Antimicrobial efficacy of natural-based endodontic solutions: a systematic review with a network meta-analysis

Abstract

This systematic review aims to provide preclinical evidence of the antimicrobial efficacy of natural endodontic solutions (NES) compared to sodium hypochlorite (NaOCl) and chlorhexidine (CHX) against Enterococcus faecalis. The study followed the PRISMA guidelines and had a registered protocol (PROSPERO – CRD42021224022). The inclusion criteria comprised ex vivo studies simulating root canal irrigation to assess the standardized mean difference of colony-forming units (CFUs). Records were systematically identified in PubMed, Scopus, EMBASE, SciELO, Web of Science, LILACS, DANS Easy, and BDTD databases in January 2024. The RoBDEMAT tool helped determine the risk of bias. As for the network meta-analysis, CFU data were collected pre- and post-irrigation protocols as an effect measure. Thirty-five studies met the eligibility criteria and were included in the qualitative analysis, and seven studies were eligible for the network meta-analysis. The total sample consisted of 1,504 root canals. Among the tested solutions, apple cider vinegar combined with 2.5% NaOCl showed the highest probability of reducing CFUs after root canal irrigation (95%CI: -0.76–0.20), followed by 2% CHX and NaOCl (considering the different concentrations [95%CI: -0.32–0.17]). The bias assessment revealed significant omissions in reporting, particularly regarding sample size calculation, sample randomization, and operator blinding. The obtained evidence demonstrated that, even though NES did not overcome NaOCl, they have antimicrobial potential against Enterococcus faecalis.

Endodontics; Plant extracts; Root canal irrigants

Introduction

Root canal anatomy represents a challenge for root canal system (RCS) disinfection due to persistent microorganisms.¹,² They can lodge in areas of difficult access, such as isthmus and dentinal tubules,³,⁴ also interfering with the chemical-mechanical disinfection of the RCS.⁴ Enterococcus faecalis, a gram-positive bacteria, is the main microorganism in primary endodontic treatment failure.⁵ E. faecalis can survive the effects of root canal treatment as a single or main constituent of bacterial flora.⁶-⁸

Several irrigation solutions may disinfect root canals, the most common being sodium hypochlorite (NaOCl) and chlorhexidine (CHX).⁹-¹¹NaOCl is the gold standard due to its tissue dissolution and antimicrobial activity.⁹ However, NaOCl shows drawbacks such as the reduction in dentin flexural strength and mainly cytotoxicity surrounding tissues when in higher concentrations.¹⁰-¹³ Considering its broad-spectrum antimicrobial activity and substantivity, CHX may represent an alternative to NaOCl; however, it cannot dissolve necrotic tissue remnants compared to NaOCl.⁹,¹¹,¹²

Recently, natural extract use as endodontic irrigation solutions has increased mainly because of their widely known medicinal properties, such as anti-inflammatory effect,¹⁴-¹⁶promising antimicrobial properties, safety, and availability.¹⁷,¹⁸ In vitro and ex vivo studies have evaluated the antifungal and antibacterial effects of natural endodontic solutions (NES), showing promising results compared to conventional chemical solutions (CCS).¹⁹-²⁴ Some of these studies suggested that the antimicrobial efficacy of NES is superior to CCS,²⁵-²⁸ while others found higher microbial reduction using NaOCl or CHX.²¹,²³,²⁹-³¹

Despite the higher number of studies comparing the antimicrobial activities of NES and CCS, convincing evidence to support NES use as an alternative to NaOCl or CHX remains unclear. Thus, this systematic review aims to provide preclinical evidence of the antimicrobial efficacy of NES compared to NaOCl and CHX against Enterococcus faecalis. The null hypothesis is that the antimicrobial efficacy of NES is similar to CCS.

METHODS

Protocol and registration

The protocol was created according to the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P)³² and recorded at the International Prospective Register of Systematic Reviews (PROSPERO) database under number CRD42021224022 (http://www.crd.york.ac.uk/PROSPERO). This systematic review was reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)³³ and conducted according to the Joanna Briggs Institute (JBI) Manual.³⁴ There was a deviation from the proposed initial protocol by substituting the risk of bias assessment tool.

Study design and eligibility criteria

The formulation of the guiding question of this review was based on the PVO (Population, Variable, and Outcome) strategy: Do natural solutions for root canal irrigation or their association with conventional chemical solutions (variable) have better antimicrobial efficacy against Enterococcus faecalis (outcome) than conventional chemical solutions alone (variable) in human permanent teeth (population)?

The inclusion criteria comprised ex vivo studies simulating root canal irrigation to obtain the standardized mean difference of colony-forming units (CFUs) of E. faecalis between natural endodontic solutions (NES) or their association with conventional chemical solutions (CCS) and conventional chemical solutions alone (NaOCl and CHX). The search had no restriction of publication year and language. The exclusion criteria were studies using dentin blocks or teeth sections immersed in antimicrobial solutions, studies on primary teeth, review articles, and clinical studies.

Sources of information, search, and study selection

Electronic searches were performed in MEDLINE (via PubMed), LILACS, SciELO, EMBASE, Scopus, and Web of Science as primary study sources. Data Archiving and Networked Services (DANS Easy) and the Brazilian Digital Library of Theses and Dissertations (BDTD) databases partially captured the “grey literature.”

The MeSH (Medical Subject Headings), DeCS (Health Sciences Descriptors), and Emtree (Emtree Subject Headings) resources aided search descriptor selection. The Boolean operators “AND” and “OR” provided several combinations between descriptors according to the syntax rules of each database. The bibliographic research occurred in February 2023, and Table 1 describes all the details. The search was updated in January 2024 to gather additional eligible studies.

Table 1
Strategies for database search.

