Azadirachta indica A. juss, Morinda citrifolia L. and Triphala as herbal endodontic irrigants: A scoping review.

Background: The success of the root canal treatment depends on the complete elimination of the microflora, biofilms and smear layer from the pulp space. A wide variety of chemical endodontic irrigants are available to achieve disinfection and thorough debridement besides mechanical means. However, detrimental properties such as allergic potential, cytotoxicity, antimicrobial resistance and safety concerns have intrigued researchers over the years to look for safer options. Aim: The review is aimed at providing comprehensive information of the studies evaluating the efficacy of Azadirachta indica A. juss (A. indica), Morinda citrifolia L. (M. citrifolia) and Triphala (fruits of Emblica officinalis Gaertn., Terminalia chebula (Gaertn.) Roxb. and Terminalia belerica Retz.) as herbal endodontic irrigants. Materials and methods: The literature review was conducted using indexed databases (PubMed, Google Scholar, Cochrane) electronically for publications in peer-reviewed journals for relevant articles evaluating the efficacy of A. indica, Triphala, M. citrifolia as endodontic irrigant from the year 1985-March 2020.
Results: A total of 58 studies were identified for full text reviewing after duplicate removal and screening title and abstracts. A total of 32 studies were included and processed for data extractions.
Conclusion: Various in-vitro/in-vivo studies utilizing these herbal irrigants have documented promising results and hold the potential to replace chemical endodontic irrigants in routine practice but more preclinical and clinical trials are needed to substantiate these results before they can conclusively be recommended as intracanal irrigating solutions. Copyright:

Introduction

In recent years, dentistry has evolved from being sacrificial to a saviour. The proponents of the dental profession have emphasized on saving a tooth diagnosed with dental caries than to resort to extraction. Root canal therapy has been the ultimate game-changer. Microorganisms have been recognized since long as the primary etiologic factors in the development of pulp and periapical lesions.[123] Elimination of microorganisms from the root canal system is a challenging task. The process of endodontic therapy involves important steps of biomechanical preparation along with effective cleaning of the root canal space to free it from bacteria and debris to allow hermetic sealing of the root canal. Various in-vitro and clinical evidence have shown that mechanical instrumentation alone is inadequate for complete elimination of bacteria from the root canal as it cannot reach every portion of the root canal system due to its complex anatomy, therefore, the use of irrigation forms an important step in this protocol[45] Irrigants can augment mechanical debridement by flushing out debris, dissolving tissue and disinfecting the root canal system thus further reducing the bacterial count, bio-films and lubricating the root canal.[6] The ideal irrigant should be bactericidal, can dissolve necrotic tissue, remove the smear layer besides lubricating the canal and not irritate to healthy tissues.[7]

Studies have shown that the use of normal saline along with mechanical preparation of the root canal space does not provide predictable disinfection.[89] The most commonly used irrigants in dentistry are sodium hypochlorite (NaOCl), chlorhexidine (CHX), ethylenediaminetetracetic acid (EDTA), citric acid, maleic acid, etc. However, they have been found to be responsible for altering the root canal microbiota, causing allergic reactions, decrease in dentinal microhardness, dentinal erosions, and biocompatibility issues.[101112] These drawbacks coupled with resistant strains have prompted researchers to look for herbal alternatives.

The ancient folk medicines have focused on the use of herbs in both eastern and western traditional medicine. Recently, dental treatment has seen a growing trend to seek natural remedies and this approach is termed phyto-therapeutics, phyto-dentistry, or ethno-pharmacology. Indian tradition has a rich heritage of medicinal plants and herbs which have been used since time immemorial. Herbal alternatives are easily available, cost-effective, have increased shelf-life, low toxicity, and exhibit lack of microbial resistance.[13] These medicinal plants and herbs are rich in phytochemicals and are known for their analgesic, anti-inflammatory, antioxidant, anxiolytic, sedative, and antibiotic properties and have been widely used as endodontic irrigants. Herbal irrigants have been found to be bio-friendly with negligible side effects and have shown promising results in the removal of the smear layer which acts as a deterrent in successful root canal therapy.[1415] This paper aimed at summarizing the existing knowledge and providing a literature review of studies evaluating the efficacy of three herbs; Azadirachta indica A. juss, Morinda Citrifolia, and Triphala as herbal irrigating solutions.

