease comprising several different symptoms amongst which in-
flammation of the bronchi, increased production of viscous mucus
and consequently cough are the most incriminating for the patient
(Verheij et al. 1989). Cough is the most frequent reason for visits to
primary care physicians and imposes a significant economic burden
Abbreviations: AA, arachidonic acid; AB, acute bronchitis; 5-LO, 5-lipoxygenase;
BALF, bronchoalveolar lavage fluid; BLT, LTB4 receptor; BRO, Bronchipret® syrup;
LT, leukotriene; COX, cyclooxygenase; DER, drug extract ratio; EtOH, ethanol; Imax ,
maximal inhibition; IC50 , calculated concentration of half-maximal inhibition; LPS,
lipopolysaccharide; RT, room temperature.
∗
Corresponding author. Tel.: +49 (0) 9181/231 309; fax: +49 (0) 9181/231 6309.
E-mail address: jan.seibel@bionorica.de (J. Seibel).
symptomatic treatment with anti- and protussive agents, mucolytics
and adrenergic beta-2 agonists, antibiotics have commonly been used
for AB therapy (Tackett and Atkins 2012) albeit recent publications in-
dicate the lack of efficacy of antibiotic treatment in AB (Smith et al.
2014). Consequently, antibiotics should only be used to prevent an
exacerbation of chronic bronchitis or in patients under the risk of de-
veloping pneumonia (Martinez 2004). Therefore, other effective and
well-tolerated treatment options without the potential for increasing
antimicrobial resistance are imperative.
Several clinical trials have demonstrated positive effects of
Bronchipret® Syrup (BRO), a fixed combination of thyme herb and ivy
leaf fluid extracts, on symptom relieve and recovery time in patients
with AB and cough (Kemmerich et al. 2006; Marzian 2007). Based on
these data BRO was included into the Guidelines of the German Res-
piratory Society for Diagnosis and Treatment of Adults Suffering from
Acute or Chronic Cough (Kardos et al. 2010).

