Endodontic infection is a polymicrobial infection currently classified as a “Biofilm-mediated disease” (1). Endodontic biofilms are usually made up of microorganisms embedded in an exopolymer matrix that can vary in composition and biology (2). This biofilm acts as a reservoir of chronic infections and a barrier to antimicrobial agents (3, 4). The common cause of endodontic treatment failure is often linked to residual micro-organisms in the root canal system, resulting from unsuccessful eradication of the biofilm (5). In some cases, this may lead to tooth extraction (6). Therefore, the major purpose of root canal treatment is to disturb the biofilm, reduce the bacterial load, and prevent microbial recolonization of the treated canal (7).

Root canal irrigation is considered a paramount step during endodontic treatment. Among multiple existing irrigants, sodium hypochlorite (NaOCl) is the most frequently used (8). Recent studies showed that NaOCl is utilized as the preferred irrigant among general dentists and endodontists in several countries, such as Australia, Turkey, and Spain (9–11). This preference is likely due to its potent antibacterial and antibiofilm properties as well as its ability to facilitate the dissolution of residual pulp tissue. However, a study conducted by Rosen et al. suggested that NaOCl might contribute to bacterial persistence by being extremely toxic to planktonic bacteria but failing to eradicate biofilm cells (12). Additionally, NaOCl can be highly toxic if accidentally extruded, and can cause allergic reactions in patients who are sensitive to chemical agents (13). Furthermore, it may reduce the mechanical properties of dentin and cause structural damage to its components, particularly collagen (14–16). Moreover, NaOCl can cause irritation to apical tissues, particularly in immature teeth with larger apices, where preserving the apical papilla is crucial for pulp regeneration therapies (17). Chlorhexidine (CHX) is another irrigant that has been proposed because of its antibacterial activity, substantivity, and lower tissue toxicity compared to NaOCl; however, it is unable to dissolve pulp tissue or efficiently eradicate biofilms (18, 19). Although, these synthetic chemicals proved efficacy in disinfection, the search for alternative irrigants continues, as no ‘ideal’ solution has yet been found. Consequently, in an attempt to avoid the limitations of these conventional synthetic irrigants, the search for potential natural adjuvants has been encouraged (20, 21).

Natural products have already been used in endodontics. For example, clove extract is regularly used as a common root canal sealer called zinc oxide eugenol (20), eucalyptol and orange extracts are also commonly used in clinics as solvents of the gutta percha for endodontic retreatment (22), also soybean and olive oil are increasingly proposed as lubricants for the removal of fractured instruments from root canals (23). Recent systematic reviews have highlighted the antimicrobial properties of plant extracts as root canal irrigants (24, 25). However, these studies did not fully address the antibiofilm efficacy of plant-based irrigants. To the best of our knowledge, there had been no previous systematic review addressing specifically the antibiofilm potential of plant extracts in combating root canal biofilms.

Hence, this systematic review provides a critical presentation of the antibiofilm effects of various plant extracts against in vitro single and multispecies biofilms compared to conventional root canal irrigants. This review also intended to explore the mechanism of action of these natural extract on biofilms, either by biofilm disruption or inhibition of biofilm formation.

This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement checklist 2020 (26).

A total of 78 studies were obtained after a search in four databases, including PubMed, Scopus, Web of Science, and Embase. After removing duplicate records, the titles and abstracts were screened. Eleven papers were included in the full-text review. Three papers were excluded because they did not meet the inclusion criteria (28–30). Eight studies were considered eligible and were included in the final review. A summary of the study selection procedure is presented in a flowchart, in accordance with PRISMA guidelines (Figure 1).

This systematic review evaluated the antibiofilm efficacy of 12 extracts, from ten plants against endodontic biofilms formed in vitro and ex vivo. The pooled data from eight studies revealed enhanced antibiofilm activity of the extracts obtained from Psidium cattleianum, Psidium guajava, Allium sativum, and Cymbopogon martinii compared to conventional root canal irrigants. However, a significant variation in the parameters related to the tested plant extracts, evaluated biofilms, and quantification methods was observed. Previous systematic reviews showed similar variability among the included articles when comparing herbal agents with sodium hypochlorite as root canal irrigants either in ex vivo studies (25) or in clinical and randomized controlled trials (24), but none of the antibiofilm parameters were investigated.