Materials and methods

The literature review was started with a defined background to review the studies involving interventions using A. indica A. juss, M. citrifolia, and Triphala as herbal endodontic irrigants. The indexed databases PubMed, Google Scholar, and Cochrane were electronically searched for articles published in English in peer-reviewed scientific journals from the year 1985-March 2020.

Inclusion criteria

In-vitro, ex-vivo and in-vivo comparative studies evaluating antibacterial activity of A. indica A. juss, M. citrifolia L., Triphala (Emblica officinalis Gaertn., Terminalia chebula (Gaertn.) Roxb. and Terminalia belerica Retz.) as herbal root canal irrigants and chemical endodontic irrigants such as CHX and NaOCl.

The studies were selected using the above-described protocol, titles, and abstracts. Reference lists of relevant studies were hand-searched to identify studies that might have been missed in the previous step. After screening title and abstract and duplicate removal a total of 58 articles were selected for full-text screening. Full-texts of pertinent studies were read independently of which 26 were excluded because they did not correspond with the inclusion criteria. Some articles were categorized as review articles [search strategy Flowchart 1].

Flowchart 1

Search Strategy

Results

Study selection

After the screening of title and abstracts and duplicate removal, a total of 58 studies were identified for full-text reviewing and assessed for eligibility. Twenty-six studies did not meet the inclusion criteria and were excluded after full-text reviewing. Thirty-two studies (13 in-vitro and 15 ex-vivo and 4 in-vivo studies) were identified for inclusion in the review.

The collected information was tabulated based on:

In-vitro studies, ex-vivo studies, in-vivo studies on A. indica A. juss

Studies evaluating Triphala and M. citrifolia as endodontic irrigant

Studies evaluating the effect of herbal irrigants on smear layer

Studies evaluating the effect of herbal irrigants on biofilms.

The studies on the comparative evaluation of methanolic and ethanolic extracts of A. indica A. juss (Neem), NaOCl and CHX as root canal irrigants were tabulated [Table 1] The in-vitro studies[16171819202122232425] compared the antimicrobial activity of ethanolic and methanolic extracts of A. indica A. juss (Neem) to varying concentrations of NaOCl (2%, 2.5%, 3%, 5%) and CHX (0.25%, 0.5%, 2%) against Enterococcus faecalis (E. faecalis) and Candida albicans (C. albicans). In all of these studies, Neem performed equally well as NaOCl without showing any toxicity as compared to NaOCl when used at lower concentrations. One study by Jose et al.[24] however differed and concluded that 2.5% NaOCl was more effective against C. albicans as compared to ethanolic extract of Neem (25 μg/ml), but the toxicity of NaOCl was found to be higher. Table 2a and b summarizes the ex-vivo and in-vivo studies evaluating A. indica A. juss as an endodontic irrigant.