Database	Search strategy (February, 2023)
PubMed (https://pubmed.ncbi.nlm.nih.gov/)	#1: ((“Dental Pulp Cavity” OR “Dentinal Tubule” OR “Dentition, Permanent” OR “Pulp Canal” OR “Root Canal” OR “Root Dentine” OR “Teeth” OR “Tooth”)
	#2: (“Irrigant” OR “Irrigating Solution” OR “Irrigation” OR “Rinsing” OR “Root Canal Irrigants” OR “Aloe” OR “Carvacrol” OR “Cetrimide” OR “Chlorhexidine” OR “Chrysopogon” OR “Cinnamomum zeylanicum” OR “Cinnamon” OR “Cymbopogon” OR “Essential Oils” OR “Ferula” OR “Green Tea” OR “Matricaria” OR “Medicinal Plant” OR “Morinda” OR “Myrtus” OR “Neem Oil” OR “Ocimum sanctum” OR “Octenidine” OR “Plant Extracts” OR “Salvia” OR “Officinalis” OR “Satureja” OR “Sodium Hypochlorite” OR “Thymus Plant” OR “Triphala”)
	#3: (“Antibacterial Activity” OR “Antibacterial Effect” OR “Antibacterial Effectiveness” OR “Antibacterial Efficacy” OR “Antibiofilm Effect” OR “Antimicrobial Effect” OR “Antimicrobial Efficacy” OR “Bacteria” OR “Bacterial Count” OR “Bacterial Counting” OR “Bacterial Growth” OR “Bacterial Load” OR “Bacterial Reduction” OR “Biofilm” OR “Colony Forming Units” OR “Disinfection” OR “Enterococcus Faecalis”))
	#4: #1 AND #2 AND #3
Scopus (http://www.scopus.com/)	#1: TITLE-ABS-KEY ( “Dental Pulp Cavity” OR “Dentinal Tubule” OR “Dentition, Permanent” OR “Pulp Canal” OR “Root Canal” OR “Root Dentine” OR “Teeth” OR “Tooth” )
	#2: TITLE-ABS-KEY ( “Irrigant” OR “Irrigating Solution” OR “Irrigation” OR “Rinsing” OR “Root Canal Irrigants” OR “Aloe” OR “Carvacrol” OR “Cetrimide” OR “Chlorhexidine” OR “Chrysopogon” OR “Cinnamomum zeylanicum” OR “Cinnamon” OR “Cymbopogon” OR “Essential Oils” OR “Ferula” ) OR TITLE-ABS-KEY ( “Green Tea” OR “Matricaria” OR “Medicinal Plant” OR “Morinda” OR “Myrtus” OR “Neem Oil” OR “Ocimum sanctum” OR “Octenidine” OR “Plant Extracts” OR “Salvia” OR “Officinalis” OR “Satureja” OR “Sodium Hypochlorite” OR “Thymus Plant” OR “Triphala” )
	#3: TITLE-ABS-KEY ( “Antibacterial Activity” OR “Antibacterial Effect” OR “Antibacterial Effectiveness” OR “Antibacterial Efficacy” OR “Antibiofilm Effect” OR “Antimicrobial Effect” OR “Antimicrobial Efficacy” OR “Bacteria” OR “Bacterial Count” OR “Bacterial Counting” ) OR TITLE-ABS-KEY ( “Bacterial Growth” OR “Bacterial Load” OR “Bacterial Reduction” OR “Biofilm” OR “Colony Forming Units” OR “Disinfection” OR “Enterococcus Faecalis” )
	#4: #1 AND #2 AND #3
LILACS (http://lilacs.bvsalud.org/)	#1: (“irrigant” OR “irrigating solution” OR “irrigation” OR “rinsing” OR “root canal irrigants” OR “aloe” OR “carvacrol” OR “cetrimide” OR “chlorhexidine” OR “chrysopogon” OR “cinnamomum zeylanicum” OR “cinnamon” OR “cymbopogon” OR “essential oils” OR “ferula” OR “green tea” OR “matricaria” OR “medicinal plant” OR “morinda” OR “myrtus” OR “neem oil” OR “ocimum sanctum” OR “octenidine” OR “plant extracts” OR “salvia” OR “officinalis” OR “satureja” OR “sodium hypochlorite” OR “thymus plant” OR “triphala”)
	#2: (“antibacterial activity” OR “antibacterial effect” OR “antibacterial effectiveness” OR “antibacterial efficacy” OR “antibiofilm effect” OR “antimicrobial effect” OR “antimicrobial efficacy” OR “bacteria” OR “bacterial count” OR “bacterial counting” OR “bacterial growth” OR “bacterial load” OR “bacterial reduction” OR “biofilm” OR “colony forming units” OR “disinfection” OR “enterococcus faecalis”)) AND ( db:(“LILACS”))
	#3: #1 AND #2
SciELO (http://www.scielo.org/)	#1: ((irrigant) OR (irrigating solution) OR (irrigation) OR (rinsing) OR (root canal irrigants) OR (aloe) OR (carvacrol) OR (cetrimide) OR (chlorhexidine) OR (chrysopogon) OR (cinnamomum zeylanicum) OR (cinnamon) OR (cymbopogon) OR (essential oils) OR (ferula) OR (green tea) OR (matricaria) OR (medicinal plant) OR (morinda) OR (myrtus) OR (neem oil) OR (ocimum sanctum) OR (octenidine) OR (plant extracts) OR (salvia) OR (officinalis) OR (satureja) OR (sodium hypochlorite) OR (thymus plant) OR (triphala))
	#2: ((antibacterial activity) OR (antibacterial effect) OR (antibacterial effectiveness) OR (antibacterial efficacy) OR (antibiofilm effect) OR (antimicrobial effect) OR (antimicrobial efficacy) OR (bacteria) OR (bacterial count) OR (bacterial counting) OR (bacterial growth) OR (bacterial load) OR (bacterial reduction) OR (biofilm) OR (colony forming units) OR (disinfection) OR (enterococcus faecalis))
	#3: #1 AND #2
Embase (http://www.embase.com)	#1: (‘dental pulp cavity’ OR ‘dentinal tubule’ OR ‘dentition, permanent’ OR ‘pulp canal’ OR ‘root canal’ OR ‘root dentine’ OR ‘teeth’ OR ‘tooth’)
	#2: (‘irrigant’ OR ‘irrigating solution’ OR ‘irrigation’ OR ‘rinsing’ OR ‘root canal irrigants’ OR ‘aloe’ OR ‘carvacrol’ OR ‘cetrimide’ OR ‘chlorhexidine’ OR ‘chrysopogon’ OR ‘cinnamomum zeylanicum’ OR ‘cinnamon’ OR ‘cymbopogon’ OR ‘essential oils’ OR ‘ferula’ OR ‘green tea’ OR ‘matricaria’ OR ‘medicinal plant’ OR ‘morinda’ OR ‘myrtus’ OR ‘neem oil’ OR ‘ocimum sanctum’ OR ‘octenidine’ OR ‘plant extracts’ OR ‘salvia’ OR ‘officinalis’ OR ‘satureja’ OR ‘sodium hypochlorite’ OR ‘thymus plant’ OR ‘triphala’)
	#3: (‘antibacterial activity’ OR ‘antibacterial effect’ OR ‘antibacterial effectiveness’ OR ‘antibacterial efficacy’ OR ‘antibiofilm effect’ OR ‘antimicrobial effect’ OR ‘antimicrobial efficacy’ OR ‘bacteria’ OR ‘bacterial count’ OR ‘bacterial counting’ OR ‘bacterial growth’ OR ‘bacterial load’ OR ‘bacterial reduction’ OR ‘biofilm’ OR ‘colony forming units’ OR ‘disinfection’ OR ‘enterococcus faecalis’)
	#4: #1 AND #2 AND #3
Web of Science (http://apps.webofknowledge.com/)	#1: “Dental Pulp Cavity” OR “Dentinal Tubule” OR “Dentition, Permanent” OR “Pulp Canal” OR “Root Canal” OR “Root Dentine” OR “Teeth” OR “Tooth” (All Fields)
	#2: “Irrigant” OR “Irrigating Solution” OR “Irrigation” OR “Rinsing” OR “Root Canal Irrigants” OR “Aloe” OR “Carvacrol” OR “Chlorhexidine” OR “Cinnamon” OR “Cymbopogon” OR “Essential Oils” OR “Green Tea” OR “Medicinal Plant” OR “Morinda” OR “Neem Oil” OR “Ocimum sanctum” OR “Octenidine” OR “Plant Extracts” OR “Salvia” OR “Officinalis” OR “Satureja” OR “Sodium Hypochlorite” OR “Thymus Plant” OR “Triphala” (All Fields)
	#3: “Antibacterial Activity” OR “Antibacterial Effect” OR “Antibacterial Effectiveness” OR “Antibacterial Efficacy” OR “Antibiofilm Effect” OR “Antimicrobial Effect” OR “Antimicrobial Efficacy” OR “Bacteria” OR “Bacterial Count” OR “Bacterial Counting” OR “Bacterial Growth” OR “Bacterial Reduction” OR “Biofilm” OR “Colony Forming Units” OR “Disinfection” OR “Enterococcus Faecalis” (All Fields)
	#4: #1 AND #2 AND #3
DANS Easy (https://easy.dans.knaw.nl)	#1: (“Dental Pulp Cavity” OR “Dentinal Tubule” OR “Dentition, Permanent” OR “Teeth” OR “Tooth”)
	#2: (“Root Canal Irrigants” OR “Essential Oils” OR “Medicinal Plant” OR “Plant Extracts”)
	#3: (“Antibacterial Activity” OR “Bacterial Reduction” OR “Colony Forming Units” OR “Enterococcus Faecalis”)
	#4: #1 AND #2 AND #3
BDTD (https://bdtd.ibict.br)	#1: (“Dental Pulp Cavity” OR “Dentinal Tubule” OR “Dentition, Permanent” OR “Teeth” OR “Tooth” OR “In Vitro Techniques” OR “In Vitro”)
	#2: (“Root Canal Irrigants” OR “Essential Oils” OR “Medicinal Plant” OR “Plant Extracts”)
	#3: (“Antibacterial Activity” OR “Bacterial Reduction” OR “Colony Forming Units” OR “Enterococcus Faecalis”)
	#4: #1 AND #2 AND #3

• DANS: Data Archiving and Networked Services; BDTD: Biblioteca Digital Brasileira de Teses e Dissertações.

