Relaxation Effect of Ethanol Extract Black (EEB) on Plain Muscle
Contraction from Insulated Guinea Pig’s Trakea by Acethyloline
Ramanathan, Ermi Girsang, Ali Napiah Nasution and I. Nyoman Ehrich Lister
Faculry of medical , Universitas Prima Indonesia, Indonesia
Keywords: Black Intersection ethanol extract, acetylcholine, guinea pig, trachea, relaxation, in vitro.
Abstract: Indonesia, asthma is a chronic disease that occurs in respiratory tract that make difficulties breathing. This is
reflected in the Household Health Survey Study data (SKRT) in various provinces in Indonesia. This study
aims to determination of mechanism of relaxation on contraction smooth muscle of guinea pig’s trachea.. This
research use in vitro model and use organ bath. The parameter measured in this research was contraction and
relaxtion of isolated animal trachea. From this reaserch informed that EEBC (0.5-4 mg/ml) concentration
effected the trachea smooth mucle and make it contract that induced by acetylcholine 1.43 x 10-4 M (r=0.982;
p<0.05). The concentration of 4mg/ml EEBC, statistically there is no different with atropine sulfate 1 x 10-6
M in decrease the contraction of guinea pig’s trachea smooth muscle induced by acetylcholine 1.43 x 10-4 M
(p>0.05). EEBC has been known have relaxing effect on smooth muscle especially trachea and has the
potency to reduce the contraction of trachea contracted with acetylcholine. The action EEBC on isolated
trachea was mediated by inhibition of the PDE enzyme.
Asthma is a disorder of chronic airway inflammation
that causes narrowing of the airways (bronchial
hyperactivity), causing recurrent episodic symptoms
like wheezing, shortness of breath, chest feeling
heavy, and coughing, especially at night or early
morning. Asthma is also a serious world health
problem that affects all age groups, ranging from
children to adults who have many adverse effects
both on themselves, family, and society. Based on the
results of the Basic Health Research (Riskesdas) in
Indonesia in 2013, the asthma prevalence was 4.5%
with the highest incidence in women was 4.6%
(Runtuwene et al, 2016). At present asthma still
shows a high prevalence. Based on data from the
World Health Organization (WHO), the number of
asthma patients in the world is estimated to reach 300
million people, and it predicted that this number
would continue to increase to 400 million sufferers by
2025. This number could be even greater considering
asthma is an underdiagnosed disease.
Asthma is a chronic inflammation of the airways
with various cell and cellular elements that play a
role. Inflammation Constant is associated with hyper-
responsive airways which results in repeated episodes
of wheezing, chest tightness, shortness of breath and
coughing, especially at night or early days. Asthma
symptoms vary, multifactorial and in a manner
potential associated with bronchial inflammation.
Proof that inflammation is a component of asthma
Eosinophil, neutrophil, cell degranulation found
mast, thickening of the sub-basement membrane, loss
of integrity epithelial cells, bronchial lumen
obstruction by mucus and Goblet cell hyperplasia at
the autopsy performed on asthma patient. Synthetic
drugs currently used in asthma pharmacotherapy
cannot act at all stages and targets of asthma, but the
herbal approach has regained its popularity in
traditional treatment systems (proven to provide
symptomatic help and help in inhibiting disease
progression). Many plants have been documented
beneficial for the treatment of various respiratory
disorders including asthma. Herbs have shown
interesting results in various specific biological
activity targets such as bronchodilation,
nephroprotective, cardioprotective, anti-
inflammatory, anticancer, immunomodulatory and
inhibiting mediators such as leukotriene,
lipoxygenase, cyclooxygenase, platelet activator,
phosphodiesterase and anti-spasmodic cytokines in
the treatment of respiratory disorders such as asthma.
Ongoing research around the world also provides
Ramanathan, ., Girsang, E., Nasution, A. and Ehrich Lister, I.
Relaxation Effect of Ethanol Extract Black (EEB) on Plain Muscle Contraction from Insulated Guinea Pig’s Trakea by Acethyloline.
DOI: 10.5220/0010292902020206
In Proceedings of the International Conference on Health Informatics, Medical, Biological Engineering, and Pharmaceutical (HIMBEP 2020), pages 202-206
ISBN: 978-989-758-500-5
2021 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
valuable clues about the exact mechanism of action
of alternative herbs (Ravindra and Avinash, 2011;
Ismail et al, 2020). Asthma can be controlled with
drugs that prevent chronic symptoms and relievers for
symptoms of asthma that spread. Education and
understanding are the keys to controlling asthma
effectively, if an asthma attack is not managed
properly it can be fatal. Asthma can actually be
prevented and treated early by always implementing
healthy living behaviors, consuming nutritious food
and drinks, exercising and getting enough rest and
avoiding things that can potentially cause asthma. For
those who have already suffered from Asthma, this
disease can be cured through modern medicine or
using natural herbal medicine.