http://dx.doi.org/10.1016/j.phymed.2015.09.001
0944-7113/© 2015 Elsevier GmbH. All rights reserved.
 J. Seibel et al. / Phytomedicine 22 (2015) 1172–1177
Although for both thyme and ivy single extracts anti-
inflammatory, smooth muscle relaxing and/or secretolytic activities
have been reported (Landa et al. 2009; Wienkotter et al. 2007; Wolf
et al. 2011) the mechanisms underlying the clinical efficacy of BRO
have not been identified, yet.
In the present study we applied a dual experimental approach to
study the anti-inflammatory efficacy of BRO in experimentally in-
duced AB in vivo and to identify potential mechanisms of action in
vitro. Intratracheal LPS instillation in rats was used as a model system
for AB to study the effects of BRO on three aspects of the disease in
vivo: (i) bronchial inflammation via mucosal and submucosal gran-
ulocyte infiltration, (ii) mobilization of granulocytes into the bron-
choalveolar fluid (BALF) and blood and (iii) the number of bronchial
goblet cells as an indirect measure of tissue remodeling and mucus
production. Due to the important role of leukotriene B4 (LTB4 ) in
neutrophil chemotaxis and of cysteinyl leukotrienes (cysLTs) in bron-
choconstriction we additionally investigated the effects of a BRO ana-
logue on 5-LO product formation in vitro.
Methods
Test items of the herbal extracts
Bronchipret® syrup was provided by Bionorica SE, Neumarkt, Ger-
many. For in vivo studies, rats were administered commercially avail-
able Bronchipret® syrup (BRO) with a final thyme/ivy extract ratio of
10:1 and an ethanol content of 7% v·v−1 . Specifically, a 10 g prepara-
tion (equivalent to 8.85 ml) of BRO contains a mixture of 1.5 g thyme
extract (1:2–2.5 drug extract ratio (DER) in ammonia solution 10%
(wt·wt−1 ), glycerol 85% (wt·wt−1 ), ethanol 90% (wt·wt−1 ) and water
(1:20:70:109)) and 0.15 g fluid extract derived from ivy leaves (DER:
1:1; extracting agent: ethanol 70% (v·v−1 )). Both extracts are stan-
dardized to the content of specific marker compounds.
Due to the ethanol content of the fluid extracts contained in BRO,
a mixture of genuine thyme herb dry extract and genuine ivy leaf
liquid extract lyophilisate with identical composition (calculated as
the ratio of the herbal substances) as contained in BRO (termed “BRO
analogue”) was used as a surrogate for studying 5-LO product syn-
thesis in vitro. This mixture was diluted with 50% ethanol (v·v−1 ) to
a concentration of 100 mg·ml−1 , homogenized by vortexing and then
incubated in an ultrasonic bath at room temperature (RT). The sus-
pension was centrifuged (3000 × g, 10 min, RT) and the supernatant
was filtrated through a disposable syringe filter (PVDF; pore size,
0.22–0.45 μm; Millipore, Billerica, MA) and immediately used for the
assays.
Bronchoalveolitis in vivo model
Animal housing
Male Wistar rats weighing 250–300 g (Rappolovo, St. Peters-
burg, Russia) were kept in polycarbonate cages (6 animals/cage) at
21 ± 1 °C and 61–75% humidity in a 12 h light/dark cycle, with ad
libitum access to water and complete pellet diet (Protein 19%; Aller
Petfood, Kuzmolovskiy, Russia). Experiments were performed accord-
ing to the recommendations and policies of the National Standard
of Russian Federation GOST R-53434-2009 and were approved by
the Ethics Committee of the Saint-Petersburg Institute of Pharmacy
JSC (Russia, Saint-Petersburg). All painful manipulations of the ani-
mals were conducted in accordance with regulatory standards (Di-
rective 2010/63/EU of the European parliament and of the council of
22 September 2010 on the protection of animals used for scientific
purposes).
Induction of bronchoalveolitis and animal treatment
Prior to experimentation 180 animals (n = 36/group, 12 ani-
mals/time point) were randomly allocated to five treatment groups
1173
by a modified method of the block randomization (Altman and Bland
1999). One additional group served as healthy control group (sham;
n = 12). Animals were acclimatized for 14 days prior to start of exper-
iments.
Bronchoalveolitis was induced by intratracheal injection of
100 μg LPS from Escherichia coli 0111:B4 (Sigma–Aldrich, St. Louis,
MO) in 200 μl 0.9% NaCl per animal 1 h prior to the first oral admin-
istration of the test compounds as described (Blackwell et al. 1999).
The animals were then treated by oral gavage with either vehicle
(7% v·v−1 ethanol) or BRO (1.7, 5.0, or 16.7 ml·kg−1 ). The doses cor-
responded to the 1-, 3- and 10-fold of the currently recommended
