japtr_313_21_R2_OA

1
Original Article 1
2 2
3
4 AQ2 Mode action prediction of catechin from Uncaria gambir 3
4
5
6 Roxb. against UDP‑N‑acetylenolpyruvyl‑glucosamine 5
6
7
8 reductase (MurB enzyme) of Streptococcus mutans: In 7
8
9
10 silico study 9
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11 11
12 12
Hendra Dian Adhita Dharsono,
13 Lydiawati Wibisono, Abstract 13
14 Ayu Trisna Hayati, Eti Apriyanti1, 14
15 Mieke Hemiawati Satari2, The prevalence of oral health problems in the global population is still high, especially 15
16 Dikdik Kurnia2 dental caries, which is considered a multifactorial disease involving the role of bacteria, 16
17 Departments of Conservative Dentistry
namely Streptococcus mutans. Gram‑positive bacteria metabolize carbohydrates and 17
18 and 2Oral Biology, Faculty of Dentistry, sugars and convert them into lactic acid, causing dental caries. The peptidoglycan (PG) 18
19 Universitas Padjadjaran, 1Department layer at the outer surface of the bacteria acts as protection. MurB enzyme is known for 19
of Chemistry, Faculty of Mathematics
20 and Natural Science, Universitas
its contribution to PG biosynthesis. Gambir (Uncaria gambir Roxb.) is famous for many 20
21 Padjadjaran, Sumedang, Indonesia efficacies. Previous studies show that catechin from herb plants such as U. gambir 21
22 J. Adv. Pharm. Technol. Res.
has antibacterial activity. This study aimed to evaluate and predict the antibacterial 22
23 activity of catechin from U. gambir against the MurB enzyme, which contributes to 23
24 forming the bacteria PG, with an in silico approach. The structure of the MurB enzyme 24
25 was collected from UniProt, and the ligands (catechin and chlorhexidine) structures 25
26 were obtained from PubChem. The AutoDock software was used to dock both ligand 26
27 and MurB enzyme visualized using PyMOL and analyzed using BIOVIA. The results 27
28 showed that catechin has a binding affinity of more than − 7 kcal/mol against the MurB 28
29 enzyme, and chlorhexidine has a higher binding affinity than catechin. Both catechin 29
30 and chlorhexidine have similar amino acids attachment by hydrogen bonds. The results 30
31 showed that catechin has competitive antibacterial activity against chlorhexidine in 31
32 inhibiting the MurB enzyme. 32
33 33
34 Key words: Catechin, dental caries, gambir, MurB enzyme, peptidoglycan 34
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INTRODUCTION
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Dental caries is the most common oral disease and is
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Address for correspondence: experienced by most people in the world. Therefore, it
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becomes a significant public health problem globally.
42 AQ3 Dr. Hendra Dian Adhita Dharsono, 42
Department of Conservative Dentistry, Faculty of Dentistry, According to the Global Burden of Disease Study 2016,
43 43
Universitas Padjadjaran, Sumedang, Indonesia. dental and oral health problems, especially dental caries,
44 E‑mail: adhita@fkg.unpad.ac.id 44
45 45
46 AQ4 Submitted: 27‑Oct‑2021 Revised: ??? This is an open access journal, and articles are distributed under the terms 46
47 Accepted: 10‑May‑2022 Published: ***
of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0
License, which allows others to remix, tweak, and build upon the work
47
48 non‑commercially, as long as appropriate credit is given and the new creations 48
49 Access this article online
are licensed under the identical terms. 49
50 Quick Response Code: For reprints contact: WKHLRPMedknow_reprints@wolterskluwer.com 50
51 Website: 51
52 www.japtr.org How to cite this article: Dharsono HD, Wibisono L, 52
53 Hayati AT, Apriyanti E, Satari MH, Kurnia D. Mode action 53
prediction of catechin from Uncaria gambir Roxb. Against
54 DOI:
UDP‑N‑acetylenolpyruvyl‑glucosamine reductase (MurB enzyme) 54
55 10.4103/japtr.japtr_313_21 of Streptococcus mutans: In silico study. J Adv Pharm Technol Res 55
56 2022;XX:XX-XX. 56

© 2022 Journal of Advanced Pharmaceutical Technology & Research | Published by Wolters Kluwer - Medknow 1
 Dharsono, et al.: Running title missing??? AQ1

1 affects almost half of the world’s population (3.58 billion Chlorhexidine has bacteriostatic properties with low 1
2 people). Gum disease (periodontal) ranks 11th most common concentrations (0.02%–0.06%) and has a high concentration 2
3 disease in the world. While in Asia‑Pacific, oral cancer is of bactericidal properties (≧0.12%).[11] Chlorhexidine is more 3