Table 1

In vitro studies on irrigant Azadirachta indica

Author	Year	Test/control/flora	Zone of inhibition (mm)			Results/conclusions
Híjar et al.[16]	2018	A. indica/CHX/saline/E. faecalis	Irrigant	24 h	48 h	A. indica demonstrated an antibacterial effect against E. faecalis without any toxicity when used at low concentration
			A. indica (25 µg/ml methanolic extract)	32-34 mm	35-42 mm
			2% CHX	17-20 mm	44-46 mm
			Saline	0	0
			P	<0.01
Sinha et al.[17]	2017	A. indica/CHX/saline/NaOCl/E. faecalis	A. indica (25 µg/ml ethanolic extract)	14.42 mm		A. indica yielded antibacterial activity equivalent to 2% CHX or 5% NaOCl against E. faecalis
			2% CHX	14.37 mm
			5% NaOCl	14.5 mm
			Saline	0 mm
			P	<0.05
Chandrappa et al.[18]	2015	A. indica/CHX/saline/E. faecalis	A. indica (25 µg/ml ethanolic extract)	26.4 mm		A. indica extract has shown significant inhibitory effect against E. faecalis compared to CHX
			2% CHX	16.9 mm
			Saline	0 mm
			P	0.001
Hegde et al.[19]	2013	A. indica/CHX/saline/NaOCl/E. faecalis/C. albicans	Irrigant	E. faecalis	C. albicans	Efficacy of A. indica extract and NaOCl was compared against E. faecalis and C. albicans and it was concluded that neem extract had significant antimicrobial activity against these organisms
			A. indica (25 µ g/ml ethanolic extract)	21.33 mm	15.33 mm
			5% NaOCl	17.67 mm	12.67 mm
			Ethanol	0	0
			P	<0.05
Mustafa et al.[20]	2016	A. indica/CHX/saline/E. faecalis	Irrigant	E. faecalis		A. indica leaf extract shows comparable zones of inhibition with that of CHX and NaOCl
			A. indica (25 µg/ml ethanolic extract)	17.19 mm
			3% NaOCl	19.22 mm
			2% CHX	20.45 mm
			Saline	0
			P	<0.05
Srinidhi et al.[21]	2014	A. indica/CHX/saline/NaOCl/E. faecalis/C. albicans	A. indica (25 µg/ml ethanolic extract)	16.33 mm		Efficacy of A. indica leaf extract against C. albicans is comparable to 3% NaOCl. Further, the action of neem extract was significantly better than that of 2% CHX
			3% NaOCl	19.66 mm
			2% CHX	8.66 mm
			Saline	0
			Ethanol	8.33 mm
			P	<0.011
Ghonmode et al.[22]	2013	A. indica/NaOCl/saline/E. faecalis	A. indica (25 µg/ml ethanolic extract)	19.57 mm		A. indica leaf extract has a significant antimicrobial effect against E. faecalis
			3% NaOCl	16.34 mm
			Saline	0 mm
			Ethanol	0
			P	<0.05
Bohora et al.[23]	2010	A. indica/CHX/saline/ethanol/E. faecalis	Irrigant	E. faecalis	C. albicans	A. indica leaf extract has a significant antimicrobial effect against E. faecalis and C. albicans
			A. indica (25 µg/ml ethanolic extract)	20.0 mm	7.1 mm
			2% NaOCl	17.0 mm	6.0 mm
			Ethanol	0	0
			P	<0.05	>0.05
Jose et al.[24]	2015	A. indica/NaOCl/E. faecalis/C. albicans	Irrigant	E. faecalis	C. albicans	A. indica leaf extract was found to be less effective against Candida and E. faecalis as compared to NaOCl but are less tissue toxic when compared with NaOCl
			A. indica (25 µg/ml ethanolic extract)	0.25 mm	0.76 mm
			2.5% NaOCl	4.3 mm	4.2 mm
			P	<0.01	<0.01
Damre et al.[25]	2015	A. indica/NaOCl/E. faecalis	Irrigant	E. faecalis		A. indica showed highest zone of inhibition against E. faecalis followed by NaOCl
			A. indica (25 µg/ml ethanolic extract)	4 mm
			5% NaOCl	3 mm
			Ethanol	0 mm

A. indica: Azadirachta indica, E. faecalis: Enterococcus faecalis, C. albicans: Candida albicans, NaOCl: Sodium hypochlorite, CHX: Chlorhexidine

Table 2a

Ex vivo studies on Azadirachta indica

Author	Irrigant	Sample	Zone of inhibition (mm)				Results/conclusions
Bhargava et al., 2015[26]	A. indica/Triphala/NaOCl/saline	60 infected pulp of primary molar from children aged 5-7 years	Quantity of irrigant	A. indica (Neem)	3% NaOCl	Triphala 60 mg/ml in 10% DMSO	3% NaOCl and A. indica showed maximum antibacterial activity against the endodontic microflora (P>0.05) followed by Triphala
			50 µl	13.8	15.8	4.6
			100 µl	15.4	17	5
			150 µl	16.6	19	6.8
Sundaram et al. 2016[27]	A. indica/NaOCl/E. faecalis/C. albicans	60 primary molars (5-7 years) indicated for pulpectomy	Irrigants		E. faecalis	C. albicans	NaOCl has maximum antimicrobial activity when compared with A. indica P<0.001
			5.25% NaOCl		19.37	19.17
			A. indica (25 µg/ml ethanolic extract)		1.6	0
Ambhore et al., 2017[28]	A. indica/NaOCl/E. faecalis/C. albicans	E. faecalis/C. albicans isolated from patients with chronic periapical infection	Irrigants		E. faecalis	C. albicans	5% NaOCl solution and A. indica leaves extract has significant antimicrobial effect against E. faecalis and C. albicans with neem being possessing a significant property of biocompatibility to oral and periapical tissues
			A. indica 10% ethanolic extract		7.2	6.5
			5% NaOCl		8.8	7.2
			Saline		0	0
			P		<0.001	<0.001

