Testing the Antibacterial Activity of Black Betel Leaf
Extract (Piper betle L. Var Nigra) on Streptococcus mutans, Porphyromonas gingivalis, and Enterococcus faecalis
Cici
Nur Aisyah Eka Putri1, Masyhudi2, Listiyawati3
Universitas Mulawarman, Indonesia
cicinuraisyahep@gmail.com1, masyhudiina@gmail.com2, listiya.lilis@ymail.com3
|
Keywords |
Abstract |
|
Antibacterial,
Black Betel Leaf Extract, Piper betle L. var nigra, Streptococcus mutans,
Porphyromonas gingivalis, Enterococcus faecalis |
Black
betel leaves (Piper betle L. var nigra) are known to have antibacterial
effects caused by secondary metabolites such as tannins, phenolic compounds,
saponins, flavonoids, alkaloids, and steroids. This study aims to assess the
antibacterial activity of ethanol extract of black betel leaves (Piper betle
L. var nigra) against S. mutans, P. gingivalis, and E. faecalis by measuring
the diameter of the inhibition zone formed. This research is a type of pure
experimental research (true experimental) with post test only control group
design. This experimental research was conducted using the disc diffusion
method in vitro, the bacteria were treated with black betel leaf extract
(Piper betle L. var nigra) with a concentration variation of 60%, 70%, 80%,
90%, and 100%. The experiment was repeated three times. The results showed
that black betel leaf (Piper betle L. var nigra) had antibacterial activity
against two of the three bacteria. This activity was observed through the
presence of distinct clear zones around the paper disks. In conclusion,
extracts derived from black betel leaf (Piper betle L. var nigra) had antibacterial
activity against S. mutans and P. gingivalis bacteria at 70%, 80%, 90%, and
100% concentrations. However, it did not have antibacterial activity against
E. faecalis bacteria. |
Corresponding Author : Cici Nur Aisyah Eka
Putri
E-mail: cicinuraisyahep@gmail.com
INTRODUCTION
Health
that needs attention apart from general body health is dental and oral health (Herawati et al., 2022) . If dental and oral health is disturbed, it will affect
bodily health, thereby affecting the quality of human resources (Septiani, Sughesti, Susanti, Sihombing, &
Novitasari, 2021) . The percentage of the population who have dental and oral
problems according to RISKESDAS in 2007, 2013 and 2018 has always increased
from 23.2% to 25.9% and in 2018 it became 57.6% (Herawati et al., 2022) . The prevalence of caries and periodontitis is very high in
Indonesia, namely caries is in first position with a percentage of 88.8% and
followed by periodontitis with a percentage of 74.1% (Riskesdas Team 2018,
2019).
The
process of caries involves a number of factors that interact with each other,
namely teeth and saliva (host), microorganisms, substrate and time (Ambarawati, Sukrama, & Yasa, 2020) . The role of microorganisms is very important in the
process of dental caries which is also supported by other factors.
Streptococcus mutans is a microorganism that causes dental caries which plays a
major role in the onset of dental caries (Restina & Warganegara, 2016) . If caries is not treated immediately, dental treatment
such as root canal treatment is necessary, however, in root canal treatment
itself, root canal infections often occur. Endodontic treatment where primary
infection or secondary infection occurs is usually caused by the colonization
of microorganisms which are dominated by anaerobic bacteria, especially
Enterococcus faecalis. (Nurbianti, Alhawaris, & Yani, 2021) .
Apart
from caries, periodontal disease is also one of the dental and oral diseases
that are often found in the world community, especially in Indonesia. (Setiawati, Robbihi, & Dewi, 2022) . Porphyromonas gingivalis is a gram-negative anaerobic
bacterium that is involved in the pathogenesis of periodontitis (Ramadhani,
Rudhanton, Diah, & Sutanti, 2022). Porphyromonas gingivalis is the most
common bacteria associated with periodontitis, from subgingival plaque periodontitis
patients, Porphyromonas gingivalis was found in 85.75% (Septiwidyati & Bachtiar, 2020) .
Current
conditions in Indonesia show an increase in oral problems every year, including
caries, periodontitis, and root canal infections which are a continuation of
the caries process. This encourages health workers to make efforts to minimize
the cause of these problems, namely oral bacteria. One of the efforts that can
be made is to use mouthwash. Long-term use of mouthwash containing alcohol can
cause dry mouth, reduce saliva production, and increase the risk of tooth decay
and bad breath. Therefore, WHO recommends and promotes the use of traditional
or herbal medicines in national health care programs because they are easily
available, cheaper, and relatively safer without harmful side effects (Alamsyah, Arma, & Hidayati, 2021).
To obtain herbal
products that are useful for treatment and prevention in the field of
dentistry, many studies have been conducted to reduce the number of
microorganisms in the oral cavity using natural ingredients. This is considered
very beneficial because people have long believed that natural ingredients can
treat various diseases with minimal side effects (Restina & Warganegara, 2016). In Indonesia,
especially in tropical climates such as East Kalimantan, there are many
biological plants that are utilized, one of which is the betel plant (Owu & Jayanti, 2020). Betel plants are
known to be effective in controlling caries and periodontal disease (Agung, Hervina, & Sandi, 2021). Known betel
varieties include green, red, and black betel (Zuraidassanaaz, 2017). Black betel
leaves have antibacterial benefits (Saputri & Rahayu, 2018). Secondary
metabolites of black betel leaf were identified to contain alkaloids,
flavonoids, tannins, saponins, phenolic compounds, carotenoids, and steroids (Owu & Jayanti, 2020).
Black betel
(Piper betle L. var nigra) is one type of betel that is widely found in
Indonesia, but has not been widely recognized by the wider community (Qhorina, Prasetya, & Ardana, 2021). Research on betel
leaf plants is quite a lot, but especially on black betel leaves (Piper betle
L. var nigra) is still small. Prasetya (2012) conducted antimicrobial tests on
black betel leaf extract and proved to be able to inhibit the growth of
microbes Streptococcus mutans, Staphylococcus aureus, Candida albicans, and
Candida utilis. Research by (Saputri
& Rahayu, 2018)
proved that 70% ethanol extract of black betel leaves has antibacterial
activity against Staphylococcus aureus. Meanwhile, research by (Aprillia, Ardana, & Kuncoro, 2021) showed that
ethanol extract of black betel leaves has antibacterial activity against the
growth of Propionibacterium acne. Recent research by Herryawan et al. (2023)
proved that black betel extract with concentrations of 25%, 50%, 75%, and 100%
was effective in inhibiting the growth of Porphyromonas gingivalis bacteria
with moderate to strong inhibition categories (Herryawan, Soerachman, & Chaeruddin, 2023).
