Comparative In vitro Evaluation of Chemical and Herbal Antimicrobial Agents for Surface Decontamination: A Pharmaceutical Perspective

INTRODUCTION

Successful endodontic treatment relies on effective chemomechanical preparation to eliminate or significantly reduce microorganisms from the root canal system. Strict aseptic protocols must be followed during this procedure to prevent contamination of instruments and obturating materials, thereby avoiding cross-infection (Voroshilov et al., 2025).

Gutta-Percha (GP) is the most widely used obturating material in endodontics. It is a dried coagulated extract obtained from plants of the Palaquium genus (family Sapotaceae), introduced to dentistry in 1847 (Bansal et al., 2020). Even when supplied in sealed packages, gutta-percha cones can acquire contamination during storage and chairside handling, resulting in loss of aseptic integrity (Bansal et al., 2020; Ramesh Chandra et al., 2017). Conventional sterilization methods using moist or dry heat cannot be employed due to the thermoplastic nature of GP, which may cause structural alterations (Ramesh et al., 2017). Therefore, rapid chairside chemical disinfection of GP cones prior to obturation is essential (Voroshilov et al., 2025).

Commonly used chemical disinfectants include sodium hypochlorite, chlorhexidine, ethyl alcohol, glutaraldehyde, and formocresol, with disinfection times ranging from 3 to 25 min (Ramesh et al., 2017). Enterococcus faecalis is frequently linked to refractory endodontic infections and treatment failure because of its resilience under unfavorable intracanal conditions (Liu et al., 2024).

Sodium hypochlorite and chlorhexidine are widely used for GP disinfection; however, sodium hypochlorite may alter the surface structure of GP cones and interfere with sealer adhesion, leading to microleakage. Chlorhexidine exhibits broad-spectrum antimicrobial activity and substantivity but also has limitations (Shailja et al., 2018). Herbal agents such as Aloe vera and neem have gained attention as eco-friendly alternatives. Aloe vera possesses antibacterial properties attributed to polysaccharides, anthraquinones, and saponins. Neem (Azadirachta indica) contains azadirachtin, which exhibits antimicrobial and anti-adherence properties (Makade et al., 2017; Kunjar and Shrivastav, 2022).

Despite their promising properties, evidence supporting the use of herbal agents for rapid chairside disinfection of gutta-percha cones remains limited. Hence, the present study was conducted to evaluate and compare the antimicrobial effectiveness of conventional chemical disinfectants with herbal alternatives, including Aloe vera and neem, for rapid gutta-percha cone decontamination.

MATERIALS AND METHODS

Following approval from the Institutional Ethics Committee (MDC_KT_D202010), this in vitro experimental study was carried out in the Department of Pedodontics and Preventive Dentistry, in collaboration with the Departments of Microbiology, Biochemistry, and Pharmacology.

Sample Size Calculation

The sample size was calculated based on previously reported antimicrobial studies evaluating disinfectant efficacy against Enterococcus faecalis. Assuming a minimum clinically significant difference of 5 mm in the mean zone of inhibition between groups, with an estimated standard deviation of 7 mm, a power of 80%, and a significance level (α) of 0.05, the minimum required sample size was determined to be 18 specimens per subgroup. To compensate for potential experimental variability, 20 gutta-percha cones were included in each subgroup, resulting in a total sample size of 80 specimens.

Eighty standardized gutta-percha cones (6% taper, size 40) were included. Enterococcus faecalis was selected as the test organism. Culture media used were brain-heart infusion broth, blood agar, and Mueller-Hinton agar. The disinfecting agents evaluated were 5% sodium hypochlorite, 2% chlorhexidine, Aloe vera extract, and neem extract. Ethanol served as the extraction solvent for herbal preparations.

Freeze-dried E. faecalis obtained from the Department of Microbiology was reactivated in brain-heart infusion broth. Sterile test tubes containing 5 mL of activated inoculum were incubated at 37ºC for 30 min. Aseptically retrieved gutta-percha cones were immersed in the inoculum and incubated under identical conditions to achieve uniform microbial contamination.