A. indica: Azadirachta indica, E. faecalis: Enterococcus faecalis, C. albicans: Candida albicans, NaOCl: Sodium hypochlorite, CHX: Chlorhexidine, DMSO: Dimethyl sulfoxide

Table 2b

In vivo studies on Azadirachta indica (Neem)

Author/Year	irrigant	Sample	Result					Result
Dutta and Kundabala 2014[29]	A. indica/NaOCl/CHX/endodontic flora	36 anterior single-rooted teeth with periapical radiolucency indicated for root canal treatment	Irrigants	Percentage reduction in microorganisms				Neem irrigant has demonstrable anti-microbial efficacy and can be considered for endodontic use. A combination of NaOCl and ethanolic neem leaf extract is synergistic antimicrobially
			2.5% NaOCl	84.86
			0.2% CHX	69.17
			A. indica (ethanolic extract)	74.09
			NaOCl+A. indica	100
			CHX+A. indica	86.83
			Saline	4.55
Podar et al., 2015[30]	MCJ/NaOCl/A. indica	32 permanent maxillary incisors, canines with aymptomatic apical periodontitis and pulpal necrosis	Irrigants	Preaerobic CFU’s	Preanaerobic CFU’s	Postaerobic CFU’s	Postanaerobic CFU’s	No difference in antimicrobial efficacy of M. citrifolia, A. indica and 3% NaOCl. P>0.05 All the test irrigants caused a significant reduction in the mean CFU counts of aerobic and anaerobic bacteria versus control P<0.05
			6% M. citrifolia	59	58.9	18.6	14.0
			A. indica (C preparation GMP Krishna)	74	62.4	43	24.2
			3% NaOCl	105.8	84.7	81.6	57.3
			Control	128	69.7	124.5	82.0
Kaur et al., 2018[31]	A. indica/NaOCl/CHX/endodontic flora	125 patients 18-45 years with definite periapical radiolucency	Irrigants	Preaerobic CFU’s	Postaerobic CFU’s	Preanaerobic CFU’s	Postanaerobic CFU’s	A. indica was better than NaOCl but it was statistically nonsignificant AI showed better results than CHX gluconate which was statistically significant
			A. indica	60.18	1.71	75.57	1.44
			3% NaOCl	79.86	0.51	83.72	0.39
			2% CHX	78.36	20.91	108.38	28.71
			Distilled water	124.86	105.21	107.10	96.48
			P	<0.001	<0.001	<0.052	<0.01

M. citrifolia: Morinda citrifolia, AI: Artificial intelligence, A. indica: Azadirachta indica, NaOCl: Sodium hypochlorite, CHX: Chlorhexidine, GMP: Good Manufacturing Practice. CFU’s: Colony Forming Units

Majority of ex-vivo studies evaluating the antimicrobial efficacy of A. indica A. juss in different solvents (ethanolic extract and 10% dimethyl sulfoxide [DMSO]) showed significant antimicrobial effect against E. faecalis and C. albicans besides being biocompatible to oral and periapical tissues.[2628] Although a single study by Sundaram et al.[27] marked 5.25% NaOCl significantly more effective than ethanolic extract of A. indica against E. faecalis. Three in-vivo studies[293031] investigating the antimicrobial efficacy of ethanolic extract and commercial preparations of A. indica A. juss (GMP Krishna, India) against endodontic aerobic and anaerobic bacteria concluded that A. indica had significant antimicrobial efficacy against endodontic microflora and was equally efficacious or better than 2.5%, 3% NaOCl and 2% CHX. Furthermore, the combination of NaOCl and ethanolic A. indica leaf extract was found to be anti-microbially synergistic.

Table 3 summarizes the in-vitro, ex-vivo and in-vivo studies evaluating the comparative efficacy of M. citrifolia and Triphala with NaOCl, CHX and A. indica A. juss. In the majority of these studies, Triphala and M. citrifolia had shown significant antibacterial effects against E. faecalis and C. albicans. Various studies showed M. citrifolia juice (MCJ) to have antimicrobial efficacy equivalent to that of 1% NaOCl and also Triphala in DMSO comparable to that of 2.5% and 5% NaOCl.[3233343536] Although in studies by Chaitanya et al.[37] 3% NaOCl scored better than 6% MCJ and Saxena et al.[38] mean zone of inhibition was found to be higher for 2.5% NaOCl than the ethanolic extract of Triphala.