This study aims
to determine the antibacterial activity of black betel leaf extract (Piper
betle L. var nigra) against the growth of the bacteria Streptococcus mutans,
Porphyromonas gingivalis and Enterococcus faecalis. The specific aim is to
determine the diameter of the inhibition zone of black betel leaf extract
(Piper betle L. var nigra) against these three bacteria. The results of this
research can be used to develop knowledge and scientific information in the
field of dentistry, as input for health institutions to maximize the use of
black betel leaves in health promotion activities, increase public information
and knowledge about the potential of black betel leaves as an antibacterial
agent, and provide practical and theoretical experience for researchers
regarding the benefits of black betel leaves in inhibiting the growth of
Streptococcus mutans, Porphyromonas gingivalis and Enterococcus faecalis
bacteria.
RESEARCH METHODS
Research Design
This
research is a type of pure experimental research (true experimental) with a
post test only control group design. This experimental research was conducted
using the disc diffusion method in vitro.
a Number
of Treatments
This
study used two different test groups, with several treatments:
The
first group (test group) is a group consisting of five treatments on each of
three bacteria namely Streptococcus mutans, Porphyromonas gingivalis and
Enterococcus faecalis with the concentration of black betel leaf extract (Piper
betle L. var nigra) which is 60%, 70%, 80%, 90% and 100%. The second group (control group) is
positive control and negative control, positive control using Chlorhexidine
gluconate 0.2%, negative control using DMSO 10%.
b Number
of repetitions
This study was conducted
three times (triplo) in the test group and also the control group.
Location and Time of Research
a Research
Location
This
research was conducted at the Pharmacology Laboratory of the Faculty of
Medicine, Mulawarman University and UPTD. Health Laboratory of East Kalimantan
Province.
b Research
Time
The
research time was conducted in February - May 2024.
Research Subjects and Sample Selection
a Test
Bacteria Subjects
The
test bacterial subjects used in this study were Streptococcus mutans,
Porphyromonas gingivalis, and Enterococcus faecalis obtained from UPTD.
Samarinda City Provincial Health Laboratory.
b Test
Plant Subjects
The
plant subjects used in this study were black betel leaves (Piper betle L. var
nigra) purchased at Jalan Mugirejo, Samarinda City, East Kalimantan. Identification
of plant subjects was carried out by the Faculty of Forestry, Mulawarman
University.
Research Tools and Materials
a Research
Tools
The
tools used in this study are stationery, handscoon, sterile masks, digital
scales, drying cabinets, blenders, maceration containers, macerate containers,
vials, erlenmeyer flasks, hot plate stirrers, petridish, autoclave, rotary
evaporator (rotav), test tubes, test tube racks, micropipettes, paper disks,
analytical scales, sterile disposable ose, sterile cotton swab, vortex mixer,
nephelometer, tweezers, anaerobic jar, incubator, vernier, Biological Safety
Cabinet (BSC).
b Research
Materials
The
materials used in this study were black betel leaves (Piper betle L var.
nigra), sterile distilled water, 70% ethanol, Nutrient Agar (NA), Mueller
Hinton Agar (MHA), 5% sheep blood, Blood Agar Base (blood agar), sterile NaCl
0.9%, Chlorhexidine gluconate 0.2%, DMSO 100%, Streptococcus mutans,
Porphyromonas gingivalis and Enterococcus faecalis bacteria.
Research
Variables
a The
dependent variable
The
dependent variable in this study is the inhibition of three oral bacteria,
namely Streptococcus mutans, Porphyromonas gingivalis and Enterococcus
faecalis.
b Free
Variable
The
independent variable in this study is the concentration of black betel leaf
extract (Piper betle L var. nigra).
c Control
variable
The
control variables in this study consisted of two, namely positive control and
negative control. Positive control is a treatment that produces an effect on
the dependent variable. This study used Chlorhexidine gluconate 0.2% as a
positive control. Chlorhexidine gluconate 0.2% is a broad-spectrum
antimicrobial agent that is one of the gold standards for plaque prevention in
dentistry. Chlorhexidine gluconate 0.2% has a bacteriostatic effect and a
bactericidal effect against all types of microbes, both gram-positive and
gram-negative bacteria. Negative control is a treatment that does not produce
an effect on the dependent variable. This study used 10% Dimethyl Sulfoxide
(DMSO) as a negative control because DMSO has no effect on bacterial inhibitory
activity and is generally only used as a solvent.
RESULTS AND DISCUSSION
Antibacterial Activity Test Results Streptococcus mutans
The antibacterial activity test was
carried out using five concentrations of black betel leaf extract, namely
concentrations of 60%, 70%, 80%, 90% and 100% and using a positive control of
0.2% Chlorhexidin gluconate and a negative control of 10% DMSO where the test
group and control group were repeated three times.
Figure 1 Results of
antibacterial activity test for treatment groups
against Streptococcus
mutans bacteria.
Source: Primary Data
The
results of the research were seen by measuring the diameter of the inhibition
zone formed around the paper discs 24 hours after being treated using a vernier
caliper in millimeters (mm) and then making a table, which then included the
data.
The measurement results are
processed and analyzed using the SPSS software application.
