INTRODUCTION:

Root canal treatment is a treatment that aims to maintain teeth in the oral cavity, that has suffered damage to the pulp tissue and periapical tissue. Root canal treatment includes biomechanical preparation, microbial control, and obturation of the root canal. The main goal is to eliminate bacteria and microorganisms in the infected root canal, heal the periapical tissue and prevent the tooth from future infections. Root canal treatment is not an easy procedure but a complex procedure that requires care and dedication to avoid root canal failure.^1,2

Setzer's study on 50 teeth that had root canal treatment and were evaluated after 5 years. There was a failure percentage of 12%, which is 8% were re-treated and 4% were extracted. Hargreaves et.al study on 44,613 teeth that underwent root canal treatment, with an evaluation after 3.5 years. The result of treatment failure was 5.6%. Chen et al conducted a study on 1,557,547 teeth that had been treated with root canals and evaluated after 5 years and the treatment failure was 7.1%. The failure of root canal treatment in several studies has varying values. Failure of root canal treatment is generally caused by poor restoration, improper root canal filling, missed canal, and resistance of microorganisms in the root canal.^3-5

One of the factors that play a role in causing the persistence of apical periodontitis lesions is the presence of microorganisms in the root canal.⁶Periodontitis is an inflammatory response to microorganisms that grow excessively and uncontrollably in the subgingival area.⁷Enterococcus faecalis is the main bacteria that cause root canal treatment failure because it has the ability to survive in extreme conditions in the root canal and has the ability to form biofilms. E. faecalis areable to survive in an environment with a high pH and low nutrients for a long time. E. faecalis often found in teeth treated with root canals and reinfection with prevalence values ranging from 30% to 90% of cases.^8-12

E. faecalis are normal commensal flora commonly found in the oral cavity, gastrointestinal tract, and genital organs. The cell wall of E. faecalis composed of three main components, namely peptidoglycan (PG), teichoic acid (TA), and polysaccharides (PSs). Peptidoglycan consists of 5-30 sub-units consisting of N-acetylglucosamine (NAG) and N-acetylmuramate (NAM) and makes up 40% of the bacterial cell wall while the rest is composed of teichoic acid and polysaccharides.^13,14

Cell walls of most bacteria are covered with peptidoglycans, or mureins, forming a mesh-like layer outlining the plasma membrane. Peptidoglycan functions to determine the shape of the bacterial cell wall, integrity, and structural strength of bacteria, as a protection against threats to bacteria and to resist cell rupture caused by high cytoplasmic osmotic pressure. The biosynthesis of peptidoglycan is assisted by several types of enzymes, one of which is the enzyme UDP-N-acetylenolpyruvyl-glucosamine reductase, or known as the enzyme Mur B.^15-16

The biosynthesis of peptidoglycan begins with the formation of enolpyruvyl-UDP-N-acetylglucosamine (EP-UDPGlcNAc) from phosphoenolpyruvate and UDP-N-acetylglucosamine via the transfer of the enol pyruvate to the C3 position of UDP-N-acetylglucosamine by the enzyme UDP-N-acetylglucosamine-1-carboxy vinyl transferase (Mur A). Furthermore, the enzyme UDP-N-acetylenolpyruvyl-glucosamine reductase (Mur B) catalyzes the reduction of the vinyl ether double bond of the enolpyruvate moiety to produce the D-lactyl ether moiety of UDP-N-acetylmuramic acid (UDPMurNAc). This lactyl ether serves as a link between peptides and glycans. The enzymes (MurC, MurD, MurE, and MurF) catalyze the gradual addition of a pentapeptide side chain to the newly reduced D-lactyl group, resulting in the formation of UDP-N-acetylmuramyl pentapeptide. Through the following mechanism, peptidoglycan is formed.^17-19

Microbial control in endodontic treatment is generally carried out by root canal irrigation and placement of intracanal medicament. Root canal irrigation can remove necrotic tissue and smear layer from the canal as well as remove microorganisms in the infected canal. For more than 40 years chlorhexidine has been the gold standard as an antimicrobial agent in dental practice. Chlorhexidine in endodontics is an irrigating agent that is bactericidal and effective against Gram-positive and Gram-negative bacteria, facultative anaerobes, and obligate anaerobes (including E. faecalis, yeasts, and fungi). Chlorhexidine in liquid form is capable of killing microorganisms within 30 seconds, while chlorhexidine in gel form takes 22 seconds (2% concentration) to 2 hours (0.2% concentration). One mechanism that can explain the effectiveness of chlorhexidine is the interaction between the positive charge of the chlorhexidine molecule and the negative charge of the phosphate group on the bacterial cell wall, which allows the chlorhexidine molecule to penetrate the bacteria with a toxic effect.^20-22

