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Abstract
Introduction: Myrtol standardized is established in the
treatment of acute and chronic bronchitis and sinusi-
tis. It increases mucociliar clearance and has muco-se-
cretolytic effects. Additional anti-inflammatory and
antioxidative properties have been confirmed for Myr-
tol standardized, eucalyptus oil, and orange oil in sev-
eral in vitro studies.
Objective: The aim of this study was to prove the abili-
ty of essential oils to reduce cytokines release and re-
active oxygen species (ROS) production derived from
ex vivo cultured alveolar macrophages.
Material and methods: Alveolar macrophages from pa-
tients with chronic obstructive pulmonary disease
(COPD, n=26, GOLD III-IV) were pre-cultured with
essential oils (10
-
3
-10
-
8
%) for 1 h and then stimulated
with LPS (1µg/ml). After 4 h and 20 h respectively a)
cellular reactive oxygen species (ROS) using 2’,7’-
dichlorofluorescein (DCF), and b) TNF-α, IL-8, and
GM-CSF secretion were quantified.
Results: In comparison with negative controls, pre-cul-
tured Myrtol, eucalyptus oil and orange oil (10
-
4
%) re-
duced in the LPS-activated alveolar macrophages ROS
release significantly after 1+20 h as follows: Myrtol -
17.7% (P=0.05), eucalyptus oil -21.8% (P<0.01) and or-
ange oil -23.6% (P<0.01). Anti-oxidative efficacy was
comparable to NAC (1 mmol/l). Essential oils also in-
duced a TNF-α reduction: Myrtol (-37.3%, P<0.001),
eucalyptus oil (-26.8%, P<0.01) and orange oil (-26.6%,
P<0.01). TNF-α reduction at 1+4 h and 1+20 h did
not vary (Myrtol: -31.9% and -37.3% respectively, P=
0.372) indicating that this effect occurs early and cannot
be further stimulated. Myrtol reduced the release of
GM-
CSF
by -35.7% and that of IL-8 only inconsiderably.
Conclusions: All essential oils tested have effective an-
tioxidative properties in ex vivo cultured and LPS-
stimulated alveolar macrophages. Additionally, Myrtol
inhibited TNF-α and GM-CSF release best indicating
additional potent anti-inflammator y activity.
Key words: essential oils, myrtol, eucalyptus oil and or-
ange oil, COPD, alveolar macrophages, cytokines, re-
active oxygen species
INTRODUCTION
Chronic bronchitis is a subset of a chronic obstructive
pulmonary disease (COPD) [1]. It is clinically diag-
nosed by the presence of cough as well as productive
sputum for at least three months during two or more
consecutive years [2]. Chronic exposure to cigarette
smoke damages the airway epithelium, leading to squa-
mous metaplasia. Epithelial layer thickness, promoted
by epithelial cell hyperplasia, hypertrophy and mucous
metaplasia increases incrementally as disease severity
worsens. Inflammatory cells are routinely observed in
the tissue and the airways of COPD patients, whereas
neutrophils are the most abundant cellular population
[3]. Airway mucus hypersecretion is a key pathophysio-
logical feature in most COPD patients. Consequently,
many drugs have been developed, either to inhibit mu-
cus hypersecretion or to reduce the viscosity to ease
mucus elimination by cough. Some have been reported
to have anti-inflammatory properties such as N-acetyl-
cysteine (NAC) and its derivatives or ambroxol [4-6].
Clinical efficacy of mucolytic drugs was discussed
somewhat controversial. Regardless of numerous pos-
itive results from the 1980s and promising metaanaly-
sis [7, 8], one newer placebo controlled and random-
ized trial with NAC is fairly disappointing [9]. As a
consequence mucolytic drugs are not generally recom-
mended for COPD treatment [10].
Phytomedicines and herbal remedies have a long
history in the treatment of COPD and of patients suf-
fering from bronchitis. Herbal medicine is very popu-
lar, but only a few studies analysed the underlying
mechanism of their efficacy [11]. Among those, Myr-
tol (CAS-No. 8002-55-9), a muco-secretolytic phy-
tomedicine containing the monoterpenes α-pinene, d-
limonene and 1,8-cineole as marker substances, pro-
vides proven efficacy in the treatment of acute and
chronic bronchitis [7, 12-14]. The aim of the present
study was the investigation of Myrtol and some other
essential oils in having anti-inflammatory properties
on human alveolar macrophages.