The results were exported to EndNote X9 (Thomson Reuters, Toronto, Canada) software, automatically excluding duplicates. The duplicates undetected by the software were manually deleted. All data were exported to Rayyan QCRI (Qatar Computing Research Institute, Doha, Qatar)³⁵ for study selection. Simultaneously, the grey literature was analyzed manually using Microsoft Word™ 2019 (Microsoft™ Ltd., Washington, USA). Two experienced reviewers (DCF and APM) underwent a calibration exercise by discussing the eligibility criteria until obtaining an adequate level of agreement (Kappa ≥ 0.81), then independently performed all study selection phases.

The two reviewers also independently analyzed the titles and abstracts of studies systematically. The excluded studies were outside the scope and did not meet the eligibility criteria. The articles whose titles agreed with the objectives of this review but did not have abstracts available were fully assessed. Any selection conflict was solved by consulting a third reviewer (FSM) for a final decision. Lastly, the two reviewers obtained and evaluated the full texts of preliminary eligible studies to verify whether they fulfilled the eligibility criteria. The third reviewer was consulted to make a final decision. The rejected articles were registered separately with the reasons for exclusion. All references from the selected studies were thoroughly assessed during the citation search stage to identify any potential eligibility.

Data collection

Two reviewers (DCF and APM) independently extracted the data. The complete assessment of eligible studies allowed the collection of the following: study identification (author, year, study design, funding sources, and country), sample characteristics (types of teeth and sample size), intervention characteristics (natural and chemical solutions and irrigation volume and time), and specific results (evaluation method, bacterial reduction values, and data processing characteristics). Studies obtained in non-English languages were translated.

Risk of bias assessment

The risk of bias assessment used the RoBDEMAT, a recent tool for critically appraising preclinical dental materials research studies.³⁶ This tool provides different bias analysis domains related to planning and allocation (D1), specimen preparation (D2), outcome assessment (D3), and data treatment and outcome reporting (D4). The answer options for each question were “sufficiently reported/adequate,” “insufficiently reported,” “not reported/ inadequate,” or “not applicable.” The two reviewers independently performed all procedures. Any conflict was solved through discussions, and when both evaluators disagreed, they referred to a third reviewer for a final decision. An overall summary risk of bias score was not provided because the tool functions solely as a checklist.

Summary measures and syntheses of results

The present study calculated the difference between the number of CFUs of E. faecalis before and after irrigation with natural and chemical solutions (NaOCl and CHX). Considering that some studies provided raw CFU values and others provided logged CFUs, all raw CFU estimates were log-transformed before calculating the difference so they would be comparable to other estimates. The CFU difference before and after irrigation was included in the network meta-analysis as the effect measure. Studies reporting only the CFU percentage decrease after treatment were excluded.

Three main assumptions of the network meta-analysis were assessed: heterogeneity, consistency, and transitivity. Studies were similar regarding their interventions, clinical characteristics of participants, comparison treatments, and outcome measures. Overall and local consistency were tested. The Walt test statistically tested overall consistency, which was insignificant at a 5% significance level. A local test on loop consistency was also performed using a node-splitting model by Dias et al. (2010),³⁷ and only three of 38 (7.8%) comparisons showed statistically significant results (p < 0.05). Thus, we assumed a consistency model to fit the network meta-analysis, considering that transitivity was present in our study.

Figure 1 depicts the geometry of the network meta-analysis, with each circle representing a different natural or chemical solution. Linked circles represent solutions that were compared. The thickness of each link represents the number of comparisons between the linked NES or CCS. The NaOCl solution, represented by the light blue circle, was the control solution in the network meta-analysis. Although the studies analyzed different NaOCl concentrations, they were grouped and considered a single control group.

Figure 1
Network geometry comparing solutions.

In the network meta-analysis, the CFU reduction of each NES and CCS was compared to the same reduction of the NaOCl solution. Thus, negative values indicate that the solution reduced more CFUs than NaOCl (control group). Considering that all studies included in the network meta-analysis had similar scales, we presented a simple mean difference combining direct and indirect comparisons with respective 95% confidence intervals. Among the advantages of fitting a network meta-analysis is the ability to estimate a comparison between two treatments if directly compared (even though the studies included in the meta-analysis did not formally compare them). Direct and indirect comparisons were organized into a forest plot for better reference. The network meta-analysis also helped estimate the cumulative probability of each natural or chemical solution being the best to reduce the number of CFUs.

All analyses used Stata 17.0 software (StataCorp LLC, College Station, USA).

Results

Study selection

The initial study selection found 16,188 studies, and 8,177 were eliminated after identifying the duplicates. The eligibility criteria were applied, retrieving 35 studies for a full-text assessment. Three studies were excluded after the full-text reading: two assessed antimicrobial activity in sectioned teeth,³⁸,³⁹ and one focused on antimicrobial activity in primary teeth.⁴⁰ Thirty-two studies¹⁴,¹⁵,²⁵-⁵⁴ remained for the qualitative synthesis. The citation search identification method found 38 additional studies. However, three articles⁵⁵-⁵⁷ were selected for the qualitative synthesis, totaling 35 studies for this systematic review. Considering that 28 studies¹⁴,¹⁵,²⁵-²⁹,³⁴-⁴⁵,⁴⁸-⁵⁵,⁵⁷ did not provide the means and standard deviations of the mean percentage (%) of CFU reduction for the irrigation solutions, only seven³⁰-³³,⁴⁶,⁴⁷,⁵⁶ were included in the quantitative synthesis. Figure 2 describes all the details.

Figure 2
PRISMA flowchart showing database searches and the study selection process.

