A Comparative evaluation of antimicrobial efficacy of Zingiber officinale, Allium sativum and Illicium verum extract against Enterococcus faecalis- An In Vitro study

Modern dentistry has undergone a significant transformation over recent decades, shifting from a predominantly extraction-based approach to one that emphasizes tooth preservation and functional rehabilitation. Contemporary clinical practice prioritizes the conservation of teeth affected by dental caries rather than their removal. A major advancement supporting this paradigm shift is the development of root canal therapy, which has revolutionized dental care by enabling the retention of natural teeth that would otherwise be lost.1

Root canal infections are polymicrobial in nature, with more than 600 microbial species identified within endodontic biofilms. Among these, Enterococcus faecalis is one of the most frequently isolated pathogens, particularly in retreatment cases and chronic apical periodontitis, with reported prevalence rates ranging from 24% to 77% as these microorganisms are notoriously difficult to eliminate due to their resistance to harsh environmental conditions and their diverse virulence mechanisms. It is a Gram- positive, facultative anaerobic coccus known for its remarkable adaptability. It can survive in both aerobic and anaerobic environments, withstand nutrient deprivation, tolerate high salt concentrations, and persist in highly alkaline conditions with pH values reaching up to 11.5, largely due to its proton pump mechanisms. This organism can penetrate dentinal tubules, adhere to collagen, form resilient biofilms, and suppress host immune responses by inhibiting lymphocyte activation.

Effective disinfection of the root canal system is essential for the long-term success of endodontic therapy. An ideal irrigating solution should possess strong antimicrobial properties, dissolve necrotic tissue, remove the smear layer, act as a lubricant during instrumentation, and be biocompatible with periapical tissues. Commonly used chemical irrigants include sodium hypochlorite (NaOCl), chlorhexidine (CHX), ethylenediaminetetraacetic acid (EDTA), and normal saline. Owing to the drawbacks and adverse effects of conventional irrigating agents, there has been a growing shift toward exploring herbal alternatives as potential root canal irrigants because of their antibacterial, anti-inflammatory, and antioxidant properties, along with their cost-effectiveness, environmental friendliness, and its tendency to reduce microbial load.1

Zingiber officinale (ginger) is a medicinal plant from the Zingiberaceae family, widely grown in Asia and valued for its antioxidant, antimicrobial, and anti-inflammatory properties while Allium sativum (garlic), belonging to the Liliaceae family, has long been used in traditional medicine because of its broad-spectrum antibacterial, antiviral, and anti-inflammatory activities. Illicium verum (star anise), a plant native to tropical and subtropical regions of Asia, demonstrates notable antibacterial and anti-inflammatory effects, largely due to the presence of the bioactive compound anethole.

Zingiber officinale, Allium sativum and Illicium verum are medicinal plants known for their antimicrobial properties. While ginger and garlic have been extensively studied, limited evidence exists regarding the antimicrobial efficacy of star anise in endodontics.

Therefore, the present in vitro study aimed to evaluate and compare the antimicrobial efficacy of extracts of Zingiber officinale, Allium sativum, and Illicium verum against Enterococcus faecalis.

The null hypothesis proposed that these herbal agents would not exhibit antimicrobial activity against the selected microorganism.

This in-vitro study was conducted in Department of Conservative Dentistry & Endodontics, Darshan Dental College and Hospital, Udaipur and at Apex Enviro Laboratory, Udaipur (Rajasthan) India.

The standard strains of Enterococcus faecalis (NCIM 5025) were obtained from the National collection of industrial microorganisms (NCIM) National Chemical Laboratory, Pune. Cultures were grown overnight in nutrient broth and adjusted to 0.5 turbidity on McFarland’s scale. The powdered extracts of Zingiber officinale (ginger), Allium sativum (garlic) and Illicium verum (Star anise) were taken from Pure origanio Pvt Ltd, (India). Aqueous extract was prepared by heating ten grams of dried powder in 400 ml distilled water for six hours at low heat. After 2 hours, it was filtered through muslin cloth and centrifuged at 5000 rpm for 15 min. The supernatant was collected and was concentrated to make the final volume one-fourth of the original volume and autoclaved at 121ºC and 15 lbs pressure.

