ISSN: 1314-6246
Patti et al.
J. BioSci. Biotechnol. 2015, SE/ONLINE: 97-101
RESEARCH ARTICLE
Jaspreet Patti 1
Iliyana Stefanova 2
Milena Draganova-Filipova 3
Plamen Zagorchev 2
Rosemary oil reduces electrical field stimulated
tetanic muscles contractility
Authors’ addresses:
ABSTRACT
1
Student of Medicine, Medical
University – Plovdiv, 4002, Plovdiv,
Bulgaria.
2
Department of Medical Physics and
Biophysics, Medical University –
Plovdiv, 4002, Plovdiv, Bulgaria.
3
Department of Medical Biology,
Medical University – Plovdiv, 4002,
Plovdiv, Bulgaria.
Correspondence:
Plamen Zagorchev
Department of Medical Physics &
Biophysics, Medical University –
Plovdiv, 4002, Plovdiv, Bulgaria.
e-mail:[email protected]
Rosemary oil is an essential oil extracted from Rosemary officinalis. It consists of
more than 50 compounds with different biological activities. Empirically it is
used in therapy for many years. Rosemary oil is analyzed in vivo and in vitro as
many of its effects - antimicrobial, antitumor, antioxidant etc. are known. We
cannot find suggested reports concerning the Rosemary oil effect on contractility
of striated muscles. The aim of this study was to investigate the influence of
Rosemary oil on maximal tetanic contraction provoked by electrical field
stimulation. We use abdominal transversal muscles strips isolated from guinea
pigs. The tetanic muscles force, was provoked by means of repeated multipulse
electrical field stimulation, square-wave pulses of supramaximal intensity (60V)
and 0.5 ms in duration were applied at frequency of 50 Hz for 3s followed by 7s
pause. The concentration effect curve of the action of Rosemary oil (1.5.10-6
M÷1.5.10-3 M) on this type electrical field stimulated - muscle activity was
plotted on a statistical graph. During our observation the effect of Rosemary oil
on contractile properties of abdominal muscles and calculated half maximal
effective concentration (EC50). Our study revealed that myorelaxant activity of
the essential oil in this concentration is time-dependent and after application in
the interval of 25 minutes the contractile ability is decreased to 5.5% within
comparison with the initial one. Our investigation is a first report on an action of
Rosemary oil on the guinea pig's striated muscles strips.
Key words: Rosemary oil, electrical field stimulation, straight muscle
contractility
Introduction
In recent years, many plant extracts were analyzed and
their positive effects on the human body were proven. The
interest of the scientist to the herbal remedies has grown
considerably, because of their harmless effect. They are
preferable as therapeutic agents to reduce inflammation and
pain. According to the European Medicines Agency (EMA)
from 2010, rosemary is a traditional herbal medical product
and can be used for symptomatic relief of dyspepsia and mild
spasmodic disorders of the gastrointestinal tract (EMA,
2010). Rosemary essential oil (RO) can be used for treating
as an adjuvant in the relief of minor muscular and articular
pain and in minor peripheral circulatory disorders (EMA).
RO consists of different active substances– some of them are
presented in high concentrations, and other compounds are
presented in traces. The effect of this natural product is a
result of the synergetic action of all substances (Faixova &
Faix, 2008). The antibacterial, antifungal and anti-oxidant
effects of RO have been described from many authors
(Santoyo et al., 2005; Bozin et al., 2007). Some rosemary
constituents have analyzed in vitro and in vivo as a potential
anticancer agents (Huang et al., 1996). Its ability to reduce
DNA damages and tumor formations determine the possible
application as the chemo preventative agent (Singletary et al.,
1996). Its anti-inflammatory and analgesic potential of RO
was proven (Takaki et al., 2008; Raskovic et al., 2015). The
application of rosemaryin folk medicine as spasmolytic was
SPECIAL EDITION / ONLINE Section “Medical Biology”
Second National Youth Conference “Biological sciences for a better future”, Plovdiv, October 30-31, 2015
97
ISSN: 1314-6246
Patti et al.
J. BioSci. Biotechnol. 2015, SE/ONLINE: 97-101
RESEARCH ARTICLE
analyzed and antispasmodic activity on the smooth muscles
contractions was proven. The authorsinduced contractions on
muscles strips from ileumby KCl and demonstrated the
involvement of calcium channels in this activity (VenturaMartínez et al., 2011). However, there is no scientific
evidence concerning the RO effect on contractility of
abdominal transversal muscles and mechanism of its action is
unknown. This determines our interest to RO as a potential
product for reduction of tetanic muscle contraction.
Materials and Methods
Ethics statement
All experiments (in vivo and in vitro) were approved by
the Bulgarian Food Safety Agency and the Ethics Committee
of the Medical University of Plovdiv, Bulgaria.