The contaminated cones were randomly assigned into two equal groups (n=40).

Group I (Chemical disinfectants)

• Subgroup IA: 5% sodium hypochlorite (n=20).
• Subgroup IB: 2% chlorhexidine (n=20).

Group II (Herbal disinfectants)

• Subgroup IIA: Aloe vera extract (n=20).
• Subgroup IIB: Neem extract (n=20).

Fresh Aloe vera leaves were washed, the gel was collected, oven-dried, powdered, and subjected to ethanol extraction, followed by filtration and solvent evaporation (Shailja, Ramesh, Anubha, and Shazia, 2018). Neem leaves were dried, pulverized, and extracted using ethanol in a 1:10 weight-to-volume ratio (Natasha et al., 2015).

Antimicrobial efficacy against E. faecalis was assessed using blood agar and Mueller-Hinton agar diffusion methods. Overnight cultures were spread onto agar plates, and standardized wells were prepared. Each well received 0.1 mL of the respective disinfectant, followed by incubation at 37ºC for 24 hr. The antibacterial effect was quantified by measuring the diameter of inhibition zones.

All observations were systematically recorded and subjected to statistical evaluation using SPSS 25.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics (mean and standard deviation) were calculated. Intergroup comparisons were performed using One-Way Analysis of Variance (ANOVA) separately for each culture medium. Post hoc pairwise comparisons were conducted using Tukey’s test. Confidence interval was set at 95%. p value <0.05 was considered statistically significant.

RESULTS

The mean zone of inhibition (in mm) of the four subgroups against E. faecalis is presented in Table 1. Among chemical agents (Group I), subgroup IB demonstrated a greater antibacterial effect than subgroup IA. The mean zone of inhibition for subgroup IB was 24.70±7.26 mm in Blood Agar (BA) and 27.90±6.23 mm in Mueller-Hinton Agar (MHA). Among herbal alternatives (Group II), subgroup IIA showed the highest mean zone of inhibition, with values of 20.10±6.66 mm in BA and 22.50±8.53 mm in MHA, which were greater than those observed for subgroup IIB.

Post hoc Tukey analysis revealed that subgroup IB demonstrated significantly greater antibacterial activity compared to subgroup IA and subgroup IIB in both culture media (p<0.05). A statistically significant difference was also observed between subgroup IIA and subgroup IIB in blood agar (p=0.018), whereas no significant difference was noted in Mueller-Hinton agar (p=0.064) (Table 2).

DISCUSSION

The persistence of microbial infection within the root canal system and periradicular tissues remains the most significant cause of endodontic failure (Gutmann and Manjarrés, 2018). Endodontic failure is multifactorial, often attributed to anatomical variations, technical errors, biological factors, and iatrogenic complications. Despite high overall success rates, absolute predictability cannot be ensured, and failures may occur before, during, or after treatment (Gulabivala and Ng, 2023). Additional complications such as nerve paresthesia, tissue emphysema, and aspiration or ingestion of instruments have also been reported (Athiban et al., 2012).

Among the microorganisms associated with endodontic failure, staphylococcus species are frequently identified as contaminants of Gutta-Percha (GP) cones during storage and handling, underscoring the need for their disinfection (Shetty et al., 2023). Enterococcus faecalis is commonly isolated from failed endodontic cases, with prevalence ranging from 24% to 77%. Its ability to survive harsh conditions, resist intracanal medicaments, and form biofilms contributes significantly to treatment failure (Fiore et al., 2019).

Since GP is heat-sensitive and cannot be sterilized using conventional thermal methods without altering its physical properties, chemical disinfection prior to obturation is essential (Kalai et al., 2020). The success of endodontic therapy is closely related to effective microbial elimination before obturation, and prevention of exogenous contamination is equally critical (Bansal et al., 2020). GP remains the most widely used obturating material due to its biocompatibility, dimensional stability, radiopacity, and minimal toxicity (Vishwanath and Rao, 2019). However, clinical handling and exposure significantly increase the risk of microbial contamination, potentially leading to reinfection (Agrawal et al., 2020).

