The Inhibition Test of Red Betel Leaves (Piper crocatum Ruiz & Pav)

Extract Against the Growth of Streptococcus pneumoniae and

Salmonella typhi

Evi Kurniawaty

1,*

, Herina Azzahra

, Intanri Kurniati

, Waluyo Rudiyanto

, Aristianto Husin

and Silvia Andriani

Faculty of Medicine, University of Lampung, Indonesia

Faculty of Health, University of Muhammadiyah Pringsewu Lampung, Indonesia

Keyword: Piper crocatum Ruiz and Pav., Streptococcus pneumoniae, Salmonella typhi.

Abstract: Infectious diseases are still a health problem in the world, both in developed and developing countries.

Infectious disease is a disease caused by pathogenic microbes (viruses, bacteria, fungi) and is the main

cause of high morbidity and mortality rates. Respiratory tract infections and typhoid fever caused by

bacteria are still the most common health problems. Pneumonia is an infection or acute inflammation of

the lung tissue caused by various Streptococcus pneumoniae bacterial microorganisms, exposure to

chemicals or physical damage to the lungs. Meanwhile, typhoid fever is a type of infection caused by the

bacterium Salmonella Enterica, especially the Salmonella Typhi derivative. The high level of resistance

to antimicrobials, which currently still causes chronic infections in humans, has led to the idea that

medical personnel must be more careful in using and finding the right antibiotics. One of the potential

medicinal plants that is known empirically to have properties for curing various diseases is red betel leaf

(Piper crocatum ruiz and Pav.). This study aims to prove that red betel leaves has an inhibitory effect

against Streptococcus pneumoniae and Salmonella typhi. This study used an experimental method. The

technique used to measure antibiotic activity is the method of well diffusion and continued by Kruskal-

Wallis test and Mann-Whitney test. The results showed that extract of red betel leaf can inhibit the growth

of Salmonella typhi at concentrations 20%, 30%, 40%, and 50% and the most effective is 50%

concentration. There is no result on Streptococcus pneumonia. Red betel leaves (Piper crocatum Ruiz &

Pav.) have an inhibitory power against Salmonella typhi but does not have an inhibitory effect on

Streptococcus pneumoniae bacteria.

1 INTRODUCTION

Infectious diseases are diseases caused by pathogenic

microbes (viruses, bacteria, fungi) and are the main

cause of high morbidity and mortality rates.

Respiratory tract infections and typhoid fever caused

by bacteria are still the most common health

problems (Syahidi HM, 2013).

Pneumonia is known to attack around 450 million

people every year. In 2018, the prevalence of

pneumonia based on health worker diagnosis was

around 2%, whereas in 2013 it was 1.8%, which

shows an increase (Riskesdas, 2018). The number of

pneumonia sufferers in Indonesia in 2013 ranged

from 23% to 27% and deaths due to pneumonia were

1.19%. In 2010 in Indonesia, pneumonia was

included in the top 10 inpatient diseases in hospitals

with a crude mortality rate (CFR) or the death rate for

a particular disease in a certain period of time divided

by the number of cases, namely 7.6% (Ministry of

Health, 2019).

Meanwhile, typhoid fever still has the highest

incidence of transmission in children, especially in

endemic countries. If you look at Indonesia's health

profile data for 2011 and 2012, typhoid fever is

included in the top 10 diseases with the most

hospitalized patients, namely in 3rd position. In 2011

there were 80,850 cases with 1,747 deaths. In 2012

the number of cases decreased to 41,081 cases with

274 deaths (Ministry of Health of the Republic of

Indonesia, 2013).

The high level of resistance to antimicrobials,

which currently still causes chronic infections in

Kurniawaty, E., Azzahra, H., Kurniati, I., Rudiyanto, W., Husin, A. and Andriani, S.

The Inhibition Test of Red Betel Leaves (Piper crocatum Ruiz & Pav) Extract Against the Growth of Streptococcus pneumoniae and Salmonella typhi.

DOI: 10.5220/0013671900003873

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Medical Science and Health (ICOMESH 2023), pages 329-334

ISBN: 978-989-758-740-5

329

humans, has given rise to the opinion that medical

personnel must be more careful in using and finding

the right antibiotics. This also encourages people,

both medical and non-medical personnel, to use

medicinal plants as an alternative healing method.