Study characteristics

Table 2 shows the main characteristics of the 35 included studies published between 2009 and 2023 and reported from 10 countries, mainly India¹⁵,³⁰,³²,³⁴,³⁵,³⁷,³⁸,⁴⁴,⁴⁵,⁵¹,⁵²,⁵⁴,⁵⁵ and Brazil.²⁷,²⁹,³¹,³³,³⁹,⁵⁰,⁵³,⁵⁷ The result analysis showed 1,504 teeth/roots exclusively considering the groups used in this review. All eligible studies used single-rooted permanent teeth. Also, all studies used NaOCl in different concentrations as CCS, and 20 used CHX.¹⁴,²⁸-³¹,³³-³⁷,³⁹,⁴²,⁴⁵,⁴⁷,⁴⁹,⁵²,⁵³,⁵⁵-⁵⁷ Forty-three natural solutions were detected during data assessment and detailed in Table 2 with respective concentrations. The most prevalent natural solutions were based on Aloe vera,³⁴,⁴⁸,⁵³,⁵⁴ Azadirachta indica,¹⁵,²⁵,⁵⁴ grape seed extract,²⁹,³¹,⁵⁰ Triphala,³⁰,³²,⁵¹ and oregano.⁴³,⁴⁷,⁵²

Table 2
Summary of the main characteristics of the included studies

Author, Year	Study design	Funding sources (grant number)	Country	Sample size*	Type of teeth	Natural solutions	Conventional solutions
Nosrat et al., 2009 46	Ex vivo experimental model**	Tehran University of Medical Sciences and health services grant (3344)	Iran	20	Single-canalled maxillary incisors and mandibular premolars	0,6% carvacrol	5.25% NaOCl
Samadi et al., 2011 49	In vitro experimental model	NR	Iran	36	Single-canalled teeth	Satureja khuzistanica Jamzad	2.5% NaOCl
							Dornelles-Morgental et al., 2011 33
Dornelles-Morgental et al., 2011 33	Ex vivo experimental model**	NR	Brazil	60	Single-canalled teeth	Apple cider vinegar	2.5% NaOCl
							2% CHX
Xie et al., 2012 56	Ex vivo experimental model**	NR	United States	60	Single-rooted anterior teeth	Berberine	5.25% NaOCl
							1% CHX
							2% CHX
Alves et al., 2013 27	Ex vivo experimental model**	FAPERJ and CNPq	Brazil	44	Maxillary	0.2% farnesol	2.5% NaOCl
					central incisors and canines	20% xylitol
Neelakantan et al., 2013 45	Ex vivo experimental model**	NR	India	24	Single-canalled mandibular premolars	2.5 mg/mL curcumin	3% NaOCl
							2% CHX
Valera et al., 2013 53	Ex vivo experimental model**	FAPESP (2006/57207-2, 2006/57206-6 and 1005/60906-7)	Brazil	60	Single-rooted teeth	Castor oil	2.5% NaOCl
						Glycolic Aloe vera extract	2% CHX gel
						Glycolic ginger extract
Vinothkumar et al., 2013 54	Ex vivo experimental model**	NR	India	72	Single-canalled mandibular premolars	Aloe barbadensis	5.25% NaOCl
						Azadiracta indica
						Curcuma longa
						Myristica fragrans
						Terminalia chebula
Sahebi et al., 2014 48	Ex vivo experimental model**	NR	Iran	40	Single-rooted teeth	80% Aloe vera	2.5% NaOCl
Somayaji et al., 2014 51	In vitro experimental model	NR	India	40	Single-canalled mandibular premolars	Triphala	5,25% NaOCl
						Withania somnifera
Ok et al., 2015 47	In vitro experimental model	NR	Turkey	60	Maxillary central incisors	Oregano extract	5.25% NaOCl
							2% CHX
Cecchin et al., 2015 29	Ex vivo experimental model**	CAPES (BEX 17910/12-9)	Brazil	30	Single-rooted teeth	6.5% grape seed extract	2.5% NaOCl
							2% CHX
Guneser et al., 2016 36	Ex vivo experimental model**	Selcuk University, Scientific Research Project Coordination Center	Turkey	40	Single-rooted teeth	Salvia officinalis	2.5% NaOCl
							5.25% NaOCl
							2% CHX
Gupta-Wadhwa et al., 2016 38	In vitro experimental model	NR	India	35	Single-canalled teeth	Ocimum sanctum	3% NaOCl
						Cinnamomum zeylanicum
						Syzygium aromaticum
Herrera et al., 2016 39	In vitro experimental model	FAPESP (13/23061-5) and CNPq (308162/2014-5)	Brazil	36	Single-rooted premolars	Uncaria tomentosa	2% CHX gel
							5.25% NaOCl
Nourzadeh et al., 2017 14	In vitro experimental model	Zanjan University of Medical Sciences (A-12-804-1)	Iran	90	Mandibular premolars	12.5 mg/mL Eucalyptus galbie	2.5% NaOCl
						6.25 mg/mL Myrtus communis L.	5.25% NaOCl
							0.2% CHX
							2% CHX
Camacho-Alonso et al., 2017 28	In vitro experimental model	NR	Spain	60	Single-rooted teeth	11% propolis	2.5% NaOCl
							0.2% CHX
Kim & Shin, 2017 41	Ex vivo experimental model**	Dankook University	Korea	16	Single-canalled teeth	10% urushiol	6% NaOCl
Zamin, 2017 57	Ex vivo experimental model	NR	Brazil	30	Single-rooted premolars	Cymbopogon martini	5.25% NaOCl
						Tetradenia riparia	2% CHX
Varman et al., 2017 55	In vitro experimental model	NR	India	30	Single-rooted mandibular premolar	Andrographis Paniculata	5.25% NaOCl
							2% CHX
Soligo et al., 2018 50	Ex vivo experimental model**	NR	Brazil	44	Mesiobuccal roots of mandibular molars	50% grape seed extract	6% NaOCl
Goud et al., 2018 34	In vitro experimental model	NR	India	30	Single-canalled mandibular premolars	100% Aloe vera	3% NaOCl
							2% CHX
Divia et al., 2018 32	In vitro experimental model	No funding	India	48	Single-rooted premolars	95% green tea polyphenols	5% NaOCl
						Morinda citrifolia
						100% triphala
Choudhary et al., 2018 30	Ex vivo experimental model	No funding	India	64	Single-rooted teeth	Morinda citrifolia	1% NaOCl
						Triphala	2% CHX
Al Qarni et al., 2019 25	Ex vivo experimental model**	NR	Saudi Arabia	40	Single-rooted anterior and premolar teeth	Azadirachta indica (neem)	2.5% NaOCl
						Salvadora persica (miswak) 15%	1.25% NaOCl
D’aviz et al., 2020 31	Ex vivo experimental model**	Self-funded	Brazil	40	Mesiobuccal roots of maxillary molars	6.5% grape seed extract	5.25% NaOCl
							2% CHX
Gupta et al., 2020 37	In vitro experimental model	NR	India	30	Single-rooted premolars	100mg/mL Vitex negundo Linn.	3% NaOCl
							2% CHX
Panchal et al., 2020 15	In vitro experimental model	No funding	India	60	Single-rooted teeth	Azadirachta indica (neem) Cinnamomum zeylanicum	3% NaOCl
Nagy-Bota et al., 2021 43	Preliminary experimental model	George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Târgu Mureș Research (294/5/14.1.2020)	Romania	7	Single-canalled mandibular premolars	Oregano oil extract	5.25% NaOCl
						Thyme oil extract
						Lemongrass oil extract
						Clove oil extract
						Melaleuca oil extract
Ibrahim et al., 2021 40	In vitro experimental model	No funding	Egypt	18	Permanent incisors or premolar teeth	8% Olea europaea extract	2.5% NaOCl
						2% Morus nigra extract
Teja et al., 2021 52	In vitro experimental model	No funding	India	120	Single-rooted teeth	0.2% oregano oil	3% NaOCl
							2% CHX
Alharbi et al., 2023 26	In vitro experimental model	NR	Saudi Arabia	30	Single-canalled mandibular premolars	Moringa oleifera extract	2.5% NaOCl
Mokhtari et al., 2023 42	In vitro experimental model	No funding	Iran	44	Single-rooted teeth	Zingiber officinale (ginger) extract	5.25% NaOCl
						Origanum majorana (marjoram) extract	2% CHX
Gulati et al., 2023 35	In vitro experimental model	No funding	India	15	Single-rooted teeth	10% Babool	2% CHX
						11% Propolis extract
Navayath et al., 2023 44	In vitro experimental model	No funding	India	16	Single-rooted mandibular incisors	Garlic extract	3% NaOCl

• *Total number of teeth considering only the groups used in this systematic review; **Categorized according to the methodological description; NaOCl: sodium hypochlorite; CHX: chlorhexidine. NR: not reported; Faperj: Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro; CNPq: Conselho Nacional de Desenvolvimento Científico e Tecnológico; Fapesp: São Paulo Research Foundation; Capes: Coordination for the Improvement of Higher Education Personnel

Table 3 summarizes the main intervention characteristics of the included studies. All irrigating procedures were performed manually without ultrasonic activating protocols. The irrigation volume varied between 1 and 30 mL, most often 5 mL.¹⁵,²⁵,²⁷,³⁰,³⁶,⁴⁶,⁵⁰ The irrigation time was between 30 seconds and 20 minutes, most often five²⁸,⁴⁶,⁴⁹ and 20¹⁵,³⁰,⁵⁴ minutes.