In vitro striated muscle experiments
Animals and tissue preparations
Fifteen male Guinea pigs (300350) g were kept under
standard laboratory conditions (temperature 22±1°C,
humidity 45% and 12-h light cycle). All in vitro experiments
were approved by the Bulgarian Food Safety Agency.
Male Guinea pigs were euthanized and transversus
abdominis striated muscles (ASM) were isolated.
Preparations were obtained while cutting the muscle tissue in
strips (20.0±1.5 mm length, 3.0±0.5 mm width). The samples
were immediately rinsed and cooled (4°C) preparation
solution. The muscle strips were isometrically fixed in
individual organ baths containing 15 mL modified Krebs’
solution (KS) with temperature 35.5±0.25°C and constantly
oxygenated with 95% O2 and 5% CO2. The preparations were
connected to an isometric force transducer (TRI 201, LSi
LETICA; Pnlabs.l., Barcelona, Spain). Preparations were
allocated to the organ baths in random manner and muscle
tension (7 mN) was applied to achieve isometrical recording.
They were allowed in an equilibration period of 20 minutes.
For inducing contractile activity, we used electrical field
stimulation (EFS) through platinum electrodes. The
electrodes were connected to both sides of each strip and to
electronic stimulator (EFS-PZ03, C-optic, Bulgaria). Tetanic
contractions were induced by pulse with intensity 60V;
duration 0.5 ms and frequency 50 Hz which determinate
conditions, similar to direct (muscle) stimulation (MS).
Shiina et al. (2010) and Su et al. (2012) conducted
experiments with EFS and similar to our parameters. The
98
mechanical muscles activity was recorded according to
experimental protocol represented previously (Ivanov et al.,
2011) with 1 ms interval of discretisation.
Concentration-response curve for Rosemary oil
The normal contractile activity was recorded when
muscle contraction (MS) was applied after the equilibration
period to preparations. RO in concentration 1,5μМ was added
to the organ baths and the change in the contractile activity
was recorded for a 5-min period. The strips were washed out
with KS before adding higher concentration. RO
concentrations 15μМ; 0.15mМ; 1.5mМ were studied and a
concentration - effect curve was obtained. The cut-off time
for each experiment was 45 minutes after muscle isolation.
Effect ofRosemary oil on the amplitude of tetanic muscle
contraction
The normal tetanic MS was obtained by 3s electrical
stimulus (constant supramaximal intensity 60V; duration 0.5
ms; frequency 50 Hz) and was compared to activity which
was achieved after 25min exposure of the strips on 0,15mМ
RO.
Drugs and solutions
Rosemary oil was purchased from Dullberg comp,
Hamburg, BRD. The preparation solution contains Na + (143
mmol/L); K+ (5.84 mmol/L) and Ca2+ (3.7 mmol/L).
Composition of KS: Na+ - 143 mmol/L; K+ - 5.84 mmol/L;
Ca2+ - 2.5 mmol/L; Mg2+ - 1.19 mmol/L; Cl− - 133 mmol/L;
HCO3− - 16.7 mmol/L; H2PO4− - 1.2 mmol/L and glucose 11.5 mmol/L.
Statistics
Skeletal muscle activity: Data are presented as mean and
standard error (SEM). Normal distribution was tested with
one sample Kolmogorov-Smirnov test. One-way analysis of
variance (ANOVA) and Tuckey post hoc test were used in
case of normal distribution. In other case non-parametric
Wilcoxon signed rank test was used. The number of tested
preparations is given as n. Results were considered
significant at P<0.05.
Results
We evaluated the effects of RO in concentrations of 1.5
μМ (n=7; P>0.05); 4.5 μМ (n=8; P>0.05); 30 μМ (n=8;
P>0.05); 0.3 mМ (n=8; P<0.05); 0.75 mМ (n=8; P<0.05);
1.05 mМ (n=9; P<0.05) and 1.5 mМ (n=7; P<0.05) on the
SPECIAL EDITION / ONLINE Section “Medical Biology”
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ISSN: 1314-6246
Patti et al.
J. BioSci. Biotechnol. 2015, SE/ONLINE: 97-101
RESEARCH ARTICLE
maximal muscle force, generated by ASM. The maximal
muscle force after MS in absence of RO was taken as 100%
and the IC50 of RO was calculated (Figure 1). The maximal
muscle force of preparations after MS was reduced with 50 %
when 0.15mМ RO was present in the medium. We found no
significant difference in the duration of muscle twitches
before and after treatment with RO (data not shown).
The application of stimuli with high frequency (50 Hz)
evokes a tetanic muscle contraction. In 10 min presence of
0.15mМ RO in the medium the maximal muscle force (10.35
± 0.54 mN) was reduced to 8.46 ±1.35 mN.