Table 3

Triphala and Morinda citrifolia

Author/year	Irrigant	Material and method	Irrigant		Results	conclusion
Satti et al. 2019[32]	Triphala/NaOCl/distilled water/E. faecalis/Ex vivo	Agar well diffusion method/M. Hilton Agar ×24 h	Triphala 50 mg/ml (aqueous extract)		20.33 mm	Triphala showed the highest antimicrobial efficacy followed by NaOCl. Triphala and showed no cytotoxic effect as compared to NaOCl mean difference of cell viability was 89.67 for human PDL fibroblasts
			NaOCl 3%		14.67 mm
			Distilled water		0
			P		<0.001
Choudhary et al.[33]	M. citrifolia/Triphala/E. faecalis/C. albicans/Ex vivo	84 single-rooted teeth inoculated with E. faecalis/C. albicans/irrigated with test solutions	Percentage reduction CFU’s after 72 h	E. faecalis	C. albicans	The overall antimicrobial effects of different irrigants were maximum for CHX, whereas MCJ and Triphala juice also showed significant reductions. 1% NaOCl was not effective in completely removing the two tested organisms. The herbal irrigants hold the promise of becoming efficient irrigants and warrant further research
			MCJ (C. preparation basic Ayurveda GZD, India)	−73.7	−78.2
			Triphala juice (C. preparartion basic Ayurveda GZD, India)	−77.7	−79.5
			1% NaOCl	−87.05	−94.4
			2% CHX	−88.2	−94.5
			Distilled water	−76.9	−77.8
			P	<0.001	<0.001
Divia et al.[34]	M. citrifolia/triphala/NaOCl/Ex vivo	60 extracted teeth inoculated with E. faecalis Irrigated with test solutions	Irrigant	Pretreatment	Posttreatment	Triphala and MC showed a significant antibacterial effect against E. faecalis. Among the tested agents, Triphala was found to be as efficacious as NaOCl
			6% M. citrifolia	940.5	158.17
			5% NaOCl	939.17	0.67
			Triphala (organic India ltd. commercial preparation)	940.92	15.92
			Distilled water	948.3	944.5
			P	<0.05	<0.001
Chandwani et al., 2017[35]	M. citrifolia/NaOCl/in vivo	60 deciduous teeth indicated for endodontic treatment	Irrigant	Preoperative	Postoperative	There was no statistically significant difference observed in the antimicrobial efficacy between 1% NaOCl and MCJ
			7% MCJ	4.78	2.39
			1% NaOCl	4.77	2.34
			P		<0.001
Shakouie et al., 2014[36]	Triphala (IMPCOPS ltd. In 10% DMSO)/NaOCl/In vitro	200 plates of BHI agar inoculated with E. faecalis×24 h	Irrigants		Zone of inhibition	Triphala exhibited better antimicrobial activity against E. faecalis compared to 0.5% and 1% NaOCl (P<0.05)
			0.5% NaOCl		4.6
			1% NaOCl		6.3
			2.5% NaOCl		7
			5% NaOCl		7.6
			5 mg/ml Triphala		7.3
Chaitanya et al., 2016[37]	M. citrifolia/NaOCl/E. faecalis/In vitro	Agar well diffusion method/M. Hilton Agar ×24 h	Irrigants		Zone of inhibition	NaOCl (3%) showed maximum antibacterial activity against E. faecalis, followed by M. citrifolia P<0.05
			6% MCJ		20.25mm
			3% NaOCl		27.33 mm
Saxena et al., 2015[38]	M. citrifolia (Vestige india)/triphala (Vindhya herbals)/A. indica (Vindhya herbals/NaOCl/E. faecalis/In vitro	Agar well diffusion method/BHI agar incubated for 24 h	Irrigants		Zone of inhibition	Mean zone of inhibition in descending order was found as NaOCl > AI > Triphala > MC > ethanol P>0.05
			M. citrifolia (20 g/25 ml of ethanol)		02.2 mm
			Triphala (20 g/25 ml of ethanol)		05.1 mm
			A. indica (20 g/25ml of ethanol)		08.8 mm
			2.5% NaOCl		22.0 mm
			Ethanol		02.2 mm
Thomas et al., 2017[39]	Triphala (Nilogram India pvt)/NaOCl/Diode laser/Randomised controlled trial/Ex vivo	49 single rooted human primary teeth reduced upto CEJ and prepared and inoculated with E. faecalis	Irrigant		CFU’s/ml	Laser was most effective against E. faecalis and Triphala showed better antibacterial efficacy than NaOCl. NaOCl can be substituted with triphala due to its herbal properties P<0.3
			Diode laser		8.00±7.87 CFUs/ml
			Triphala in 10% DMSO		58.60±16.63 CFU’s/ml
			3% NaOCl		69.80±19.57 CFU’s/ml

