Antibacterial efficacy and molecular docking analysis of Zingiber offinale and Allium sativum against Fusobacterium nucleatum - An in vitro study

Abstract

Background:

Fusobacterium nucleatum is a keystone organism associated with secondary endodontic infections. Numerous virulence traits are displayed by this bacterium, which coaggregates with other species and is speculated to function as a “supportive bridge” between primary and secondary invaders in the formation of root canal biofilms.

Aim and Objectives:

To evaluate the antimicrobial efficacy of Zingiber officinale (ZO) (Ginger) and Allium sativum (AS) (Garlic) against F. nucleatum.

Materials and Methods:

Extracts were prepared by powdering freshly dried ginger and garlic, and the extraction process was done using a Soxhlet apparatus. Broth microdilution assay and microtiter plate assay of the ginger and garlic extracts were done for evaluating antibacterial activity. Gas chromatography-mass spectrometry analysis was done to identify phytochemical constituents present in extracts responsible for antibacterial action. Molecular docking was done to evaluate the interaction between phytoconstituents and the target protein (NanA) of F. nucleatum.

Statistical Analysis:

Statistical analysis was done by IBM SPSS version 30.0. One-way ANOVA test was used for intergroup comparison.

Results:

The minimum inhibitory concentration was determined to be 0.9 μg/mL for ZO and AS extracts. Both Ginger and Garlic extract showed a dose-dependent activity on F. nucleatum growth, with the highest inhibition at 500 μg/mL. Docking showed N-amyl isovalerate in ginger and furaneol in garlic exhibited the strongest binding affinity with the target protein NanA.

Conclusion:

Garlic and ginger extracts both showed better antibacterial activity against F. nucleatum than 3% sodium hypochlorite, indicating that they could potentially utilized as a natural alternative irrigant.

INTRODUCTION

The elimination of microorganism from the root canal system and preventing the risk of re-infection are essential for the success and outcome of endodontic treatment.[1] A bacteria-free root canal is difficult to obtain because of anatomical complications, organic debris, and microbes that reside deep within the dentinal tubules. Bacteria in the root canal can be found as free-floating planktonic cells or in complex biofilms.[2,3] Currently, mechanical debridement and chemical disinfection are used to treat endodontic infections. However, present disinfection methods have few downsides. As a result, the necessity for novel alternatives has been extensively researched.[4,5]

A recent approach is the use of phytochemicals, which are plant-based antibacterial agents that are being extensively explored. These phytochemicals are gaining popularity due to their ease of availability, lack of antimicrobial resistance development, and other therapeutic qualities. The primary mode of action of these phytochemicals ranges from blockage of efflux pumps in bacteria to suppression of bacterial cell adhesion and motility.[6,7] Zingiber officinale, i.e., ginger belongs to the Zingiberaceae family, and its primary antibacterial properties may be ascribed to the presence of phenolic compounds called gingerol and shogaol.[8] On the other hand, the bulbous perennial herb Allium Sativum (AS), i.e., garlic is a member of the Alliaceae family. The main antimicrobial action is due to the presence of allicin, ajoene, and various aliphatic sulfides in garlic.[9]

In recent years, substantial study has determined that Fusobacterium nucleatum is the key bacteria responsible for secondary and persistent endodontic infections. This organism exhibits a lot of virulence characteristics and is regarded a “supportive bridge” between primary and secondary invaders in root canal biofilm formation as it coaggregates with multiple species.[10] There is limited evidence showing that the extracts of ginger and garlic have antibacterial activity against F. nucleatum. In addition, molecular docking analysis was done to evaluate the interplay between phytochemical component and protein in F. nucleatum. Molecular docking, a “molecular modeling technique,” is utilized to analyze how a protein in bacteria will interact with small molecules or ligands in herbal extracts. The binding energy and interaction characterization of several phytocompounds in plant extracts with bacterial proteins using molecular docking gives important insights into their potential as antibacterial agents.[11] Therefore, this study aimed to investigate the antimicrobial efficacy and molecular docking analysis of ginger and garlic extract against F. nucleatum.