Study design

In vitro antimicrobial activities of the tested herbal extracts against resistant endodontic pathogen (E. faecalis) were determined using the agar well diffusion method. In this study, a total of twenty Mueller–Hinton agar plates (n-20) were prepared. Aliquots of the Enterococcus faecalis bacterial suspension were uniformly spread on each agar plate using an L-shaped spreader. Subsequently, four wells of 6 mm diameter were prepared at equidistant points on each of the twenty agar plates using a sterile borer. Each plate was divided into four sections corresponding to the test materials. All test materials were freshly prepared and aseptically dispensed into the respective wells, ensuring that each plate contained all four tested materials. The following groups were prepared for the study:

 Group 1 (n-20)- Allium Sativum (Garlic), Group 2 (n-20)- Zingiber Officinale (Ginger), Group 3 (n-20)- Illicium Verum (Star-anise) and Group 4 (n-20)- Normal saline.

After 24-48 hours of incubation, well defined zones of inhibition were observed. The zone of inhibition refers to the size of the clear circular zone without bacterial growth around the wells. The diameter of inhibition zones around each well was measured in millimeter using a digital vernier caliper. Three Measurements were taken for microorganism in each plate, and all measured values were registered for statistical analysis to determine the difference between the groups. Descriptive (Mean ± SD) and comparative statistics were used to compare and illustrate the results. One Way Analysis of Variance (ANOVA) followed by Tukey’s Post Hoc Test was done with SPSS 16.0 software. The confidence interval was taken as 95% for the analysis (P < 0.05).

Groups	Sample size(N)	Mean	SD	SD error
Group 1 (2.5% A. Sativum)	20	23.527	2.464	0.551
Group 2 (2.5% Z. officinale)	20	22.997	2.771	0.620
Group 3 (2.5% I. verum)	20	28.849	3.595	0.804
Group 4 (Normal Saline)	20	0.000	0.000	0.000

 

Table 1: Descriptive analysis of mean zones of inhibition of all the irrigants against Enterococcus faecalis.

 

	Sum of Squares	Df	Mean Square	F	Sig
Between Groups	9887.402	3	3295.801	494.266	0.000
Within Groups	506.773	76	6.668
Total	10394.176	79

Table 2: One-way ANOVA showing comparison of mean zones of inhibition of all the irrigants against Enterococcus faecalis.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Graph    1:Bar     graph     depicting mean   zone of  inhibition indicating antimicrobial efficacy of tested irrigants against E. Faecalis

 

Table 3: Post hoc Tukeys analysis depicting intragroup comparison all the irrigants against Enterococcus Faecalis

*. The mean difference is significant at the 0.05 level.

Photograph 1: Measurement of the inhibition zones in MHA by digital vernier caliper:

(A) 2.5% Allium Sativum, (B) 2.5% Zingiber officinale and (C) 2.5% Illicium Verum.

Endodontic infections originate from a highly complex and diverse microbial ecosystem. The primary objective of endodontic therapy is the complete elimination of these microorganisms from the root canal system to prevent persistent infection and ensure long-term treatment success. Among the microorganisms frequently isolated from infected and failed root canals, Enterococcus faecalis has been strongly associated with endodontic treatment failure and recurrent periapical disease.2
It possesses exceptional survival capabilities that allow it to persist under adverse conditions such as nutrient deprivation, extreme pH levels, and exposure to intracanal medicaments. These characteristics contribute significantly to their role in persistent infections and the development of chronic apical periodontitis. Studies have reported that E. faecalis is one of the most prevalent species detected in both extraradicular infections and cases of persistent endodontic infection. Importantly, this organism rarely exists in isolation.
Owing to its marked persistence, virulence potential, and resistance to conventional intracanal procedures, this microorganism was selected for in vitro antimicrobial evaluation. Accordingly, a standard strain of Enterococcus faecalis (NCIM 5025) was employed in the present study to assess the antimicrobial efficacy of the tested agents.
Among the various laboratory techniques available, the agar well diffusion method is one of the most employed for antimicrobial testing. Its popularity is attributed to its simplicity, reproducibility, cost-effectiveness, and minimal equipment requirements. This method allows direct comparison of multiple antimicrobial agents against the same microorganism under standardized conditions. The formation of distinct zones of inhibition provides clear visual evidence of antimicrobial activity, which can be accurately measured using a ruler or digital caliper.12
However, the agar well diffusion method has certain limitations. It is suitable only for substances capable of diffusing through agar media, making it less effective for agents with poor solubility or limited diffusibility. Additionally, zone size does not always correlate with minimum inhibitory concentration, providing primarily qualitative or semi-quantitative data. Variables such as agar composition, medium thickness, inoculum density, pH, and incubation, conditions can also influence the results.12