A. indica: Azadirachta indica, E. faecalis: Enterococcus faecalis, C. albicans: Candida albicans, NaOCl: Sodium hypochlorite, CHX: Chlorhexidine, M. citrifolia: Morinda citrifolia, AI: Artificial intelligence, MCJ: M. citrifolia juice, DMSO: Dimethyl sulfoxide, PDL: Periodontal ligament, BHI: Brain-heart infusion, CEJ: Cemento-enamel junction, CFU’s: Colony forming unit

The study by Thomas et al.[39] compared effectiveness of Diode laser in primary teeth as compared to 3% NaOCl and Triphala in 10% DMSO; in this randomized controlled trial, laser was found to be the most effective followed by Triphala which showed better antibacterial efficacy than NaOCl.

Table 4 summarizes the efficacy of herbal irrigants in removing the smear layer. Sebatni and Kumar[40] in their study is proved that the aqueous extract of A. indica A. juss was an effective irrigating solution for the removal of the smear layer. Sowjanyaa et al.[42] concluded that Triphala in DMSO (1:1) could be used as an adjunct to EDTA as final rinse for better removal of the smear layer. Susan et al.[41] observed that combination of aqueous extract of Triphala and 5% NaOCl with ultrasonic activation and 10% Triphala in 10% DMSO were as effective as 17% EDTA and 5% NaOCl when used in combination as irrigating solutions for the removal of the smear layer. Murray et al.[43] found that when used in combination with 17% EDTA 6% MCJ was as effective as 6% NaOCl and more effective than 2% CHX in the removal of E. faecalis smear layer.

Table 4

Herbal irrigants and smear layer

Author/year	Sample	Material & method	Irrigant		Results	Conclusion
Sebatni and Kumar 2017[40]	A. indica/NaOCl/smear layer/SEM/Ex vivo	40 healthy permanent extracted teeth due to orthodontic/periodontal reasons prepared and irrigated with test solutions sectioned and observed - SEM	Test solution		Smear layer removal efficacy score	A. indica leaf extract showed significant smear layer removal followed by NaOCl
			NaOCl		2.2
			A. indica (aqueous extract 15 g/150 ml)		1.3
			P		0.001
Sowjanyaa et al.[41]	Triphala (1:1 DMSO Imcops, Chennai)/EDTA/SEM/Ex vivo	60 unhealthy due periodontal/prosthetic reasons prepared and irrigated with 2.5% NaOCl and saline followed by test irrigant; sectioned-SEM	Irrigant	17% EDTA	Triphala	Triphala showed good cleaning efficacy in removing the smear layer but EDTA was found to be more effective (P<0.005) The most effective removal of the smear layer occurred with the use 17% EDTA as a final rinse followed by the use of Triphala
			Coronal	1.1	1.15
			Middle	2.0	1.9
			Apical	2.9	2.95
				P	<0.005
Susan et al., 2019[42]	Triphala/EDTA/NaOCl (SHC)/SEM/Ex vivo	74 permanent molars with 25-35 degree root curvature prepared and irrigated with 5% SHC	Normal saline	5% NaOCl + 5% triphala in 10% DMSO		Triphala was very effective and was nearly as efficient in the removal of smear when compared to groups II at the coronal and apical thirds whereas in the middle third it was comparable to groups II and IX. On statistical comparison and analysis, there was no significant difference between group II, V, and VIII (P>0.05)
			5% NaOCl + 17% EDTA	5% NaOCl + 10% triphala in 10% DMSO
			5% NaOCl + triphala	10% citric acid in 10% DMSO
			5% NaOCl + triphala + sonic activation	10% DMSO
			5% NaOCl + triphala + ultrasonic activation
			5% NaOCl + 3% triphala in 10% DMSO
Murray et al., 2008[43]	6% M. citrifolia/6% NaOCl/2% CHX/smear layer/17% EDTA/0.9%saline/SEM/Ex vivo	60 permanent single rooted teeth inoculated with E. faecalis for 30 days	Irrigant	Percentage reduction in smear layer		Both MCJ and NaOCl treatments were similarly effective with a rinse of 17% EDTA (P<0.2471) to completely remove up to 80% of the smear layer from some aspects of the root canal. MCJ was more effective than CHX for removing the smear layer (P<0.0085) and saline as the negative control (P<0.0001). The efficacy of MCJ was similar to NaOCl in conjunction with EDTA as an intracanal irrigant
			6% MCJ (coronal)	60
			Middle	70
			Apical	40
			2% CHX (coronal)	10
			Middle	30
			Apical	0
			6% NaOCl (coronal)	80
			Middle	80
			Apical	60