human daily dose after allometric conversion to human equivalent
doses (U.S. Food and Drug Administration 2005). Treatment com-
prised up to 3 administrations at 1, 25 and 49 h post LPS injection.
Dexamethasone (Dex, 5 mg·kg−1 ) was also administered at 1, 25 and
49 h post LPS into the femoral muscle as a positive control to demon-
strate an anti-inflammatory effect. All compounds (including vehicle)
were diluted to a final ethanol content of 7% v·v−1 to assure equal
ethanol uptake.
BALF, blood and tissue collection and analysis
For the analysis of BALF 6 rats/group were sampled at 24, 48
and 72 h post intratracheal LPS-instillation. Rats were anesthetized
by intravenous injection of Zoletil® (tiletamine/zolazepam; Virbac,
France) at 0.7 mg·kg−1 . The right main bronchus was ligated and a
catheter was inserted from the trachea into the left lung. Warm saline
(37 °C) was repeatedly run through the catheter and the resulting
BALF (ca. 200 μl) was passed through a mesh (Pharmaceutical com-
pany Volga Manufactory, LLC) to remove mucus, followed by centrifu-
gation (1500 × g, 15 min, 4 °C).
Resulting supernatants were stored in liquid nitrogen until anal-
ysis. Pellets were resuspended and total leukocyte counts were mea-
sured with a veterinarian hematological analyzer (Abacus Junior Vet,
Diatron, Hungary). Differential leukocyte counts of the BALF were
performed in May-Grünwald/Giemsa stained slides using light mi-
croscopy. A minimum of 100 cells/slide were counted.
For collecting blood leukocytes, venous blood was withdrawn
from the tail vein (6 animals/group/time point) into a plastic tube
containing heparin (20 ED·ml−1 ; FGUP “Moscow endocrine factory”,
Russia) and leukocytes were counted in an Abacus Junior Vet hema-
tological analyzer (Diatron, Hungary).
Lung histology was performed in hematoxylin and eosin stained
formalin-fixated tissue slices from the right lung. Inflammation in the
bronchial tissue was evaluated by semi-quantitative analysis of mu-
cosal and submucosal granulocyte infiltration using a scoring system
ranging from normal = 0 to severe = 3. For histochemical calcula-
tion of goblet cell numbers samples were taken from medium-sized
bronchi obtained from central regions of the lungs. Three slides from
each animal were counter-stained with Alcian blue at pH 2.5 and gob-
let cells within 1 mm of the epithelial layer were counted. All goblet
cells in medium-sized bronchi per slide were counted. Morphologi-
cal examination was performed using light-optical microscopy (Le-
ica DC320 (Leica Microsystems, Germany)) at 200-fold magnification
(Harris et al. 2007).
Blood cells and cell isolation
Neutrophils and monocytes were isolated from buffy coats from
healthy adult volunteers obtained at the Institute of Transfusion
Medicine, University Hospital Jena. Neutrophils were immediately
isolated by dextran sedimentation and centrifugation on Nycoprep
cushions (PAA Laboratories, Linz, Austria) and hypotonic lysis of ery-
throcytes as described (Pergola et al. 2008). Monocytes were sep-
arated from peripheral blood mononuclear cells by adherence to
culture flasks as described (Pergola et al. 2011). Cells were finally re-
suspended in PBS pH 7.4 containing 1 mg·ml−1 glucose and 1 mM
CaCl2 (PGC buffer).
1174 J. Seibel et al. / Phytomedicine 22 (2015) 1172–1177
Analysis of cytotoxicity
Neutrophil and monocyte viability was analyzed by MTT assay as
described before (Koeberle et al. 2008). Test compounds or vehicle
(0.375% EtOH, final concentration) were added to the cells seeded in
96-well plates and samples were incubated for 30 min at 37 °C; posi-
tive control for cell lysis: 1% triton or 16.6% ethanol.
LTB4 formation in intact human neutrophils
Neutrophils (5 × 106 cells·ml−1 PGC buffer) were pre-incubated
with the compounds or vehicle (0.025% EtOH) for 10 min at 37 °C and
then stimulated for another 10 min with ionophore A23187 (2.5 μM).
The reaction was stopped with 1 ml of methanol and 30 μl 1 N HCL
and 500 μl PBS and 200 ng prostaglandin B1 (PGB1 , internal standard)
was added. The samples were subjected to solid-phase HPLC extrac-
tion on C18-columns (100 mg, UCT, Bristol, PA, USA) and analyzed by
RP-HPLC as described (Werz et al. 2002). LTB4 quantities were calcu-
lated on the basis of PGB1 . Amounts of the cysLTs C4 , D4 and E4 were
below the detection limit.
CysLT formation in intact human monocytes
Monocytes (1 × 106 cells·ml−1 PGC buffer) were incubated for