Table 1 Antibacterial
Activity Test Results for Treatment Groups
against Streptococcus
mutans
|
Treatment group |
Diameter zone
resistor (mm) |
|
Average (mm) ±SE |
One
Way ANOVA test |
|
|
R1 |
R2 |
R3 |
|||
|
60% |
0 |
0 |
0 |
0.00±0.000 |
|
|
70% |
1.00 |
0.52 |
0 |
0.50±0.288 |
|
|
80% |
1.1 |
1.56 |
0 |
0.88±0.462 |
|
|
90% |
2.05 |
2.58 |
0 |
1.54
± 0.786 |
0,000 |
|
100% |
2.88 |
2.00 |
0 |
1.62±0.852 |
|
|
CHX 0.2 ( K +) |
6.22 |
6.48 |
5.38 |
6.02±0.331 |
|
|
DMSO 10% (K - ) |
0 |
0 |
0 |
- |
|
Information: R
= Repetition (repetition); - = cannot inhibit
Table
1 presents the results of the average diameter of the inhibition zone (mm) for Streptococcus
mutans obtained from treatment with each extract concentration in three
repetitions where these results have been reduced by the diameter of the paper
disc (6 mm). The results of treatment of the test groups respectively at
concentrations of 60%, 70%, 80%, 90% and 100% showed an average diameter of the
inhibition zone of 0.00 ± 0.000 mm, 0.50 ± 0.288 mm, 0.88 ± 0.462 mm, 1.54 ±
0.786 mm , and 1.62 ± 0.852 mm. Meanwhile, the results of the positive control
treatment, namely CHX 0.2 or Chlorhexidine gluconate 0.2%, had an average
inhibitory zone diameter of 6.02 ± 0.331 mm, and the negative control DMSO 10%
did not produce a clear zone or an inhibitory zone equal to 0 mm.
The
Shapiro-Wilk normality test (Appendix 8) shows that all treatments have a
significance value of p>0.05, while the Lavene's homogeneity test has a
significance value of p<0.05, so in this case the research data can be said
to be normally distributed and not homogeneous, but to test Differences between
several treatment groups can still be used using the One-Way ANOVA Test. The
p-value in the Sig column. shows a value of 0.000, where the value of 0.000
< 0.05 which means there is a significant difference in the treatment group,
to find out which group has a significant difference, it is carried out Post-Hoc test analysis (Appendix 8).
Because the variants used are not the same, the analysis used is the
Games-Howell test.
Table
2 Post-hoc test results (Games Howell)
|
Games Howell |
60% |
70% |
80% |
90% |
100% |
K+ |
|
60% |
|
,610 |
,556 |
,542 |
,558 |
.011* |
|
70% |
,610 |
|
,971 |
,802 |
,799 |
.002* |
|
80% |
,556 |
,971 |
|
,967 |
,958 |
.007* |
|
90% |
,542 |
,802 |
,967 |
|
1,000 |
,075 |
|
100% |
,558 |
,799 |
,958 |
1,000 |
|
,097 |
|
K+ |
.011 |
,002 |
,007 |
,075 |
,097 |
|
Note: * = has a
significant difference (p<0.05)
In Table
2 and also in Appendix 8, the results of the Post-Hoc test on Streptococcus
mutans bacteria are presented, where the Sig value. The positive control had a
significant difference with concentrations of 60%, 70% and 80% (p<0.05).
This shows that Chlorhexidine gluconate 0.2% is better at inhibiting
Streptococcus mutans bacteria than this concentration. However, at
concentrations of 90% and 100% there was no significant difference (p>0.05)
with the positive control, so it can be said that its antibacterial power is
almost as strong as Chlorhexidine gluconate 0.2%.
Porphyromonas gingivalis
The
antibacterial activity test was carried out using five concentrations of black
betel leaf extract, namely concentrations of 60%, 70%, 80%, 90% and 100% and
using a positive control of 0.2% Chlorhexidin gluconate and a negative control
of 10% DMSO where the test group and control group were repeated three times.
Figure 2 Results of antibacterial activity test
for treatment groups
against Porphyromonas
gingivalis bacteria.
Source: Primary Data
The
results of the research were seen by measuring the diameter of the inhibition
zone formed around the paper discs 24 hours after being treated using a vernier
caliper in millimeters (mm) and then making a table, and then the measurement
data was processed and analyzed using the SPSS software application.
Table
3 Antibacterial Activity Test Results for Treatment Groups
against
Porphyromonas gingivalis
|
Treatment group |
Diameter zone resistor (mm) |
|
Average (mm) ±SE |
Kruskal Wallis test |
|
|
R1 |
R2 |
R3 |
|||
|
60% |
0 |
0.3 |
0 |
0.10±0.100 |
|
|
70% |
0.68 |
0.65 |
0 |
0.44±0.221 |
|
|
80% |
1.4 |
1.4 |
0 |
0.93±0.466 |
|
|
90% |
2.1 |
1.6 |
0 |
1.23±0.633 |
0.079 |
|
100% |
2.78 |
2.62 |
0 |
1.80±0.901 |
|
|
CHX 0.2 (K +) |
8.28 |
8.35 |
3.75 |
6.79±1.521 |
|
|
DMSO 10% (K - ) |
0 |
0 |
0 |
- |
|
Information: R
= Repetition (repetition); - = cannot inhibit
Table 3 presents the results of the average
diameter of the inhibition zone (mm) against Porphyromonas gingivalis which obtained from behavior each extract
concentration in three repetitions where the results were reduced by the
diameter of the paper disc (6 mm). The results of the treatment of the test
groups respectively at concentrations of 60%, 70%, 80%, 90% and 100% showed an
average diameter of the inhibition zone of 0.10 ± 0.100 mm, 0.44 ± 0.221 mm,
0.93 ± 0.466 mm, 1.23 ± 0.633 mm , and 1.80 ± 0.901 mm. Meanwhile, the results
of the positive control treatment, namely CHX 0.2 or Chlorhexidin gluconate
0.2%, had an average inhibitory zone diameter of 6.79 ± 1,521 mm, and the
negative control DMSO 10% did not produce a clear zone or an inhibitory zone
equal to 0 mm.
The
Shapiro-Wilk normality test (Appendix 9) shows that several treatments have a
significance value of p<0.05, and the Lavene's homogeneity test has a
significance value of p<0.05, so in this case the research data can be said
to be not normally distributed and not homogeneous, so that To test differences
between several groups using an alternative test, namely the Kruskal Wallis
Test. The p-value in the Asymp column. Sig. shows a value of 0.079, where 0.079 > 0.05, which means there is no
significant difference in each treatment group. This shows that each extract
concentration with the positive control is almost equivalent in its
antibacterial strength.
Enterococcus faecalis
The antibacterial activity test was
carried out using five concentrations of black betel leaf extract, namely
concentrations of 60%, 70%, 80%, 90% and 100% and using a positive control of
0.2% Chlorhexidin gluconate and a negative control of 10% DMSO where the test
group and control group were repeated three times.
