Artemisinin as a novel endodontic irrigant: In vitro assessment of antimicrobial, antioxidant, and Fourier transform infrared-based structural characterization

Abstract

Context:

Artemisinin is a sesquiterpene lactone extracted from Artemisia annua that is best known for its potent antimalarial activity. Recent evidence highlights its additional antimicrobial and antioxidant properties. These features are particularly relevant in endodontics, where safer and more biocompatible alternatives to conventional irrigants are needed.

Aims:

To evaluate the in vitro antimicrobial and antioxidant effects of artemisinin against Enterococcus faecalis and Candida albicans and characterize its molecular design using Fourier transform infrared (FTIR) spectroscopy.

Subjects and Methods:

The antimicrobial activity was determined using the ditch plate method and the antioxidant capacity was evaluated using the total antioxidant assay with optical density measured at 695 nm, while FTIR spectroscopy analysis revealed the functional groups present in artemisinin extract.

Results:

Artemisinin exhibited significant inhibitory activity, against both the test microrganisms. The antioxidant analysis revealed a concentration-dependent increase in activity, with the highest effect at 10 mg/mL (2.08 mm/L). FTIR spectra showed multiple absorption bands corresponding to hydroxyl, phenolic, and aliphatic groups, indicating complex molecular characteristics.

Conclusions:

Artemisinin demonstrates dual antimicrobial and antioxidant properties, suggesting its potential as a safer, biologically compatible endodontic irrigant. To validate the in vitro findings, further research for defining its efficacy, optimum concentration, and safety parameters are required under in vivo as well as clinical conditions.

INTRODUCTION

The success of endodontic therapy hinges on thorough microbial elimination from the pulp space for establishing an environment conducive to healing. Despite advancements in root-canal instrumentation and irrigation techniques, persistent and recurrent infections remain significant causes of treatment failure. The Enterococcus faecalis (E. faecalis) and the Candida albicans (C. albicans) are considered to be two of the most challenging microorganisms, particularly due to their ability to invade the dentinal tubules, form resistant biofilms, and withstand harsh environmental conditions. These pathogens are strongly associated with secondary infections and unsuccessful root-canal therapies.[1,2] Conventional irrigants such as sodium hypochlorite (NaOCl) and later chlorhexidine (CHX) were widely used due to their broad antimicrobial spectrum and tissue-dissolving properties.[3] However, their use is limited by several drawbacks, including cytotoxicity, unpleasant taste, allergic potential, and the risk of damaging surrounding tissues when extruded beyond the apex. Moreover, microbial resistance and biofilm tolerance further reduce their effectiveness.[4] These limitations underscore the urgent need for safer, biocompatible, and equally effective alternatives in root canal disinfection.

Artemisinin is a sesquiterpene lactone extracted from Artemisia annua, medicinal plant that garnered global attention for its potent antimalarial effects. Since its discovery, research has revealed a broader pharmacological profile, including properties of antimicrobial, antioxidant, as well as anti-inflammatory and anticancer effects.[5,6] Artemisinin exerts its biological effects primarily from the generation of the reactive oxygen species (ROS) via its endoperoxide bridge, disrupting cellular functions in microorganisms and malignant cells. These mechanisms, combined with its natural origin and relative safety, make it an appealing candidate for applications beyond malaria therapy.[7]

In dentistry, the antimicrobial potential of artemisinin is of particular interest in addressing persistent endodontic infections. The E. faecalis is a Gram-positive facultative anaerobe that is highly resistant to many conventional agents and is a leading cause of posttreatment disease.[8] Similarly, C. albicans, an opportunistic fungal pathogen, can colonize the dentinal surfaces and survive harsh endodontic environments, contributing to recurrent infections.[9] Effective control of these pathogens is therefore central to improving treatment outcomes.