4 the 3rd most common type of cancer. Dental and oral health effective in killing  (bactericidal) Gram‑positive bacteria 4
5 can reflect the overall health of the body, including if there while having a low bactericidal effect on Gram‑negative 5
6 is a lack of nutrition and symptoms of other diseases in bacteria. Although chlorhexidine provides several benefits, 6
AQ6
7 the body. In particular, severe dental caries leads to tooth some common side effects, for example, causing tooth 7
8 infections which can cause pain and chronic systemic staining, tongue staining, composite and glass‑ionomer 8
9 infections.[1] restoration staining, burning sensation, mucosal irritation, 9
10 and taste disturbances.[10‑13]  Chlorhexidine can provide a 10
11 As a multifactorial disease, dental caries is caused by cytotoxic effect on gingival fibroblasts, periodontal ligament 11
12 interrelated conditions. Notably, there are five factors that cells, and osteoblasts. Karpiński and Szkaradkiewicz[11] 12
13 are showing prominent influence to the formation of caries stated that chlorhexidine could stimulate apoptosis and 13
14 lesions, namely retention and accumulation of plaque, necrotic cell death. 14
15 frequency of carbohydrate intake, frequency of exposure 15
16 to acidic foods, protective factors for pellicle and saliva, as Paul et al.[14] stated that natural sources could be used as an 16
17 well as fluoride and other substances that can control caries alternative to antimicrobials, which helps to reduce side 17
18 development.[2] Streptococcus mutans is a Gram‑positive effects. In combination with natural sources, antimicrobial 18
19 bacteria that has the ability to metabolize carbohydrates and agents can be used as synergically in therapies or as an 19
20 sugars and further convert them into lactic acid (known as auxiliary to delay the development of bacterial resistance.[14] 20
21 lactic acid bacteria), which causes tooth demineralization One of the medicinal plants is gambir (Uncaria gambir Roxb.) 21
22 and leads to the formation of dental caries.[3‑5] The cell which has long been used by locals and used for maintaining 22
23 wall of Gram‑positive bacteria is composed of complex teeth and gum health.[15] The active compound of U. gambir 23
24 AQ5 macromolecules . The macromolecules of which consist of is catechin, either in form of pure catechin or catechol. 24
25 peptidoglycans (PGs), which resembles a sac that surrounds Catechin had been proven in preventing the formation 25
26 the cytoplasmic membrane and other glycopolymers such as of extracellular glucan, which attaches S. mutans to the 26
27 teichoic acid or polysaccharides and proteins.[4] PG consists of tooth surface, while catechol can inhibit the activity of the 27
28 a glycan chain formed from N‑acetylglucosamine (GlcNac) glucosyltransferase enzyme. This enzyme is related to the 28
29 and N‑acetylmuramic acid (MurNAc) with various formation of dental plaque.[16] U. gambir has astringent, 29
30 attached amino acids.[3,4] The initial stage in the cytoplasm antibacterial, and pharmacological properties. [17] U. 30
31 is the formation of N‑acetylglucosamine‑N‑acetylmuramyl gambir, which is used as an antibacterial in mouthwash 31
32 pentapeptide, which is catalyzed by several Mur (MurA–F) preparations, is expected to be able to terminate or inhibit 32
33 enzymes. UDP‑N‑acetylenolpyruvylglucosamine the growth of bacteria that cause dental plaque. The use of 33
34 reductase (MurB) catalyzes the reduction of enolpyruvate gambir as a mouthwash preparation is an effort to explore 34
35 to D‑lactate to produce UDP‑N‑acetylmuramate, which has the benefits of U. gambir.[15,17] This research aimed to observe 35
36 a role in the formation of PG.[3] the interaction of the active compound catechin from U. 36
37 gambir against UDP‑N‑acetylenol pyruvylglucosamine 37
38 Clinically, one of the treatment modes carried out to reductase (MurB enzyme) which plays role in the formation 38
39 prevent and treat caries is the Caries Management by Risk of PG in S. mutans through the in silico approach using a 39
40 Assessment (CAMBRA) approach, introduced by Young molecular docking modeling. 40
41 and Featherstone.[6] CAMBRA is a tool used to assess the risk 41
42 factors of dental caries for diagnosis, preventive measures, MATERIALS AND METHODS 42
43 and dental care management. Caries risk assessment is 43
44 carried out through history taking, bacterial tests, and Materials for in silico analysis 44
45 caries preventive planning through several measures, such UDP‑N‑acetylenolpyruvylglucosamine reductase (MurB 45