Figure
3 Results of antibacterial activity test for treatment groups
against
Enterococcus faecalis bacteria.
Source:
Primary Data
The results of the research were seen by
measuring the diameter of the inhibition zone formed around the paper discs 24
hours after being treated using a vernier caliper in millimeters (mm) and then
making a table, and then the measurement data was processed and analyzed using
the SPSS software application.
Table 4 Antibacterial Activity Test Results for Treatment
Groups
against Enterococcus faecalis
|
Treatment Group |
Diameter zone resistor (mm) |
|
Average (mm) ±SE |
Kruskal Wallis test |
|
|
R1 |
R2 |
R3 |
|||
|
60% |
0 |
0 |
0 |
0.00±0.000 |
|
|
70% |
0 |
0 |
0 |
0.00±0.000 |
|
|
80% |
0 |
0 |
0 |
0.00±0.000 |
|
|
90% |
0 |
0 |
0 |
0.00±0.000 |
0.005 |
|
100% |
0 |
0 |
0 |
0.00±0.000 |
|
|
CHX 0.2 (K +) |
4.88 |
5.68 |
5.05 |
5.20±0.243 |
|
|
DMSO 10% (K - ) |
0 |
0 |
0 |
0.00±0.000 |
|
Information: R
= Repetition (repetition); - = cannot inhibit
Table
4 presents the results of the average diameter of the inhibition zone (mm) against Enterococcus faecalis obtained from the
treatment of each concentration extract
in three repetitions where the results have been reduced by the diameter of the
paper disc (6mm). The results of the treatment of the test group at all
concentrations, namely 60%, 70%, 80%, 90% and 100%, showed an average diameter
of the inhibition zone of 0.00 ± 0.000 mm, which means there was no inhibition
zone at all. Meanwhile, the results of the positive control treatment, namely
CHX 0.2 or Chlorhexidine gluconate 0.2%, had an average diameter of the
inhibition zone of 6.79 ± 1.521 mm, and
the negative control 10% DMSO did not produce a clear zone or the zone of
inhibition was equal to 0 mm.
The
Shapiro-Wilk normality test and the Lavene's homogeneity test (Appendix 10)
both show that the treatment group has a significance value of p<0.05, so in
this case the research data can be said to be not normally distributed and not
homogeneous, so as to test the differences between several groups using an
alternative test, namely the Kruskal Wallis Test. The p-value in the Asymp
column. Sig. shows a value of 0.005, where the value 0.005 <0.05, which
means there is a significant difference in the treatment group. This difference
can be seen from (Appendix 10) visualization dun test, that the inhibition zone
of the positive control Chlorhexidine gluconate 0.2% has a significant
difference to all test groups.
The test plant used in this research was the black
betel leaf plant (Piper betle L. var nigra). Black betel leaves were determined
to ensure that the plants used were truly black betel leaves (Piper betle L.
var nigra). Determination was carried out at the Tropical Forest Ecology and
Biodiversity Conservation Laboratory, Faculty of Forestry, Mulawarman
University. Antibacterial activity testing in this study was carried out using
the disc diffusion method to see the diameter of the inhibition zone. In this
study, three bacteria were used, namely Streptococcus mutans ATCC 35668,
Porphyromonas gingivalis and Enterococcus faecalis ATCC 29212. Preparation of
black betel leaf simplicia powder was carried out at the Pharmacology
Laboratory, Faculty of Medicine, Mulawarman University. The antibacterial
activity test was carried out at the East Kalimantan Provincial Health
Laboratory UPTD using Bio Safety Cabinet Class II (BSC II) to prevent
contamination.
Interpretation
and Discussion of Results
The results of
this research prove that black betel leaf extract (Piper betle L. var nigra)
can inhibit two of the three bacteria tested, namely Streptococcus mutans and
Porphyromonas gingivalis, as indicated by the presence of a clear zone or
inhibition zone that is formed. This inhibition zone is evidence that there is
antibacterial activity in the sample of black betel leaf extract (Piper betle
L. var nigra) used by researchers. Antibacterial activity can be influenced by
several factors which are divided into biological factors and technical
factors. Technical factors can mostly be controlled by researchers but
biological factors cannot be controlled by researchers. Technical factors that
influence the size of the inhibition zone in the disc diffusion method include:
inoculum concentration, disc installation time, incubation temperature,
incubation time, plate size, agar media thickness and media composition. (Nor, Indriarini, & Koamesah, 2018) . The turbidity
of the bacterial suspension used has been adjusted to the McFarland standard of
0.5 or the equivalent of 1x108 bacteria/mL which has been confirmed using a
nephelometer (Aprillia et al., 2021) . The medium used
to test antibacterial activity on the bacteria Streptococcus mutans and
Enterococcus faecalis is Mueller Hinton Agar + 5% sheep blood which has been
adapted to the latest CLSI standards. Meanwhile, the test medium for
Porphyromonas gingivalis bacteria is Blood Agar, which is adapted to previous
research, namely (Nabila, Purnamasari, & Alhawaris, 2021) which also used
the disc diffusion method for Porphyromonas gingivalis bacteria. The
temperature and incubation time in this study have been adjusted to 1x24 hours
with a temperature of 35 ± 20C (CLSI, 2020; Hudzicki, 2016). Meanwhile,
biological factors consist of persisters and resistance. Persisters come from
cells that are dormant or replicate slowly so they cannot be killed by
antibacterial agents. The persister’s factor has been controlled by using an
inoculum that does not exceed 24 hours or an inoculum in the logarithmic phase.
Resistance cannot be controlled in research because it is an adaptation of
bacteria to survive (MOJA, 2015) .