10 min at 37 °C with test compounds or vehicle (0.025% EtOH) and
then stimulated for 10 min with ionophore A23187 (2.5 μM). The re-
action was stopped on ice, samples were centrifuged at 500 × g for
10 min at 4 °C and supernatants were analyzed for cysLT levels using
an enzyme immunoassay kit which detects LTC4 , D4 and E4 according
to the manufacturer’s instructions (Sapphire Biosciences, Waterloo,
NSW, Australia).
Activity of purified 5-LO
Human recombinant 5-LO was expressed in E. coli Bl21 (DE3)
cells, transformed with pT3-5LO and purified by ATP-agarose column
(Fischer et al. 2003). 5-LO (0.5 μgml–1 PBS, pH 7.4, 1 mM EDTA,
1 mM ATP) was pre-incubated for 10 min at 4 °C with test compounds
or vehicle (0.25% EtOH), pre-warmed for 30 s at 37 °C and 2 mM CaCl2
and 20 μM AA were added. The reaction was stopped after 10 min at
37 °C by addition of ice-cold methanol and PGB1 was added. Formed
metabolites (all-trans isomers of LTB4 and 5-H(P)ETE) were extracted
by solid phase extraction on C18-columns and analyzed by HPLC as
described above.
Statistics
In vivo study data are presented as mean ± standard error of
the mean (SEM). Statistical data evaluation versus LPS/vehicle control
was performed by one-way analysis of variance (ANOVA) followed
by Dunnett’s Multiple Comparison Test (of log-transformed data in
case of heteroscedasticity). In case of unequal variances and for the
Fig. 1. BRO reduces LPS-induced mucosal and submucosal infiltration of granulocytes in bronchi of rats. (A) Mucosal and (B) submucosal granulocyte infiltration as rated on
a 0 to 3 scale from stained histological sections. N = 4–6. ∗ p < 0.05, ∗∗ p < 0.01, Kruskal–Wallis test, followed by Dunn’s test versus LPS/vehicle group. § p < 0.05; Mann–Whitney
U-Test versus sham group. n.d., not detectable (mean score = 0 ± 0).
histology analyses non-parametric Kruskal–Wallis test, followed by
Dunn’s Multiple Comparison Test versus the LPS/vehicle group was
used. Comparison of the sham group versus LPS/vehicle control was
assessed by either Mann–Whitney U-test (histology data) or student’s
t-test (all other data) featuring Welch’s correction in case of unequal
variances.
For in vitro experiments results are expressed as arithmetic
means ± SEM of at least three independent experiments, each col-
lecting single data points. IC50 values were calculated by non-linear
regression. Statistical analysis was done with GraphPad Prism 5 soft-
ware (GraphPad Software Inc., San Diego, CA). A p value < 0.05 was
considered statistically significant.
Results
BRO suppresses granulocyte infiltration in bronchial tissue
Intratracheal instillation of LPS (100 μg/rat) caused morphologi-
cal and histological changes in both bronchi and alveoli. Within 24 h
animals of the LPS/vehicle group developed acute catarrhal purulent
bronchitis or macrofocal pneumonia (data not shown). Granulocyte
infiltration was used as a surrogate parameter to measure inflamma-
tion of the bronchial tissue. The animals of the LPS/vehicle group ex-
hibited mean scores of 2.0 (mucosal) and 2.5 (submucosal) after 24 h
which remained elevated after 72 h (1.5 and 1.3, respectively; Fig. 1A,
B). This was accompanied by accumulation of an exudate containing
neutrophils, lymphocytes and macrophages in the bronchial lumen
and the alveoli (not shown).
Repeated administration of all three doses of BRO to LPS-treated
animals once daily up to 72 h resulted in a numerical decrease both
in the mucosal and submucosal granulocyte infiltration beginning at
24 h, with mean scores at that time point ranging from 1.0 to 1.2
(mucosal) and from 1.0 to 0.5 (submucosal, Fig. 1A, B). Maximum
effects of BRO were obtained with 16.7 ml·kg−1 at 48 h (0.3, mu-
cosal) and 72 h (0.2, submucosal). Treatment with dexamethasone
(5 mg·kg−1 ) attenuated granulocyte infiltration with significant ef-
fects only at 72 h post LPS.
BRO reduces leukocytes in BALF
Administration of LPS led to a significant increase of leukocytes
(Fig. 2A), specifically of granulocytes (Fig. 2B) in BALF. This increase
was maximal after 24 h and continuously decreased until 72 h. Even
at 72 h post LPS treatment, leukocyte and granulocyte counts in BALF
were still markedly increased versus sham-treated animals. Treat-
ment with BRO reduced the number of leukocytes and granulocytes
in BALF at virtually all time points and doses with significant effects
 J. Seibel et al. / Phytomedicine 22 (2015) 1172–1177 1175