One of the potential medicinal plants which is known

empirically to have properties for curing various

diseases is Red Betel Leaf (Piper crocatum Ruiz and

Pav.). In society, red betel is famous for its properties

in treating diseases such as diabetes mellitus,

hepatitis, kidney stones, lowering cholesterol,

preventing stroke, gout, hypertension, coronary heart

disease, uterine cancer, breast cancer, hemorrhoids,

tuberculosis, medicine. toothache, canker sores, body

odor, venereal disease, liver inflammation, prostate

inflammation, eye inflammation, vaginal discharge,

ulcers, fatigue, joint pain, softening of the skin, ear

inflammation, cough medicine, pneumonia, sore

throat, gingivitis, inflammation breasts, nose bleeds,

and coughing up blood. Apart from that, there has

been a lot of research on antimicrobial plants that can

fight infection, one of which is red betel leaf (Piper

crocatum Ruiz and Pav.) and green betel leaf (Piper

betle Linn) (Julistiara, 2017).

2 METHOD

This research is an experimental study using a Post-

test Only Control Group design. In this study, the

experimental group was given treatment for a certain

period of time, then the dependent variable was

measured in the two groups, and the results were

compared for differences. The test microbes used

were gram-positive bacteria (+) Streptococcus

pneumoniae and gram-negative bacteria (-)

Salmonella typhi obtained from the Bandar Lampung

Regional Health Laboratory (LABKESDA).

The research test material used was red betel

leaves (Piper crocatum Ruiz & Pav.) which were

washed using clean water, then dried using an oven at

a temperature of 105°C. Extraction was carried out by

the Organic Chemistry Laboratory, Faculty of

Mathematics and Natural Sciences (FMIPA),

University of Lampung using the soxhletation

method with 96% ethanol solvent at a temperature of

70°C. Then the red betel leaf extract is diluted using

saline to obtain varying concentrations.

The culture medium used in this study was blood

agar plates in 10 cm petri dishes. This medium is used

to culture Streptococcus pneumoniae and Salmonella

typhi. After culturing, Mueller-Hinton Agar (MHA)

media was used as a medium for testing the diameter

of the inhibition zone for Streptococcus pneumoniae

and Salmonella typhi bacteria.

The population in this study, namely pure culture

Streptococcus pneumonia and Salmonella typhi

bacteria, was divided into seven research groups. In

this research, various levels of red betel leaf extract

will be tested, namely at a level of 10%; 20%; 30%;

40%; and 50%, as well as with Erithromycin as a

positive control for Streptococcus pneumoniae

bacteria, Ciprofloxacin as a positive control for

Salmonella typhi bacteria, and distilled water as a

negative control. The research results were then

tested for statistical analysis using a statistical

program.

3 RESULTS

3.1 Phytochemical Test Results of Red

Betel Leaves (Piper crocatum Ruiz

and Pav.)

Based on the phytochemical test, the results showed

that red betel leaves were identified as containing

saponin, terpenoid, tannin and flavonoid compounds.

3.2 Streptococcus pneumoniae Bacterial

Inhibition Zone Test

The results of measuring the inhibition zone of red

betel leaf (Piper crocatum Ruiz & Pav.) extract

against Streptococcus pneumonia bacteria with 4

repetitions are as shown in the table 1.

Based on the data obtained, it is known that there

is no inhibition zone formed from five concentrations

of red betel leaf extract against Streptococcus

pneumoniae in 4 repetitions. Meanwhile, in the K (+)

group, the average inhibition zone was 19.625 mm

and the K (-) group it was 0 mm.

Table 1: Results of measuring the diameter of the inhibitory

zone of red betel leaf (Piper crocatum ruiz and Pav.) extract

against Streptococcus pneumoniae.

Treament Inhibition Zone

Measurement Streptococcus

pneumoniae (mm)

Inhibition

Zone

average

(mm)

I II III IV

K (+) 10,6 22,74 22,58 22,58 19,625

K (-) 0 0 0 0 0

P1 0 0 0 0 0

P2 0 0 0 0 0

P3 0 0 0 0 0

P4 0 0 0 0 0

P5 0 0 0 0 0

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330

3.3 Salmonella typhi Bacterial

Inhibition Zone Test

The results of measuring the inhibition zone of red

betel leaf (Piper crocatum Ruiz & Pav.) extract

against Salmonella typhi bacteria with 4 repetitions,

the measurement results obtained are presented in the

table 2.