Although mechanical debridement is essential in root canal therapy, chemical irrigation remains the cornerstone of root canal disinfection. Sodium hypochlorite has long been considered the gold standard irrigant due to its strong antimicrobial and tissue-dissolving properties. Despite these advantages, sodium hypochlorite has several drawbacks, including unpleasant taste, cytotoxicity to periapical tissues, risk of severe inflammatory reactions
 
following apical extrusion, and negative effects on the mechanical properties of dentin.13
These limitations, combined with the global rise in antibiotic resistance emphasized by the World Health Organization, have renewed interest in natural, plant-derived antimicrobial alternatives. India’s rich heritage of herbal medicine has contributed significantly to growing research interest in phytochemicals for dental and endodontic applications. Herbal irrigants are known for their antimicrobial, anti-inflammatory, antioxidant, and antiseptic properties, along with favorable biocompatibility, cost-effectiveness, and reduced risk of microbial resistance.
Zingiber officinale (ginger) has been valued for over 4000 years for its medicinal properties. The antimicrobial activity of ginger is attributed primarily to its phenolic compounds, including gingerols, shogaols, and paradols. along with other bioactive constituents such as quercetin and zingerone. Studies have demonstrated significant antimicrobial efficacy of ginger derivatives, particularly against E. faecalis.14
Allium sativum (garlic) is another extensively studied medicinal plant known for its antibacterial, antifungal, antiviral, and anti-inflammatory properties.
The primary bioactive compound, allicin, is formed when garlic is crushed and exhibits potent antimicrobial activity by interfering with vital microbial enzymes and disrupting cell membrane integrity.
Illicium verum (star anise) has gained attention for its antimicrobial, anti-inflammatory, and anti-quorum sensing properties. Its principal bioactive component, anethole, along with other phytochemicals, disrupts bacterial membranes, inhibits respiration, interferes with iron transport, and suppresses quorum sensing mechanisms essential for biofilm formation. Several studies
have reported significant antibacterial and antifungal activity of Illicium verum extracts against drug-resistant pathogens.16

In the present study, aqueous extracts of garlic, ginger, and star anise were evaluated to mimic their natural mode of consumption. Aqueous extraction was selected based on evidence that water-based extracts can exhibit antimicrobial activity comparable to organic solvent extracts. Powdered herbal extracts with high purity were used to ensure standardization and reproducibility.
Among the tested herbal agents, Illicium verum demonstrated the highest antimicrobial efficacy, exhibiting the largest zones of inhibition against both Enterococcus faecalis . This enhanced activity is attributed to its rich content of phenolic compounds and flavonoids that interfere with quorum sensing and biofilm formation. Allium sativum and Zingiber officinale also showed notable antimicrobial activity, though to a lesser extent. Normal saline showed no antimicrobial effect and served as a negative control.
The null hypothesis was rejected, as all tested herbal irrigants exhibited antimicrobial efficacy against the selected microorganism. These findings highlight the clinical potential of herbal irrigants, particularly Illicium verum, as effective and biocompatible alternatives for endodontic disinfection and warrant further biochemical and clinical investigations.

It is possible to draw the following conclusions based on the results of this study that Illicium Verum extract possesses strong antimicrobial activity against Enterococcus Faecalis, suggesting its potential as a root canal irrigant or therapeutic agent. Allium sativum and Zingiber Officinale also showed antimicrobial effects though less pronounced compared to Illicium Verum. However, due to the study’s limitations their clinical application in infected human canals remains uncertain. Further clinical trials are necessary to confirm their definitive role in endodontic treatment.

 Financial support and sponsorship

 Nil.

 Conflicts of interest

 There are no conflicts of interest.

1.      Agnihotri A, Jhamb S, Shrama U, Rohtagi S. Azadirachta indica A. juss, Morinda citrifolia L. and Triphala as herbal endodontic irrigants: A scoping review. Ayu. 2020 Jul-Sep;41(3):148-158.

2.      Choudhary E, Indushekar KR, Saraf BG, Sheoran N, Sardana D, Shekhar A. Exploring the role of Morinda citrifolia and Triphala juice in root canal irrigation: An: ex vivo: study. Journal of Conservative Dentistry and Endodontics. 2018 Jul 1;21(4):443-9.

3.      Haripriya V, Mahalaxmi S, Vidhya S, Purushothaman PV. Antibacterial efficacy of curcumin, allicin, gingerol and cinnamon against Enterococcus faecalis: An in vitro study. Journal of Herbmed Pharmacology. 2023 Aug 10;12(4):560-6.

4.      Shahi Ardakani A, Afrasiabi S, Sarraf P, Benedicenti S, Solimei L, Chiniforush N. In vitro assessment of SWEEPS and antimicrobial photodynamic therapy alone or in combination for eradicating Enterococcus faecalis biofilm in root canals. Pharmaceutics. 2023 Nov 15;15(11):2628.