Table 3
Summary of the main intervention characteristics and specific results of the included studies.

Author, Year	Intervention groups*	Irrigation volume	Irrigation time	Evaluation method	Results (bacterial reduction)	Data processing characteristics
Nosrat et al., 2009 46	(1) 0.6% carvacrol	5 mL	5 min	Mean percentage of CFU reduction	No significant difference	Kruskal-Wallis and Mann-Whitney U tests (p<0.05)
	(2) 5.25% NaOCl
Samadi et al., 2011 49	(1) 0.31 mg/mL SKJ	+	5 min	Mean percentage of CFU reduction	No significant difference	Kruskal-Wallis test (p<0.05)
	(2) 0.62 mg/mL SKJ
	(3) 2.5% NaOCl
	(4) 2% CHX
Dornelles-Morgental et al., 2011 33	(1) ACV	2 mL	+	Mean number of CFU	(1) ACV < (2), (3), (4)	ANOVA and post hoc Tukey’s tests (p<0.05)
	(2) 2.5% NaOCl
	(3) 2.5% NaOCl + ACV
	(4) 2% CHX
Xie et al., 2012 56	(1) 2 mg/mL BBr	6 mL	2 min	Mean number of CFU	(1) 2 mg/mL BBr < (3) = (2), (4), (5)	One-way analysis and Scheffé test (p<0.05)
	(2) 1 mg/mL BBr + 1% CHX				(2) 1 mg/mL BBr + 1% CHX = (4)
	(3) 5.25% NaOCl
	(4) 1% CHX
	(5) 2% CHX
Alves et al., 2013 27	(1) 0.2% farnesol	5 mL	4 min	Mean number of CFU	(4) 2.5% NaOCl > (1), (2), (3)	Mann-Whitney U test (p<0.05)
	(2) 20% xylitol
	(3) 0.2% farnesol + 20% xylitol
	(4) 2.5% NaOCl
Neelakantan et al., 2013 45	(1) Curcumin	1 mL	30 min	Mean number of CFU	(1) Curcumin = (2), and > (3)	Kruskal-Wallis test (p<0.05)
	(2) 3% NaOCl
	(3) 2% CHX
Valera et al., 2013 53	(1) Castor oil	+	+	Mean number of CFU	(1) Castor oil = (3), (4), (5)	Kruskal-Wallis and Dunn’s tests (p<0.05)
	(2) A. vera				(2) A. vera = (3), and < (1), (4), (5)
	(3) Ginger
	(4) 2.5% NaOCl
	(5) 2% CHX gel
Vinothkumar et al., 2013 54	(1) A. barbadensis	6 mL	20 min	Mean percentage of remaining bacteria	(2) A. indica = (6), and > (3), (4), (5), (1)	One-way ANOVA/Kruskal-Wallis and post hoc Tukey’s HSD tests (p<0.05)
	(2) A. indica
	(3) C. longa
	(4) M. fragrans
	(5) T. chebula
	(6) 5.25% NaOCl
Sahebi et al., 2014 48	(1) A. vera	8 mL	15-20 min	Mean number of CFU	(1) A. vera < (2)	One-way-ANOVA
	(2) 2.5% NaOCl					and t-tests
Somayaji et al., 2014 51	(1) 5.25% NaOCl	5 mL	+	Mean number of CFU	(1) = (4)	One-way ANOVA and Bonferroni’s post hoc test (P<0.001)
	(2) Triphala				(1) > (2), (3)
	(3) Withania somnifera
	(4) Triphala + Withania somnifera
Ok et al., 2015 47	(1) 1% oregano extract	6 mL	2 min	Mean number of CFU	(2) 2% OE = (3), (5) > (4)	Kruskal-Wallis and Mann-Whitney U tests (p<0.05)
	(2) 2% oregano extract				(1) 1% OE < (5)
	(3) 5% oregano extract
	(4) 5.25% NaOCl
	(5) 2% CHX
Cecchin et al., 2015 29	(1) 6.5% GSE	20 mL	2 min	Mean number of CFU	(1) 6.5% GSE = (3) > (2)	One-way ANOVA and Tukey post hoc tests (p<0.05)
	(2) 2.5% NaOCl
	(3) 2% CHX
Guneser et al., 2016 36	(1) S. officinalis	5 mL	3 min	Mean number of CFU	(1) S. officinalis < (2), (3), (4)	Kruskal-Wallis and Mann-Whitney U test (p<0.05)
	(2) 2.5% NaOCl
	(3) 5.25% NaOCl
	(4) 2% CHX
Gupta-Wadhwa et al., 2016 38	(1) O. sanctum	20 mL	6.5 min	Mean number of CFU	(4) 3% NaOCl > (1), (2), (3)	Kruskal-Wallis test (p<0.05)
	(2) C. zeylanicum
	(3) S. aromaticum
	(4) 3% NaOCl
Herrera et al., 2016 39	(1) U. tomentosa	1 mL	+	Percentage and median values of CFU reduction	(1) U. tomentosa = (2), (3)	Friedman, Wilcoxon and Kruskal-Wallis tests (p<0.05)
	(2) 2% CHX gel
	(3) 5.25% NaOCl
Nourzadeh et al., 2017 14	(1) E. galbie	2 mL	+	Mean percentage of CFU reduction	(1) E. galbie < (4)	Kruskal-Wallis and Mann-Whitney U test (p<0.05)
	(2) M. communis L.				(2) M. communis L. < (3), (4), (6)
	(3) 2.5% NaOCl
	(4) 5.25% NaOCl
	(5) 0.2% CHX
	(6) 2% CHX
Camacho-Alonso et al., 2017 28	(1) 11% propolis	3 mL	5 min	Mean number of CFU	No significant difference	ANOVA and Tukey’s test (p<0.05)
	(2) 2.5% NaOCl
	(3) 0.2% CHX
Kim & Shin, 2017 41	(1) 10% urushiol	4 mL	30 sec	Mean number of CFU	No significant difference	Kruskal-Wallis and Mann-Whitney U tests (p<0.05)
	(2) 6% NaOCl
Zamin, 2017 57	(1) 0.5 mg/mL CTB	15 mL	+	Mean percentage of CFU	(5) ≥ (4) ≥ 0.5 mg/mL CTB	Kruskal-Wallis and Dunn (p<0.05)
	(2) 2.5 mg/mL CTB				(5) ≥ (4) ≥ 2.5 mg/mL CTB
	(3) 5.0 mg/mL CTB				(5) ≥ (4) ≥ 5.0 mg/mL CTB
	(4) 5.25% NaOCl
	(5) 2% CHX
Varman et al., 2017 55	(1) 2% CHX	+	+	Mean number of CFU	(3) A. Paniculata = (1) > (2)	One-way ANOVA +
	(2) 5.25% NaOCl					Kruskal-Wallis with Tukey’s post hoc tests
	(3) 0.5% A. Paniculata
Soligo et al., 2018 50	(1) 50% GSE	5 mL	+	Mean percentage of CFU reduction	No significant difference	ANOVA and Tukey HSD post hoc tests (p<0.05)
	(2) 6% NaOCl
Goud et al., 2018 34	(1) 100% Aloe vera	3 mL	1 min	Mean number of CFU	(1) 100% Aloe vera = (2) < (3)	One-way ANOVA and post hoc Games-Howell tests (p<0.001)
	(2) 3% NaOCl
	(3) 2% CHX
Divia et al., 2018 32	(1) 95% green tea	+	+	Mean number of CFU	(3) 100% triphala = (4) > (1), (2)	Kruskal–Wallis and Duncan’s Multiple Range tests (p<0.05)
	(2) Morinda citrifolia				(1) 95% green tea > (2)
	(3) 100% triphala
	(4) 5% NaOCl
Choudhary et al., 2018 30	(1) Morinda citrifolia	5 mL	20 min	Mean number of CFU	(1) Morinda citrifolia < (4), (3)	One-factor Kruskal-Wallis test (p<0.05)
	(2) Triphala				(2) Triphala < (4), (3)
	(3) 1% NaOCl
	(4) 2% CHX
Al Qarni et al., 2019 25	(1) A. indica	5 mL	10 min	Mean number of CFU	(1) A. indica > (2)	Kruskal-Wallis and Mann-Whitney U tests (p<0.05)
	(2) S. persica				(2) S. persica < (4), (1), (3)
	(3) 2.5% NaOCl				(4) 1.25% NaOCl + A. indica > (2)
	(4) 1.25% NaOCl + A. indica
D’aviz et al., 2020 31	(1) 6.5% GSE solution	10 mL	4 min	Mean percentage of CFU reduction	(3) 5.25% NaOCl > (4), (2) > (1)	Two-way ANOVA and Tukey HSD post hoc tests (p<0.05)
	(2) 6.5% GSE gel
	(3) 5.25% NaOCl
	(4) 2% CHX
Gupta et al., 2020 37	(1) V. negundo Linn.	10 mL	+	Mean number of CFU	(2) 3% NaOCl > (1) > (3)	Student’s paired t-test, one-way ANOVA and post hoc Tukey tests (p<0.05)
	(2) 3% NaOCl
	(3) 2% CHX
Panchal et al., 2020 15	(1) A. indica	5 mL	20 min	Mean number of CFU	(2) C. zeylanicum = (3) > (1)	Kruskal-Wallis and Mann-Whitney tests (p<0.05)
	(2) C. zeylanicum
	(3) 3% NaOCl
Nagy-Bota et al., 2021 43	(1) Oregano oil extract	3 mL	+	Mean number of CFU	(1) Oregano, (2) Thyme, (4) Clove = (6), and > (3) > (5)	Semi-quantitative method
	(2) Thyme oil extract
	(3) Lemongrass oil extract
	(4) Clove oil extract
	(5) Melaleuca oil extract
	(6) 5.25% NaOCl
Ibrahim et al., 2021 40	(1) 8% Olea europaea extract	30 mL	7.5 min	Mean and median number of CFU	No significant difference	Kruskal–Wallis One-way
	(2) 2% Morus nigra extract					ANOVA and Dunn’s tests
	(3) 2.5% NaOCl
Teja et al., 2021 52	(1) 3% NaOCl	+	15 min	Zone of inhibition (mm)	(3) 0.2% oregan oil > (1), (2)	One-way ANOVA and post hoc Tukey test (p<0.05)
	(2) 2% CHX
	(3) 0.2% oregan oil
Alharbi et al., 2023 26	(1) 2.5% NaOCl	+	+	Mean number of CFU	(3) 100% M. oleifera extract + 1.25% NaOCl > (1) = (2)	Kruskal-Wallis and Student’s t/ Mann–Whitney
	(2) 100% M. oleifera extract					U-tests (p<0.05)
	(3) 100% M. oleifera extract + 1.25% NaOCl
Mokhtari et al., 2023 42	(1) Ginger oil extract	1 mL	2 min	Descriptive median number of CFU	(1) Ginger extract = (2) Marjoram extract < (3) = (4)	Kruskal-Wallis and Mann-Whitney tests (p<0.05)
	(2) Marjoram chloroform extract
	(3) 5.25% NaOCl
	(4) 2% CHX
Gulati et al., 2023 35	(1) 11% Propolis extract	3 mL	+	Mean number of CFU	(1) 11% Propolis > (3) > (2)	One-way ANOVA and non-related post hoc test (p<0.05)
	(2) 10% Babool
	(3) 2% CHX
Navayath et al., 2023 44	(1) Garlic extract	+	(1) 5 min	Mean number of CFU	(1) Garlic extract < (2)	Chi-square test (p<0.05)
	(2) 3% NaOCl		(2) 2 min

