Keywords
Enterococcus faecalis
-
Abrus precatorius Linn - root canal treatment - well diffusion - antibacterial
Introduction
Pulp necrosis and irreversible pulpitis are the most common cases found in dentistry.
They can be prevented and treated with root canal treatment.[1 ]
[2 ] Root canal treatment is a dental procedure that aims to maintain or improve the
condition of infected tooth to be accepted biologically by the tissues. Successful
root canal treatment depends on the ability to eliminate microorganisms from the root
canal system and the reinfection prevention.[3 ]
[4 ]
The most common root canal microorganisms isolated from infections are anaerobic bacteria,
one of which is Enterococcus faecalis bacterium. It is considered to be the main cause of root canal abnormalities with
a prevalence value of 77%.[5 ]
[6 ]
Enterococcus faecalis is the dominant species of facultative anaerobic gram-positive cocci that exist in
pairs, singles, short chains, oval or rounded egg shaped.[7 ]
[8 ]
Enterococcus faecalis can survive and multiply in the root canal with poor nutrient, high pH (alkaline)
up to 11.5, and any help from other bacteria.[9 ] The resilience of E. faecalis to survive in an unfavorable root canal environment causes this bacterium to be a
pathogen that leads to root canal treatment failure. The failure can be prevented
by using an appropriate irrigation with low toxicity solution.[7 ] An ideal irrigation material should be nontoxic, able to dissolve organic and inorganic
tissues, prevent smear layers formation during root canal preparation, and have antimicrobial
properties.[4 ]
Two percent chlorhexidine (CHX) was suggested as a root canal irrigation agent because
its antimicrobial effect can effectively protect the root canal after root canal treatment
at that concentration. However, 2% CHX is not recommended as the first option for
irrigation agents since it causes discoloration and allergic reactions when used repeatedly
over a long period of time.[10 ]
[11 ] The damaging effects of these irrigation agents have encouraged people to search
alternative treatments, such as herbal medicine. One herbal medicinal plant often
utilized as traditional medicine by the community is saga leaves.[8 ]
Abrus precatorius Linn is known as cough medicine and treatment for stomatitis, pharyngitis, and tonsillitis.[12 ] According to research, it has been reported that saga leaves (A. precatorius Linn) have preclinical anti-inflammatory properties. Saga leaf extract contains flavonoids,
terpenoids, tannins, alkaloids, and saponin compounds that are effective as antibacterials
by inhibiting cyclooxygenase and lipooxygenase.[13 ]
[14 ] The antibacterial activity of saga leaves has been tested and proven effective against
Streptococcus mutans bacteria, a gram-positive facultative anaerobic bacteria.[15 ] This study was conducted to determine the effect of different saga leaf concentrations
extract in inhibiting E. faecalis bacteria growth.
Materials and Methods
This was a laboratory experimental study with in vitro posttest only control group
design by giving different treatments to E. faecalis bacteria with saga leaf extract (A. precatorius Linn) in various concentrations with the well diffusion test method. The formation
of inhibition zone was calculated in each treatment. This research was conducted at
Aretha Medika Utama Laboratory, Bandung.
Plant Determination
The saga leaves were obtained from a local herbal plantation in South Bogor subdistrict,
Bogor city, West Java, which was then identified at the Biology Laboratory, Padjadjaran
University, Jatinangor, West Java, to obtain the determination of A. precatorius Linn.
Concentration Series Preparation
This study was treated with a positive control in the form of 2% CHX, negative control
in the form of 100% dimethyl sulfoxide (DMSO), and saga leaf extract (A. precatorius Linn) with concentrations of 3.125, 6.25, 12.5, 25, 50, and 100%. Calculation of
dilution of saga leaf extract solution and 100% DMSO were performed to make series
of concentrations. The varying concentration of extracts used are as follows:
100% saga leaf extract: stock solution.
50% saga leaf extract: 500 µL stock solution + 500 µL DMSO 100% (Solution A).
25% saga leaf extract: 500 µL solution A + 500 µL DMSO 100% (Solution B).
12.5% saga leaf extract: 500 µL solution B + 500 µL DMSO 100% (Solution C).
6.25% saga leaf extract: 500 µL solution C + 500 µL DMSO 100% (Solution D).
3.125% saga leaf extract: 500 µL of solution D + 500 µL of 100% DMSO.