Beyond antimicrobial activity, oxidative stress can play a crucial part in the progress of oral and periapical disease. Free radicals generated during inflammation can impair healing and damage host tissues. Antioxidants counteract these harmful effects by neutralizing ROS, thereby supporting tissue repair. Artemisinin’s reported antioxidant activity suggests it could reduce oxidative burden in periapical tissues following root-canal treatment, offering a dual therapeutic benefit.[6]

Despite promising evidence from pharmacological and microbiological studies, limited research has examined artemisinin’s utility in dental sciences, particularly in endodontics. This gap warrants investigation into its antimicrobial and antioxidant effects within a controlled experimental setting. This study was designed to be conducted in accordance to the Checklist for Reporting in vitro Studies guidelines to ensure methodological rigor and reproducibility.[10]

The study objectives were threefold: (1) to evaluate antimicrobial efficacy of artemisinin against the E. faecalis and the C. albicans; (2) to assess its antioxidant activity using a total antioxidant capacity assay; and (3) to characterize the chemical structure of artemisisnin through Fourier transform infrared (FTIR). By exploring these properties, the study aims to establish a foundation for considering artemisinin as a potential alternative or adjunctive agent in endodontic therapy.

SUBJECTS AND METHODS

Preparation of artemisinin extract

Dried Artemisia annua L. (15 g) was subjected to solvent extraction using 80% concentration of methanol, ethanol, and acetone each under the reflux conditions for a duration of 24 h and the resultant extract, filtered through Whatmann filter paper was subsequently concentrated into viscous residues in a rotary evaporator using reduced pressure.[5]

Antimicrobial (ditch plate) method

The antimicrobial activity of artemisinin was evaluated using the ditch plate method. A sterile Mueller–Hinton (MH) agar plate was prepared, and a ditch (15 mm × 70 mm) was cut into the medium. The artemisinin extract was incorporated by adding it to 7 mL of molten MH Agar maintained at a temperature of 40°C and then poured into a ditch to allow solidification. Care was taken to ensure the ditch surface was level with the surrounding agar. The ditch-plate technique is a recognized method for testing antimicrobial agents, especially those that may have limited solubility.

Fresh cultures of E. faecalis and C. albicans in the plate were streaked in a perpendicular direction to the ditch by using a sterile nichrome loop and incubated at 37°C for a 24-h period. The antimicrobial activity was studied by observing the inhibition of microbial growth within and adjacent to the ditch.[11,12]

Total antioxidant assay

A phosphor-molybdenum reduction assay was used to estimate the total antioxidant capacity of artemisinin. Briefly, 100 μL of artemisinin extract was pipetted into a clean test tube, followed by the addition of 5% trichloroacetic acid to precipitate proteins. After standing for 5 min, the mixture was centrifuged, and the supernatant was collected. The reaction mixture was incubated in a water bath at 90°C for 90 min. A blank was prepared by substituting distilled water in the place of the sample. After cooling, the formation of a bluish-green complex was measured using the spectrophotometer at 695 nm against the blank. Antioxidant activity was calculated by plotting the absorbance values against a standard calibration curve, with results noted in mm/L.[13,14,15]

Fourier transform infrared spectroscopy analysis

The structural features of artemisinin were characterized using FTIR. The extract was analyzed with an Alpha II FTIR spectrometer (Experiment ATR_ZnSe1.XPM, resolution: 4 cm⁻¹). Spectra were recorded to identify the functional groups, with particular attention to carbonate and phosphate bands. Absorbance peaks were analyzed within the fingerprint region (600–1500 cm⁻¹) and broader ranges (2500–4000 cm⁻¹) to detect hydrogen bonding, hydroxyl groups, and aliphatic compounds.[16,17,18]

RESULTS

Antimicrobial activity

Artemisinin demonstrated inhibitory effects against both test organisms. Growth of E. faecalis and C. albicans was suppressed around areas adjacent to the artemisinin-containing ditch, indicating antimicrobial activity in vitro. The inhibition pattern suggested a direct interaction between artemisinin and the microbial cultures [Figure 1].