Table 2: Results of measuring the diameter of the inhibitory

zone of red betel leaf extract (Piper crocatum Ruiz & Pav.)

against Salmonella typhi bacteria.

Treatment

Inhibition Zone Measurement

Salmonella typhi (mm)

Inhibition

Zone

average

(mm)

I II III IV

K (+) 37,68 38,7 42,42 42,92 40,43

K (-) 0 0 0 0 0

P1 0 0 0 0 0

P2 5,4 8,72 11,8 6,7 8,155

P3 8,50 8,42 8,42 9,38 8,68

P4 12,72 13,42 11,46 11,58 12,295

P5 15,98 14,92 13,98 13,04 14,48

Based on the table 2, it shows that there is an

inhibitory power of red betel leaf extract against

Salmonella typhi at concentrations of 20%, 30%.

40%, 50%. Based on the research results, the group

that produced the largest inhibitory zone was the

group with a concentration of 50%. This shows that

the greater the concentration of red betel leaf (Piper

crocatum Ruiz & Pav.) extract, the greater the

inhibition zone produced.

Next, the Shapiro-Wilk normality test was carried

out to determine whether the data was normally

distributed or not. The Shapiro-Wilk test was chosen

because the data was less than 50. The results of the

normality test are presented in the table 3.

Table 3: Normality test results of the inhibition zone of red

betel leaf (Piper crocatum Ruiz & Pav.) extract against

Salmonella typhi.

Based on the table 3, in the normality test the

significance results obtained for the K (+) group were

0.259, for the P2 group 0.789, for the P3 group 0.009,

for the P4 group 0.381, and for the P5 group 0.977.

Meanwhile, P1 and K (-) have no data. In the

normality test, p > 0.05 was obtained, which means

that all data was normally distributed.

Table 4. Results of the Levene homogeneity test of the

inhibition zone of red betel leaf (Piper crocatum Ruiz &

Pav.) extract against Salmonella typhi.

Levene's homogeneity test

Sig.

Inhibitory zone of red betel leaf (Piper

crocatum Ruiz & Pav.) extract against

Salmonella typhi

0,006

Based on the table 4, the homogeneity test obtained

0.006 (p < 0.05), which means the data is not

homogeneous. Due to the non-homogeneity of the

data, the requirements for carrying out the One- Way

ANOVA Test were not met.

Table 5: Results of the Kruskal-Wallis test for the inhibition

zone of red betel leaf (Piper crocatum Ruiz & Pav.) extract

against Salmonella typhi

Kruskal-Wallis test Sig.

Inhibitory zone of red betel leaf (Piper

crocatum Ruiz & Pav.) extract against

Salmonella typhi

0,0001*

Based on the results of the analysis, p value <0.05

was obtained which indicates that each treatment

given with various concentrations of 10%, 20%, 30%,

40%, 50% and the positive control with erythromycin

and the negative control using distilled water showed

a significant difference in the zone. barriers are

formed. Next, further analysis was carried out using

the Mann-Whitney Post hoc Test to find out which

groups were significantly different.

The Mann Whitney post hoc multicomparison test

aims to determine whether there is a difference in the

mean results of the inhibition zones formed in the 7

types of treatments. The difference in inhibition zone

values is considered significant if p < 0.05. Based on

the table above, it shows that there are significant

differences in the inhibition zone at each

concentration with the antibiotic Ciprofloxacin 500

mg. This shows that the inhibitory power of each

concentration is not greater than that of the antibiotic

used.

Treatment Si

(

)

0,259*

(-) -

P1 -

P2 0,789*

P3 0,009*

P4 0,381*

P5 0,977*

The Inhibition Test of Red Betel Leaves (Piper crocatum Ruiz & Pav) Extract Against the Growth of Streptococcus pneumoniae and

Salmonella typhi

331

Table 6: Post hoc Mann-Whitney test of red betel leaf (Piper crocatum Ruiz & Pav.) extract against Salmonella typhi.