MATERIALS AND METHODS

Ethical clearance

Before the commencement, the study received ethical approval from the Institutional Ethical Committee in accordance with national and institutional ethical norms (IHEC-CDCRI/2024/FAC-0042).

Preparation of ginger and garlic extract

Ginger and Garlic (Zingiber officinale [ZO] and Allium sativum [AS]) were obtained from Organic India Ltd., India. Freshly dried ginger and garlic were coarsely powdered with an electric grinder. The coarse powder was then exhaustively extracted in a Soxhlet apparatus.[12] In this extraction procedure, 500 mL of ethanol was used to extract 60 g of precisely weighed powdered substances. Ethanol was vaporized using a rota evaporator, and the extracts were dried under partial vacuum at 80°C, leaving behind a thick, semi-solid residue. The extracts were kept in a refrigerator until it could be used after being dissolved in 2 mL of 10% dimethyl sulfoxide to achieve an equivalent concentration.

Microbiological assays

Minimum inhibitory concentration

Nutrient broth and F. nucleatum (ATTC-23726) were acquired from HiMedia, India. The minimum inhibitory concentration (MIC) for F. nucleatum susceptibility was evaluated using the broth microdilution technique in sterile, disposable, flat-bottomed 96-well microtiter plates. Group I - saline, Group II - 3% NaOCl, Group III - Zingiber officinalis, and Group IV - Allium sativum. Briefly, 0.5 McFarland inoculum suspensions were further diluted in nutrient broth at a ratio of 1:100. In a 96-well plate with 50 μL of test sample (garlic extracts and ginger extract) at serial concentrations, 50 μL of the bacterial suspension was added. Approximately 5 × 10⁵ CFU/mL was the final bacterial inoculum.

The extracts of garlic and ginger had final concentrations of 500, 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.9, and 0.9 μg/mL. The plates were then incubated for 18–24 h at 37°C. Using a microplate reader, the results were read at 600 nm. Variations in color were noted and documented. All the tests were done in triplicates.

Microtiter plate assay

The Microtiter plate assay (MTP) assay, as described by Christensen et al.,[13] was used to evaluate a drug’s effectiveness in preventing biofilm growth by using 96 well-flat bottom polystyrene titer plates. 180 μL of BHI broth was added to each well, 10 μL of an overnight bacterial culture was added as an inoculant. This was incubated for 24 h at 37°C, after which 10 μL of test agents (Ginger and Garlic extracts) were added from the generated stock solution of 500, 250, 125, 62, 5, 31.25, 15.6, 7.8, 3.9, 1.9, and 0.9 μg/mL, respectively. After the incubation period, free-floating bacteria were removed by washing with 0.2 mL of phosphate buffer saline at pH 7.2. Crystal violet (0.1%, w/v) was used to stain the adhesion of sessile bacteria after it had been fixed with sodium acetate (2%). The excess stain was washed with deionized water and allowed to dry. Further, dried plates were cleaned with 95% ethanol, and optical density (OD) was measured at 600 nm using a microtiter plate reader. The percentage of biofilm inhibition was determined using the given formula. The assay was done in triplicates.

Molecular docking

Gas chromatography-mass spectrometry analysis of the extracts

The Gas chromatography-mass spectrometry (GC-MS) analysis[14] (GC-MS-QP2010SE, Shimadzu) of ethanol extracts of Zingiber officinalis and Allium sativum recorded a total of 20 peaks for Zingiber officinalis and 14 peaks for Allium sativum corresponding to the compounds that were recognized by relating their peak retention time, peak area, height, and mass spectral fragmentation patterns.

Selection of target protein in Fusobacterium nucleatum

The NanA gene encodes N-acetylneuraminate lyase (NanA),[15] a critical enzyme in F. nucleatum’s sialic acid metabolic pathway, and was selected as the target protein for the present study. Sialic acids are nine-carbon sugars that have a role in bacterial adhesion, colonization, and immune evasion. Inhibiting NanA function impairs sialic acid metabolism, reducing bacterial growth and pathogenicity. The protein data bank (PDB) format of the target protein (3D structure) was retrieved from Research Collaboratory for Structural Bioinformatics PDB (PDB id: 5ZKA). AutoDock 4.2.6 (Scripps Research Institute, United States) software was used to convert the target protein from PDB to PDBQT format. The optimized target protein was subjected to the molecular docking study.