Antibacterial
activity is also influenced by several factors such as the content of
antibacterial compounds, extract concentration, extract diffusion power, and
the type of bacteria being inhibited (Fitriani, 2014) . The content of
antibacterial compounds extracted from a plant is influenced by the solvent so
that the choice of solvent is important in the diffusion power of the extract (MOJA, 2015) . The choice of
solvent is based on its ability to have large polarity or be semi polar so that
it can dissolve various chemical components in samples that are polar to
nonpolar in maximum amounts (Handoyo, 2020) . The solvent
chosen for this research was 70% ethanol solvent, because the antibacterial
compounds thought to be found in black betel leaves (Piper betle L. var nigra)
include tannins, phenolic compounds, saponins, flavonoids, alkaloids, steroids
(Prasetya & Angga, 2013) , and terpenoids (Maharani & Fernandes, 2021) . These compounds
are polar, semi-polar and non-polar compounds (steroids and terpenoids). Polar
compounds will dissolve in polar filter solutions and nonpolar compounds will
dissolve or disperse in nonpolar solvents. 70% ethanol is more polar than 96%
ethanol and more non-polar than 50% ethanol, so compounds that are polar and
non-polar will tend to dissolve more in 70% ethanol. The choice of 70% ethanol
solvent is expected to optimize the content of the compounds contained in the
extract so that it has antibacterial power (Kamaruddin & Arnov, 2023) . Several studies
that also used 70% ethanol solvent to extract antibacterial compounds in black
betel leaves (Piper betle L. var nigra) are (Aprillia et al., 2021) .
Compounds that
may play a role in causing the antibacterial activity of black betel leaf
extract (Piper betle L. var nigra) are active compounds in the phenol group and
their derivatives, especially tannins and flavonoids (Owu & Jayanti, 2020) . Phenolic
compounds are polar and act as antibacterials. The mechanism of action of phenol
compounds in killing bacterial cells is by denaturing bacterial cell proteins.
As a result of the denaturation of bacterial cell proteins, all bacterial cell
metabolic activities stop because all bacterial cell metabolic activities are catalysed
by enzymes which are proteins. At high concentrations, phenol content can
penetrate and disrupt bacterial cell walls and precipitate proteins in
bacterial cells, while at lower concentrations phenol inactivates important
enzyme systems in bacterial cells (Marfuah, Dewi, & Rianingsih, 2018) .
Tannin is a
derivative of phenol compounds. Tannins have antibacterial activity by
precipitating proteins, inactivating enzymes, and destroying or inactivating
genetic material. The antibacterial properties of tannins depend on their
chemical structure and molecular weight. Low molecular weight tannins have
better activity than higher molecular weight tannins (Kurniasari, 2022) . Tannins can be
classified into condensation tannins and hydrolysis tannins. Previous research
showed that only hydrolysed tannin showed antibacterial activity, hydrolysed
tannin was found to have much better antibacterial activity than condensed
tannin or a mixture of the two (Fitriani, 2014) .
Flavonoids are
the largest group of phenolic compounds (Marfuah et al., 2018) . Flavonoids are
antibacterial by binding to proteins via hydrogen bonds, causing the protein
structure to be damaged. Most cell wall structures and cytoplasmic membranes
contain proteins and fats. Flavonoids also break the bonds between N-Acetyl
glucosamine and N-Acetylramic acid which is found in the peptidoglycan layer of
cell membranes. Damage to the peptidoglycan layer which is the framework of the
cell membrane will result in instability of the cell membrane and the bacterial
wall, causing the selective permeability function, active transport function,
control of the protein structure of the bacterial cell to be disturbed which
will result in the escape of macromolecules and ions from the cell, so that the
bacterial cell becomes loses shape and lysis occurs (Ernita Sari, Rahmawan, & Sahara, 2021) . Phenol is able
to cause coagulation of cell proteins and lyse cells at high levels, whereas at
low concentrations phenol protein complexes are formed with weak bonds and
immediately decompose so that the antibacterial effect becomes weak (Fitriani, 2014) .
Other compounds
which are also thought to act as antibacterial include saponins, alkaloids,
steroids and terpenoids. Saponins, which are detergents, have amphipathic
molecules (containing hydrophilic and hydrophobic parts) which can dissolve
membrane proteins. The hydrophobic end of saponin binds to the hydrophobic
region of cell membrane proteins by displacing some of the bound lipid
elements, resulting in bacterial cell lysis. The mechanism of action of
alkaloids as antibacterial is by disrupting the peptidoglycan components in
bacterial cells so that the cell wall layer does not form completely and causes
cell death. Apart from that, alkaloids also inhibit the formation of protein
synthesis so that they can disrupt bacterial metabolism (Fajrina, Bakhtra, Eriadi, Putri, & Wahyuni, 2021)
.
Steroids have antibacterial activity through
interaction with cell phospholipid membranes which are permeable to lipophilic
compounds, causing membrane integrity to decrease and cell membrane morphology
to change which can cause bacterial cells to become brittle and lyse (Saputri & Rahayu, 2018) . Meanwhile,
terpenoid compounds have antibacterial activity through reactions with porins
or Tran’s membrane proteins in the outer membrane of bacterial cell walls,
forming strong polymer bonds, resulting in the destruction of poring. Damage to
poring, which is the gateway for compounds to enter and exit, will reduce the
permeability of bacterial cell walls. This cell wall permeability will disrupt
the transport of nutrients and other compounds, so that bacterial growth is
hampered or dies (Ernita Sari et al., 2021) . Flavonoids,
tannins, saponins and steroids are known to work synergistically in inhibiting
bacterial growth.
Antibacterial
Activity of Black Betel Leaf Extract Against Streptococcus mutans Bacteria
The results of
research testing the antibacterial activity of black betel leaf extract (Piper
betle L. var nigra) were proven to inhibit the growth of Streptococcus mutans
by forming a clear zone or the diameter of the inhibitory zone around the paper
disc. This research is in accordance with research conducted by previous
researchers that betel leaves can inhibit the growth of gram-positive bacteria,
namely Streptococcus mutans bacteria (Owu & Jayanti, 2020) . This is because
gram-positive bacteria only have a single layer in their cell walls, little
lipids and lots of echoic acids. Echoic acid is a polymer that is soluble in
water and is polar. Most of the antibacterial compounds in black betel leaves
are polar compounds, so these compounds can easily penetrate the peptidoglycan
layer of gram-positive bacteria which are also polar. (Magvirah, Marwati, & Ardhani, 2020) .