Treatment K (+) K (-) P1 P2 P3 P4 P5

K (+) - 0,014* 0,014* 0,021* 0,020* 0,021* 0,021*

K (-) 0,014 - 1,000 0,014* 0,013* 0,014* 0,014*

P1 0,014* 1,000 - 0,014* 0,013* 0,014* 0,014*

P2 0,021* 0,014* 0,014* - 0,772 0,083 0,021*

P3 0,020* 0,013* 0,013* 0,772 - 0,020* 0,020*

P4 0,021* 0,014* 0,014* 0,083 0,020* - 0,043*

P5 0,021* 0,014* 0,014* 0,021* 0,020* 0,043* -

4 DISCUSSIONS

Red betel leaf extract has an inhibitory response

against Salmonella typhi bacteria but does not have

an inhibitory effect against Streptococcus

pneumoniae bacteria. This is caused by differences in

the cell wall structure of the two bacteria.

Streptococcus pneumoniae bacteria are gram-positive

bacteria whose cell walls are composed of PG

(peptidoglycan) so that the cell walls formed are quite

thick and stiff. Meanwhile, Salmonella typhi bacteria

are gram-negative bacteria whose cell walls contain

much smaller amounts of PG (Peptidoglycan) than

Streptococcus pneumoniae, but on the outside there is

an outer membrane which is composed of

lipoproteins and phospholipids and contains

lipopilysaccharides. These bacteria have a thick cell

wall in the form of peptidoglycan, which is located

between the inner membrane and the outer

membrane. This difference makes Salmonella typhi

bacteria more susceptible to being destroyed by

antibacterial agents than Streptococcus pneumoniae,

so that in this study the inhibition zone for Salmonella

typhi was larger than for Streptococcus pneumoniae

(Pratiwi I, 2011)

Various factors such as bacterial population,

temperature, incubation period, antimicrobial

concentration, and the environment around the

microbe can influence antimicrobial activity in vitro.

Researchers are trying to control factors that can be

controlled, for example, choosing the age and

condition of the red betel leaves taken, the process of

making extractions of red betel leaves (Piper

crocatum Ruiz and Pav.) using ethanol as a solvent so

that chemicals that have an antibacterial effect on the

leaves Red betel (Piper crocatum Ruiz and Pav.) can

be polluted. Then, the bacterial population was

standardized by comparing the turbidity with 0.5%

Mac Farland solution. The researchers adjusted the

temperature, culture media and incubation time

according to theory (Kurniawan, 2015).

The antibacterial effectiveness test of red betel

leaf (Piper crocatum Ruiz and Pav.) extract showed

the presence of an inhibition zone which acts as an

indicator of the antibacterial effect. It can be

concluded that the compounds resulting from the

extraction of red betel leaves (Piper crocatum Ruiz

and Pav.) which diffuse into the agar from the well

method are able to inhibit the growth of Salmonella

typhi bacteria but not Streptococcus pneumoniae

bacteria. Erythromycin 500 mg was used as a positive

control and showed an inhibitory zone for the growth

of Streptococcus pneumoniae bacteria and

Ciprofloxacin 500 mg was used as a positive control

for Salmonella typhi also showed an inhibitory zone

for the growth of the bacteria. Meanwhile, the

negative control, namely distilled water, did not show

any inhibition zone on the Mueller Hinton Agar

media.

In Salmonella typhi, the results of the inhibition

zone formed prove that the greater the concentration

of red betel leaf (Piper crocatum Ruiz & Pav.)

extract, the greater the antibacterial activity. This is in

line with research by Rahmayanti Y in 2014 which

stated that the higher the concentration of red betel

leaf (Piper crocatum Ruiz & Pav.) extract, the more

antibacterial active ingredients it contains so that the

inhibition zone formed will be larger. Increasing the

concentration of antibacterial compounds is expected

to increase the penetration of antibacterial

compounds into microbial cells.

In this study, there were no inhibitory zone results

for red betel leaf (Piper crocatum Ruiz & Pav.)

extract against Streptococcus pneumoniae bacteria.

This research is not in line with previous research

conducted by Pratiwi I in 2011 which stated that there

had been results in the form of a reduction in the

number of bacterial colonies by 47.1%. The MIC

result of a concentration of 6.25% also indicates that

this concentration is active in inhibiting

Streptococcus pneumoniae. This may be because the

concentration of the extract used must be higher and

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the extract is stored for too long so that some of the

active antibacterial compounds begin to decrease.