The application of stimuli with high frequency and long
lasting duration of 0.5 ms evokes a tetanic muscle
contraction. In the background of 0.15 mМ RO, applied for
20 min, the maximal muscle force (10.35±0.54 mN) was
reduced to 5.76±0.40 mN (Figure 3 - top). If 0.15mМ RO
presences in the medium for more than 25 min we can detect
only a little alteration of the normal muscular tonic activity.
The muscle response to the electrical stimulus 500µs 60V
50Hz can be calculated as a 3.5% - rise of the tonic striated
muscle force (Figure 3 - bottom).
To check striated muscle condition after application of
RO we studied effect of MS on the maximal muscle force
generated by AMS after 3h application of 0.15 mМ RO and 6
times tissue-bath solution replacement. Results on Figure 4
showed relative the same maximal striated muscle force,
which is an indication for unaffected muscle tissue in case of
long treatment with RO - Figure 4.
Figure 1. Concentration-response curve of RO on the
generated muscle force after MS. * P˂0.05 vs. controls
(Tukey post-hoc test).
Figure 2. Effect of 10 min 0.15mМ RO action on the maximal
muscle force generated by AMS after MS (↑). Tetanic muscle
contractions (60V; 0.5ms; 50 Hz) before and after addition of
RO to the organ baths. Arrows (↑) indicate the initial moment
of MS.
Figure 3. Effect of 20 min and 25 min action of 0.15m М RO
on the maximal muscle force generated by AMS after MS (↑).
Tetanic muscle contractions (60V; 0.5ms; 50 Hz) before and
after addition of RO to the organ baths. Arrows (↑) indicate
the initial moment of MS.
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J. BioSci. Biotechnol. 2015, SE/ONLINE: 97-101
RESEARCH ARTICLE
Figure 4. Effect of MS (↑) tetanic muscle contractions (60V;
0.5ms; 50 Hz)on the maximal muscle force generated by AMS
after 3h application of 0.15mМ RO and 6 times tisue-bath
solution replasement. Arrows (↑) indicate the initial moment
of MS.
Discussion
Rosemary essential oil is one of the most popular
essential oils for its wide array of health benefits. In applying
the RO in high concentrations on smooth muscles registered
spasmolytic effect which is reduced at low concentrations in
vitro (Sagorchev et al., 2010). This gave us reason to presume
that rosemary affects the contractile properties which is
confirmed in our experiments tracking the nature of tonic
contractions on striated muscle after application of electrostimulation in the absence and presence of Rosemary
essential oil.
It is known that the muscle tissue is controlled with
electric stimulation under the nerve system in vivo. From a
biophysical point of view possible stimuli is the electrical
stimulation (Thelen et al., 1997). The tetanic contractions on
the abdominal transversal muscles were induced by impulses
with supramaximal intensity 60V, duration 0.5 ms and
frequency 50Hz. The motor-induced responses were studied
in implementing the EFS on controls on such pre-treated with
RO (1.5.10-6 M1.5.10-3 M). As controls in these experiments
were used motor responses induced electrical charges from
field stimulation. The provoked mechanical responses have
unique nature - rapid contraction with peak immediately
followed by relaxation. The maximal muscle force of
preparations after MS was reduced with 50 % when 0,15mМ
RO was present in the medium. Our study revealed that
myorelaxant activity of the RO in concentration 0,15.10 -3M is
time-dependent and after application in the interval of 25
minutes the contractile ability is decreased to 5,5% within
comparison with the initial one.
100
Increasing the time duration of RO action at the same
other conditions lead to a reduction of the response
amplitudes, which could be explained by intensifying the
repolarization and the release of various mediators of
intramural nerve structures. It is known that a fundamental
prerequisite for muscle relaxation is the reduction of
intracellular Ca2+ concentration. This reduction is possible in
the inhibition of transmembrane Ca2+ transport, decreased
release of Ca2+ from intracellular calcium stores, activation of
different types of Ca2+ pumps, as well as the synchronized
activation of several of the said processes (Widmaier et al.,
2010).
Due to the main application of essential oils in
aromatherapy massage it will be important to prove their
ability to penetrate through the skin and the existence of the
local effect. But action on skeletal muscles in vitro is
different for different oils (Lis-Balchin, 2005). The chemical
diversity of the RO constituent determines the diversity of
their potential to affect various biological structures and
processes. Since there are no literature data RO to penetrate
muscle cells probably the described phenomenon is
connected with influence on the intramural nerve structures
or the membrane receptors. Such interference could explain
the change in the power of electrostimulation responses
observed following administration of RO - responsive Ca2+
current.