5.      Zehnder M. Root canal irrigants. Journal of endodontics. 2006 May 1;32(5):389- 98.

6.      Ahmed N, Karobari MI, Yousaf A, Mohamed RN, Arshad S, Basheer SN, Peeran SW, Noorani TY, Assiry AA, Alharbi AS, Yean CY. The antimicrobial efficacy against selective oral microbes, antioxidant activity and preliminary phytochemical screening of Zingiber officinale. Infection and Drug Resistance. 2022 Jan 1:2773- 85.

7.      Bhatwalkar SB, Mondal R, Krishna SB, Adam JK, Govender P, Anupam R. Antibacterial properties of organosulfur compounds of garlic (Allium sativum). Frontiers in microbiology. 2021 Jul 27;12:613077.

8.      Assiry AA, Karobari MI, Bhavikatti SK, Marya A. Crossover Analysis of the Astringent, Antimicrobial, and Anti‐inflammatory Effects of Illicium verum/Star Anise in the Oral Cavity. BioMed Research International. 2021;2021(1):5510174

9.      Jose J, Krishnamma S, Peedikayil F, Aman S, Tomy N, Mariodan JP. Comparative evaluation of antimicrobial activity of QMiX, 2.5% Sodium Hypochlorite, 2% Chlorhexidine, Guava Leaf extract and Aloevera extract against Enterococcus faecalis and Candida albicans–An in-vitro Study. Journal of clinical and diagnostic research. 2016 May 1;10(5):ZC20.

10.   Dioguardi M, Di Gioia G, Illuzzi G, Arena C, Caponio VC, Caloro GA, Zhurakivska K, Adipietro I, Troiano G, Lo Muzio L. Inspection of the microbiota in endodontic lesions. Dentistry Journal. 2019 May 1;7(2):47.

    11.   Fiallos ND, Aguiar AL, Nascimento da Silva B, Rocha MF, Sidrim JJ, Castelo Branco de Souza Collares Maia D, Cordeiro RD. Enterococcus faecalis and         Candida albicans dual-species biofilm: establishment of an in vitro protocol and characterization. Biofouling. 2022 Apr 21;38(4):401-13.

12.   Du Q, Yuan S, Zhao S, Fu D, Chen Y, Zhou Y, Cao Y, Gao Y, Xu X, Zhou X, He J. Coexistence of Candida albicans and Enterococcus faecalis increases biofilm virulence and periapical lesions in rats. Biofouling. 2021 Nov 28;37(9-10):964-74.

13.   Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: A review. Journal of pharmaceutical analysis. 2016 Apr 1;6(2):71-9.

14.   Gulati RK, Jain N, Singh A, Jain A, Jindal A, Kumawat MK, Paiwal K, Jain Sr A, Kumawat Sr MK. Comparative Evaluation of the Antimicrobial efficacy of Chemical and Phytomedicinal agents when used as Intracanal Irrigants: An In Vitro Study. Cureus. 2023 Nov 13;15(11).

15.   Kale PP, Raut AW. A proposed classification system for herbal endodontic irrigants. Journal of Conservative Dentistry and Endodontics. 2021 May 1;24(3):293-5.

16.   Giriraju A, Yunus GY. Assessment of antimicrobial potential of 10% ginger extract against Streptococcus mutans, Candida albicans and Enterococcus faecalis: An: in vitro: study. Indian journal of dental research. 2013 Jul 1;24(4):397-400.

17.   Venâncio PC, Figueroba SR, Nani BD, Ferreira LE, Muniz BV, Del Fiol FD, Sartoratto A, Rosa EA, Groppo FC. Antimicrobial activity of two garlic species (Allium sativum and A. tuberosum) against Staphylococci infection. in vivo study in rats. Advanced pharmaceutical bulletin. 2017 Apr 13;7(1):115

18.   Eswar K, Venkateshbabu N, Rajeswari K, Kandaswamy D. Dentinal tubule disinfection with 2% chlorhexidine, garlic extract, and calcium hydroxide against Enterococcus faecalis by using real-time polymerase chain reaction: In vitro: study. Journal of Conservative Dentistry and Endodontics. 2013 May 1;16(3):194-8.

19.   Noumi E, Ahmad I, Adnan M, Patel H, Merghni A, Haddaji N, Bouali N, Alabbosh KF, Kadri A, Caputo L, Polito F. Illicium verum L. (Star anise) essential oil: GC/MS profile, molecular docking study, in silico ADME profiling, quorum sensing, and biofilm-inhibiting effect on foodborne bacteria. Molecules. 2023 Nov 21;28(23):7691.