SEM: Scanning electron microscopy, A. indica: Azadirachta indica, E. faecalis: Enterococcus faecalis, NaOCl: Sodium hypochlorite, CHX: Chlorhexidine, M. citrifolia: Morinda citrifolia, MCJ: M. citrifolia juice, EDTA: Ethylenediaminetetracetic acid, DMSO: Dimethyl sulfoxide, SHC: Sodium Hypochlorite

Table 5 summarizes the studies on the use of herbal irrigants and their effect on biofilms. Tyagi et al.[44] investigated the antimicrobial efficacy of MCJ, ethanolic extracts of A. indica A. juss and propolis as compared to 5% NaOCl and found propolis to be equally effective as NaOCl against C. albicans biofilm followed by A. indica A. juss and MCJ. Garg et al.[45] in an ex-vivo study evaluating Triphala in 10% DMSO, 6% MCJ and A. indica A. juss (aqueous extract) found Triphala to be as efficacious as 5.25% NaOCl followed by A. indica and MCJ against E. faecalis biofilm. Rosaline et al.[46] found that ethanolic extract of A. indica A. juss achieved maximum reduction in adherence of E. faecalis to dentin (9.30%) followed by 5.25% NaOCl (12.50%) and least by MCJ (44.2%) and saline (86.7%). VinothKumar et al.[47] found ethanolic extract of A. indica (1:1) to provide promising results against both E. faecalis and C. albicans biofilms when compared to 5.25% NaOCl.