• *Only the groups used in this systematic review were mentioned; +Not mentioned by the author; = not statistically significant difference; < significantly less bacterial reduction; > significantly greater bacterial reduction; NaOCl: sodium hypochlorite; CHX: chlorhexidine; SKJ: Satureja khuzistanica Jamzad; ACV: apple cider vinegar; BBr: berberine; OE: oregano extract; GSE: grape seed extract; CTB: Cymbopogon martini + Tetradenia riparia blend.

Risk of bias assessment

Table 4 demonstrates the critical appraisal of the studies included in this systematic review. Regarding the planning and allocation domain, six of the 35 studies reported a sample size calculation,²⁶,³⁰,⁴⁰,⁴²,⁴⁴,⁵⁴ and no study described sample randomization for experimental procedures. Furthermore, four articles did not sufficiently report the inclusion of a control.¹⁵,⁴⁰,⁵¹,⁵² Two studies showed biases related to sample standardization.⁴⁰,⁵⁶ None of the included articles reported operator blinding. Moreover, relevant information was missing in the data treatment and outcome domain in a high prevalence of studies regarding statistical analysis¹⁴,¹⁵,²⁵,²⁷,³²,³⁴,³⁸,⁴³,⁴⁵,⁴⁶,⁴⁸,⁵¹,⁵³-⁵⁵ and correct outcome reporting.³⁷,⁴⁰,⁴⁶,⁴⁸,⁴⁹,⁵¹,⁵²

Table 4
Risk of bias assessment of included studies.