Black Intersection is known to have many benefits
and pharmacological activities, including
bronchodilators, anti-cancer, antidiabetic,
antihyperlipidemic, and neutralizing free radicals.
Black Intersection is easily found in many tropical
regions including Indonesia, Malaysia, and Thailand
traditionally used as herbal medicines which are
consumed for managing body health (Curcuma
aeruginosa Roxb).
Based on the high incidence of asthma that occurs
in all age groups and the anti-oxidant and relaxation
effects of ethanol extract Black Curcuma (EEBC) are
very good, researchers are interested in conducting
the benefits of EEBC on tracheal smooth muscle
contraction in male guinea pigs induced by
acetylcholine in vitro and their role in muscarinic
receptors, and their effects in inhibiting the enzyme
phosphodiesterase. From several literature studies,
research with EEBC is still rarely found.
This research conducted experimentally including the
collection of plant material, manufacture of simplicia,
characteristics of simplicia, phytochemical screening
of simplicia and extraction of the preparation of the
test material and the stages of testing the EETH
relaxation effect on the contraction of trachea animal
induced by an organ organ. In this study there are two
variables, namely the independent variable and the
dependent variable; independent variables are ethanol
extract black methamphetamine (EEBC) with a
concentration of 0.5; 1; 1,5; 2; 2.5; 3; 3.5; and 4 mg /
ml, while the dependent variable is relaxation of the
isolated trachea contracted by acetylcholine (Figure
1). Sample Preparation and Extraction: The leaves of
Curcuma aereginosa (Figure 2) collected from old
deli market, Dairi Regency, North Sumatra Province,
Indonesia. Simplisia weighed 500 grams put into a
container. The first maceration of simplicia was
soaked with ethanol as much as 5 L for 6 hours while
stirring occasionally, then allowed to stand for 18
hours. The filtrate was separated from the residue.
Then the residue was dried in an oven at 500C. After
the dry, residue was macerated again with 5 L.
ethanol solvent soaked for 6 hours while stirring
occasionally, then let stand for 18 hours. The filtrate
was separated from the residue and the residue was
dried, then maserat evaporated with a rotary
evaporator so that a thick extract was obtained.
Figure 1. Research conceptual framework
Figure 2. Black Intersection tree (Borobudur Herbal
medicine industry).
Relaxation Effect of Ethanol Extract Black (EEB) on Plain Muscle Contraction from Insulated Guinea Pig’s Trakea by Acethyloline
The animals used in this study were male guinea
pigs (Cavia porcellus), weighing between 300-500
grams with ages 3-4 months. This animal was
acclimatized for one week with the aim to
homogenize its food and life so that it is deemed
eligible for research. Before being used in research,
animal experiments were fasted for 24 hours so that
the tracheal contraction used was not influenced by
other substances. The research procedure used first
was submitted to the Ethical clearance for research to
obtain ethical approval that the procedures performed
meet the ethical requirements in experimental
The guinea pig trachea which has been normalize
for 45 minutes was contracted by giving acetylcholine
solution so as to obtain the maximum submaxative
concentration in the organ bath. After obtaining the
maximum stable contraction conditions, the graded
concentration of EEBC was performed. The response
that occurs will be recorded on the order. Testing the
mechanism of action through inhibition of muscarinic
receptors is done by comparing the contraction
pattern of acetylcholine that has been incubated
EEBC with the contraction pattern of acetylcholine
incubated atropine sulfate and the pattern of
acetylcholine contraction without incubation.
The thracea that has been balanced for 45 minutes
is incubated for 20 minutes with EEBC in the organ
immersion. Then the trachea is contracted by giving
acetylcholine chloride solution. Responses to
contractions that occur will be recorded. The
acetylcholine chloride solution is given until the
maximum submaxative concentration is reached.
After obtaining a stable maximum contraction
condition, EEBC is given with multilevel
concentration. The response that occurs will be
recorded on the order. The correlation curve of EEBC
concentration and % relaxation response compared to
% relaxation response due to the administration of
EEBC without incubation with theophilin.
The data obtained in this study are % contraction
and % relaxation of tracheal smooth muscle on a
computer. The relationship graph between the
logarithm of concentration and % response is made.
EC80 value (concentration of agonist that can
produce a response of 80% of the maximum
response) receptor agonist, calculated based on the
graph of the relationship of concentration to%
response. EC80 is calculated based on the equation
Based on phytochemical tests on methanol extracts of
Black Intersection Rhizome (Curcuma aeruginosa
Roxb.) With Thin Layer Chromatography (TLC)
method to determine the contentthe types of
secondary metabolites such as alkaloids, flavonoid,
steroid, saponin and tannin. The relaxation effect of
the EEBC by increasing the concentration (0,5-4
mg/ml). The administration of the EEBC
concentration series produced a relaxing effect on the
contractions induced acetylcholine.