Concerning the plant extracts, the type of extracts and solvent seemed to play a determining role, since a total biofilm elimination was generally observed with the ethanolic extracts, while only biofilm reduction was induced with the aqueous extracts of the same plant at the same concentration. This was observed in our study with P. cattleianum extracts at a concentration of 5× MLC against a single-species biofilm of E. faecalis (33). Similar observations have been reported for other plants and biofilms. For instance, the ethanolic extract of Mangifera indica L showed higher efficacy than the aqueous extract in reducing Staphylococcus spp. biofilms (39). Moreover, the ethanolic extracts of Cytinus hypocistis and Cytinus ruber had not only the strongest antibacterial effect on Staphylococcus epidermidis but also total eradication, while the aqueous extracts showed only antibacterial effects at higher concentrations (40). Therefore, it can be proposed that the type of plant extract is strongly related to the resulting antibiofilm efficacy. In our study, different solvents were used for plant extract suspension. Prabhakar et al. (36) and Fiallos et al. (35) used DMSO for Triphala and V.vinifera extracts, while Marinković et al. (34) employed ethanol for C.martinii and T.zygis EOs to assess their antibiofilm effectiveness. These solvents possess inherent antimicrobial properties that could potentially bias the results (41). To address this, an independent evaluation of the solvent activity should have been included to determine an optimal concentration that dissolves the plant extract without significantly affecting tested microbial strains. Consequently, DMSO and ethanol should serve as controls. Otherwise, the absence of such controls may compromise the validity of findings, as antibiofilm efficacy might be partially attributed to the solvent. Additionally, in our study, Miglani and Tani-Ishii utilized guava leaves extract in the form of selenium nanoparticles. The outcomes showed superior antibiofilm efficacy compared to NaOCl at a concentration of 5.25% and CHX at a concentration of 2% (37). In fact, similar favorable outcomes have been observed in the literature when using nanotechnology-based delivery systems (42–44). Marinkovic et al. explored the potential of Cymbopogon citratus EO nanoemulsion as an auxiliary root canal disinfectant within the root canals of extracted teeth against E. faecalis biofilms. The results demonstrated the significant potential of the nanoemulsion form of EO as an irrigant compared with the use of the pure form of EO (45). These advantages may be attributed to the increase in surface area and enhanced bioactivity of natural compounds when used at the nanoscale (46–48).

Several variations were also observed in the tested biofilms, including age, number of species in the biofilm, composition, origin, and the substratum used for biofilm cultivation. Regarding the age of the biofilm, our study confirmed that the bacteria in mature biofilms are more resistant to plant extracts than those in young biofilms. Prabhakar et al. showed that Triphala extract eliminated the biofilm completely at three weeks of growth, while only reducing it at six weeks of growth (36). Similar findings were reported for Streptococcus aureus and P. aeruginosa biofilms, as young biofilms of 24 h were more easily eradicated than older biofilms of 72 h, given that the biofilm matrix thickness increases with age (49, 50). Thus, it is essential to standardize the biofilm age when comparing the antibiofilm activity of different plant extracts due to the influence of biofilm age on biofilm biomass, thickness, cell count, and antibiofilm resistance. Stojicic suggested a three-week period of maturation when evaluating the antimicrobial activity of irrigants in in vitro or ex vivo experiments (51). In our study, the biofilms were composed of single species. Only one study described a multispecies biofilm (34). An in vitro or ex vivo biofilm model should be designed so that the findings can be extrapolated to clinical practice. A relatively simple model, such as a single species biofilm, may offer the advantage of simplicity and reproducibility; however, multispecies biofilms provide greater complexity, virulence, resistance, and more a more realistic representation of the biological systems in infected root canals. Therefore, it is recommended that future studies evaluate the antibiofilm effect of plant extracts in vitro using multispecies endodontic-like biofilm models to more closely simulate clinical reality (52). The included studies in this systematic review explored different bacteria typically found in primary or persistent root canal infections. E. faecalis was the most frequently tested species, either alone or in combination with other bacteria due to its popular pathogenicity in endodontology (53, 54). Only Massunari et al. tested a Candida albicans biofilm (33), as it is the most involved fungus in persistent and refractory root canal infections (55, 56). In the included studies, laboratory reference strains were mostly used to form biofilms, which offers high reproducibility because of their well-known genome sequences and properties. However, these strains present significant genetic and phenotypic differences compared to their clinical counterparts, primarily due to adaptative changes after being sub-cultured several times since their first isolation (57, 58). In contrast, three studies reported the use of wild clinical isolates (31, 33, 35) and one study used both types of strains (30). Furthermore, to study the antibiofilm efficacy of plant extracts, various substrate models have been used to grow biofilms. Polystyrene microtiter plates were used as synthetic surfaces (31–33). For natural surfaces, dental substrates have been described in three different forms: dentinal disks (35), complete roots (34), and half of roots (36) (Figure 3). While human dentin represents the natural environment of the biofilm, the extracted teeth used in these experiments usually originate from different individuals, introducing variability in dentin structure and composition (anatomy and age). This variability is not the case when nonbiological materials are used as substrates (52). However, the latter could alter the initial stage of biofilm formation since receptors on dentin are essential for bacterial adherence. To limit bias, standardization of the substrates could be proposed either for the synthetic ones using hydroxyapatite pretreatment or the use of comparable dental specimens, such as bovine root incisors (49).