MATERIAL AND METHODS
STUDY DESIGN
The study was approved by the Saxonian Ethics Com-
mittee in Dresden, Germany (EK-BR-27/05-2). Myr-
tol, eucalyptus oil and orange oil were tested in an
open, single-center and case-controlled study to re-
duce certain pro-inflammatory parameters in isolated
alveolar macrophages. The cells were derived from
EUROPEAN JOURNAL OF MEDICAL RESEARCHDecember 7, 2009
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Eur J Med Res (2009) 14(Suppl. IV): 205-209 © I. Holzapfel Publishers 2009
ANTI-INFLAMMATORY EFFECTS OF MYRTOL STANDARDIZED AND OTHER
ESSENTIAL OILS ON ALVEOLAR MACROPHAGES FROM PATIENTS WITH
CHRONIC OBSTRUCTIVE PULMONARY DISEASE
U. Rantzsch, G. Vacca, R. Dück, A. Gillissen
S
t. George Medical Center, Robert-Koch-Hospital, Leipzig, Germany
S. I-X, 1-284:Layout 1 24.11.2009 10:43 Uhr Seite 205
COPD patients (n = 26) by bronchoscopy and bron-
choalveolar lavage (BAL), using a standard method as
reported earlier [15, 16]. The patients were all recruit-
ed from the Robert-Koch-Hospital, an academic
teaching hospital and integral part of the St. George
Medical Center, in Leipzig, Germany, specialized in
pulmonary medicine including lung cancer. COPD
was defined according to the Global Initiative for
Lung Disease [10]. Only patients with GOLD stage
III-IV were eligible to ensure high cellular activity due
to persistent inflammation in the airways. All patients
gave their informed consent prior to inclusion. They
were all on regular treatment with inhaled long-acting
β2-agonists, tiotropium bromide, and theophylline and
inhaled corticosteroids. None received systemic corti-
costeroids or mucolytics. Baseline characteristics of
the population studied and of the BAL content are
shown in Table 1.
EXPERIMENTAL PROTOCOL
The purity of isolated alveolar macrophages was veri-
fied using an inverse fluorescence microscope (Nikon
Eclipse TS100, type 120) after pre-incubation in which
viable cells adhere to the bottom of the tissue culture
plate. Alveolar macrophages viability, before and after
the incubation with essential oils, was determined by
trypan blue exclusion (<95%). The highest concentra-
tion of essential oils used had no adverse effects on
cell viability. alveolar macrophages were pre-incubated
in 96-well tissue culture plates (10
5
per well filled with
1 ml RPMI, fetal calf serum [FCS, 10%], 1%w/v
penicillin/streptomycin/amphotericin/1% L-gluta-
mine) for up to 20 h. Myrtol, orange oil, eucalyptus oil
(10
-3
-10
-8
%, all a generous gift from G. Pohl-Boskamp
GmbH & Co. KG, Hohenlockstedt/Germany) diluted
in RPMI were than added for 1 h. After washing, the
cells were further incubated in RPMI/1%FCS/peni -
cillin/streptomycin/amphotericin/L-glutamin and
LPS
(1µg/ml) for the next 4 h (= 1+4 h) and 20 h (= 1+20
h) respectively. Conditioned media were than used for
further analysis (below). The concentrations of essen-
tial oils and LPS were determined in pre-experiments.
They also were adapted to known concentrations in
blood [14].
ANTIOXIDATIVE CAPACITY
Antioxidative properties of essential oils were tested
as a function of inhibited release of cellular reactive
oxygen species (ROS). ROS were quantified using the
fluorescence marker 2’,7’-dichlorofluorescein (DCF,
Merck, Darmstadt, Germany). The intensity of fluo-
rescence measured corresponds to ROS concentration
as reported earlier [17]. ROS generation was estimated
as the maximal DCF mediated fluorescence of 2 min-
utes at 37°C with the excitation and emission wave-
lenghts at 488 and 520 nm respectively (fluoropho-
tometer FLUOstar Optima, BMG Labtech, Offen-
burg, Germany). In pre-experiments serial dilutions of
reagent DCF were monitored on the fluorometer to
obtain a standard curve of fluorescence per nanomole
DCF. ROS produced by alveolar macrophages were
expressed as picomoles DCF per 10
5
cells and - in
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December 7, 2009
Table 1. Baseline data and cellular broncho-alveolar lavage (BAL) content from the study population.