A A
# Sham
Leukocytes in BALF (10 . ml )

Dex 5 mg .kg-1 BRO-TE 5.0 ml .kg-1

-1

Leukocytes in blood (10 . ml )

20 Sham

-1
Vehicle
10 Dex 5 mg .kg-1 Vehicle BRO-TE 1.7 ml .kg-1 BRO-TE 16.7 ml .kg-1
6

6
BRO 1.7 ml .kg-1 15
# #
means ± SEM

means ± SEM
BRO 5.0 ml .kg-1 #
#
BRO 16.7 ml .kg-1
10
5 # * *
*** *** * * **
** * * ***
*** 5
* *
*** ** ***

0 0
24h p.a. 48h p.a. 72h p.a. 24h p.a. 48h p.a. 72h p.a.

LPS LPS

B B
Granulocytes in BALF (10 . ml )

Granulocytes in blood (10 . ml )

-1

-1
# #
6

6
10 10
means ± SEM

means ± SEM
# #
5 5
# *
**
** **
*** ** *** ***
*** *** ***
*** ***
0 0
24h p.a. 48h p.a. 72h p.a. 24h p.a. 48h p.a. 72h p.a.

LPS LPS

Fig. 2. BRO decreases leukocyte and granulocyte counts in bronchial fluid after LPS
stimulation. (A) Total leucocyte and (B) granulocyte counts in BALF after treatment
with vehicle, dexamethasone (dex), or BRO. Control animals (sham) received only ve-
hicle without LPS. N = 6. ∗ p < 0.05, ∗∗∗ p < 0.001; ANOVA, followed by Dunnett’s test
versus LPS/vehicle group. # p < 0.05; student’s t-test + Welch’s correction versus sham
group.
Fig. 3. BRO decreases leukocyte and granulocyte counts in peripheral blood af-
ter LPS stimulation. (A) Total leucocyte counts and (B) granulocyte counts in periph-
eral blood after treatment with vehicle, dexamethasone (dex), or BRO. Control animals
(sham) received only vehicle without LPS. N = 6. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001;
ANOVA, followed by Dunnett’s test versus LPS/vehicle group. # p < 0.05; student’s
t-test (A) + Welch’s correction (B) versus sham group.

at the medium and high dose at 48 h (total cells and granulocytes)

and 72 h (total cells), respectively, post LPS challenge. Dexametha-
sone significantly reduced leukocyte and granulocyte counts in BALF
at all time points compared to LPS/vehicle.

Effects of BRO on leukocytes and granulocytes in peripheral blood

LPS-administration significantly increased leukocyte (Fig. 3A) and

granulocyte counts (Fig. 3B) in peripheral blood compared to the
sham group. Treatment with BRO at all doses caused significant de-
creases of leukocyte counts at 48 and 72 h compared to LPS/vehicle
with similar efficacy as dexamethasone.