The results of
the treatment in the test group, precisely at a concentration of 60%, did not
show a clear zone forming around the disc, indicating that at this
concentration black betel leaf extract (Piper betle L. var nigra) could not
inhibit Streptococcus mutans bacteria at all. Meanwhile, at concentrations of
70%, 80%, 90% and 100%, a clear zone can be seen forming around the disc and
has an average diameter of the inhibition zone respectively 0.50 ± 0.288 mm,
0.88 ± 0.462 mm, 1.54 ± 0.786 mm, and 1.62 ± 0.852 mm which shows that at this
concentration black betel leaf extract (Piper betle L. var nigra) can inhibit
Streptococcus mutans bacteria. Then the results were interpreted based on the
categories of Davis & Stout (1971), and it was found that black betel leaf
extract (Piper betle L. var nigra) at concentrations of 70%, 80%, 90% and 100%
had weak strength in inhibiting the growth of Streptococcus bacteria. mutants.
The results of
this research are different from previous research conducted by Ningsih in
2013, she proved that red betel leaf extract (Piper crocatum) at a
concentration of 100% had an inhibitory zone diameter of 11.78 mm which was
classified as strong. This can occur due to differences in the types of betel
leaves used, where similar plants may contain the same secondary metabolites
but in different quantities (Qhorina et al., 2021) . In accordance
with Hasanah's research in 2020, red betel leaf extract (Piper crocatum) has
higher antibacterial effectiveness in inhibiting bacterial growth, because
there are active compounds in red betel leaves (Piper crocatum) which are
stronger than black betel leaves (Piper betle L). . var nigra). Apart from the
type of plant, different results can occur due to differences in the test
methods used. In Ningsih's (2013) research, he used the well diffusion method,
where the antibacterial activity using the well method had a larger zone
compared to the disc method. This is thought to be because the isolate is
active not only on the surface of the agar but also down to the bottom,
resulting in a more homogeneous and efficient osmosis process in inhibiting
bacterial growth (Nurhayati, Yahdiyani, & Hidayatulloh, 2020) . The method in
this research, namely disc diffusion, was chosen because it is practical, easy
to do, does not require special equipment and is relatively cheap. However, the
size of the inhibition zone formed depends on the incubation conditions, inoculum,
prediffusion and preincubation as well as the thickness of the medium (Rizki, Latief, Fitrianingsih, & Rahman, 2022) .
In Appendix 8,
multiple comparisons of Streptococcus mutans bacteria show that the positive
control inhibition zone has a significant difference with concentrations of
60%, 70%, 80% (p<0.05). This shows that Chlorhexidine gluconate 0.2% is
better at inhibiting Streptococcus mutans bacteria than this concentration.
However, at concentrations of 90% and 100% there was no significant difference
(p>0.05) with the positive control, so it could be said that its
antibacterial power was almost equivalent to Chlorhexidine gluconate 0.2%. The
positive control in this study, namely Chlorhexidine gluconate 0.2%, had an
average inhibition zone diameter of 6.02 ± 0.331 mm. Chlorhexidine gluconate
0.2% was chosen as a positive control because this compound has significant
antibacterial ability against gram-positive bacteria, one of which is
Streptococcus mutans, in accordance with the test bacteria in this study (Pambudi, Wasiaturrahmah, & Aspriyanto, 2021) . Chlorhexidine
contains phenol which has a bacteriostatic effect at levels of 0.2-1%, is
bactericidal at levels of 0.4-1.6%, and is fungicidal at levels above 1.3%. The
basic ingredient chlorine is a high level disinfectant because it is very
active on all bacteria, viruses, fungi, parasites and some spores. (Pradayani, Pertiwi, & Ambarawati, 2021) . The mechanism
of action is to disrupt the cell membrane transportation process and bacterial
metabolism, so that the cell wall becomes lysed. The process begins with the
0.2% Chlorhexidine gluconate compound binding the Streptococcus mutans
bacteria, which is caused by ionic bonds in the form of attracting cations from
the Chlorhexidine molecule and anions of the Streptococcus mutans cell wall.
This ionic bond will cause a selective increase in the permeable peptidoglycan
of Streptococcus mutans so that the cell membrane becomes damaged, the
cytoplasm leaks, ultimately causing the death of the bacteria (Pambudi et al., 2021) .
The negative
control used in this study, namely 10% DMSO, did not show any inhibition zone
at all, which proves that 10% DMSO as an extract solvent does not have antibacterial
activity and does not affect the test results in this study. DMSO was chosen
because it can dissolve almost all compounds, both polar and non-polar. DMSO is
a compound that has low toxicity, has anti-inflammatory and analgesic effects (Rahmi & Putri, 2020) . DMSO has the
ability to penetrate cell membranes, however, when using DMSO as a solvent, the
final concentration of DMSO should not exceed 10% because it can cause cell
membrane rupture. The 10% DMSO solvent is an organic solvent and is not
bactericidal so it cannot affect the test results (Manarisip, Fatimawa, & Rotinsulu, 2020) .
The results of this research also show that each
increase in the concentration of black betel leaf extract (Piper betle L. var
nigra) given will result in an increasingly larger diameter of the inhibition
zone. This research is in accordance with Owu's research in 2020 which used
betel leaf extract (Piper betle L) against Streptococcus mutans bacteria and
Saputri & Rahayu's research in 2018 which used black betel leaf extract
(Piper betle L. var nigra) against Staphylococcus aureus bacteria (Owu & Jayanti, 2020) . This research
also shows that the diameter of the inhibition zone is directly proportional to
the concentration of betel leaf extract. The greater the concentration, the
greater the active substance dissolved, because the less diluent is used. So a solution
with a higher concentration will have a more concentrated active substance (Amanda, Mastra, & Sudarmanto, 2018) .
Antibacterial
Activity of Black Betel Leaf Extract Against Porphyromonas gingivalis Bacteria
The results of
the antibacterial activity test of black betel leaf extract (Piper betle L. var
nigra) were proven to inhibit the growth of Porphyromonas gingivalis by forming
a clear zone or the diameter of the inhibitory zone around the paper disc. This
research is in accordance with research conducted by Herryawan in 2023 &
Sendy in 2014 that green, red and black betel leaves can inhibit the growth of
gram-negative bacteria, namely Porphyromonas gingivalis bacteria (Herryawan et al., 2023) . This can happen
because gram-negative bacteria have thin peptidoglycan cell walls, only about
10% of the dry mass of their cell walls. Gram-negative bacteria contain a lot
of lipids and have porin proteins which act as channels for the entry of active
substances into bacterial cells. The entry of this active substance damages
enzyme activity in cells and causes cell damage. High lipid levels in cells
will increase the permeability of active substances into cells (Rompas, Wewengkang, & Mpila, 2022) . One of the
compounds that is thought to play an important role in these bacteria is
steroids, where steroids can interact with cell phospholipid membranes which
are permeable to lipophilic compounds, causing membrane integrity to decrease
and cell membrane morphology to change causing cells to become brittle and
lysed (Nisa, 2019).