Preparation of ligands (phytocompounds)

For docking purpose, 20 phytocompounds of ginger extract and 14 phytocompounds of garlic extract have been collected from the PubChem database in SDF format and were later converted from SDF to PDB format using BIOVIA Discovery Studio. Preparation of the ligand was performed using AutoDock 4.2.6 software. AutoDock 4.2.6 software was used for converting PDB format of all phytocompounds (both ginger and garlic) to PDBQT format.[16]

Molecular docking

Bacterial target protein (NanA) was successfully docked with a total number of 34 reported compounds, i.e., 20 phytocompounds of Ginger and 14 phytocompounds of garlic extracts using AutoDock vina software. BIOVIA Discovery Studio was used to analyse the docked complex’s topmost conformation.[16]

Statistical analysis

Descriptive and Inferential statistics were analyzed by IBM SPSS version 30.0 (IBM Corp. Released 2024. IBM SPSS Statistics for Windows, Version 30.0. Armonk, NY, USA: IBM Corp). Mean and SD were used to summarize the OD values. One-way ANOVA was used for intergroup comparison of OD values between four groups. P <0.05 was considered as statistically significant difference.

RESULTS

Minimum inhibitory concentration

The MIC was determined to be 0.9 μg/mL for ZO and AS extracts. However, the MIC-50 was found to be 77.18 μg/mL and 24.94 μg/mL for ZO and AS extracts.

Microtiter plate assay

The OD values, which indicate bacterial growth inhibition, were compared among groups treated with Saline (control), Sodium Hypochlorite (NaOCl), ZO, and AS across a dilution range from 500 μg/ml to 0.9 μg/ml using One-Way ANOVA. At all concentrations, OD values for Saline and NaOCl remained high (1.556 ± 0.062), reflecting no significant antibacterial effect. In contrast, both ZO and AS showed a marked concentration-dependent decrease in OD values, signifying antimicrobial activity. ZO consistently showed lower OD values than AS at higher concentrations (e.g., 0.284 at 500 μg/mL and 0.301 at 250 μg/mL), indicating superior efficacy. The ANOVA results revealed statistically significant differences (P < 0.001) at all concentrations, confirming that the type of agent used significantly affects bacterial biofilm inhibition [Table 1].

Table 1.

Intergroup comparison of optical density values

	Mean±SD	95% CI for mean		F	P
		Lower	Upper
500 µg/ml
Saline	1.556±0.062	1.402	1.710	796.03	<0.001
NaOCl	1.556±0.062	1.402	1.710
ZO	0.284±0.033	0.202	0.367
AS	0.183±0.001	0.182	0.185
250 µg/ml
Saline	1.556±0.062	1.402	1.710	585.13	<0.001
NaOCl	1.556±0.062	1.402	1.710
ZO	0.301±0.045	0.189	0.413
AS	0.248±0.039	0.153	0.344
125 µg/ml
Saline	1.556±0.062	1.402	1.710	409.001	<0.001
NaOCl	1.556±0.062	1.402	1.710
ZO	0.547±0.064	0.388	0.706
AS	0.362±0.015	0.325	0.400
62.5 µg/ml
Saline	1.556±0.062	1.402	1.710	393.44	<0.001
NaOCl	1.556±0.062	1.402	1.710
ZO	0.589±0.061	0.437	0.740
AS	0.417±0.002	0.412	0.422
31.25 µg/ml
Saline	1.556±0.062	1.402	1.710	424.51	<0.001
NaOCl	1.556±0.062	1.402	1.710
ZO	0.620±0.045	0.508	0.732
AS	0.451±0.015	0.415	0.487
15.6 µg/ml
Saline	1.556±0.062	1.402	1.710	294.58	<0.001
NaOCl	1.556±0.062	1.402	1.710
ZO	0.624±0.046	0.510	0.739
AS	0.478±0.063	0.321	0.636
7.8 µg/ml
Saline	1.556±0.062	1.402	1.710	333.35	<0.001
NaOCl	1.556±0.062	1.402	1.710
ZO	0.633±0.048	0.514	0.752
AS	0.503±0.042	0.398	0.609
3.9 µg/ml
Saline	1.556±0.062	1.402	1.710	343.87	<0.001
NaOCl	1.556±0.062	1.402	1.710
ZO	0.708±0.044	0.600	0.817
AS	0.585±0.009	0.563	0.607
1.9 µg/ml
Saline	1.556±0.062	1.402	1.710	189.52	<0.001
NaOCl	1.556±0.062	1.402	1.710
ZO	0.765±0.055	0.629	0.901
AS	0.596±0.076	0.408	0.785
0.9 µg/ml
Saline	1.556±0.062	1.402	1.710	123.36	<0.001
NaOCl	1.556±0.062	1.402	1.710
ZO	0.899±0.104	0.639	1.158
AS	0.659±0.044	0.551	0.767