Preparation of Saga Leaf Extract
This research was conducted at the Central Laboratory, Padjajaran University, Jatinangor,
West Java. A total of 1 kg of saga leaves was dried and pulverized using blender to
form fine powder; 287 g of saga leaf powder were used in the first maceration process
by soaking it with 1 L of 96% ethanol solvent in a tightly closed container. The soaking
process was performed for 72 hours and mixed occasionally. The saga leaf solution
then proceeds to the filtering stage using a filter paper to separate the solution.
The filtrate was separated and placed in a glass bottle. The filtrate was then evaporated
using vacuum rotary evaporator with a temperature of 45 to 50°C; 50 rpm speed; 170–180
mbar pressure for roughly 5 hours until a thick saga leaf extract was obtained. Furthermore,
the dilution process was performed using 100% DMSO at a concentration of 3.125, 6.25,
12.5, 25, 50, and 100%. Finally, the ethanol extract of saga leaves with various concentrations
were placed into closed sterile bottles and stored in the refrigerator at −20°C.
Phytochemical Analysis
Phytochemical analysis test was conducted to test the presence of bioactive compounds,
such as tannins, flavonoids, alkaloids, steroids, triterpenoids, phenolics, and saponins
from the saga leaf extract. Alkaloids were identified using Dragendorff reagent. Flavonoids
were identified using HCl + Mg, H2 SO4 , and NaOH 10%. Saponins were detected by heating method. Tannins and phenolics were
identified using 1 and 5% FeCl3 reaction, and steroids and triterpenoids were analyzed with H2 SO4 + CH3 COOH. Qualitative results were expressed as (+) for presence and (−) for absence of
phytochemicals.
Culture of Enterococcus faecalis Bacteria
The sample in this study was E. faecalis bacteria (ATCC 29212) obtained from Aretha Medika Utama Laboratory, Bandung. The
study used 19 g of Mueller–Hinton agar (MHA) and 10.5 g of Mueller–Hinton broth (MHB)
as the growth media which were measured using an analytical balance. Microwave was
used to help dissolve the growth media in 500 mL of ddH2 O. The next step was the sterilization process using an autoclave at 121°C with a
pressure of 1.5 atm for 20 minutes under anaerobic conditions. Colony suspension method
was used to prepare the bacterial inoculum by inoculating E. faecalis colonies that had been cultured for 18 to 24 hours on MHA medium, into MHB medium.
The solution's turbidity was adjusted according to the McFarland 0.5 standard solution's
turbidity to produce an inoculum with a bacterial count of ∼1 to 2 × 108 CFU/mL. Pure
culture of bacteria was taken as much as 1 ose and implanted on MHA by swab method.
Diffusion Test
The inoculation process on the test agar plates was performed using the swab method
by dipping a sterile cotton swab into the prepared bacterial suspension. The cotton
swab was pressed against the tube wall to remove excess suspension, which was then
applied to the surface of the MHA medium evenly. The inoculation was allowed to rest
at room temperature for 3 to 5 minutes until the suspension was absorbed into the
agar. After that, holes were made in the MHA medium using 8-mm-diameter tips. Each
hole was filled with 50 µL of different concentrations of saga leaf extract, starting
with 3.125, 6.25, 12.5, 25, 50, and 100%, the positive control 2% CHX, and the negative
control DMSO 100%, respectively. In this study, the test agar wells were made for
three repetitions. The agar plates were first incubated at 37°C for 24 hours before
measuring the diameter of the inhibition zone formed using a caliper.
Statistical Analysis
Data from the measurement of inhibition zone diameter were obtained and tested statistically
using the normality test (Shapiro–Wilk), homogeneity test (Levene), parametric test
(one-way analysis of variance), and further test (post hoc test).
Results
Phytochemical Examination
The extracted saga leaf plants were then subjected to qualitative phytochemical testing
with the following test results.
Based on the results of phytochemical tests, only secondary metabolite compounds flavonoids
appeared in low amounts (+) with 10% NaOH testing, while saponins, tannins, steroids,
and phenolic appeared in moderate amounts (++).
Measurement of Inhibition Zone Diameter of Saga Leaf Extract against Enterococcus faecalis
In this study, antibacterial measurements were calculated from the inhibition zone
diameter. The inhibition area measured is a clear zone where E. faecalis bacteria did not grow. The clear zone area was measured using a caliper in millimeters
(mm). The results are shown in [Tables 1 ] and [2 ], and [Figs. 1 ] and [2 ].
Fig. 1 The zone of inhibition of saga leaf extract against Enterococcus faecalis shown by a clear area.
Fig. 2 Comparison of inhibition zone diameter of saga leaf extract against Enterococcus faecalis bacteria.