Table 1. The % EEBC relaxation data in the tracheal smooth
muscle contracted with 1.43 x 10-4 M acetylchoin
Based on the regression correlation test on the
percentage of the effect of relaxation with the EEBC
concentration is a positive correlation with a
correlation value of 0.970 (the correlation is close to
1) and an R Square value (R2) of 0.940. It can be
stated that about 94.0% the percentage of the relaxing
effect of extracts on tracheal smooth muscle increases
with increasing EEBC concentration. Testing the
mechanism of action through inhibition of muscarinic
receptors was done by comparing the contraction
strength of acetylcholine (1.43 x 10-4 M) which has
been incubated with EEBC at a concentration of 3.2
M with the contraction strength of acetylcholine (1.43
x 10-4 M) without incubation and the contraction
strength of acetylcholine induced with atropine
sulfate (1 x 10-6 M) as shown in Figure 3.
HIMBEP 2020 - International Conference on Health Informatics, Medical, Biological Engineering, and Pharmaceutical
Figure 3. Relaxation effect% EEBC on tracheal guinea pigs
contracted using 1.43 x 10-4 M acetylchoin.
The contraction effect of acetylcholine incubated
with EEBC compared to acetylcholine without
incubation showed statistically significant differences
(p <0.05). Statistical results of the comparison of the
contraction of acetylcholine (1.43 x 10-4 M)
incubated EEBC at a concentration of 3.2 M with
acetylcholine (1.43 x 10-4 M) incubated atropine
sulfate (1 x 10-6 M) also showed results significantly
different (p <0.05). EEBC and atropine sulfate both
have a relaxing effect on acetylcholine-induced
contractions, but the contraction force incubated with
atropine sulfate was smaller, because atropine sulfate
used is a pure compound, whereas at EEBC it is not a
pure compound but is a plant chemical compound in
which it is smaller. There are still some other plant
chemical compounds. The results of testing the
mechanism of the EEDC's relaxing effect on the
tracheal smooth muscle can be seen in Table 2 and
Figure 4.
Based on statistical data it can be indicated that
EEBC relaxes the guinea pig's smooth muscle by
inhibiting muscarinic receptors, this can be proven by
looking at the statistical comparison of the results of
acetylcholine without incubation and acetylcholine
incubated by EEBC which shows significantly
different contraction results between the two (p <0,
05). EEBC inhibits the increase in contractions by
acetylcholine which has been described above that
acetylcholine works to stimulate muscarinic receptors
which increase smooth muscle contraction. The
increase in contractions by acetylcholine decreases
because the muscarinic receptors are already largely
inhibited by EEBC so that the muscarinic receptors
do not all interact with acetylcholine to produce
contractions. Inhibiting the release of acetylcholine
causes reduced muscarinic receptor activity which
mediates smooth muscle contraction [13].
Table 2. % EEBC relaxation data in the tracheal smooth
muscle contracted with acetylcholine 1.43 x 10-4 M after
incubating theophylline 10-4 M
Figure 4. Relaxation effect% EEBC on guinea pigs tracheal
contracted using 1.43 x 10-4 M acetylchoin after
theophylline incubation.
The initial incubation of the tracheal smooth
muscle with theophylline aims to inhibit the activity
of phosphodiesterase (PDE). Inhibition of PDE will
increase cellular cAMP levels so that it will cause
bronchodilation. Testing the mechanism of the EETH
relaxation effect on tracheal smooth muscle showed
that at initial incubation with theophylline 10-4 M
there was no significant difference in the EEBC
relaxation effect compared to the relaxing effect on
the treatment without theophylline incubation
(control) (p> 0.05; independent t-test). This result is
due to the inhibition of PDE by theophylline causing
EEBC no longer relaxes through this mechanism, so
the resulting relaxation effect is no different from
control. It can be concluded that the relaxing effect of
EEBC is related to the inhibitory effect of
phosphodiesterase activity.
Relaxation Effect of Ethanol Extract Black (EEB) on Plain Muscle Contraction from Insulated Guinea Pig’s Trakea by Acethyloline
Black Intersection has a relaxing effect on the isolated
tracheal smooth muscle of guinea pigs contracted
with acetylcholine, Black Intersection has the ability
that is not different from atropine sulfate 1 x 10-6 M
in reducing the contraction of isolated guinea pig
tracheal smooth muscle induced by acetylcholine, the
strength of acetylcholine without incubation
contraction compared with acetylcholine with EETH
incubation showed statistically significantly different
results (p <0.05). This shows that EETH works by
inhibiting M-3 receptors. The mechanism of the
relaxing effect of EETH on isolated guinea pig
smooth muscle is mediated through inhibition of the
enzyme PDE.
