In Vitro Evaluation of Antacid and Anti-flatulent Activity in Ethanol

Extract Syrup of Clove Leaves (Eugenia Caryophyllata Thunberg)

Nurul Widi, Retno Widyowati, Herra Studiawan, Sukardiman*

Department of Pharmacognosy and Phytochemistry,

Faculty of Pharmacy

Universitas Airlangga, Surabaya

Keywords: Eugenia caryophyllata Thunberg, antacid, anti-flatulent, in vitro

Abstract: Objective: Peptic ulcer has high prevalence in the world that caused by high level of gastric acid. The

sample of this study was ethanol extract syrup of clove leaves (Eugenia caryophyllata Thunberg) that had

gastro-protective and anti-ulcer activities. Methods: The pH of ethanol extract syrup of clove leaves and

their neutralizing effects on artificial gastric acids were determined and compared with negative

(aluminium hydroxide) and positive controls (magnesium hydroxide). A modified model of Vatier’s

artificial stomach was used to determine the duration of consistent neutralization effect on artificial gastric

acids. The neutralization capacity in vitro was determined with the titration method of Wu model, while

Rezak method was used to test the anti-flatulent activity by creating foam from sodium lauryl sulphate. The

syrup was subsequently added and the remaining height of foam formed was calculated. Results: The

results showed that ethanol extract syrup of clove leaves had antacid and anti-flatulent in vitro. Compared

to the water group, ethanol extract syrup of clove leaves was found to possess significant gastric acid

neutralizing effects. The duration for consistent neutralization of ethanol extract syrup of clove leaves was

significantly longer than that of water group. Also, there are anti-flatulent effects by decrease of foaming in

vitro model. Conclusion: Ethanol extract syrup of clove leaves was consistently active in the artificial

stomach model and suggested to have antacid and anti-flatulent effects similar to the positive control.

1 INTRODUCTION

Peptic ulcer (PU) is one of the most common

gastrointestinal disorders. The goals of treatment for

PU are to relieve pain, enhance ulcer healing,

prevent complications, and prevent ulcer recurrence.

Current drug therapy for PU is oriented primarily

toward neutralizing (e.g. antacids) or reducing the

amount of acid secreted (e.g. H2 receptor

antagonists, proton pump inhibitors) or protecting

the gastric mucosa from the effects of acid (e.g.

sucralfate). As the role of Helicobacter pylori is

becoming better understood, treatment with

antibiotics is becoming an important part of PU

therapy and recurrence prevention (Holle, 2010).

Although effectiveness can be obtained with these

clinical drugs, their potential side effects and drug

interactions represent a major problem in therapy.

Moreover, newer drugs introduced for the treatment

of PU are expensive and the cost-effectiveness is

also an important consideration. Clinically, many

people cannot use chemosynthetic drugs because of

the potential side effects. Therefore, traditional

herbal drugs possessing fewer side effects should be

investigated as an ideal alternative for the treatment

of PU (Blume et al., 2006)

Eugenia caryophyllata Thunb. is known as clove

which belongs to the family Myrtaceae and esteemed

as a flavouring agent, used as a spice for scenting,

chewing tobacco, an ingredient of betel chew and

control nausea, vomiting, cough, diarrhoea,

dyspepsia, flatulence, stomach distension and gastro

intestinal spasm, relieve pain, cause uterine

contractions and stimulate the nerves (Okasha et al.,

2007). The pharmacological activities of the plant

reported as anti-microbial, anti-viral, anti-oxidant,

anti-diabetic, anti-inflammatory, anti-platelet, anti-

stress, anti-pyretic, chemopreventive,

hepatoprotective, anaesthetic, aphrodisiac, and

insecticidal (Khanna & Parle, 2011).

