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.

1 INTRODUCTION

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

202

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

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.

2 MATERIALS AND METHOD

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.

Organpreparation

thetracheais

separated

Organbathkreb's

solution

Isometric

transducer

Acetylcholine

EEBC

TrachealContraction

Theopholine

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

203

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

animals.

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

(1).

3 RESULTS AND DISCUSSIONS

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

204

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

205

4 CONCLUSIONS

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.

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