Despite this risk, many clinicians do not routinely disinfect GP cones prior to obturation, highlighting the importance of rapid chairside disinfection followed by aseptic handling (Jain et al., 2019). Rapid sterilization techniques, such as those proposed by Senia et al., remain simple, economical, and reliable (Frank and Pelleu, 1983). Chemical disinfectants are preferred, but the choice of agent is crucial, as certain disinfectants may adversely affect GP surface characteristics (Bansal et al., 2020). Sodium hypochlorite, commonly used at concentrations between 0.5% and 5%, exhibits potent antimicrobial activity but may compromise GP integrity at higher concentrations (Kulkarni and Desai, 2019). Chlorhexidine has been proposed as a safer alternative, demonstrating broad-spectrum antimicrobial activity and substantivity without damaging GP cones (Kalai et al., 2020).

In recent years, herbal agents have gained attention as eco-friendly alternatives. Aloe vera and neem (Azadirachta indica) extracts exhibit significant antimicrobial activity against Escherichia coli, Staphylococcus aureus, and E. faecalis (Shenoi et al., 2014; Athiban et al., 2012; Kulkarni and Desai, 2019). Neem leaf extracts, rich in azadirachtin and tannins, demonstrate broad-spectrum antimicrobial efficacy, including effective decontamination of GP cones (Devi and Sharma, 2023). Studies further confirm that ethanolic extracts of neem and Aloe vera, alone or in combination, are effective in disinfecting GP cones (Natasha et al., 2015).

While these findings are promising, most studies are in vitro and do not fully replicate the clinical oral environment involving saliva, biofilm interactions, and dentinal tubule architecture. Additionally, the effects of herbal disinfectants on GP cone integrity remain underexplored. Future research should include in vivo validation, testing against polymicrobial biofilms, and assessment of long-term antimicrobial substantivity. Standardization of concentrations, exposure times, and formulations of herbal extracts will be essential to establish clinically applicable chairside disinfection protocols.

CONCLUSION

Chlorhexidine was found to be an effective chemical disinfectant for rapid decontamination of gutta-percha cones. Herbal agents also demonstrated significant antimicrobial activity, with Aloe vera showing the highest efficacy within a short contact time. Neem extract exhibited promising results, supporting the potential of herbal alternatives in endodontic practice. These findings suggest that both chemical and herbal disinfectants can be reliably used for chairside sterilization of gutta-percha cones.

ACKNOWLEDGEMENT

The authors would like to thank the Department of Microbiology and Pharmacology for their technical support and assistance in conducting the microbiological analysis. We also acknowledge the institutional support provided for carrying out this study.

ABBREVIATIONS

GP

Gutta-percha

BA

Blood agar

MHA

Mueller-Hinton agar

ANOVA

Analysis of variance

NaOCl

Sodium hypochlorite

CHX

Chlorhexidine.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

FUNDING

This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

AUTHORS CONTRIBUTIONS

A - Research concept and design, B - Collection and/or assembly of data, C - Data analysis and interpretation, D - Writing the article, E - Critical revision of the article, F - Final approval of the article. Preethi Vem: A-F; Sathwika Rao Nadipelli: A-F; Daneswari V: A-F; Vaishnavi Julakanti: A-F; Shivani Thoom: A-D, F; Deekshita KV: A-D; Naseemoon Shaik: A-F.

SUMMARY

This in vitro study evaluated the antimicrobial efficacy of chemical and herbal agents used for gutta-percha cone decontamination. Among chemical agents, 2% chlorhexidine demonstrated the highest antimicrobial activity against Enterococcus faecalis. Among herbal agents, Aloe vera showed superior efficacy compared to neem. The findings suggest that while chemical disinfectants remain highly effective, herbal alternatives possess promising antimicrobial potential and may serve as safe, eco-friendly chairside disinfectants.