Cloves leaves were used as a sample in this study

because clove leaves contain eugenol that has ability

to neutralize gastric acid and reduce bloating

stomach. The syrup dosage form was chosen because

syrup is suitable to cover the bitterness of extract. In

addition, the syrup preparation was faster than any

272

Widi, N., Widyowati, R., Studiawan, H. and Sukardiman, .

In Vitro Evaluation of Antacid and Anti-ﬂatulent Activity in Ethanolic Extract Syrup of Clove Leaves (Eugenia Caryophyllata Thunberg).

DOI: 10.5220/0008361102720276

In Proceedings of BROMO Conference (BROMO 2018), pages 272-276

ISBN: 978-989-758-347-6

other dosage forms (Oliveira et al., 2014; Oliveira,

2014).

The secretion of gastric acid (HCl) is intimately

related to PU disease. Antacids heal ulcers through

elimination of gastric acid by neutralization and

have been used in the treatment of PU for many

years. Common antacid preparations include sodium

bicarbonate (SB), calcium carbonate, and salts of

aluminium and magnesium. Since some people

cannot use chemosynthetic drugs because of their

side effects, ethanol extract syrup of clove leaves

should be considered as an alternative for the

treatment of PU. To reveal the anti-ulcer effects of

ethanol extract syrup of clove leaves, therefore, the

present study aimed to assess their antacid effects

on gastric acid neutralization compared to water and

positive controls (aluminium and magnesium). The

antacid effects were assessed in vitro using the

titration method Wu modified model of Vatier’s

artificial stomach. It was utilized to determine the

effects on gastric acid secretion and emptying (Wu

et al., 2010).

2 MATERIALS AND METHODS

2.1. The Preparation of Cloves Leaves

Ethanol Extract Syrup

Clove leaves ethanol extract at 1, 2 and 3% was

mixed with 15% propylene glycol. Then, 65%

sucrose was added into the mixture and stirred until

it was dissolved. Water was subsequently added into

that mixture.

2.2. Chemicals and Reagents

Sodium chloride and pepsin were purchased from

Sigma (St. Louis, MO, USA) and 1 mol/L

hydrochloric acid was obtained from Merck

(Darmstadt, Germany), propylene glycol, sucrose,

aluminium hydroxide and magnesium hydroxide as

positive control drugs of antacid were purchased

from PT First Medipharma (Indonesia),

dimethylpolysiloxane as positive control of anti-

flatulent was purchased from Pharos (Indonesia),

and sodium lauryl sulphate (Germany).

2.3. Instruments

The experimental instruments consisted of an

adjustable electrode stand, a series flatbed recorder,

a micro tubing pump, a standard pH meter, a

stirrer/hot plate, and a series multi-functional

temperature controller.

2.4. Preparation of Artificial Gastric Acid

Two grams of sodium chloride and 3.2 mg of pepsin

enzymes were dissolved in 500 mL water, then 7.0

mL hydrochloric acid and adequate water were

added to make a 1000 mL solution of the artificial

gastric acid at pH 1.2.

2.5.Determination of the Neutralizing

Effects on Artificial Gastric Acids

Ninety mL of each test solution was added to 100

mL artificial gastric juices at pH 1.2. The pH values

were determined to examine the neutralizing effect.

2.6. Determination of the Duration of

Consistent Neutralization Effect on

Artificial Gastric Acids using the

Modified Model of Vatier’s

Artificial Stomach

Ninety mL of each test sample was added to 100 mL

of artificial gastric juice at pH 1.2 in the beaker glass

of the artificial stomach at 37℃ and continuously

stirred (30 rpm) with magnetic stirring apparatus.

Artificial gastric juice at pH 1.2 was pumped at 3

mL/min into the container of the artificial stomach,

and it was pumped out at 3 mL/min at the same time.

A pH meter was connected to continuously monitor

of pH meter in the container of artificial stomach.

The duration of neutralization effect was determined

when the pH value was returned to its initial value

(pH 1.2).