Chlorhexidine as an irrigation solution in root canal treatment also has drawbacks in terms of its toxicity which can cause damage to human cells. Chlorhexidine has toxicity to periapical tissue. The degradation of chlorhexidine produces para-chloroaniline and free radicals that are harmful to vital tissues. Chlorhexidine inhibits protein synthesis in periodontal ligament (PDL) cells and inhibits mitochondrial activity in human periodontal ligament (PDL) cells, thereby triggering inflammation and impairing tissue repair. However, the use of antibiotics is limited because some bacteria have a drug-resistant effect, thereby reducing the effectiveness of antibiotics.²⁴ Chlorhexidine has also been reported to have a resistant effect on Staphylococci and Pseudomonas stutzeri bacteria. To reduce or even eliminate the detrimental effects of currently existing root canal irrigation materials, it is necessary to find safer alternative irrigation materials such as using botanical materials.^25-28

Secondary metabolites such as flavonoids are known to contain several biological activities such as anti-viral, antimicrobial, anti-carcinogenic, and anti-inflammatory.^29,30Gambir extract contains one which is tannin with a content of about 20-50%. Tannins are polyphenolic biomolecules that can interact and form macromolecules such as proteins, gelatin, polysaccharides, and alkaloids. Tannin compounds are found in leaves, flowers, seeds, roots, and the bark of trees. Carvalho's study stated that tannins were effective in inhibiting cocci, with MICs of 250g·mL-1 in Staphylococcus epidermidis and 500g·mL-1 in Staphylococcus aureus and Streptococcus agalactiae.^31-33

The antimicrobial mechanism of tannins is also obtained through: i. The astringent properties of tannins can induce complexation with enzymes and substrates so that many microbial enzymes are inhibited when they bind to tannins such as cellulases, pectinases, xylanases, peroxidases, lactases, or glycosyltransferases. Tannins also have the ability to block essential microbial metabolic enzymes such as proteolytic maceration enzymes ii. The toxicity of tannins is related to their action on the cell membranes of microorganisms. iii. Complexation of metal ions by tannins so that bacterial growth is disrupted. Bacteria such as Bacteroides fragilis, Clostridium perfringens, Escherichia coli, and Enterobacter cloacae require iron ions as a cofactor in their metabolism, iron ions are also needed for the reduction of ribonucleotide precursors from DNA, formation of haem and several other important functions of bacteria to survive in an anaerobic environment. Tannins have the ability to chelate macronutrient minerals such as iron ions, making iron ions unavailable to microorganisms.^34-36

Research in the discovery of new antibiotics began to be introduced with the in silico method. A Recent study on the mode action prediction of phenolic compound isolated from U. gambir had been shown to have similarities in amino acid attachments and hydrogen bonds as chlorhexidine against MurB enzyme.³⁷In silico is a method of approaching a real condition or situation in a computer simulation using a certain program. The in-silico method is widely used in research that searches for new drugs. The in-silico method promises that the identification of new compounds can be done faster and at a lower cost. This in silico method covers a broad field, including: (i) docking studies, computationally studying a ligand or drug during binding to a specific target protein;^38,39 (ii) chemistry, linking activity and structure using statistical means; or (iii) bioinformatics, by deriving drug targets from genomic data. However, in clinical implementation, in vitro tests are also needed to confirm the discovery of a compound's activity.⁴⁰

Tannins are known to have antibacterial effects, so this study describes the in-silico study to see the activity and interaction of tannins with the UDP-N-acetylenolpyruvoyl-glucosamine reductase (MurB enzyme) by simulating molecular docking between tannin compounds and the MurB enzyme.

MATERIALS AND METHOD:

Materials for In silico:

UDP-N-acetylenolpyruvoyl-glucosamine reductase (MurB enzyme)as the protein with code P08373 that was obtained from https://www.uniprot.org/. Tannin and chlorhexidine as control ligands were obtained from https://www.pubchem.ncbi.nlm.nih.gov/. with Tannin compound code CID: 108065 and Chlorhexidine compound code CID: 9552079.

Methods:

Preparation of the structure of the tannin and chlorhexidine compounds was carried out by changing the data format from the SDF format to the PDB format on the Tannin and chlorhexidine compounds downloaded from https://www.pubchem.ncbi.nlm.nih.gov/. Compound data format changes were performed using OPEN BABEL GUI software with a 3D compound structure.