Table 1
Phytochemical test results on saga leaf extract (Abrus precatorius Linn)
No.
Metabolite compounds
Test method
Test results
1
Alkaloid
Dragendorff reagent
−
2
Flavonoid
Concentrated HCl and Mg reagent
−
2N H2 SO4 reagent
−
10% NaOH reagent
+
3
Saponin
Heated
++
4
Tannin
1% FeCl3 reagent
++
5
Steroid
Concentrated H2 SO4 reagent and anhydrous CH3 COOH
++
6
Triterpenoid
Concentrated H2 SO4 reagent and anhydrous CH3 COOH
−
7
Phenolic
5% FeCl3 reagent
++
Table 2
Results of measurement of the diameter of the zone of inhibition of saga leaf extract
against Enterococcus faecalis bacteria
Treatment
Inhibition zone diameter (mm)
Mean
Standard Deviation
RSD
P 1
P 2
P 3
Positive control
(2% chlorhexidine)
17.50
17.06
15.10
16.55
1.28
7.72
Negative control
(dimethyl sulfoxide 100%)
0.00
0.00
0.00
0.00
0.00
0.00
100% saga leaf extract
9.12
9.69
9.57
9.46
0.30
3.18
50% saga leaf extract
8.08
7.59
7.50
7.72
0.31
4.04
25% saga leaf extract
5.50
4.56
5.06
5.04
0.47
9.33
12.5% saga leaf extract
3.45
3.01
4.09
3.52
0.54
15.44
6.25% saga leaf extract
2.12
2.15
2.19
2.15
0.04
1.63
3.125% saga leaf extract
1.43
1.33
1.08
1.26
0.25
19.72
Abbreviation: RSD, relative standard deviation.
Based on the tables and figures above, it can be inferred that saga leaf extract shows
antimicrobial activity against E. faecalis bacteria characterized by the presence of inhibition zone diameter at 100, 50, 25,
125, 6.25, and 3.125% concentrations of saga leaf extract. The largest inhibition
zone was with 100% concentration with an average diameter of 9.46 mm, and the lowest
inhibition zone was with 3.125% concentration with an average diameter of 1.26 mm.
The level of inhibition shown by saga leaf extract is directly proportional to the
level of concentration where higher extract concentration produces higher inhibition
area.
Discussion
Inferring from the data, saga leaf extract shows an effect in inhibiting E. faecalis bacteria growth. The diameter of the inhibition zone grows from using the smallest
to the largest concentration where the smallest inhibition zone happened at 3.125%
concentration with 1.26 mm, whereas the largest inhibition zone was at 100% concentration
at 9.46 mm diameter. The result can be explained due to higher concentration of saga
leaf extract has more bioactive compounds contained. These bioactive compounds cause
the inhibition zone to appear on the bacterial culture media.
The ability of saga leaf extract to inhibit the growth and kill E. faecalis bacteria is related to bioactive compounds that has antibacterials properties. The
results of qualitative phytochemical tests on saga leaf extract used in this study
showed the presence of phenolic compounds, tannins, saponins, steroids in moderate
amounts; flavonoids in small amounts; and no alkaloid compounds were detected. There
are factors that causes failure to detect alkaloid compounds such as soil type, soil
pH, organic matter content, air temperature, and rainfall in the area where the plant
grows. These factors can cause content in the plants of each region to have different
cadences.[16 ]
[17 ]
Phenolic compounds are said to prevent systemic inflammation by restoring redox balance
by modulating the inflammatory response through mitigating the cytokine pathway to
degrade oxidative stress. However, the actual mechanism of phenolic acid itself in
inhibiting bacteria is not fully understood because it has a complex chemical structure.
In some studies, it is hypothesized that phenolic acids can damage the electrochemical
gradient of the mitochondrial membrane and prevent systemic inflammation.[18 ]
[19 ]
Saponin compounds are able to react with porins (transmembrane proteins) on the outer
membrane of the bacterial cell wall to form strong polymer bonds that causes porin
damage.[20 ] Damage to porins will reduce the permeability of the bacterial cell membrane since
it is the entrance and exit of the compound. It causes bacterial cells to lack nutrients,
inhibited its growth and die.[20 ]
[21 ] This helps the entry of tannin and flavonoid compounds to enter bacterial cells.