Next, the Kruskal-Wallis test was carried out, this

was done to evaluate whether there was a significant

difference in the mean number of colonies between

groups with variations in extract concentration. There

were significant results in the antibacterial activity

test of red betel leaf (Piper crocatum Ruiz & Pav.)

extract against Salmonella typhi bacteria. The

significance was 0.001 (p < 0.050), which means the

results were significant (there was a difference in the

mean number of colonies in the effect of red betel leaf

(Piper crocatum Ruiz & Pav.) extract on Salmonella

typhi bacteria).

Post hoc tests using Mann-Whitney were also

carried out again in the study of the inhibitory power

of red betel leaf (Piper crocatum Ruiz & Pav.) extract

against Salmonella typhi bacteria and obtained p

values <0.05 for most concentrations. This shows that

in the post hoc test using Mann-Whitney, a significant

difference in the diameter of the inhibition zone was

obtained between the control group (positive control

and negative control) and the test group (10%, 20%,

30%, 40% and 50% concentration groups). Based on

the Mann-Whitney Post hoc test, the positive control

group had a significant difference from the 50%

concentration group, where the positive control group

had a stronger inhibitory effect. The 40%

concentration group had a significant difference from

the 50% concentration group, where the 50%

concentration group had a stronger inhibitory power.

The 30% concentration group had a significant

difference from the 40% concentration group, and the

40% concentration group had a stronger inhibitory

power. The 20% concentration group had a

significant difference from the 30% concentration

group, where the inhibitory power was stronger in the

30% concentration group. The 10% concentration

group had a significant difference from the 20%

concentration group, where the 20% concentration

group had a stronger inhibitory power.

The positive controls used in this study were

Erythromycin on Streptococcus pneumoniae bacteria

and Ciprofloxacin on Salmonella typhi bacteria. From

the research results, the positive control had the

highest inhibitory effect.

Based on research, the antibacterial substances

contained in red betel leaf extract can inhibit the

growth of Salmonella typhi bacteria, namely

saponins, terpenoids, tannins and flavonoids. This is

in line with research by Agustina S. Beon Karol

Geovani Batista Leki (2016) regarding the analysis of

the active compound content contained in red betel

leaf (Piper crocatum Ruiz & Pav.) extract.

Based on the results of phytochemical tests, red

betel leaf extract contains saponins, terpenoids,

tannins and flavonoids. Saponins work by denaturing

proteins. The surface actives substances of saponin is

similar to detergent, therefore saponin can reduce the

surface tension of bacterial cell walls and damage

membrane permeability in bacteria. Saponin will then

diffuse through the cytoplasmic membrane and

disrupt the stability of the membrane. Flavonoids

work as antibacterials by forming complex

compounds with extracellular proteins that have the

integrity of bacterial cell membranes. Flavonoids are

temporary phenolic compounds that act as protein

coagulators (Fadlilah M, 2015)

Terpenoids have antibacterial activity by reacting

with porins which are transmembrane proteins in the

outer membrane of bacterial cell walls. The

terpenoids then form strong polymer bonds and cause

damage to the porins. Damage to these porins will

reduce the permeability of bacterial cell walls which

will result in a number of bacterial cells lacking

nutrition. Then the bacteria will experience growth

barriers or die (Sudarmi et al., 2017).

The tannin compounds contained in red betel leaf

extract also act as antibacterials. The antibacterial

mechanism of action of tannins is by lysing bacterial

cells. Tannins target bacterial polypeptide walls

which cause cell wall formation to be less than perfect

and cause bacterial cell death. Apart from that,

tannins can also inactivate bacterial enzymes and

disrupt the passage of proteins in the inner layers of

cells (Sapara et al., 2016).

5 CONCLUSIONS

Red betel leaf (Piper crocatum Ruiz & Pav.) extract

has inhibitory power against Salmonella typhi

bacteria but not against Streptococcus pneumoniae

bacteria. In research conducted on Salmonella typhi

bacteria, the average inhibition zone formed at a 10%

concentration was 0 mm, a 20% concentration was

8.155 mm, a 30% concentration was 8.68 mm, a 30%

concentration was 12.295 mm, and the concentration

100% is 14.48 mm.

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