Astuti, P., Yunus, F., Antariksa, B., dan Ratnawati. (2011).
Prevalensi dan Gejala Klinis Obstructive Sleep Apnea
(OSA) pada Pasien Asma. Journal Indonesia Medical
Association. 61(7): 273-279.
Barnes, P.J. (1995). Cyclic Nucleotides and
Phosphodiesterases and Airway Function. European
Respiratory Journal. 8(1): 457-462.
Boskabady, M.H., Keyhanmanesh, R., dan Saadatloo,
M.A.E. (2008). Relaxant Effects of Different Fractions
from Nigella sativa L. on Guineapig Tracheal Chains
and Its Possible Mechanism(s). Indian Journal of
Experimental Biology. 46(12): 805-810.
D. Satria, J. Silalahi, G. Haro, S. Ilyas, P. A. Z. Hasibuan.
Chemical Analysis and Cytotoxic Activity of N-
Hexane Fraction of Zanthoxylum acanthopodium DC.
Fruits. Rasayan J Chem. 12(2), 803-808 (2019). DOI:
10.31788/RJC. 2019.1225180
Depkes RI. (2013). Farmakope Herbal. Edisi I. Jakarta:
Departemen Kesehatan Republik Indonesia. Halaman.
Huntley, A., Ernst, E. (2000). Herbal Medicines for
Asthma: A Systemic Review. Thorax 55: 925–929.
Husori, D.I., Riyanto, S., dan Nugroho, A.E. (2012).
Relaxation Effect of Marmin on Guinea pig Tracheal
Smooth Muscle via NO-independent Mechanism.
Asian Pacific Journal of Tropical Disease. 1(1): 154-
Husori, D.I. (2011). Peranan Epitelial Terhadap Efek
Relaksasi Senyawa Marmin dari Aegle marmelos
Correa pada Otot Polos Trakea Marmut Terisolasi.
Tesis. Yogyakarta: Fakultas Farmasi Universitas
Gadjah Mada. Hal. 16, 27, 30, 33.
Ikawati, Z. (2006). Pengantar Farmakologi Molekuler.
Yogyakarta: Gajah mada University Press. Hal.61-63,
Ismail, A., Nainggolan, M.F., Turnip, A., Innovationto
speed up the Developmentof Rose Picking Agro-
Tourism in Gunung Sari, IOP Conference Series: Earth
and Environmental Science, Majalengka, 2020.
Karaman M, Firinci F, Cilaker S, Uysal P, Tugyan K,
Yilmaz O, Uzuner N, Karaman O. (2012). Anti-
inflammatory effects of curcumin in a murine model of
chronic asthma. Allergol Immunopathol (Madr) 40:
Kelly ,H.W.,Sorkness, C.A., (2005). Asthma dalam Dipiro,
J.T., Talbert, R.L., Yee, G.C., Matzke, G.R., Wells, B.J.
dan Posey, L.M.,Pharmacotherapy A Pathophysiologic
Approach, Sixth Edition, 517, The McGraw-Hill
Companies, USA.
Kumar, G., Rout, P., Mohanty, R., Dileep, C. (2013).
Emerging Trends of Herbal Care in Dentistry. Journal
of Clinical and Diagnostic Research. 2013 Aug, Vol
7(8): 1827-1829.
E. Harahap, U., Husori, D.I., Marianne. Yuliasmi, S.,
Patilaya, P., Laila, L., et al. (2017). Inhibitory Effect of
Ethanolic Extract of Curanga Fel-Terrae (Pugun
Tano) Leaves on Acetylcholine Muscarinic-3
Receptors Induced on Isolated Guinea Pig Tracheal.
Asean Journal of Pharmaceutical and Clinical
Research. Vol 10, Issue 1, 2017. Hal. 2-3.
Ramadhani, D. (2014). Efek relaksasi ekstrak etanol daun
pugun tanoh (Curanga fel-terrae (Lour.) Merr.)
terhadap otot polos trakea marmut terisolasi dan
pengaruhnya pada fosfodiesterase. Skripsi. Medan:
Fakultas Farmasi USU.Hal. 31,33
Sari, P.D. (2015). Efek Ekstrak Etanol Daun Belimbing
Wuluh (Averrhoa bilimbi L.) Terhadap Kontraksi Otot
Polos Ileum Marmut Jantan (Cavia porcellus)
Terisolasi. Skripsi. Medan: Universitas Sumatera Utara.
Hal. 21,26.
Setiadi, Adi dkk. (2016). Keragaman Beberapa Aksesi
Temu Hitam (Curcuma aeruginosa Roxb.)
Berdasarkan Karakter Morfologi. 45 (1):71-78.
HIMBEP 2020 - International Conference on Health Informatics, Medical, Biological Engineering, and Pharmaceutical