2.7. Anti-flatulent Test in Removing

Foam

One hundred mL of gastric acid was put into the

artificial hull and 0.625 g sodium lauryl sulphate was

subsequently added into it. The mixture was heated

at 37 ℃ with stirrer speed of 30 rpm to form 150 mL

foam. The height of foam formed was measured.

Then, 10 mL of clove leaves ethanol extract syrup

and water was added until 90 mL. The mixture was

observed within 20 minutes and the remaining height

foam was measured.

In Vitro Evaluation of Antacid and Anti-ﬂatulent Activity in Ethanolic Extract Syrup of Clove Leaves (Eugenia Caryophyllata Thunberg)

273

2.8. Statistical Analysis

The statistical calculations were performed using

the software IBM SPSS Statistic 23. The

experimental data were expressed as mean ± SEM

where SEM is Standard error of Mean; Comparison

between the groups were analyzed by One-way

Analysis of Variance (ANOVA) using Dunnett

Multiple Comparisons Test by considering Test Vs

control. The differences were considered to be

statistically significant when **P<0.05.

3 RESULTS

3.1. Antacid Activity Test

The durations for consistent neutralizing effects of

clove leaves ethanol extract syrup at 1, 2 and 3 %

solutions were 80.14 ± 3.63, 104.51 ± 6.08 and

140.78 ± 5.33 minutes, respectively. Those of

positive control and negative control solutions were

268.31 ± 10.68 and 0.02 ± 0.00 minutes,

respectively. The action duration of positive control

was the longest, followed by clove leaves ethanol

extract syrup at 3% (Table 1 and Figure 1).

Table 1. Duration of antacid effect for consistent

neutralization of gastric acid

Drug

Time (minute)

Positive control

268.31 ± 10.68

Negative control

0.02 ± 0.00

Formula 1

80.14 ± 3.63

Formula 2

104.51 ± 6.08

Formula 3

140.78 ± 5.33

Data were presented as mean ± SEM *P < 0.05 when

compared to water group

Figure 1. The duration of antacid effect for consistent neutralization of gastric acids

Ninety mL of test sample was added to 100 mL

of artificial gastric juices at pH 1.2 in an artificial

stomach. The duration was determined as the pH

value was returned to 1.2.

3.2. Anti-flatulent Activity Test

Based on the results of anti-flatulent activity test, the

data analysis showed that positive control treated

with dimethylpiloxane was able to remove foam

with 0.2 ± 0.0 cm. While the height foam in negative

control treated with syrup containing only carrier

0.2

0.4

0.6

0.8

1.2

1.4

1.6

1.8

2.2

2.4

2.6

2.8

3.2

3.4

3.6

3.8

4.2

4.4

0 25 50 75 100 125 150 175 200 225 250 275 300

t (minute)

Kontrol Positif Kontrol negatif Formula 1 Formula 2 Formula 3

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274

without clove leaves ethanol extract was 1.8 ± 0.1

cm. The height foam formed in the treatment group

of clove leaves ethanol extract syrup of 1, 2 and 3%

were 0.90 ± 0.0 cm, 1.2 ± 0.3 cm and 1.2 ± 0.3 cm,

respectively. The results of clove leaves ethanol

extract syrup were significantly different compared

to water (table 2 and figure 2).

Table 2. Remaining high foam within 20 minutes

Drug

Height foam (cm)

Positive control

0.2 ± 0.0

Negative control

1.8 ± 0.1

Formula 1

0.9 ± 0.0

Formula 2

1.2 ± 0.3

Formula 3

1.2 ± 0.3

Data were presented as mean ± SEM *P < 0.05 when

compared with water

Figure 2 Remaining high foam within 20 minutes

Ninety mililiters of test sample was added to 100

mL of artificial gastric juices at pH 1.2 in an

artificial stomach. Remaining high foam within 20

minutes.