Mean Range Median Median SD
Patients (n) 26 ---
M
ale/female (n) 17/9 ---
Age (years) 63.9 41-79 62.4 8.5
BMI (kg/m
2
) 26.5 19-34 24.5 2.5
Smoker-current (%) 21.9 ---
Former-smoker (%) 41.7 ---
S
moker (pack-years) 19.2 10-35 17.5 7.9
Former smoker (pack-years) 21.0 10-35 20.0 8.8
Lung function
FEV1 (l) 2.2 0.6-4.1 2.1 1.1
FEV1 (%predicted) 73.5 29.7-124.6 70.5 26.8
V
C max (l) 2.8 0.83-5.1 2.8 1.2
VC max (%predicted) 73.7 43.20-126.0 73.2 22.8
FEV1/VC max 78.6 45.39-98.6 81.4 12.0
RV (l) 2.4 1.08-5.4 2.1 1.0
RV (%predicted) 112.4 61.5-226.6 107.0 40.5
BAL
Total cells (10
6
/100ml) 29.7 10-85 28.8 16.9
Macrophages (%) 61.2 28-90 63.5 19.2
Neutrophils (%) 12.4 1-50 6.0 12.7
Eosinophils (%) 2.0 0-14 1.0 3.2
Lymphocytes (%) 17.2 4-62 15.0 11.8
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terms of antioxidative capacity of the oils used - pre-
sented in % reduction. Because of its already proven
antioxidative properties NAC (1 mmol/l; Sigma
Chemicals) was used as a positive control [4]. The neg-
a
tive control consisted of cell medium only.
MEDIATOR RELEASE
Mediator release from cultured alveolar macrophages
was quantified using enzyme linked immunosorbent
assays according to the manufacturer's protocols
(ELISA). We measured TNF-α (Immuno Tools,
Friesoythe, Germany), IL-8 and GM-CSF (IBL, Ham-
burg, Germany).
STATISTICAL METHODS
The effects of potential interacting variables (essential
oils with different concentrations and incubation
times) on outcomes were assessed using two-way
ANOVA. Univariate comparisons were made using the
Student's t-test. Log transformation was used for sta-
bilization of variance, as required. SigmaStat 3.1 soft-
ware (SPSS cooperation) was used for all statistical cal-
culations. Error bars in graphs represent the standard
deviation of the mean.
RESULTS
EFFECT OF ROS REDUCTION
In comparison with the negative control, pre-cultured
Myrtol, eucalyptus oil and orange oil (10
-
4
%) reduced
ROS in LPS-activated alveolar macrophages signifi-
cantly: Myrtol (P<0.05), eucalyptus oil and orange oil
(P<0.01; Fig. 1).
EFFECT OF TNF-α REDUCTION
Myrtol reduced cellular TNF-α release in a 0.0001%
dilution (incubation time 1+20 h) by 37.3% (P<0.01;
Fig. 2). Again, there was no statistical difference be-
tween Myrtol, eucalyptus and orange oil. Maximal
TNF-α reduction by Myrtol was already reached after
1+4 h: 31.9% reduction (P<0.01, compared with
medium alone; P=0.37 compared with 1+20 h, Fig. 3).
EFFECT OF IL-8 AND GM-CSF REDUCTION BY
MYRTOL
Due to low cell number and limited amount of cell
medium per well, we focused on Myrtol to be tested
for IL-8 and GM-CSF reduction. Myrtol (0.0001%,
1+20 h) reduced cellular IL-8 release by 12.3% and
GM-CSF-secretion by 35.8%.
DISCUSSION
In the present study, we confirmed anti-inflammatory
properties of essential oils. Our results demonstrate
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Fig. 1. Reactive oxygen species (ROS) reduction in LPS (1
µg/ml)-stimulated alveolar macrophages from COPD pa-
tients with various essential oils (0.0001% dilution) at 1+20 h
incubation time (see text for details). Data presented as
means ±SD, NAC - N-acetylcysteine (1 mmol/l), orange - or-
ange oil, eucalyptus - eucalyptus oil. #P<0.05, *P<0.01 com-
pared with cell medium/LPS alone, non-significant differ-
ences among essential oils and NAC.
Fig. 2. Reduction of TNFα release from LPS (1 µg/ml-stim-
ulated alveolar macrophages from COPD patients at 1+20 h
incubation time (see text for details). TNFα - tumor necrosis
factor alpha, NAC - N-acetylcysteine (1 mmol/l), orange -
orange oil, eucalyptus - eucalyptus oil. *P<0.01 compared
with cell medium/LPS alone.
Fig. 3. Reduction of TNFα release from LPS (1 µg/ml)-
stimulated alveolar macrophages derived from COPD pa-
tients. Incubation time: 1 h pre-incubation with myrtol
0.0001% solution, followed by a washing step and 4 h incu-
bation (in RPMI/fetal calf serum/LPS). Differences non-sig-
nificant between myrtol incubation times, but P<0.01 (*)
compared with cell medium alone. TNFα - tumor necrosis
factor alpha.
S. I-X, 1-284:Layout 1 24.11.2009 10:43 Uhr Seite 207
that particularly Myrtol at known therapeutic blood con -
centrations was a surprisingly strong inhibitor of TNF-
α
and GM-CSF, but was also able to reduce the con-
c
entration of the chemotactic mediator IL-8 and ROS.
