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The Antispasmotic Activity
of Thymus vulgaris Extract
Original Paper
Abstract: The effect of an extract of Thymus vulgaris
on induced spasms was investigated on guinea-pig trachea preparations. By the
experimental setup used, effects of ethanol as the vehicle could be
differentiated from the activity of the herbal constituents. The extract
reversibly and concentration-dependently antagonized the contraction of the Musculus transverses tracheae, provoked by four different spasmogens (BaCl2, carba-
chol, histamine, prostaglandin F2a). The degree of the
antispasmodic activity was dependent on the individual spasmogen
with prostaglandin F2 a being most efficiently antagonized.
Key words: Thymus vulgaris, Lamiaceae, guinea-pig
trachea, antispasmodic activity, ethanol.
Introduction
Thymus vulgaris and preparations are reported to have
a broad spectrum of biological effects, e.g., antimicrobial (1), antiviral (2)
and expectorating (3). Thyme preparations are traditionally used internally as
well as externally, for instance, against whooping cough and bronchitis. The
isolated guinea-pig trachea is a well accepted pharmacological model for the
study of bronchospasmolytics (4). In the literature
there is one publication about the test of ethanolic
extracts of Thymus vulgaris on this organ preparation (5). Only 6 out of 14
different extracts significantly reduced the tone of guinea-pig trachea.
The extracts were only tested versus carbachol as the spasmogen (5).
In a later paper the investigation of flavones as putative active components of
Thymus vulgaris on the guinea-pig trachea versus carbachol
was described (6). Ethanol may also contribute to the total effect of the
Thymus vulgaris extracts and the biological response may depend on the spasmogen used. Therefore, we have studied the
antispasmodic action of thyme extract and ethanol alone versus BaCl2, carbachol, histamine, and prostaglandin F2 a in order to discriminate between drug and solvent effects
in the presence of different spasmogenic stimuli.
Materials and Methods
Chemicals unless otherwise indicated, all chemicals
were of p.a. grade and were purchased from Merck (
For the preparation of thyme extract BNO 1018, the
comminuted herb of Thymus vulgaris (Plantamed Lot
500181) was percolated with a mixture of water/96% ethanol 3/7 (v/v). After
filtration the resulting extract had a residual dry mass of
16.2 mg/g or, respectively, 14.47 mg/ml and an
ethanol concentration
of 66.9% (v/v) (GC). The concentrations of thymol and carvacrol were 0.072%
and 0.005%, respectively, as determined by capillary gas chromatography. An
HPLC profile of the intermediate polar range of the constituents of BNO 1018
which is mainly represented by polyhydroxy
compounds was recorded at 256 and 332nm (Fig.1A). The separation was performed on
a HP1090 (series 2) HPLC system equipped with an integrated diode array
detector. The column (250 . 3 mm) was filled with Eurospher 100 (C18/5mm) RP material. The solvent flow was
adjusted to 0.3 ml/min at 60 8C. BNO 1018 was diluted with twice the volume of
60% (m/m) ethanol and 5 ml were injected. A gradient formed of 0.85% phosphoric
acid (= solvent A) and acetonitrile (= solvent B) was
used as
mobile phase (gradient: 0 min 93% A + 7% B, 40 min 70% A + 30%
B). The UV-Vis spectra of the peaks 2±5 are shown in Fig.1B).
Equipment fo pharmacological
experiments An IOA-5301 isolated organ bath assembly with 10 ml thermostatic jacketed
chambers was from Föhr Medical Instruments
(Seeheim/Ober-Beerbach,
Antispasmodic Activity of Thymus vulgaris Extract on
the Isolated Guinea-Pig Trachea:
Discrimination Between Drug
and Ethanol Effects
Adolf Meister1, Günther
Bernhardt1, Volker Christoffel2, and Armin
Buschauer1,*
1 Institut für Pharmazie, Universität Regensburg,
2 Plantamed Arzneimittel GmbH, Neumarkt/Opf.,
Revision accepted:
Planta Medica 65 (1999) 512±516
_ Georg Thieme
Verlag
·
ISSN: 0032-0943
Received:
512 Preparation of the guinea-pig trachea
A breeding
stock of Dunkin Hartley Crl:(HA)BR guinea-pigs was obtained from
Guinea-pigs (400±700 g) of both sexes were killed by decapitation,
the thoracic part of the trachea was rapidly removed and immersed in bathing
solution at 37 8C. The bathing solution (136.9mM NaCl,
11.9mM NaHCO3, 8.3mM glucose,
2.7mM KCl, 1.8mM CaCl2,
1.1mM MgCl2 and 0.4mM NaH2PO4) was gassed with carbogen_
(95% O2, 5% CO2, Linde, Höllriegelskreuth,
Germany) to maintain a pH-value of 7.4. The trachea was dissected free of
adhering adipose and connective tissue under a stereomicroscope and cut into
eight rings, each containing 2 or 3 adjoining cartilages. A cotton thread was
tied to the cartilage of each tubular segment onboth
sides adjacent to the Paries membranaceus
before the rings were opened ventrally by cutting the cartilage opposite to the
Musculus transversus
tracheae. This preparation allows the measurement of the activity of the
tracheal smooth muscle uninfluenced by the elasticity of the cartilage (7).