Author, Year	D1: Bias in planning and allocation			D2: Bias in sample or specimen preparation		D3: Bias in outcome assessment		D4: Bias in data treatment and outcome reporting
	Control group	Randomization of samples	Sample size reporting	Standardization of samples	Identical experimental conditions	Adequate and standardized testing procedures	Operator blinding	Statistical analysis	Correct reporting of outcomes
Nosrat et al., 2009 46	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Not adequate	Adequate	Not reported	Insufficiently reported	Insufficiently reported
Samadi et al., 2011 49	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Insufficiently reported
Dornelles-Morgental et al., 2011 33	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Xie et al., 2012 56	Sufficiently reported	Insufficiently reported	Not reported	Not reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Alves et al., 2013 27	Sufficiently reported	Not reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Insufficiently reported	Sufficiently reported
Neelakantan et al., 2013 45	Sufficiently reported	Not reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Insufficiently reported	Sufficiently reported
Valera et al., 2013 53	Sufficiently reported	Not reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Insufficiently reported	Sufficiently reported
Vinothkumar et al., 2013 54	Sufficiently reported	Not reported	Sufficiently reported	Sufficiently reported	Adequate	Adequate	Not reported	Insufficiently reported	Sufficiently reported
Sahebi et al., 2014 48	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Insufficiently reported	Insufficiently reported
Somayaji et al., 2014 51	Insufficiently reported	Not reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Insufficiently reported	Insufficiently reported
Ok et al., 2015 47	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Cecchin et al., 2015 29	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Guneser et al., 2016 36	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Gupta-Wadhwa et al., 2016 38	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Not adequate	Not reported	Insufficiently reported	Sufficiently reported
Herrera et al., 2016 39	Sufficiently reported	Sufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Nourzadeh et al., 2017 14	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Insufficiently reported	Sufficiently reported
Camacho-Alonso et al., 2017 28	Sufficiently reported	Sufficiently reported	Not reported	Sufficiently reported	Insufficiently reported	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Kim & Shin, 2017 41	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Zamin, 2017 57	Sufficiently reported	Not reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Varman et al., 2017 55	Sufficiently reported	Not reported	Not reported	Sufficiently reported	Insufficiently reported	Adequate	Not reported	Insufficiently reported	Sufficiently reported
Soligo et al., 2018 50	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Goud et al., 2018 34	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Insufficiently reported	Sufficiently reported
Divia et al., 2018 32	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Insufficiently reported	Insufficiently reported	Not reported	Insufficiently reported	Sufficiently reported
Choudhary et al., 2018 30	Sufficiently reported	Insufficiently reported	Sufficiently reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Al Qarni et al., 2019 25	Sufficiently reported	Not reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Insufficiently reported	Sufficiently reported
D'aviz et al., 2020 31	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Gupta et al., 2020 37	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Insufficiently reported
Panchal et al., 2020 15	Not reported	Sufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Insufficiently reported	Sufficiently reported
Nagy-Bota et al., 2021 43	Sufficiently reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Insufficiently reported	Sufficiently reported
Ibrahim et al., 2021 40	Insufficiently reported	Insufficiently reported	Sufficiently reported	Insufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Insufficiently reported
Teja et al., 2021 52	Not reported	Insufficiently reported	Not reported	Sufficiently reported	Adequate	Not adequate	Not reported	Sufficiently reported	Insufficiently reported
Alharbi et al., 2023 26	Sufficiently reported	Insufficiently reported	Sufficiently reported	Sufficiently reported	Adequate	Sufficiently reported	Not reported	Sufficiently reported	Sufficiently reported
Mokhtari et al., 2023 42	Sufficiently reported	Insufficiently reported	Sufficiently reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Gulati et al., 2023 35	Sufficiently reported	Not reported	Not reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported
Navayath et al., 2023 44	Sufficiently reported	Insufficiently reported	Sufficiently reported	Sufficiently reported	Adequate	Adequate	Not reported	Sufficiently reported	Sufficiently reported

Specific study results

Table 3 summarizes the parameters and specific results collected from the included studies. Overall, 21 studies did not present a significant difference in bacterial reduction between NES and CCS.¹⁵,²⁸,²⁹,³¹,³²,³⁴,³⁹-⁴¹,⁴³,⁴⁵-⁴⁷,⁴⁹-⁵¹,⁵³-⁵⁷ NES (oregano, grape seed extract, and Andrographis Paniculata) had better antimicrobial activity against NaOCl in four studies.²⁹,⁴⁷,⁵²,⁵⁵ Two studies showed higher antimicrobial efficacy for curcumin⁴⁵ and propolis³⁵ compared to CHX. When comparing CCS, NaOCl had better antimicrobial activity than CHX in three studies.³¹,³⁷,⁴⁵ Meanwhile, CHX was more satisfactory than NaOCl in five studies.²⁹,³⁴,⁴⁷,⁵³,⁵⁵ Overall, eight studies showed no difference in effectiveness between NaOCl and CHX.¹⁴,²⁸,³³,³⁶,⁴²,⁴⁹,⁵³,⁵⁶

Network meta-analysis

Only seven studies provided sufficient information for inclusion in the network meta-analysis, jointly comparing directly and indirectly 16 types of NES or CCS. The thickness of the lines evidenced that most studies compared 2% CHX with NaOCl (Figure 1). There was no indication of inconsistency in the analyzed mapping.

Eight of the 15 NES or CCS compared to NaOCl were significantly less effective than the control solution – presenting positive mean difference values. CFUs increased from 1.04 (95%CI: 0.77–1.31) for Triphala to 1.91 (95%CI: 0.69–3.14) for green tea polyphenols compared to NaOCl. The remaining seven NES or CCS had statistically similar CFU reductions to NaOCl, as these included the null value in their 95% confidence intervals (Figure 3). The highest CFU reduction compared to NaOCl occurred with 0.6% carvacrol (mean difference = -0.47) but without evidence of a statistically significant difference compared to the reference group (95%CI:-6.55–5.61). Table 5 provides a detailed description of the mean differences between each solution and the reference treatment.

Figure 3
Mean difference of log (CFU/mL) reduction between NES or CCS and NaOCl (control group).

Table 5
Mean difference of CFUs and respective 95% confidence intervals between each solution and NaOCl.

Solution	MD	95%CI
NaOCl	Ref.	-
0.6% Carvacrol	-0.47	-6.55–5.61
ACV + 2.5% NaOCl	-0.28	-0.76–0.20
2% CHX	-0.07	-0.32–0.17
Berberine + 1% CHX	0.12	-0.34–0.58
2% oregan extract	0.17	-0.52–0.86
5% oregan extract	0.34	-0.35–1.02
1% CHX	0.48	-0.35–1.31
Triphala	1.04	0.77–1.31
1% oregan extract	1.45	0.25–2.66
6.5% GSE solution	1.47	0.74–2.20
Morinda citrifolia	1.48	1.16–1.80
ACV	1.80	0.79–2.81
6.5% GSE gel	1.84	1.38–2.30
Berberine	1.89	1.53–2.26
Green tea polyph.	1.91	0.69–3.14

• *Negative values indicate reduction, positive values indicate increase compared to NaOCl.
• NaOCl: sodium hypochlorite; CHX: chlorhexidine; ACV: apple cider vinegar; GSE: grape seed extract.

Based on the cumulative probability plot in Figure 4, apple cider vinegar combined with 2.5% NaOCl (ACV + 2.5% NaOCl) was the fastest to reach 100%, presenting the highest probability of being the best solution to reduce CFUs after irrigation. The second-best solution was 2% CHX, followed by NaOCl (control).

Figure 4
Ranked probability of each solution being the best/worst to reduce the number of CFUs.

Discussion

This systematic review assessed the antimicrobial efficacy of natural endodontic solutions (NES) and conventional chemical solutions (CCS) against Enterococcus faecalis because that is the most prevalent microorganism in persistent root canal infections.³⁷ The null hypothesis of this review was partially accepted, as carvacrol and oregano extracts had statistically similar CFU reductions to sodium hypochlorite (NaOCl). Likewise, apple cider vinegar and berberine showed equivalent results to NaOCl when associated with CCS. Nevertheless, other NES assessed in this study were less effective than NaOCl.³⁰-³³,⁴⁶,⁴⁷,⁵⁶ Although not comprising the primary outcome of this systematic review, the authors suggest that combining NES with CCS might be beneficial as a complementary solution for eliminating E. faecalis.