46 as fluoride application, chlorhexidine mouthwash, dental enzyme) as the protein with code P08373 was obtained 46
47 sealants, and use of xylitol.[6] from UniProt (https://www.uniprot.org/). The ligands of U. 47
48 gambir content were catechin and chlorhexidine, determined 48
49 Chlorhexidine has been long used as an antiseptic and is as control ligands, obtained from PubChem (https://www. 49
50 commonly used as a mouthwash in the field of dentistry.[7,8] pubchem.ncbi.nlm.nih.gov/). 50
51 Chlorhexidine‑contained mouthwash has long been used 51
52 and recognized as a regimen to clean teeth mechanically.[9,10] In silico of Uncaria gambir Roxb compounds 52
53 Chlorhexidine gluconate with a concentration of 0.12% is The chemical structure of catechin and chlorhexidine was 53
54 considered to have the highest efficacy as a mouthwash in obtained from PubChem using the three‑dimensional (3D) 54
55 preventing dental plaque formation and has become the gold structure with Open Babel 3.1.1 Protein Data Bank (PDB) 55
56 standard of antimicrobial mouthwash against S. mutans.[10,11] format program. UDP‑N‑acetylenolpyruvyl glucosamine 56

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1 reductase (MurB enzyme) structure was obtained from hydrogen donor and receiver are in one molecule, and they 1
2 AQ7 UniProt with the PDB format. The AutoDock version 1.5.6 are both closely spaced between molecules. Atoms that act as 2
3 software removed the H2O groups and natural ligands hydrogen acceptors are electronegative atoms of a molecule 3
4 in the UDP‑N‑acetyl enol pyruvylglucosamine reductase or ion that consist of lone pairs of electrons that play a role in 4
5 protein (MurB enzyme), then saved it in PDB format. hydrogen bonds. Due to their electronegative solid charge, 5
6 The docking results were visualized using PyMOL and hydrogen donors (N, O, and F) attract the covalently bonded 6
7 then analyzed using BIOVIA, which showed the docking electron pair closer to the nucleus of the molecule and away 7
8 position and ligand–residue interactions in 3D molecules. from the hydrogen atom. The donors to hydrogen bonds are 8
9 often powerfully and electronegatively charged atoms such 9
10 RESULTS as N, O, or F, covalently bonded with hydrogen bonds. The 10
11 hydrogen atom is allowed to have a partially positive charge 11
12 The binding affinity of ligands against the MurB enzyme which forms an attraction between the hydrogen atoms and 12
13 was determined based on the docking results between both binds to the donor and the lone pair acceptor. The result of this 13
14 ligands against the MurB enzyme, and it was observed interaction forms hydrogen bonds.[18] 14
15 that the binding affinity of catechin was lower than 15
16 chlorhexidine. The binding affinity of each ligand to the An intermolecular hydrogen bond is a bond that occurs 16
17 MurB enzyme is presented in Table 1. The two ligands between separate molecules. This bond occurs since there 17
18 have different interaction positions with the MurB enzyme, is an interaction between the hydrogen donor and the 18
19 as shown in Figure 1. Details of amino acid interactions hydrogen acceptor. However, hydrogen bonding cannot 19
20 between ligands (catechin and chlorhexidine) and the MurB occur without a significant electronegative difference 20
21 enzyme are shown in Table 2 and Figure 2. between hydrogen and bonding atoms.[18] The present 21
22 study showed that catechin and chlorhexidine ligands form 22
23 DISCUSSION amino acid attachments with the same hydrogen bond, 23
24 Ser50, Asn226, Asn51, and Ser229. The attachment of amino 24
25 Based on data analysis, this research discovered that acids with the same hydrogen bonds between catechins and 25
26 catechin has a binding affinity of more than  −  7 kcal/ chlorhexidine indicates that catechins have antibacterial 26
27 mol against the MurB enzyme, while chlorhexidine has a activity as an inhibitor of the MurB enzyme with a binding 27
28 higher binding affinity than catechin. Chlorhexidine as a affinity of more than  −  7 kcal/mol, which is predicted to 28
29 control ligand had the highest binding affinity of − 9.6 kcal/ have an antibacterial activity or similar mechanism as 29
30 chlorhexidine and is competitive against chlorhexidine in 30
mol on the MurB enzyme. However, the position of each
31 31
ligand (catechin and chlorhexidine) on the MurB enzyme
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was not the same, with different amino acid interactions
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been shown in Figure 1 and Table 2.