Determination of antispasmodic activity
Each preparation was mounted isometrically
in a 10ml organ
bath, filled with bathing solution (containing 1mm indomethacin) which was continuously gassed at 37 8C with carbogen_. The organ preparations were given an initial
resting tensionof 8mN and were allowed to equilibrate
for 60±80 minutes. When a stable baseline was obtained, the smooth muscles were
contracted by addition of the spasmogen under investigation
at a concentration provoking submaximal contractions.
The spasmogen was washed out, when the force reached
a plateau, i.e., after about 5±15 min, and the isolated organ preparations were
allowed to relax until the baselines had stabilized again. The whole procedure
was repeated (usually 3±5 times) until the difference in the amplitudes of two
subsequent contractions was less than 10%. Before cumulative
concentration-response curves (8) were recorded for the individual
spasmogen, the organ preparations were allowed to reequilibrate for 30±50 min. These curves, which were
obtained with the spasmogen alone, were taken as
internal controls. After removal of the spasmogen,
when the stable basal tension
of the tracheal strip had been reached, the test compound
or extract was added to the bath, 5 min prior to the construction of cumulative
concentration-response curves of the respective spasmogen.
In order to distinguish between the effect of the vehicle ethanol and changes
in contractility induced by the active compound(
s) of Thymus vulgaris,
control experiments were always performed in parallel with the respective
ethanol concentrations, corresponding to the ethanol content of the different dilutions
of the extract.
Data analysis and statistics
For each individual tracheal preparation the maximal
contraction
of the first concentration-response curve, obtained for
the respective spasmogen
alone, was set as 100%, whereas
Fig. 1 HPLC fingerprint of extract BNO 1018. (A)
Chromatograms
of the intermediate polar range recorded at 256 nm
(solid line) and
332 nm (dotted line). Peak 1 (16.6 min) was
identified as chloro-
genic acid. (B) Normalized UV-Vis
spectra of constituents 2
(24.9 min), 3 (29.2 min), 4 (32.7
min) and 5 (34.1 min).
Fig. 2 Original charts of the isometric contraction
of the M. trans-
versus tracheae in an isolated guinea-pig organ
preparation. (A; in-
set) The untreated tracheal preparation is spontaneously
active. (B)
The spontaneous contraction is inhibited by addition
of 1
m
M indo-
methacin into the organ bath (arrow), resulting in baseline stabiliza-
tion.
Antispasmodic Activity of Thymus vulgaris Extract on
the Isolated Guinea-Pig Trachea Planta Med. 65 (1999)
513
maximal relaxation (observed during the whole experiment)
was defined as 0%. Means SEM were plotted as
concentration-
response curves. In Figure 8 the effect of ethanol was
subtracted from the effect of the ethanolic
thyme extract and
maximal total errors were considered according to the laws
of
error propagation.
Results and Discussion
Trachea contraction: stabilization of the baseline
In vitro the isometrically
mounted untreated trachea preparations
were spontaneously active (cf. Fig. 2A). Due to the
irregular
contractions and relaxations it was very difficult to distinguish
between these spontaneous activities and drug related
effects. As shown in Figure 2B, addition of 1
m
M indomethacin
to the bathing medium resulted in a stable baseline of
the tone of the tracheal muscle, a prerequisite for
precise and
reproducible measurements. At this concentration, the cyclooxygenase
inhibitor indomethacin produces a
long-lasting
depression of the intrinsic tone of the tracheal preparation
(9), (10). Therefore, all experiments were performed
in the
presence of 1
m
M indomethacin.
Fig. 4 Inhibition of BaCl2-stimulated smooth muscle
contraction
on the isolated guinea-pig trachea preparation.