The structure of Muramidase B (MurB) enzyme was downloaded from https://www.uniprot.org/ with code P08373 which is in the form of UDP-N-acetylenolpyruvoyl glucosamine reductase (MurB) protein and prepared using AutoDockTools-1.5.6 software to remove H₂0 groups and natural ligand, then save in PDB format. Docking is done by directing the molecular model of the ligand (tannin compound, chlorhexidine) to the active site of the target protein (MurB) with a maximized binding area. Then the calculation of the binding between the ligand and the target protein at various poses will appear as a scoring value through the PyRx software.⁴¹Visualization of the bond between receptor and ligand molecules using PYMOL tools. Furthermore, to determine the docking position and the interactions formed, an analysis was carried out with BIOVIA.

RESULTS:

Binding affinity of ligands against MurBEnzyme:

The results showed that tannins have a lower binding affinity than chlorhexidine which can be seen in Table 1. Tannins and chlorhexidine have different binding positions as shown in Figures 1 and 2. The two ligands interact with amino acids in the MurB enzyme which are presented in Table 2.

Table 1: Binding affinity of tannin and chlorhexidine against MurB enzyme

Binding affinity of ligand against MurB Enzyme
Position	Tannin	Position	Chlorhexidine
Mode 0	-8.2	Mode 0	-10.6
Mode 1	-8.0	Mode 1	-10.6
Mode 2	-8.0	Mode 2	-10.0
Mode 3	-7.9	Mode 3	-9.6
Mode 4	-7.9	Mode 4	-9.4
Mode 5	-7.7	Mode 5	-9.3
Mode 6	-7.6	Mode 6	-9.2
Mode 7	-7.5	Mode 7	-9.1
Mode 8	-7.4	Mode 8	-9.1

Mode 0 (A) Mode 0 (B)

Figure 1: Position of the ligand to the MurB enzyme, mode 0-1, (A) tannins, (B) chlorhexidine

Mode 2(A) Mode 2(B) Mode 3(A) Mode 3(B)

Mode 4(A) Mode 4(B) Mode 5(A) Mode 5(B)

Mode 6(A) Mode 6(B) Mode 7(A) Mode 7(B)

Mode 8(A) Mode 8(B)

Figure 2: Position and hydrogen bonding of the ligand to the MurB enzyme, mode 2-8, (A) tannins, (B) chlorhexidine

8=Tabel 2. Interaction of ligand - MurB enzyme

Attachment of amino acids by hydrogen bonds
Position	Tannin	Position	Chlorhexidine
Mode 0	PRO 324, SER 336, ALA 337, GLU 334	Mode 0	ILE 45, GLN 120, PRO 111, SER 50, ALA 227, ASN 51
Mode 1	ASN 233, GLN 288, LYS 217, TYR 190, ARG 214	Mode 1	ILE 45, PRO 111, SER 50, ASN 51, ALA 227
Mode 2	VAL 335, ALA 337, GLN 168	Mode 2	PRO 111, SER 50, ALA 227, ASN 226, PRO 219, LEU 218
Mode 3	GLU 48, PRO 7, LYS 222	Mode 3	ARG 159, SER 50, SER 116, ILE 45, PRO 111
Mode 4	LYS 262, ASN 233, PRO 255, GLN 256	Mode 4	SER 116, ILE 45, GLU 325
Mode 5	PRO 255, GLN 256, THR 189, GLU 128, LYS 262, ARG 159, GLY 123	Mode 5	SER 229, PRO 111, SER 50
Mode 6	GLU 128	Mode 6	GLY 123, SER 50, PRO 111
Mode 7	ARG 159, THR 189, LYS 262	Mode 7	ASN 233, SER 229, ASN 226, PRO 219
Mode 8	PRO 7	Mode 8	ILE 45, SER 116, ASN 51, SER 50, PRO 111, GLU 325

Figure 3. Ligand-MurB enzyme interaction mode 0: (a) tannin, (b) chlorhexidine

DISCUSSION:

Molecular docking is a molecular modeling method that can predict intermolecular bond interactions to form stable complexes.^42-44In molecular docking two stages must be carried out, namely autogrid and autdock. Autogrids are used to determine the area of the protein that will interact with the ligand. While AutoDock is used to calculate the interaction of amino acids at the active site with ligand molecules.⁴⁵The result of molecular docking is the binding affinity and hydrogen bonds formed. Binding affinity is used to indicate the strength of the bonds between compounds and proteins. The smaller (minus) the value of the binding affinity, the stronger and more stable the bonds formed. The hydrogen bonds formed are used to analyze the mechanism of interaction that occurs between compounds and proteins. Based on the results of the study, the binding affinity value of tannins was -8.2 in mode 0 and -7.4 in mode 8. This binding affinity value was lower than chlorhexidine, which was -10.6 in mode 0 and -9.1 in mode 8, which can be seen in Table 1. Although the binding affinity value of tannins is lower than that of chlorhexidine, tannins still have strong and stable bonds because they have a negative binding affinity value of -7.4 to -8.2.