Effects of BRO on goblet cells in bronchial tissue

The number of bronchial mucosa goblet cells was determined

histologically at 72 h post LPS challenge and was expressed as
cells·mm−1 of the epithelial layer of the bronchi (Fig. 4). Goblet cell
numbers increased significantly in response to LPS administration
(63.7 ± 1.7 cells·mm−1 ) when compared to the numbers in sham-
treated controls (28.0 ± 1.2 cells·mm−1 ). BRO at all doses caused a
significant reduction in goblet cell numbers compared to LPS/vehicle-
treated animals, with an efficacy that was similar to dexamethasone. Fig. 4. BRO reduces the number of goblet cells in bronchioles after LPS stimula-
tion. Goblet cell numbers at 72 h post LPS after treatment with vehicle, dexamethasone
(dex), or BRO. Paraffin sections of lung tissue samples of rats were stained with hema-
BRO analogue inhibits cellular leukotriene production and 5-LO
toxylin and eosin to assess morphology. The slides were counter-stained with Alcian
enzyme activity blue for histochemical calculation of goblet cell numbers. Cell numbers were counted
in medium-sized bronchi, taken at the central regions of the lungs within 1 mm of the
We used neutrophils and monocytes from human periph- epithelial layer. N = 5–6. ∗∗ p < 0.01, ∗∗∗ p < 0.001; ANOVA, followed by Dunnett’s test
versus LPS/vehicle group. # p < 0.05; student’s t-test correction versus sham group.
eral blood for analysis of the effects of BRO analogue on major
1176 J. Seibel et al. / Phytomedicine 22 (2015) 1172–1177
Fig. 5. BRO analogue suppresses the production of 5-LO products in vitro. Forma-
tion of LTB4 and of cysLTs was analyzed in intact human neutrophils or monocytes,
respectively, after stimulation with A23187 (2.5 μM) for 10 min. The activity of human
recombinant 5-lipoxygenase (5-LO, purified enzyme) was analyzed by incubation of
the enzyme with 20 μM AA as substrate. BRO analogue at the indicated concentra-
tions or vehicle (0.025% ethanol) was added 10 min prior to initiating 5-LO product
formation. Data are given as means ± SEM, n = 3.
mediators of inflammation and bronchoconstriction, i.e. LTs, because
mechanistic studies on LT biosynthesis with leukocytes from rats are
laborious due to the limited amounts of blood obtainable. BRO ana-
logue inhibited A23187-stimulated biosynthesis of cysLTs in mono-
cytes with an IC50 = 10 μg·ml−1 and a maximum inhibition (Imax )
of 98% at 100 μg·ml−1 (Fig. 5). Similarly, A23187-stimulated biosyn-
thesis of LTB4 in human neutrophils was inhibited by BRO analogue
(IC50 = 36 μg·ml−1 ), with almost total suppression at 100 μg·ml−1 .
BRO analogue furthermore inhibited the catalytic activity of isolated
human recombinant 5-LO with an IC50 = 19 μg·ml−1 and an Imax of
81% at 100 μg·ml−1 . The concentrations of BRO analogue used re-
vealed no significant impact on cell viability (MTT assay, data not
shown). Thus, BRO analogue potently inhibited 5-LO and effectively
suppressed the agonist-induced formation of LTs from monocytes
and neutrophils.
Discussion
The purpose of this study was to evaluate the anti-inflammatory
efficacy of BRO, a fixed combination of thyme herb and ivy leaf fluid
extracts, in vivo and to obtain insights into the underlying modes of
action. This was approached by applying (i) an in vivo model of bron-
choalveolitis in rats and (ii) experiments addressing 5-LO product for-
mation in vitro. We found that oral administration of BRO in a model
of moderate-to-severe bronchoalveolar inflammation in rats attenu-
ated the inflammatory response by reducing both systemic leukocy-
tosis as well as local infiltration of granulocytes into lung tissue and
alveolar space.
With the current data we cannot discriminate at which level BRO
affects LPS-induced inflammation. Both inhibition of LPS-induced
signaling in epithelial cells as well as suppression of chemotaxis and
infiltration of pro-inflammatory granulocytes in the lung are com-
patible with our data. We observed no clear dose-dependency for
the anti-inflammatory effects suggesting that either maximum ef-
ficacy conferrable by BRO ingredients was already achieved at 1–3-
fold of the HED or that relevant active ingredients were not absorbed
completely at the higher doses. Future studies employing a wider
dose-range or more frequent dosing intervals could help to further
address this question. One potential mode of action underlying the
anti-inflammatory activity of BRO in vivo might be suppression of 5-
LO activity in human leukocytes along with reduced LT production.
LTB4 is a potent chemoattractant for neutrophils and reduction of
LT release could potentially contribute to attenuation of granulocyte
infiltration.
In addition, BRO significantly attenuated tissue remodeling evi-
denced as an inhibition of goblet cell metaplasia in bronchial ep-
ithelium after LPS challenge. This finding is of particular interest
since hypersecretion of viscous mucus by goblet cells is one of
the major clinical manifestations of patients suffering from various
pulmonary diseases including AB (Kim et al. 2003). Whether BRO
reduced goblet cell metaplasia by acting directly on goblet cell
precursors or whether attenuation was secondary to reduced LPS-
induced inflammation remains unclear. However, the observation
that BRO showed comparable efficacy on goblet cell metaplasia as the