Concentration-re-
sponse curves of BaCl2 alone (*) and in presence of (A) 3
(n), 10
(H) and 100 (!)
m
M papaverine;
(B) 3 (n), 10 (H) and 100 (!)
m
M
papaverine plus ethanol at a final concentration of 2.0% (v/v);
(C)
0.2 (n), 0.7 (~) and 2.0 (H) % ethanol (v/v); (D) 0.3
(n), 1.0 (~)
and 3.0 (H) % (v/v) of an ethanolic
extract of Thymus vulgaris, yield-
ing final ethanol concentrations of 0.2 (n), 0.7 (~) and
2.0 (H) %
(v/v) in the organ bath. Means of 3±4 experiments SEM.
Fig. 5 Inhibition of carbachol-stimulated
smooth muscle contrac-
tion on the isolated guinea-pig trachea preparation.
Concentration-
response curves of carbachol alone
(*) and in presence of (A) 3
(n), 10 (H), 30 (l) and 100 (!)
m
M papaverine; (B) 3 (n), 10
(H),
30 (l) and 100 (!)
m
M papaverine plus ethanol
at a final concentra-
tion of 2.0% (v/v); (C) 0.2 (n), 0.7 (~) and 2.0 (H) %
ethanol (v/v);
(D) 0.3 (n), 1.0 (~) and 3.0 (H) % (v/v) of an ethanolic extract of
Thymus vulgaris, yielding final ethanol
concentrations of 0.2 (n), 0.7
(~) and 2.0 (H) % (v/v) in the
organ bath. Means of 3±5 experi-
ments SEM.
Fig. 3 Representative registration of a cumulative
concentration-re-
sponse curve of a typical spasmogenic
agent (BaCl2) in the presence
of 1
m
M indomethacin. The isolated guinea-pig trachea preparation
was mounted isometrically
before indomethacin and BaCl2 were ad-
ded. The arrows indicate the actual concentration of the
spasmo-
gen.
Planta Med. 65 (1999) Adolf
Meister, Günther Bernhardt, Volker Christoffel, and Armin Buschauer 514
Response of isolated trachea preparation to spasmogen
Spasmolytics are commonly screened by cumulative addition
of the test compounds to a precontracted
organ preparation.
The activity of such compounds is determined by their
ability
to decrease the tone of smooth muscle (11), (12).
However, as
shown in Figure 3, the amplitude of the spasmogen induced
contraction decreases with time, with vast interindividual
differences
in the fading pattern. For this reason the above
described
procedure is prone to errors, especially false positive
results, since fading may be interpreted as spasmolytic activity.
Therefore, we decided to evaluate antispasmodic
activity
by comparing cumulative concentration-response curves
of
the spasmogen in the absence
and the presence of the extract
or the reference compound (cf. Figs. 4±7). Although
this protocol
is rather time consuming, this approach provides
reproducible
results and more detailed information, e.g., by the
shape and position of the curves (competitive or non-compettive
antagonism).
Effect of ethanol on the contractility of the
tracheal smooth
muscle
To evaluate the effect of ethanol on the trachea
preparation,
the solvent was used in concentrations that correspond
to the
final ethanol concentrations in the organ bath [i.e., 0.2
%, 0.7 %,
2.0% (v/v)] when the ethanolic
extract of Thymus vulgaris was
investigated. Within the accuracy of the method, for the highest
ethanol concentration of 2.0% (v/v) relaxation of the
tracheal
preparation was observed, when the contractions were
provoked with BaCl2, carbachol and
histamine, respectively,
whereas for prostaglandin F2
a
ethanol was ineffective
(Figs. 4C±7C). To decide, whether the observed antispasmodic
effects resulted from an active principle present in the
extract,
different concentrations of the standard musculotropic
spasmolytic
papaverine were combined with a fixed concentration
of ethanol [2.0% (v/v)] (Figs. 4B±7B). The effects of
these
combinations were compared with the effects of the respective
concentrations of papaverine (Figs.
4A±7A) and of ethanol
alone (Figs. 4C±7C). From these experiments, there was
no indication for a superadditive
effect.
Effect of an ethanolic
extract of Thymus vulgaris on the
contractility of the tracheal smooth muscle
To investigate the potential antispasmodic activity
of the
ethanolic extract of Thymus vulgaris, the tracheal
preparations
were incubated with different concentrations of the extract,
prior to the cumulative addition of various spasmogens. For
the provocation of the contraction, the musculotropic spasmogen
BaCl2 as well as the receptor agonists
carbachol, histamine
and prostaglandin F2
a
were selected. As a positive con-
Fig. 6 Inhibition of histamine-stimulated smooth
muscle contrac-
tion on the isolated guinea-pig trachea preparation.