3 DISCUSSION

Stomach is an organ that undergoes propulsion,

mixing of food, digestion and absorption of food

along with secretary functions. The parietal cells of

stomach secrete about 2500 mL of gastric juice

daily. The acid in this gastric juice kills many

bacteria and provide a low pH for pepsin to start

protein digestion. Mucosal erosions or ulcerations

take place when aggressive factors overwhelm the

defensive factors of the gastrointestinal mucosa. It

leads to the arrival of gastritis, peptic ulcer and

gastroesophageal reflux disease (Katharinne et al.,

2010). The aggressive factors well established for

several decades are acid and pepsin. Hence peptic

ulcer diseases are mostly treated with antacids, H2

receptor antagonists and proton pump inhibitors.

Among them, antacids have been widely used in the

treatment of ulcer.

Antacids are generally inorganic salts which

dissolve in acid gastric secretions which release

anions that partially neutralize gastric hydrochloric

acid. They generally react chemically to neutralize

or buffer existing quantities of stomach acid but do

not have direct effect on its output. This action

results in increased pH value of stomach contents

and thus provide relief of hyperacidity symptoms.

These medications also reduce acid concentration

within the lumen of the esophagus which causes an

increase in intra-esophageal pH and a decrease in

pepsin activity (Katzung, 2010). These medicaments

do not decrease the volume of gastric secretions.

Most of the antacids available in the market are

efficient but is often unacceptable because of the

common side effects, especially altered bowel

functions.

0.2

0.4

0.6

0.8

1.2

1.4

1.6

1.8

2.2

2.4

2.6

2.8

0 5 10 15 20 25

height foam (cm)

t (minute)

Kontrol Positif Kontrol Negatif Formula 1

Formula 2 Formula 3

Positive control

Negative control

Formula 1

Formula 2

Formula 3

In Vitro Evaluation of Antacid and Anti-ﬂatulent Activity in Ethanolic Extract Syrup of Clove Leaves (Eugenia Caryophyllata Thunberg)

275

Antacids that contain aluminium contribute

aluminium to the diet but may cause constipation or

lead to phosphorous deficiency where as on long

term or inappropriate use can lead to aluminium

toxicity. Calcium containing antacids contribute

calcium to diet and may produce constipation.

Magnesium containing antacids contribute

magnesium to diet and produce a side effect of

diarrhea on prolonged use may even lead to

magnesium toxicity (Gunawan et al., 2009). It is

reported that SB should be avoided even though it is

a potent neutralizer of acid as it contains significant

amounts of sodium and may alter the systemic pH.

One major fact that should be considered while

selecting an antacid for the treatment is its drug

interactions. Significant interactions occur with

quinolone antibiotics, tetracycline and iron sulphate

(Tsung-Hsiu et al., 2010). Hence considering the side

effects and drug interactions of antacids, the herbal

drugs having fewer side effects should be identified

as an alternative for the treatment of peptic ulcers. It

is widely understood that herbal medicines have

recently generated an increased interest in the

treatment of gastritis. Hence in our present study we

had applied the titration method of Wu the modified

model of Vatier’s artificial stomach, which mimic

the regular physiological functioning of a human

stomach, to explore the antacid effects of clove

leaves that were well known for its potency to cure

all types of wounds.

Clove leaves ethanol extract syrup has antacid

and anti-flatulent activity. Eugenol inside this

formula was expected to have antacid and anti-

flatulent. It included in polyphenol group that has

pKa of 10.19, thus it was considered as a weak base.

So, it had the same mechanism of action as antacid

drugs which are also considered as weak base by

reacting with hydrochloric acid to form salts and

neutralize gastric acid. In comparison to negative

controls, Remington stated that if the pH has

reached 2.3 then it has been able to neutralize gastric

acid by 90% (Hoover & John, 2005).

Anti-flatulent test was also performed in this

study due to the ability of high gastric acid to cause

flatulence. Thus, it can be concluded that good

antacids are the one that has high efficacy, few side

effects, and able to reduce bloating stomach.

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