Based on the results of docking in mode 0 to mode 8, tannins have hydrogen bonds with the MurB enzyme on 21 amino acids: PRO 324, SER 336, ALA 337, GLU 334, ASN 233, GLN 288, LYS 217, TYR 190, ARG 214, VAL 335, GLN 168, GLU 48, PRO 7, LYS 222, PRO 255, GLN 256, THR 189, ARG 159, GLY 123, GLU 128, LYS 262.Chlorhexidine in mode 0 to mode 8 has a hydrogen bond with MurB enzyme at 15 amino acids: ILE 45, GLN 120, PRO 111, SER 50, ALA 227, ASN 51, ASN 226, PRO 219, LEU 218, ARG 159, SER 116, GLU 325, SER 229, GLY 123, ASN 233, which can be seen in Table 2.

Analyzing through the hydrogen bonds formed, the amino acids in the MurB enzyme that binds to tannins are more than those in the chlorhexidine bond with the MurB enzyme because the position of the tannin bonds to the MurB enzyme from mode 0 to mode 8 has more varied locations. This can be seen in Figures 1 and 2. The position of the tannin bond from mode 0 to mode 8 is always on the outside of the MurB enzyme surface compared to the chlorhexidine binding to the MurB enzyme which is on the inside of the MurB enzyme with almost the same position in several bonding modes.

One of the factors that affect the biological activity of compounds is the hydrogen bonds formed to amino acids in enzymes. Tannins and chlorhexidine have the same hydrogen bonds at the active site of the MurB enzyme, namely the amino acids ARG 159, GLY 123, and ASN 233. This shows that tannins have activity against the MurB enzyme as well as chlorhexidine, although the binding affinity value of tannins to the MurB enzyme is lower than chlorhexidine.

Chlorhexidine has a binding affinity of more than -9,1 kcal.mol^-1 as a control ligand. Chlorhexidine has been widely used in endodontic treatment because chlorhexidine has an inhibitory effect on bacteria both gram-positive and gram-negative bacteria. Chlorhexidine can enter the bacterial cell wall through the positive charge on chlorhexidine and the negative charge on the phosphate complex in the bacterial cell wall. This gives a good toxic effect on chlorhexidine. The effectiveness of chlorhexidine has been demonstrated against the bacteria Enterococcus faecalis, Actinomyces israelii, Streptococcus mutans, Staphylococcus aureus, Porphyromonasendodontalis, Porphyromonas gingivalis and Prevotella intermedia and Candida albicans.

Tannins are able to synergize with other antimicrobial ingredients and their synergism often has a greater effect. Tannins directly attack cellular organelles and in cell membranes of various microorganisms, and inhibit their growth.^46,47Other studies have shown that tannins have great potential for binding proteins and adhesins, interfering directly with the availability of media essential for bacterial metabolism and growth.⁴⁰Tannins are oligomers and polymers capable of making tannin-protein complexes and/or polysaccharides in bacterial cell membranes. The formation of these complexes causes a reduction in protein solubility, increases the aggregation and deposition of proteins present on the membrane, and inhibits the action of enzymes from microbes, thereby removing important substrates for microbial growth causing plasma membrane lysis.^40,47The complexation effect of tannins with proteins and other compounds results in inhibition of bacterial extracellular enzymes and loss of substrate availability for bacterial metabolism, which will lead to the inactivation of microorganisms and cause the death of microorganisms.^48-50

CONCLUSIONS:

Tannins can be recommended as a competitor material that can replace chlorhexidine as an antibacterial agent for root canal irrigation. Compounds obtained from medicinal plants (medicinal plants) Uncaria gambir (Roxb), which are effective against microorganisms resistant to conventional drugs. They can minimize side effects and are more affordable. This research found that tannin has the same amino acid attachments with the same hydrogen bond compared with chlorhexidine which is the gold standard for the antibacterial agent. However, further studies are still needed to clarify and discover the activity of the tannin compound through in vitro studies so it can be implemented clinically.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 05.10.2022 Modified on 01.03.2023

Research J. Pharm. and Tech 2023; 16(10):4568-4574.

DOI: 10.52711/0974-360X.2023.00744