reference drug dexamethasone which completely blocked leukocyte
increase in BALF suggests that the effects of BRO on inflammation
versus goblet cell metaplasia could result from different biological
activities.
The LPS-induced lung inflammation model in rats was chosen be-
cause it exhibits many hallmark features observed during AB, that is,
acute pulmonary inflammation characterized by a prominent local
infiltration of leukocytes, edema, production of inflammatory media-
tors, and bronchial histological changes such as metaplasia of mucus-
producing goblet cells. Thus, the model is suitable to study pharmaco-
logical activities relevant for the treatment of AB (Harris et al. 2007)
including local and systemic anti-inflammatory properties and the
normalization of excessive mucus production.
Our findings complement previous studies by others (Fachini-
Queiroz et al. 2012) who analyzed the beneficial effects of Thy-
mus vulgaris essential oil (TEO) and its terpene components thy-
mol and carvacrol using in vivo (carrageenan-induced pleurisy in
rats) and in vitro (chemotaxis of leukocytes from rat pleural cav-
ity) models of inflammation. In vivo, oral administration of TEO re-
duced the exudate volume as well as the number of total leukocytes
and neutrophils in the exudate. Mechanistically, the observation that
BRO analogue inhibits 5-LO product synthesis is consistent with re-
ports on anti-inflammatory activities of thymol, carvacrol and thy-
moquinone (Fachini-Queiroz et al. 2012). In addition, thyme com-
ponents most probably exhibit further pharmacological activities as
evidenced by data on activation of PPAR-γ (Hotta et al. 2010) and
inhibition of the generation of various inflammatory mediators such
as PGE2 (Skold et al. 1998), neutrophil elastase (Braga et al. 2006)
or reactive oxygen species and peroxynitrite (Jukic and Milos 2005;
Youdim and Deans 1999).
Also, certain components from ivy extracts may exert anti-
inflammatory effects via mechanisms complementing inhibition of
the LT pathway. For example, saponins such as hederasaponin-C, -E
and -F can inhibit bradykinin and prostaglandin pathway signaling
(Gepdiremen et al. 2005) wheras α -hederin sustains beta-2 adrener-
gic signaling (Wolf et al. 2011).
It is tempting to speculate that the inhibitory effect on mucus-
producing goblet cell numbers demonstrated here together with
the known stimulating effects of thyme on ciliary beat frequency
(Wienkotter et al. 2007) could normalize mucociliary clearance and
thus underlie the observed reduction of cough and Bronchitis Sever-
ity Score in patients with AB after BRO therapy (Kemmerich et al.
2006; Marzian 2007). In addition, attenuation of LPS-induced neu-
trophil influx to the BALF indicates anti-inflammatory activity of BRO,
which could contribute to the observed reduction of bronchial in-
flammation and bronchitis symptoms in patients.
Taken together, it appears that the numerous compounds found
in thyme/ivy extracts act through multiple targets and mechanisms
to concomitantly affect inflammation and excessive mucus produc-
tion and thus effectively reduce acute cough and bronchitis symp-
toms. In particular, the marked inhibitory effects on LT biosynthe-
sis at low BRO analogue concentrations suggest that interference
with these potent pro-inflammatory (LTB4 ) and bronchoconstrict-
ing (cysLTs) lipid mediators with strong relevance for the patho-
physiology of AB is likely to contribute to the beneficial features of
BRO in vivo. Therefore, assessing the effect of BRO on LT levels in
 J. Seibel et al. / Phytomedicine 22 (2015) 1172–1177
humans where elevated concentrations of LTs in bronchitis have been
reported (Crooks et al. 2000) is challenging.
Conclusion
The fixed combination of thyme and ivy extracts marketed as
Bronchipret® syrup (BRO) exhibits potent anti-inflammatory prop-
erties in vivo in a rat model of bronchitis primarily through suppres-
sion of leukocytosis, granulocyte migration to the lung and reduction
of goblet cells metaplasia that are responsible for the production of
viscous mucus. Results from in vitro studies obtained with a thyme
and ivy extracts mixture as contained in BRO (calculated as the ra-
tio of the herbal substances) suggest a reduction of LT synthesis via
inhibition of 5-LO as a potential mode of action. These results pro-
vide a potential explanation for the mechanisms of action underlying
the clinically-observed therapeutic action of BRO. Future experiments
using a viral infection-based experimental model for bronchitis may
further support the efficacy of BRO, and the use of computational
techniques, such as the in silico model for 5-LO inhibitors (Steinhilber
and Hofmann 2014) could help to identify its active pharmaceutical
ingredients.
Conflict of interest
J. Seibel, K. Wosikowski, M. D. Lehner and J. Haunschild are
employees of Bionorica SE, Neumarkt, Germany which markets
Bronchipret® Syrup.
C. Pergola, O. Werz and K. Kryshen received funding from Bionor-
ica SE.
The contributions of the medical writers M. Kaszkin-Bettag and S.
O’Shea to the manuscript were paid by Bionorica SE.
Acknowledgments
We thank medical writers Prof. Dr. Marietta Kaszkin-Bettag and
Dr. Sean O’Shea (PharmaLex GmbH, Mannheim, Germany) for their
contribution to the preparation of the manuscript and helpful discus-
sions.
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