Figure 1.

Representative ditch plate assay showing inhibition zones for Enterococcus faecalis and Candida albicans. Artem: Artemisinin, DMSO: Dimethyl sulfoxide, Amp: Ampicillin

Total antioxidant assay

The antioxidant activity of artemisinin was concentration-dependent. As the concentration of the extract increased, antioxidant capacity also rose, reaching the highest value at 10 mg/mL (2.08 mm/L) [Table 1]. These findings confirm that artemisinin exhibits measurable antioxidant potential, with increased effectiveness at higher concentrations.

Table 1.

Total antioxidant activity of artemisinin

Concentration (mg/mL)	Antioxidant activity (mm/L)
0.3	0.47
0.6	0.50
1.0	0.67
1.3	0.79
2.5	1.21
5.0	1.54
10.0	2.08

Fourier transform infrared analysis

FTIR spectroscopy of artemisinin revealed multiple absorption bands, confirming the molecular complexity of the compound [Figure 2]. The key features included.

Figure 2.

Fourier transform infrared spectrum of artemisinin showing key absorption peaks

• Fingerprint region (600–1500 cm⁻¹): Vinyl-related peaks at 900–990 cm⁻¹ and double olefinic bonds near 890 cm⁻¹

• Single Bond region (2500–4000 cm⁻¹): A broad hydrogen-bonded band between 3650 and 3250 cm⁻¹, consistent with hydroxyl groups

• Hydroxyl compounds: Peaks between 1600 and 1300 cm⁻¹ range, 1200–1000 cm⁻¹ and 800–600 cm⁻¹

• Phenolic groups: Distinct peaks at 3670 and 3550 cm⁻¹

• Aliphatic compounds: Absorbance at 2935 and 2860 cm⁻¹, with characteristic peaks at 1470 and 720 cm⁻¹.

These spectral findings confirm the presence of the hydroxyl, phenolic, and aliphatic functional groups evidently pointing to the bioactive nature of artemisinin.

DISCUSSION

The findings of this in vitro study provide compelling evidence that artemisinin possesses both antimicrobial and antioxidant properties, supporting its potential as a novel adjunct in endodontic therapy. Persistent root canal infections continue to pose a major clinical challenge, with E. faecalis and C. albicans frequently implicated in treatment failures. These microorganisms can invade dentinal tubules, form resilient biofilms, and withstand harsh chemical and environmental conditions, rendering conventional irrigants less effective.[19] The observed inhibition of their growth by artemisinin in this study highlights its promise as an alternative or complementary agent to current endodontic disinfectants.

The minimum inhibitory concentration of ethanolic artemisinin extract reported for C. albicans and other artemisinin extracts for E. faecalis is 125 mg/mL and 0.156 mg/mL, respectively, that reflects the strong broad-spectrum antimicrobial effect of Artemisia annua derivatives against diverse bacterial and fungal pathogens.[20,21,22] Its mechanism is attributed to cleaving of the endoperoxide bridge occurring amidst intracellular iron that generates ROS known to cause membrane disruption, protein oxidation, and nucleic acid damage.[23] This unique oxidative mechanism may account for its effectiveness against resistant species such as E. faecalis, which can persist despite treatment with NaOCl or CHX. In contrast to these agents, artemisinin is naturally derived and demonstrates a more favorable safety profile, suggesting a combination of potent antimicrobial efficacy and enhanced biocompatibility – an essential requirement for irrigants that may contact periapical tissues.[24]

Beyond its antimicrobial potential, artemisinin demonstrated concentration-dependent antioxidant activity, further enhancing its therapeutic value. Oxidative stress is a recognized contributor to periapical inflammation and delayed posttreatment healing, as excess ROS can damage host tissues and impede repair.[25] The antioxidant assay performed in this study revealed a marked capacity of artemisinin to neutralize free radicals, with maximal activity observed at 10 mg/mL. Functional groups identified via FTIR – particularly hydroxyl and phenolic moieties – correlate with this antioxidant effect, supporting the structural basis for its free-radical-scavenging properties.[20] The combination of antimicrobial and antioxidant functions distinguishes artemisinin from conventional irrigants, suggesting a dual mechanism that may simultaneously facilitate disinfection and promote periapical healing.