The results of
treatment at concentrations of 60%, 70%, 80%, 90% and 100% showed the average
diameter of the inhibition zone respectively, namely 0.10 ± 0.100 mm, 0.44 ±
0.221 mm, 0.93 ± 0.466 mm, 1.23 ± 0.633 mm, and 1.80±0.901 mm. These results
were interpreted based on the categories of Davis & Stout (1971), and it
was found that black betel leaf extract (Piper betle L. var nigra) at all concentrations
had weak power in inhibiting the growth of Porphyromonas gingivalis bacteria.
The results of
this research are slightly different from research conducted by Herryawan in
2023, where he proved that black betel leaf extract (Piper betle L. var nigra)
based on the category of David & Stout (1971), at a concentration of 100%
is classified as strong (mean: 19.65mm) in inhibiting the growth of
Porphyromonas gingivalis bacteria (Herryawan et al., 2023) . This is thought
to occur due to many factors, including differences in the origin of the plant
and the extraction process, considering the different geographical conditions
and climate changes which result in variations in the chemical compound content
of a plant. The black betel leaf plant (Piper betle L. var nigra) used in this
research comes from Samarinda, which is planted on vines on tree stands as a
natural stake in the yard, protected by shade, without fertilizer application
since planting and aged at harvest ± 3 years. Where these factors can influence
the quality and content of black betel leaves (Piper betle L. var nigra)
produced, so that they can cause different results from other studies even
though they use the same type of plant. In the book "Cultivation and
benefits of betel for health" by Widiyastuti, Rahmawati and Mujahid
(2020), it is stated that in conditions under shade, with light intensity
around 50%, betel is still able to grow with morphological changes in the form
of wider and thinner leaves. However, betel will produce better quality leaves
if planted in an open area with full sunlight. Harvesting betel must also be done
at the right age and time (1-2 years after planting) to obtain the most optimal
quality. Planting betel in less fertile soil requires additional (organic)
fertilizer so that the soil in which it grows can better hold water.
In Appendix 9,
the results of the Kruskal Wallis test on Porphyromonas gingivalis bacteria,
the P-value in the Asymp column. Sig. shows a value of 0.079, where 0.079 >
0.05, which means there is no significant difference in each treatment group.
This shows that each treatment in this study has an inhibition zone that is not
significantly different or is almost equivalent in its antibacterial strength
against Porphyromonas gingivalis bacteria. This shows that the positive control
used, namely Chlorhexidin gluconate 0.2%, is almost equivalent in antibacterial
strength to the entire concentration of black betel leaf extract (Piper betle
L. var nigra) in inhibiting the growth of Porphyromonas gingivalis bacteria.
The positive
control in this study, namely Chlorhexidine gluconate 0.2%, had an average
inhibition zone diameter of 6.79 ± 1,521 mm. Chlorhexidine gluconate 0.2% was
chosen as a positive control because it is the gold standard mouthwash which
has broad spectrum antimicrobial properties and is effective against various types
of gram-positive, gram-negative bacteria and fungi, and is a mouthwash that is
often used in chronic periodontitis therapy. Chlorhexidine molecules have a
positive charge (cation) and most bacterial molecular charges are negative
(anions). This causes strong attachment of Chlorhexidine to the bacterial cell
membrane. Chlorhexidine will cause changes in the permeability of bacterial
cell membranes, causing the release of cell cytoplasm and low molecular weight
cell components from inside the cell through the cell membrane, causing
bacterial death.
The negative
control used in this study, namely 10% DMSO, did not show any inhibition zone
at all, which proves that 10% DMSO as an extract solvent does not have
antibacterial activity and does not affect the test results in this study. DMSO
was chosen because it can dissolve almost all compounds, both polar and
non-polar. DMSO is also a compound that has low toxicity, has anti-inflammatory
and analgesic effects (Rahmi & Putri, 2020) . DMSO has the
ability to penetrate cell membranes, however, when using DMSO as a solvent, the
final concentration of DMSO should not exceed 10% because it can cause cell
membrane rupture. The 10% DMSO solvent is an organic solvent and is not
bactericidal so it cannot affect the test results (Manarisip et al., 2020) .
The results of this research also show that each
increase in the concentration of black betel leaf extract (Piper betle L. var nigra)
given will result in an increasingly larger diameter of the inhibition zone.
This research is in accordance with Herryawan's research in 2023, which used
extracts of red betel leaves (Piper crocatum) and black betel leaves (Piper
betle L. var nigra) against Porphyromonas gingivalis bacteria (Herryawan et al., 2023) . Supported by
Sendy's research in 2014, which used red betel leaf extract (Piper crocatum)
against Porphyromonas gingivalis bacteria. This research also shows that the
diameter of the inhibition zone is directly proportional to the concentration
of betel leaf extract. The greater the concentration, the greater the active
substance dissolved, because the less diluent is used. So a solution with a
higher concentration will have a more concentrated active substance (Amanda et al., 2018) .
Antibacterial
Activity of Black Betel Leaf Extract Against Enterococcus faecalis Bacteria
The results of
the antibacterial activity test of black betel leaf extract (Piper betle L. var
nigra) in this study proved that it could not inhibit the growth of
Enterococcus faecalis bacteria. This research is in accordance with research
conducted by Wastri in 2021 that basil leaf extract (O. basilicum) has no
inhibition zone against Enterococcus faecalis and also Wijaya's research in
2021 that there is no antibacterial effectiveness of Virgin Coconut Oil (VCO)
extract against Enterococcus faecalis. In 2009, Limsuwan also carried out
extensive screening of 31 plant species with 12 pathogenic bacteria, and proved
that betel leaves (Piper betle) showed significant activity against almost all
species, except for one bacteria, namely Enterococcus faecalis.