F
or 1,8-cineole, one of the main monoterpenes and
k
ey biological marker substance of Myrtol, a reduction
of cellular TNF-α release in PMA (12-myristate 13-ac-
etate, 10 nM)-activated lymphocytes and in LPS (10
µg/ml)-treated monocytes has been shown earlier [18].
Due to differences in the experimental setup and me-
diators measured, different cell population and cell
source, and varying information on the dosage of the
essential oils tested, it seems impractical to compare
their data with ours. Nonetheless, both studies clearly
indicate anti-inflammatory properties of terpenoids,
despite the differences.
Graßmann et al. [14] reported a Myrtol and euca-
lyptus oil-driven reductions of hypochlorous acid
(HOCl) in a system based on the degranulation of ac-
tivated neutrophilic granulocytes isolated from the
whole blood. Myrtol and eucalyptus oil also inhibited
peroxynitrite (ONOO
-
) formation indicating that
both oils interact with aggressive oxygen radicals from
the OH•-type [14].
In an arachidonic model, oral administration of
Myrtol results in a dose-dependent reduction in the
leukotriene concentration (LTC4/D4/E4) [19]. A
marked reduction in the rise in PGE2 induced by an
inflammatory stimulus (TPA application) further con-
firms the anti-inflammatory properties of Myrtol in an
isolated perfused bovine udder model. In supplemen-
tary investigations on basophilic leukaemia cells in
rats, Myrtol inhibited 5-lipoxygenase, another key en-
zyme in the inflammatory cascade [19]. Our data are
therefore in good accordance with earlier reports, al-
though we used an alveolar macrophages based system
measuring fluorescence as a non-selective method to
compute ROS, rather than a cell free system using gas
chromatography as Graßmann et al. did [14]. Antiox-
idative properties of Myrtol were comparable with
those of orange oil, eucalyptus oil, but also with NAC
which we used as a positive control. NAC is well
known for its direct (through oxidation of its molecu-
lar SH-group), but also for its indirect (precursor func-
tion of cellular glutathione formation) antioxidative
potential [20-24]. The exact mechanism how essential
oils exert this function on inflammatory cells is still
unknown.
Our experimental design did not allow direct inhibi-
tion of oxygen radicals, e.g., via oxidation as known
from NAC or glutathione. So, we assume that Myrtol,
eucalyptus oil, and orange oil interfere with pro-in-
flammatory cell metabolism, eventually leading to a
drop in cellular ROS and cytokine release. Because this
effect is shown in alveolar macrophages from COPD
patients, who are well known to suffer from high ROS
burden and whose airways are further characterized by
elevated cytokine levels [25-27], Myrtol may function
as a logical therapeutic choice. However, it still needs
to be investigated to what extent essential oils given to
patients reveal anti-inflammatory function in the air-
ways, and if so, whether this effect directly correlates
with clinical measurable benefits for the patients, be-
side its proven mucolytic efficacy.
Earlier studies indicate that Myrtol standardized
protects against COPD exacerbations [7, 12, 13]. The
authors stated that mucolytic ability may have been re-
sponsible for this effect. Considering our results and
e
arlier reports [14, 19], it can be assumed that anti-in-
f
lammatory properties of Myrtol may have con-
tributed to the reduction of exacerbation rates in
COPD as reported from earlier trials [13].
The present study has various disadvantages. Our
single data point varied considerably as indicated by
large standard deviation, being predominately related
to the cell source. The quality of lavaged alveolar
macrophages is not always uniform, due to various
reasons related to the individual patients, their smok-
ing status, medication taken, disease stage, co-morbidi-
ty, and the patient's performance during flexible bron-
choscopy. To cope with these problems, we measured
our samples in triplicates, causing a shortage of avail-
able cells. Due to a limited number of alveolar
macrophages, we were unable to perform all assays si-
multaneously with the three essential oils. For that rea-
son, we tested only Myrtol for IL-8 and GM-CSF re-
duction, but not eucalyptus oil and orange oil. All in
all, we were able to confirm previous reports that es-
sential oils and, particularly, Myrtol have anti-inflam-
matory properties.
Acknowledgments: Supported by a research grant from G.
Pohl-Boskamp GmbH & Co. KG, Hohenlockstedt, Ger-
many.
Conflicts of interest: The authors reported no conflicts of in-
terest in relation to this article.
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Corresponding author :
Adrian Gillissen, MD PhD
St. George Medical Center
Robert-Koch-Hospital
Nikolai-Rumjanzew-Str. 100
D-04207 Leipzig, Germany
Phone: +49 341 4231201
Fax: +49 341 4231201
E-mail: adrian.gillissen@sanktgeorg.de
www.rkk-leipzig.de
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