Significant difference in OD values is seen between the groups in all the concentrations. OD: Optical density, CI: Confidence interval, SD: Standard deviation, ZO: Zingiber officinale, AS: Allium sativum, NaOCl: Sodium hypochlorite

Results of molecular docking

The docking results showed “isovaleric acid or N-amyl isovalerate” a phytocompound in ginger extract revealed a stable interaction with NanA protein of F. nucleatum [Figure 1]. The binding affinity was −3.2 Kcal/mole (forming two hydrogen bonds). For garlic, a phytocompound named “Furaneol” revealed interaction with NanA protein of the bacteria [Figure 2]. The binding affinity was −3.8 Kcal/mole, indicating a stable and favourable interaction (forming five hydrogen bonds) [Table 2].

Figure 1.

(a) Three-dimensional (3D) structure of target protein N-acetylneuraminate lyase (NanA), (b) 3D structure of Phytocompound named N-Amyl Isovalerate, (c) Topmost conformation of Docked complex (NanA + N-Amyl Isovalerate)

Figure 2.

(a) Three-dimensional (3D) structure of target protein N-acetylneuraminate lyase (NanA), (b) 3D structure of Phytocompound named Furaneol, (c) Topmost conformation of Docked complex (NanA + Furaneol)

Table 2.

Comparing the binding energy of target protein and phytocomponent through molecular docking

Protein-Ligand complex	Binding affinity	Bonds	Amino acid residues
5ZKA_n-amyl isovalerate	−3.2 Kcal/mole	2 hydrogen bonds	LYS (B) 3-2.84 Å
			LYS (B) 2-2.72
		2 hydrophobic alkyl	LYS (B) 221-4.87 Å
			ILE (A) 225-5.48 Å
5ZKA_ Furaneol	−3.8 Kcal/mole	5 hydrogen bonds	LYS (B) 3-2.07 Å
			LYS (B) 2-2.91 Å
			LYS (B) 2-2.81 Å
			GLY (B) 197-2.30 Å
			ASP (B) 199-2.59 Å

DISCUSSION

All periapical and pulpal infections are largely caused by microorganisms. Recently, F. nucleatum has been identified as a possible candidate for posttreatment endodontic infections hence was chosen for this study.[17] Pereira et al.[18] emphasized the importance of F. nucleatum as the most common bacterium (71.6%) in teeth with posttreatment infections, whereas Barbosa-Ribeiro et al.[19] found that F. nucleatum was present in 75% of failed endodontically treated teeth.