Tannins activates reverse transcriptase, the adhesions of the microbial cell, DNA
topoisomerase enzymes that will interfere with bacterial DNA synthesis, and also attack
cell wall polypeptides, causing damage to bacterial cell wall.[22 ] All of this is possible because tannins have a target on the polypeptide wall of
bacterial cells, resulting in incomplete cell wall formation and then bacterial cells
will die.[20 ]
It is reported that steroids act as antibacterial due to lipid membrane correlation
and sensitivity to steroid compounds that show leakage in liposomes. Interaction between
steroids and cell phospholipid membranes that are permeable to lipophilic compounds
causes integrity of the membrane to decrease and changes to the morphology of the
cell membrane, causing bacterial cells to become fragile and lysed.[23 ]
[24 ]
Flavonoids function as antibacterial by inhibiting bacterial growth by forming complex
compounds against extracellular proteins that disrupt the integrity of the bacterial
cell membrane. The compound denatured the bacterial cell proteins and damaged the
cell membrane to prevent reparation process.[25 ] Furthermore, bacterial mobility is also inhibited by flavonoids due to the presence
of flavonoid's hydroxyl groups that cause alterations in organic components and nutrient
transport that lead to harmful effects on bacteria.[26 ]
Abrus precatorius contains the lethal toxin Abrin, a toxalbumin that inhibits protein synthesis leading
to cell death and tissue damage.[3 ] It is necessary to investigate further the toxicity of its active constituents and
side effects.
Abrin is a type-II ribosome-inactivating protein from A. precatorius seeds that is similar in structure and properties to ricin. It is classified as a
Category B bioterrorism warfare agent. Abrin is 75 times more toxic than ricin, and
is classified as a Category B biological warfare agent in the United States due to
its widespread source, ease of preparation, and lethality. Abrin has a molecular weight
of ∼63 kDa, consisting of A chain with N-glycosidase activity and a galactose-specific
lectin B chain. The structure of the abrin B chain consists of lectin that binds to
the β-D-galactoside portion of the cell surface and mediates the internalization/endocytosis
of the entire toxin into the host cell. There are four isoforms of abrin: abrin-a,
abrin-b, abrin-c, and abrin-d. Abrin-a and abrin-d are the most toxic. Abrin-b and
abrin-c show weak B-chain lectin activity.[1 ]
[2 ]
After entering the cell, the A chain can hydrolyze the N-glycosidic bond of the 28S
ribosomal RNA of eukaryotic cells and catalyze depurination, causing ribosome inactivation
and inhibits protein synthesis, therefore causing cell apoptosis. Due to its high
toxicity, consumption by human will cause death due to multiple organ failure. Abrin
is highly toxic, with an estimated fatal dose of 0.1 to 1 µg/kg. It has caused death
after intentional or accidental poisoning. Clinically, abrin is considered an immunotoxin
that can target cancer cells and can be delivered to the tumor site after combining
with antitumor drugs.[1 ]
[2 ]
[3 ]
[4 ]
At the cellular level, abrin inhibits protein synthesis, leading to cell death. Many
of the features observed in abrin poisoning can be shown by abrin-induced endothelial
cell damage, which leads to increased capillary permeability. This results in fluid
and protein leakage and tissue edema (vascular leak syndrome). The most reported cases
of human poisoning involve the consumption of jequirity beans, most of which cause
gastrointestinal toxicity.[2 ]
In previous studies, saga leaf extract (A. precatorius Linn) was shown to have antibacterial activity by inhibiting several bacteria, both
gram-negative and gram-positive bacteria, such as S. mutans bacteria which are included in the gram-positive facultative anaerobic bacteria group.
Saga leaf extract (A. precatorius Linn) was proven to effectively work as an antibacterial at concentrations of 50,
25, 12.5, and 6.25%. It was also proven at 50% concentration of saga leaf extract
(A. precatorius Linn) that the inhibition zone was formed to inhibit Streptococcus mutans bacteria at a diameter of 10.3 mm. Therefore, the results of this research are comparable
to the results of previous studies conducted that shows ethanol extract of saga leaves
(A. precatorius Linn) works effectively as an antibacterial.
Conclusion
Based on the results of the study, it can be concluded that saga leaf extract (A. precatorius Linn) is able to show antimicrobial activity against E. faecalis bacteria characterized by the presence of inhibition zones formed in different concentrations
of saga leaf extract, starting at the smallest concentration of 3.125% to the largest
concentration of 100%. The level of inhibition shown by saga leaf extract is directly
proportional to the level of concentration, which means that the higher the concentration
of the extract, the higher the inhibition produced. The level of inhibition of the
extract is lower than that of CHX solution and is significantly different based on
the statistical tests conducted.