The results of
this research are different from the results of Armianty & Mattulada's
research in 2014 which proved that betel leaf extract could inhibit the growth
of Enterococcus faecalis bacteria at a concentration of 20%, as well as Pasril
& Yuliasant's research in 2022 which proved that red betel leaf extract
(Piper crocatum) able to inhibit the growth of Enterococcus faecalis bacteria.
This can occur due to differences in the types of betel leaves used, where
similar plants may contain the same secondary metabolites but in different
quantities (Qhorina et al., 2021) . This is also in
accordance with Hasanah's research in 2020 that red betel leaf extract (Piper
crocatum) has higher antibacterial effectiveness in inhibiting bacterial
growth, because there are active compounds in red betel leaves (Piper crocatum)
which are stronger than black betel leaves (Piper betle L. var nigra). Each
type of betel leaf has a different quantity of secondary metabolite content, so
that at the same concentration you can get different results. The content of
secondary metabolites in a plant is influenced by several factors, both
internal and external. Internal factors such as genes and external factors
include light, temperature, humidity, pH, nutrient content in the soil and
altitude. Different altitudes will produce different temperatures. A series of
metabolic processes in plants will be disrupted so that the compounds produced
from these processes will be different at each altitude. This can affect the
amount of antibacterial substances contained in the sample and can cause the
inhibition zone not to form due to its inability to damage cell membranes and
disrupt cell physiological processes (Nabila et al., 2021) .
Apart from the
type of betel leaf used, this difference could be caused by the bacteria being
inhibited, where the bacteria used in this study were pure culture bacteria
obtained from the Regional Health Laboratory and it cannot be known how many
times the bacteria have been cultured. Yuasa et al., (2003) in (Huyyirnah & Fitriyani, 2020) stated that
sub-culture that has been carried out many times can cause the possibility of
bacterial contamination, as well as decreasing or losing the pathogenicity of
bacteria. The CLSI troubleshooting guide also states several things that can
cause the inhibition zone to be too small or even too small, namely
contamination, using magnification to read the zone, too heavy an inoculum,
errors in preparing the inoculum and media thickness. Differences in the
thickness of the agar media can influence the diffusion of the test substance
into the agar, thereby affecting the diameter of the inhibition zone. The
thicker the media used, the smaller the diameter of the inhibition zone that
occurs. Agar media that complies with CLSI guidelines is 4 mm. In this
research, the media should not be measured.
In Appendix 10,
the results of the Kruskal Wallis test on Enterococcus faecalis bacteria, the
P-value in the Asymp column. Sig. shows a value of 0.005, where the value 0.005
<0.05 which means there is a significant difference in the treatment group.
This difference can be seen from the results of the Dunn test (Appendix 10)
that there is a significant difference between Chlorhexidine gluconate 0.2% and
all test groups. The positive control in this study, namely Chlorhexidine
gluconate 0.2%, had an average inhibitory zone diameter of 5.20 ± 0.243 mm
against Enterococcus faecalis bacteria. Chlorhexidine gluconate 0.2% was chosen
as a positive control because it is a broad spectrum and low toxic root canal
irrigation material, where its use at high concentrations (>2%) can be
bactericidal which causes precipitation of bacterial cell structures, whereas
its use at low concentrations (0 .2%) can be bacteriostatic, namely inhibiting
the growth and development of bacteria including Enterococcus faecalis (Dini Permata Sari, Nahzi, & Budiarti, 2019) . Supported by
research by Armianty & Mattulada in 2014, which proved that Chlorhexidine
gluconate 0.2% has an antibacterial effect against Enterococcus faecalis
bacteria.
The negative
control used in this study, namely 10% DMSO, was proven to have no
antibacterial activity and did not affect the test results. DMSO was chosen
because it can dissolve almost all compounds, both polar and non-polar. DMSO is
also a compound that has low toxicity, has anti-inflammatory and analgesic
effects (Rahmi & Putri, 2020) . DMSO has the
ability to penetrate cell membranes, however, when using DMSO as a solvent, the
final concentration of DMSO should not exceed 10% because it can cause cell
membrane rupture. The 10% DMSO solvent is an organic solvent and is not
bactericidal so it cannot affect the test results (Manarisip et al., 2020) .
Research
Limitations
The plants used
in this research were obtained from black betel farmers in Samarinda, whose
growth from planting to harvest cannot be controlled directly by researchers,
and no phytochemical tests were carried out to confirm the content or secondary
metabolites contained in the leaf extract.
Black betel
(Piper betle L. var nigra) was used so it cannot be determined with certainty
which compounds played an important role in the results of this study and how
much secondary metabolites were absorbed by the solvent, and the bacteria used
in this study were cultured bacteria obtained from Samarinda Regional Health
Laboratory, where it cannot be known for certain how many times the bacteria
have been cultured, while sub-cultures that have been carried out many times
can cause the possibility of bacterial contamination, as well as decreasing or
losing the pathogenicity of the bacteria.
CONCLUSION
Based
on the results of the research that has been carried out, it can be concluded
that: there is antibacterial activity of black betel leaf extract (Piper betle
L. var nigra) against the growth of Streptococcus mutans bacteria at
concentrations of 70%, 80%, 90% and 100% with an average diameter of the
inhibition zone respectively 0.50 ± 0.288 mm, 0.88 ± 0.462 mm, 1.54 ± 0.786 mm,
and 1.62 ± 0.852 mm. There is also antibacterial activity of black betel leaf
extract (Piper betle L. var nigra) against the growth of Porphyromonas
gingivalis bacteria at concentrations of 70%, 80%, 90% and 100% with an average
diameter of the inhibition zone respectively 0.44 ± 0.221 mm, 0.93 ±0.466 mm,
1.23 ± 0.633 mm, and 1.80 ± 0.901 mm. However, there was no antibacterial
activity of black betel leaf extract (Piper betle L. var nigra) against the
growth of Enterococcus faecalis bacteria. Further research needs to be carried
out to determine the antibacterial activity of black betel leaf extract using
the dilution method, as well as the antibacterial activity against bacteria
other than Streptococcus mutans, Porphyromonas gingivalis, and Enterococcus
faecalis. Comparative research regarding the secondary metabolite content of
various types of betel leaves also needs to be carried out to understand
differences in antibacterial activity test results.
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