Several NaOCl concentrations have been used for root canal irrigation. The most used concentration is 2.5% NaOCl because it achieves an adequate bacterial effect while cytotoxicity remains low.²⁸ However, considering that volume and frequency of irrigation influence the antimicrobial efficacy of NaOCl more than its concentration,⁵⁸,⁵⁹ this review considered in the meta-analysis all NaOCl concentrations as a single control solution, although higher NaOCl concentrations might increase cytotoxicity.²⁸ Chlorhexidine (CHX) has represented an alternative endodontic solution due to drawbacks from NaOCl. The advantage of CHX is the substantivity that ensures a durable effect even if the solution concentration decreases in the oral environment.²⁸,⁵³ CHX concentrations of 0.2%-2% show antimicrobial effects but cannot dissolve the organic tissue.²⁸,²⁹,⁵³,⁵⁸ This systematic review corroborates the literature data on NaOCl and CHX being the preferred solutions for root canal disinfection.⁵⁸

Natural extracts have been extensively used in endodontics due to their antibacterial and antifungal properties.⁶⁰ Studies have demonstrated that some of these extracts are effective against microorganisms commonly found in the oral cavity, including E. faecalis.⁶⁰,⁶¹ Their action mechanism is based on their lipophilic components, which inhibit ATPase bacterial activity and increase the non-selective permeability of the bacterial cell membrane. Besides inhibiting bacterial colonization, this mechanism makes bacteria more sensitive to antimicrobial agents. Moreover, NES may disrupt the lipidic membrane, causing the death of microorganisms.⁴⁶,⁶²

Among all natural solutions included in this review, 0.6% carvacrol and 2% and 5% oregano extracts were the NES with similar or superior antimicrobial potential to NaOCl when evaluated without association with CCS.⁴⁶,⁴⁷ Carvacrol has a broad spectrum of antibacterial activity and inhibits ATPase activity, favoring permeability to microbial agents.⁴⁶ It also shows anti-inflammatory effects by suppressing prostaglandin production and inhibiting neutrophilic elastase enzyme due to a hydroxyl group in its chemical composition, representing the most bioactive molecule to inhibit this enzyme activity.⁴⁶,⁶³ However, 0.6% carvacrol should not be considered similar to NaOCl due to the uncertainty of data from the study included in the meta-analysis,⁴⁶ showing a high standard deviation. The literature describes that carvacrol is the main constituent of oregano essential oil, corresponding to 68% of its composition.⁶⁴ However, it is impossible to assure that the similar antibacterial efficacy of oregano with NaOCl is due to carvacrol in its composition because the authors did not describe the extract vehicle.⁴⁷ Otherwise, the qualitative analysis of this review demonstrated superior or similar results for oregano essential oil based on carvacrol compared to NaOCl and CHX.⁴³,⁵²

The meta-analysis showed that apple cider vinegar + 2.5% NaOCl and berberine + 1% CHX were the NES with similar or higher CFU count reduction than NaOCl. Apple cider vinegar is an acid solution that potentially removes the smear layer and improves immunologic response. Maleic acid (C₄H₆O₅) is the main constituent of apple cider vinegar and may enhance the immune system by being present in the Krebs cycle.⁶⁵ Berberine is an alkaloid extracted from medicinal plants, and it possesses a broad antimicrobial spectrum against bacteria, fungi, and viruses with low toxicity.⁵⁶ However, the antimicrobial effect of apple cider vinegar + 2.5% NaOCl and berberine + 1% CHX as root canal irrigants may be due to the association of these extracts with NaOCl or CHX. When evaluated without an association, those natural solutions did not show similar antimicrobial efficacy to CCS in both studies. Even isolated berberine and apple cider vinegar showed the potential to induce cell death by reducing CFU count and were significantly less effective than other solutions (Figure 3).

Associating NaOCl with acidic solutions is not necessarily beneficial due to the need for high NaOCl concentrations to reach a neutral pH solution.³³ However, the quantitative analysis of this review showed that apple cider vinegar combined with 2.5% NaOCl (ACV + 2.5% NaOCl) was the fastest solution to achieve a 100% probability of CFU count reduction. Considering that apple cider vinegar has adequate properties for root canal cleaning, such as smear layer removal and antibacterial potential, the authors encourage further studies to use this substance, possibly associating apple cider vinegar with a solution used for endodontic treatment. Conversely, apple cider vinegar alone showed some of the worst probability of reaching total microorganism elimination.

Curcumin showed similar antimicrobial efficacy to NaOCl and superior to CHX in young biofilms. In mature biofilms, curcumin exhibited higher bacterial elimination than CHX.⁴⁵ This enhanced efficacy may stem from curcumin’s ability to penetrate and dismantle the extracellular polysaccharide matrix and bacteria akin to NaOCl. This matrix is a nutrient source and conducive surface for increased cell growth, crucial for bacterial biofilm maintenance.⁶⁶,⁶⁷ Although apple cider vinegar, berberine, and curcumin have demonstrated similar or even superior antimicrobial activity, it is worth noting that these compounds may not necessarily have an intervention effect comparable to NaOCl or CHX. Despite significant differences, their relevance regarding antimicrobial effects may not be conclusive and requires a more consistent evaluation.

The limitation of this review process stems from the assessment of preclinical studies. As NaOCl and CHX are extensively used and established as standard irrigant solutions in the literature,⁵⁹ clinical studies evaluating the antimicrobial effect of natural solutions as endodontic irrigants are scarce. This information allows us to consider natural-based irrigant solutions as complementary agents instead of substitutes in combating endodontic microbiota. The present review reinforces this perspective, as the combination of apple cider vinegar + 2.5% NaOCl exhibited superior efficacy against E. faecalis compared to 2.5% NaOCl alone.

The results obtained in this network meta-analysis should be interpreted with caution, primarily due to the limitations of the gathered evidence. Although RoBDEMAT is designed for assessing bias in preclinical studies, it should be used carefully. This tool combines bias assessment with reporting issues, potentially hindering the distinction between study limitations from reporting deficiencies and related to study design biases. This integration might create confusion in decision-making. Information such as sample size calculation, operator and assessment blinding, allocation concealment, and sample randomization might introduce biases. The risk of bias assessment demonstrated a lack of reporting, mostly of sample size calculation, sample randomization, and operator blinding. The latter may be limited in this study type mainly due to the odor and color of irrigating solutions, such as NaOCl and root canal irrigants based on alcoholic vehicles.¹⁵ Editors, peer-reviewers, and authors of research articles must endorse these limitations, collaboratively aiming at adherence to reporting guides.

Additionally, individual biases make it challenging to reach a definitive conclusion regarding the antimicrobial efficacy of natural endodontic irrigants. This systematic review only examined monospecies biofilms, thus failing to capture the complexities of diverse microbiomes and intricate interactions associated with endodontic infections. Consequently, extrapolating the findings to clinical practice is limited. However, our results encourage the formulation of new studies, including randomized clinical trials, to confirm our findings regarding CFU bacterial count after irrigation with NES and provide reliable data for clinical application.

Conclusion

The obtained evidence shows that, although natural endodontic solutions (NES) did not overcome NaOCl, they have antimicrobial potential against Enterococcus faecalis. Although the evidence was based on ex vivo studies, this review provides the preclinical antibacterial efficacy of NES. All studies exhibited reporting flaws regarding bias analysis, so further standardized studies must be conducted to confirm or refute our findings.

Acknowledgments

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001. We are thankful for the support of Conselho Nacional de Desenvolvimento Científico e Tecnológico - Brazil (CNPq) and of Fundação de Amparo à Pesquisa do Estado de Minas Gerais - Brazil (FAPEMIG).

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Publication Dates

• Publication in this collection
  20 Dec 2024
• Date of issue
  2024

History

• Received
  20 Sept 2023
• Accepted
  29 Apr 2024
• Received
  10 June 2024