The FTIR analysis provided additional insights into the molecular characteristics of artemisinin, revealing prominent absorption bands corresponding to hydroxyl, phenolic, and aliphatic groups. These structural attributes are consistent with its biological activity: Hydroxyl and phenolic groups contribute to antioxidant capacity, while aliphatic and olefinic bonds facilitate the generation of reactive intermediates responsible for antimicrobial effects.[26] Such findings confirm that artemisinin’s chemical architecture underlies its multifunctional behavior.

When compared with conventional irrigants, the potential advantages of artemisinin become apparent. NaOCl remains the gold standard by virtue its strong antimicrobial and well known tissue-dissolution property, yet its cytotoxicity and potential for severe periapical injury limit its safety.[27] CHX, although less toxic, lacks tissue-dissolving capacity and is less effective against biofilms, with concerns regarding long-term resistance. Artemisinin, being naturally derived and biocompatible, offers a unique balance of safety and efficacy, along with the added benefit of antioxidant protection. While extrapolation from in vitro to clinical scenarios must be approached cautiously, these findings provide a foundation for further translational research.

Clinically, an artemisinin-based irrigant or medicament could enhance root canal disinfection by targeting resistant organisms such as E. faecalis while simultaneously promoting periapical tissue healing. Such dual functionality aligns with current trends favoring biologically compatible, multifunctional agents in endodontic care. However, practical challenges – such as optimizing solubility, stability, and delivery within the root canal system – must be addressed before clinical application.

This study has several limitations. Being in vitro in nature, it does not replicate the complex oral environment, where factors such as saliva, host immunity, and multispecies biofilms influence efficacy. Moreover, the antimicrobial evaluation was restricted to E. faecalis and C. albicans; future investigations should include additional pathogens relevant to endodontic infections, such as Porphyromonas gingivalis and Fusobacterium nucleatum. Although the antioxidant capacity was well demonstrated, its biological impact on periapical tissue healing requires validation in animal and clinical models. Further, while FTIR confirmed characteristic functional groups, advanced spectroscopic and pharmacokinetic analyses would deepen the understanding of artemisinin’s mechanisms in oral environments.

Future research should therefore focus on in vivo assessments, including cytotoxicity testing on periapical fibroblasts and osteoblasts and formulation studies to develop stable irrigation or sustained-release delivery systems. Controlled clinical trials will ultimately be necessary to determine whether the promising in vitro outcomes translate into tangible clinical benefits.

In summary, artemisinin demonstrates significant antimicrobial and antioxidant properties, underscoring its potential as a multifunctional adjunct in endodontic therapy. Its efficacy against resistant microorganisms, coupled with its ability to counter oxidative stress, positions it as a promising candidate for the development into a safe and biocompatible irrigant or intracanal medicament. Although further investigation is recommended, these results add to the collective body of evidence that contribute toward strategies integrating natural bioactive compounds into contemporary endodontic practice.

CONCLUSIONS

This in vitro study demonstrated that artemisinin exhibits significant antimicrobial activity against E faecalis and C. albicans species, along with notable antioxidant potential. FTIR analysis confirmed the presence of functional groups – such as hydroxyl and phenolic moieties – responsible for these bioactivities. Collectively, the results suggest that artemisinin may serve as a safe and biologically compatible alternative or adjunct to conventional root canal irrigants. Its dual antimicrobial and antioxidant actions not only enhance infection control but may also promote periapical tissue healing, underscoring its promise as a multifunctional agent in endodontic therapy.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.