Concentration-
response curves of histamine alone (*) and in presence of (A)
3
(n), 10 (H) and 30 (l)
m
M papaverine; (B) 3 (n), 10
(H) and 30 (l)
m
M papaverine plus ethanol
at a final concentration of 2.0% (v/v);
(C) 0.2 (n), 0.7 (~) and 2.0 (H) % ethanol (v/v); (D)
0.3 (n), 1.0 (~)
and 3.0 (H) % (v/v) of an ethanolic
extract of Thymus vulgaris, yield-
ing final ethanol concentrations of 0.2 (n), 0.7 (~) and
2.0 (H) %
(v/v) in the organ bath. Means of 3±4 experiments SEM.
Fig. 7 Inhibition of prostaglandin F2
a
-stimulated smooth muscle
contraction on the isolated guinea-pig trachea preparation. Concen-
tration-response curves of prostaglandin F2
a
alone (*) and in pres-
ence of (A) 1 (~), 3 (n), and 10 (H)
m
M papaverine; (B) 1 (~), 3
(n), and 10 (H)
m
M papaverine plus ethanol
at a final concentration
of 2.0% (v/v); (C) 0.2 (n), 0.7 (~) and 2.0 (H) %
ethanol (v/v); (D)
0.3 (n), 1.0 (~) and 3.0 (H) % (v/v) of an ethanolic extract of Thy-
mus vulgaris, yielding final ethanol concentrations of
0.2 (n), 0.7
(~) and 2.0 (H) % (v/v) in the
organ bath. Means of 4
experiments
SEM.
Antispasmodic Activity of Thymus vulgaris Extract on
the Isolated Guinea-Pig Trachea Planta Med. 65 (1999)
515
trol, different concentrations of the musculotropic
spasmolytic
papaverine were used (Figs. 4A±7A).
Usually, pA2 values are determined to describe the
potencies
of competitive antagonists, whereas pD
¢
2 values are given for
non-competitive antagonists (13). However, none of these
terms is useful for the pharmacological characterization
of
the ethanolic thyme extract,
as can be seen from the shape of
the concentration-response curves of the different spasmogens.
To convey the full information, complete concentrationresponse
curves are presented in Figures 4±7. As becomes obvious
from Figures 4D±7D, the ethanolic
extract of Thymus
vulgaris inhibited the spasmogen-induced
contractions in a
concentration-dependent manner. The inhibitory effect on
the contraction of the Musculus
transversus tracheae was reversible
upon washing (data not shown). Based on the assumption
that the effects of ethanol and the active compound(
s) of Thymus vulgaris on the
isolated guinea-pig trachea
were additive, the effect of 434
m
g/ml Thymus vulgaris on
a submaximal contraction
caused by the different spasmogens
was calculated. The results are shown in Figure 8. The
highest antispasmodic activity was found, when the
contraction
of the smooth muscle was provoked with prostaglandin
F2
a
(89% inhibition), followed by the stimulators
histamine
(73%) inhibition), and BaCl2 (49%
inhibition). The inhibitory
effect of the thyme extract on tracheal contraction was
lowest
(42%) when carbachol
was used for stimulation.
In summary, the pharmacological investigations of the
ethanolic
thyme extract on guinea-pig trachea preparations revealed
that the marked dose-dependent antispasmodic activity
found under the experimental conditions described
can definitely be attributed to the constituents of
Thymus
vulgaris.
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Prof. Dr. A. Buschauer
Institut für Pharmazie
Universität Regensburg
Universitätsstr. 31
D-93053
E-mail: armin.buschauer@chemie.uni-regensburg.de
Fax: +49 941-943 4820
Fig. 8 Antispasmodic effect of Thymus vulgaris extract
on the
isolated guinea-pig trachea preparation (concentration in the
organ
bath: 3.0% (v/v) ethanolic
extract corresponding to 434
m
g/ml dry
extract). Inhibition of the response to carbachol
(3
m
M), BaCl2
(3
m
M), histamine (30
m
M) and prostaglandin F2
a
(1
m
M) in concen-
trations producing submaximal
contraction (cf. Figs. 4±7). The am-
plitude after addition of the respective spasmogen
was taken as
100% (i.e., 0% inhibition). Values were corrected for the effect of
the vehicle (2.0% v/v of ethanol in the organ bath).
Mean values of
3±4 experiments SEM.
Planta Med. 65 (1999) Adolf
Meister, Günther Bernhardt, Volker Christoffel, and Armin Buschauer 516