Conclusion
We characterized the contractile properties of abdominal
transversal muscles. The present results suggest that
Rosemary essential oil depresses force development,
probably acting as a calcium channel blocker. It is the first
report on a new mechanism of action of Rosmarinus
officinalis on the guinea pig striated muscles. We speculated
that the RO may offer powerful and effective therapies for
muscular spasms, tissue repair and myorelaxation.
References
Bozin B, Mimica-Dukic N, Samojlik I, Jovin E. 2007. Antimicrobial
and antioxidant properties of rosemary and sage (Rosmarinus
officinalis L. and Salvia officinalis L., Lamiaceae) essential oils.
J. Agric Food Chem., 55 (19): 7879–7885.
European Medicines Agency. Community herbal monograph on
Rosmarinus officinalis L., aetheroleum, 2010. Available from:
ehttp://www.ema.europa.eu/docs/en_GB/document_library/Herb
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J. BioSci. Biotechnol. 2015, SE/ONLINE: 97-101
RESEARCH ARTICLE
al_Community_herbal_monograph/2009/12/WC500018312.pdf
Faixova Z, Faix S. 2008. Biological effects of rosemary
(Rosmarinas officinalis) essential oil. Folia Vet., 52(3-4): 135–
139.
Huang M, Ho CT, Wang ZY. 1994. Inhibition of skin tumorigenesis
by rosemary and its constituents carnosol and ursolic acid.
Cancer Res., 54(3): 701–708.
Ivanov I, Nikolova S, Aladjov D, Stefanova I, Zagorchev P, 2011.
Synthesis and contractile activity of substituted 1,2,3,4
Tetrahydroisoquinolines. Mol., 16: 7019–7049.
Lis-Balchin M. 2005. Aromatherapy Science: A Guide for
Healthcare Professionals. Pharmacological study of essential
oils: in vivo and in vitro, Pharmaceutical Press, London, p. 4557.
Raskovic A, Milanovic I, Pavlovic N, Milijasevic B, Ubavic M,
Mikov M. 2015. Analgesic effects of rosemary essential oil and
its interactions with codeine and paracetamol in mice. Eur. Rev.
Med. Pharmacol. Sci., 19 (1): 165–172.
Sagorchev P, Lukanov J, Beer AM. 2010. Investigations into the
specific effects of rosemary oil at the receptor level.
Phytomedicine, 17(8-9):693–697.
Santoyo S, Cavero S, Jaime L, Ibanes E, Senorans FJ, Reglero G.
2005. Chemical composition and antimicrobial activity of
Rosmarinus officinalis L. essential oil via supercritical fluid
extraction. J. Food Prot., 68 (4): 790–795.
Shiina T, Shima T, Masuda K, Hirayama H, Iwami M, Takewaki T,
Kuramoto H, Shimizu Y. 2010. Contractile properties of
esophageal striated muscle: comparison with cardiac and
skeletal muscles in rats. J. Biomed. Biotechnol., p. 7.,
doi:10.1155/2010/459789.
Singletary K, MacDonald C, Wallig M. 1996. Inhibition by
rosemary and carnosol of 7,12-dimethylbenz[a]anthracene
(DMBA)-induced rat mammary tumorigenesis and in vivo
DMBA-DNA adduct formation. Cancer Lett., 104 (1): 43–48.
Su T, Zei W, Su C, Hsiao G, Lin M. 2012. The effects of the KCNQ
openersretigabine and flupirtine on myotonia in mammalian
skeletal muscle induced by a chloride channel blocker.
Evidence-Based
Complem.
Altern.
Med.,
p.
9,
doi:10.1155/2012/803082.
Takaki L, Bersani-Amado E, Vendruscolo A, Sartoretto SM, Diniz
SP, Bersani-Amado CA, Cuman RKN. 2008. Anti-inflammatory
and antinociceptive effects of Rosmarinus officinalis L.
Essential oil in experimental animal models. J. Med. Food.,
11(4): 741–746.
Thelen MH, Simonides WS, Van Hardeveld C. 1997. Electrical
stimulation of C2C12 myotubes induces contractions and
represses thyroid hormone-dependent transcription of the fattype sarcoplasmic reticulum Ca2+-ATPase gene. Biochem. J.,
321: 845–848.
Ventura-Martínez R, Rivero-Osorno O, Gómez C, GonzálezTrujano ME. 2011. Spasmolytic activity of Rosmarinus
officinalis L. involves calcium channels in the guinea pig ileum.
J. Ethnopharmacol., 137 (3): 1528–1532.
Widmaier ER, Hersel R, Strang KT. 2010. Muscle. Vander's Human
Physiology: The mechanisms of body function, 12th ed.,
McGraw-Hill, New York, p. 250–291.
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