34 34
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Hydrogen bonds are formed by an attractive intermolecular force
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when hydrogen atoms are strongly bonded to electronegative
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atoms. Hydrogen bonds consist of intramolecular and
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intermolecular hydrogen bonds.[18,19] Intramolecular hydrogen
39 A B C 39
bonds are bonds that are formed in a single molecule. The
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41 41
42 Table  1: The binding affinity of catechin and 42
43 chlorhexidine against the MurB enzyme 43
44 The binding affinity of ligand against the MurB 44
45 enzyme 45
46 Position Catechin Position Chlorhexidine 46
47 in mode in mode 47
D E F
48 0 −8.5 0 −9.6 48
49 1 −8.3 1 −9.5 49
50 2 −8.1 2 −8.9 50
51 3 −8 3 −8.9 51
52 4 −7.7 4 −8.8 52
53 5 −7.7 5 −8.7 53
54 6 −7.5 6 −8.7 G H I 54
55 7 −7.3 7 −8.6
Figure 1: Positions of catechin (green chain) and chlorhexidine (purple 55
AQ10
56 8 −7.2 8 −8.6 chain) against MurB enzyme in nine positions or modes 56

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1 Table  2: Interaction of ligand‑MurB enzyme 1

2 Attachment of amino acids by hydrogen bonds 2
3 Position in mode Catechin Position in mode Chlorhexidine 3
4 0 Ser116, Ser50, Asn51 0 Pro111, Ser50 4
5 1 Ser116, Arg327, Asn226 1 Ser229, Asn226, Pro219 5
6 2 Ser50, Asn51, Arg327 2 Ser116, Ile45, Asn51 6
7 3 Ser229, Gly123 3 Ser229, Asn226, Pro219
7
8 4 Asp169 4 Gly123, Asn226, Pro219
8
9 5 Arg327, Asp169, Ile45 5 Asn226, Pro219, Pro111, Ser50
9
10 6 Asp220, Asn226, Ser229 6 Val43, Phe171, Gln168, Phe163
10
11 7 Asn224, Pro221, Asn9, Asn294 7 Ser50, Pro219
11
12 8 Ser50, Arg159, Ser229 8 Gly123, Ser50
12
13 13
14 14
15 15
16 16
17 17
18 18
19 19
20 20
21 21
22 22
23 a b 23
24 Figure 2: Ligand‑MurB enzyme interaction: (a) catechin; (b) chlorhexidine 24
25 25
26 inhibiting the MurB enzyme. Catechin can be used as an from U. gambir has antibacterial by inhibiting the MurB 26
27 alternative to the MurB enzyme inhibitor. enzyme. This research found that U. gambir has similar 27
28 amino acid attachments with a similar hydrogen bond to 28
29 Catechin is a complex flavonoid compound from the chlorhexidine, the gold standard for the antibacterial agent. 29
30 polyphenol group with antioxidant and antibacterial However, further analysis is still needed to clarify and find 30
31 properties.[20] Previous research discovered that phenols could the activity of compounds using in vitro methods for clinical 31
32 change cell membrane permeability, changes in an intracellular implementation. 32
33 function triggered by hydrogen bonds of phenol compounds 33
34 to enzymes, and changes in rigidity of cell wall with loss Financial support and sponsorship 34
35 Nil. 35
of cell wall integrity due to the interaction of phenol with
36 36
cell membranes.[21] The docking results found in the current
37 Conflicts of interest 37
research indicated that catechins are competitive inhibitors
38 There are no conflicts of interest. 38
of the MurB enzyme and thus can be used as an alternative
39 39
material with antibacterial properties with a binding affinity
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28 Author Queries??? 28
29 AQ1: Kindly provide the running title. 29
30 AQ2: Kindly check the article title provided in new system “Mode Action Prediction of Catechin from Uncaria gambir 30
31 Roxb. Against UDP‑N‑Acetylenolpyruvoyl‑Glucosamine Reductase (MurB enzyme) of Streptococcus mutans: In 31
32 Silico Study” Please confirm anyone article title. 32
33 AQ3: Kindly confirm the correspondence author name, details and E‑mail id were taken from the xml. 33
34 AQ4: Kindly provide the revised date. 34
35 AQ5: Kindly review the sentence. 35
36 AQ6: Kindly review the text part as it seems incomplete. 36
37 AQ7: Kindly provide complete manufacturer details such as company name, city, state and country name. 37
38 AQ8: Please provide publisher name. 38
39 AQ9: Kindly provide author initial. 39
40 AQ10: Kindly check the panel 0‑8 changed as A‑I and also provide panel A‑J caption for Figure 1. 40
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