F. nucleatum serves as a “bridge organism” linking secondary colonizers (mainly anaerobic) like Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans to primary colonizers like Streptococcus species.[20] The virulence factors of F. nucleatum are numerous. Aid1, CmpA, Fap2, FomA, FadA, and RadD are fusobacterial adhesins that mediate invasion and essential for bacterial coaggregation and dissemination. It also contains an enzyme called N-acetylneuraminate lyase, or NanA. This enzyme enables the bacteria to convert sialic acid (N-acetylneuraminic acid) into pyruvate and N-acetylmannosamine, effectively on the host’s mucosal glycoproteins by using the easily accessible sialic acid as a source of nutrients. This ability influences the colonization and potential pathogenicity of F. nucleatum in root canal biofilms; hence, Nan-A was chosen for molecular docking in this study.[21,22] This is the novel study to demonstrate the antibacterial efficacy and molecular docking analysis of ginger and garlic extract against F. nucleatum.

The MTP assay was used in this study to assess how well an irrigant disrupted the biofilms, 500, 250, 125, 62.5 μg/mL concentrations of ginger and garlic extract surpassed the antimicrobial efficacy of 3% sodium hypochlorite. Ginger has numerous active ingredients, including terpene and phenolic chemicals that have antimicrobial properties. The main polyphenols in ginger are called gingerols, and they include 6-gingerol, 8-gingerol, and 10-gingerol, as well as quercetin, zingerone, gingerenone-A, and 6-dehydrogingerdione. Ginger also contains a hemiterpenoid called N-amyl isovalerate (isovaleric acid) as well as a number of terpene components, including β-bisabolene, α-curcumene, zingiberene, α-farnesene, and β-sesquiphellandrene.[23]

A study by Abdollahi-Mansoorkhani[24] HR, revealed that the ethanolic ginger extract provided comparable and analogous results to 2.5% NaOCl against E. Faecalis. In another research by Huang et al.[25] where the evaluation of short, medium, and long-chain fatty acids’ antibacterial efficacy against a range of oral microbes was tested. The Results exhibited that isovaleric acid (short-chain fatty acid) showed activity against F. nucleatum. This suggests that ginger extract can be a potent antimicrobial agent against F. nucleatum. The docking results of this study also showed isovaleric acid (n-amyl isovalerate), a phytocompound in ginger extract revealed good interaction with NanA protein of F. nucleatum.

Organosulfur compounds, saponins, phenolic compounds, and polysaccharides are among the many bioactive substances found in garlic. Organosulfur compounds, including allicin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, ajoene, and S-allyl-cysteine are the main active ingredients in garlic. In addition to sulfur-containing chemicals, garlic contains important aroma-active molecules such as acetic acid, furaneol, allyl methyl trisulfide, (E, Z)-2, 6-nonadien1-ol, and 5-heptyldihydro-2 (3H)-furanone and 3-methylbutanoid.[26] A study by Mehta et al.[27] found that 3% NaOCl, aqueous ozone, diode laser, and AS extract all exhibited strong antibacterial activity against both aerobic and anaerobic bacteria. Schwab[28] through his research in in vivo models of Pseudomonas infection, furaneol was found to be beneficial by decreasing Pseudomonas aeruginosa quorum sensing and improving bacterial clearance from the mouse lung. Molecular docking in the present study also revealed “Furaneol” a phytocomponent in garlic exhibited interaction with NanA protein of the bacteria.

The study’s findings demonstrated that extracts of ginger and garlic could be a viable, affordable, and easily obtainable natural substitute for conventional irrigants. They are a desirable option for clinical usage due to their biocompatibility and higher antibacterial activity against F. nucleatum. The findings of this study show that ginger and garlic both has superior antibacterial efficacy to sodium hypochlorite, a commonly used irrigant. Despite the study’s encouraging findings, there are few limitations in the study. Clinical infections are usually polymicrobial; however, all of the investigations in the present study were carried out in vitro and assessed against a single strain of bacteria. In conclusion, to better replicate clinical settings and evaluate the potential of ginger and garlic extracts as the next-generation endodontic irrigant, future research should concentrate on extensive in vivo studies and multispecies biofilm models.

CONCLUSION

Ginger and garlic extract demonstrated superior antibacterial efficacy compared to 3% sodium hypochlorite against F. nucleatum, suggesting its potential as an effective herbal alternative irrigant that can be used for endodontic disinfection.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.