Since biofilm removal is the preferred outcome in endodontic therapy, it is crucial to distinguish antimicrobial activity from antibiofilm activity. The antimicrobial activity could be considered a component of antibiofilm activity, as some plant extracts may exhibit potent activity against planktonic cells but little or no activity against biofilms (59, 60). For example, in our study, Prabhakar showed that extracts of Triphala and GTP exhibited similar antibacterial activity against E. faecalis strains with comparable MICs but showed different antibiofilm effects even at the same extract concentration and biofilm age (36). This difference in activity could be attributed to the presence of a polymeric matrix barrier in biofilms that could be selective according to the plant extract (2, 61) or to the presence of a specific compound in the plant extract with specific biofilm-related responses, resulting in a higher antibiofilm activity. These findings highlight the importance of considering not only antibacterial assays, but also antibiofilm assays when assessing the effectiveness of plant extracts. To assess the efficacy of plant extracts against biofilms, some studies have explored their potential in inhibiting biofilm formation, whereas others have focused on their ability to disturb established biofilms. It is important to evaluate the ability of plant-based root canal extracts to inhibit biofilm formation and disrupt biofilms to better understand their mechanism of action (60, 62). Furthermore, various quantitative and qualitative techniques have been employed to assess the antibiofilm effectiveness of different plant extracts, with culture and microscopy techniques being the most commonly used. However, it is recommended to use a combination of two or more complementary methods to draw conclusions about antimicrobial or antibiofilm effects (49, 63).

This systematic review disclosed various shortcomings and limitations in the studies investigating the effectiveness of plant extracts against endodontic biofilms. When employing the JBI checklist for risk of bias assessment, essential details, including the time of contact with the irrigant, the volumes utilized, and the appropriate controls were absent in several studies. Additionally, the findings of this systematic review were limited by the heterogeneity of the parameters employed, such as the type of extract, biofilm models, and the quantification methods. In addition, the overall quality of the studies reviewed was variable, with a limited number of high-quality studies meeting the inclusion criteria. Thus, this review emphasizes the importance of conducting well-designed studies and stresses the need for more rigorous protocols in future research.

This systematic review qualitatively synthesized eight heterogeneous studies to assess the antibiofilm effectiveness of plant extracts against single and multispecies biofilms. The study showed that while all plant extracts exhibited either a biofilm disruption and/or inhibition of biofilm formation, they were less effective compared to the control group except for P. cattleianum and P.guajava. The results described should be carefully analyzed and compared due to high variety in methodological parameters. This review emphasizes the importance to improve both on the reporting and the methodological aspects of the studies in order to elevate the level of certainty in evidence, thereby contributing significantly to clinical applications.