Table 5

Studies on herbal irrigants and biofilms

Author/year	Sample	Material & method	Irrigant			Result
Tyagi et al., 2013[44]	M. citrifolia/A. indica (ethanolic extract)/11% propolis (ethanolic extract/NaOCl/Saline/C. albicans biofilm/Ex vivo	50 extracted mandibular premolars incubated with C. albicans culture 2 days, treated with test solutions for 10 min	Irrigant	CFU’s		NaOCl and propolis exhibited highest antimicrobial efficacy against C. albicans. It was followed by the A. indica. M. citrifolia had limited antifungal action followed by the negative control group of saline P<0.001
			6% M. citrifolia	92.0±0.82
			A. indica Ethanolic extract 25 µg/ml	3.8±0.92
			5% NaOCl	0.20±0.42
			11% Propolis	0.20±0.42
			Saline	133.2±1.03
Garg et al., 2014[45]	M. citrifolia/A. indica/Triphala/NaOCl/E. faecalis biofilm/Ex vivo	70 single rooted premolars with fully formed apex prepared and irrigated with5% NaOCl sectioned and placed in tissue culture ×6 weeks then immersed in test solution ×10 mins	Irrigant	CFU’s		Triphala was found to be as efficacious as NaOCl. The use of herbal alternatives as root canal irrigation solutions might prove to be advantageous considering several unfavorable properties of NaOCl NaOCl ~_ Triphala > Neem > MCJ>Saline P<0.001
			6% M. citrifolia	64.6
			A. indica (aqueous extract15 g/100 ml)	50.1
			Triphala (60 mg/ml 10% DMSO	1.4
			5.25% NaOCl	0.3
			Saline	93.0
Rosaline et al., 2013[46]	A. indica (ethanolic extract)/M. citrifolia (ethanolic extract)/NaOCl/Saline/E. faecalis/Confocal laser scanning microscope/Ex vivo	50 noncarious single rooted teeth specimens were treated with 5.25% NaOCl and 5 mmol EDTA followed by final irrigant	Irrigant	Percentage of adherent E. faecalis to dentin		AI treatment produced the maximum reduction in adherence of E. faecalis to dentin (9.30%) followed by NaOCl (12.50%), M. citrifolia (44.20%) and saline (86.70%) No statistical difference was seen between AI and NaOCl
			Saline	86.70
			5.25% NaOCl	12.5
			M. citrifolia (1.25 mg/ml)	44.20
			A. indica (ethanolic extract 0.33 mg/ml)	9.3
Vinothkumar et al., 2013[47]	A. indica (ethanolic extract 1:1)/NaOCl/saline/C. albicans/E. faecalis/Polymerase chain reaction/Ex vivo	84 extracted teeth single rooted mandibular premolars prepared and irrigated with 5.25% NaOCl and 17% EDTA followedby contamination by E. facealis C. albicans ×21 days, test irrigants, PCR	Percentage of bacteria remaining	E. faecalis	C. albicans	0.033% AI was equally efficient to 5.25% NaOCl in reducing E. faecalis within the root canals 0.033% AI was highly efficient to 5.25% NaOCl in reducing C. albicans within the root canals when compared with other extracts. P<0.005
			A. indica (ethanolic extract)	13.1	8.2
			5.25% NaOCl	11.5	11.5
			Saline	70.4	65.5

A. indica: Azadirachta indica, E. faecalis: Enterococcus faecalis, C. albicans: Candida albicans, NaOCl: Sodium hypochlorite, M. citrifolia: Morinda citrifolia, AI: Artificial intelligence, MCJ: M. citrifolia juice, EDTA: Ethylenediaminetetracetic acid, PCR: Polymerase chain reaction, DMSO: Dimethyl sulfoxide, CFU’s: Colony forming units

Discussion

The major objective of root canal treatment is to disinfect the entire root canal system. Cleaning, shaping, and use of antimicrobial medicaments are effective in reducing the bacterial load to some extent, but some bacteria do remain behind and multiply causing reinfection. Taking the ineffectiveness, potential side-effects and safety concerns of synthetic drugs into consideration, herbal alternatives for endodontic usage might prove to be advantageous. Over the past decade, interest in drugs derived from medicinal plants has markedly increased. This review aimed at compiling the studies evaluating the efficacy of irrigants utilizing herbs in cleaning root canal of its debris in the form of bacterial biofilm and smear layer.

A. indica A. juss (Neem) has been described for its value in traditional Indian medicinal texts.[48] Neem tree is also named as “village dispensary” as every part including the seeds, leaves and bark has a medicinal value.[49] In majority of studies reviewed, the A. indica extract used was prepared from the leaf part of the plant utilizing various solvents including ethanol, distilled water, and methanol. Several techniques are used for making the neem leaf extract utilizing different solvents.[50] Our search screened several studies that evaluated the antimicrobial properties of neem extract as an irrigant during root canal treatment and compared it with NaOCl and CHX irrigants. E. faecalis was chosen as one of the test microorganisms in most of these experiments due to its persistence in endodontically treated teeth with apical periodontics, its resistance to NaOCl and its rapid growth along the entire wall of the dentinal tubules.[515253] In majority of in vitro studies, A. indica A. juss demonstrated significant efficacy against E. faecalis and C. albicans and proved to be as efficacious as NaOCl at various concentrations (2.5%, 3%, 5%).[262728293031]

Ex-vivo studies also showed biocompatibility with oral tissues and significant antimicrobial efficacy against E. faecalis and C. albicans equivalent to that of NaOCl. In comparative clinical studies evaluating of A. indica A. juss demonstrable reduction of aerobic and anaerobic flora was seen equivalent to that of CHX and NaOCl.

Dutta and Kundabala et al.[29] investigated the synergistic action between the herbal and chemical irrigants, which potezntiated their individual anti-microbial efficacy and they hypothesized that using the NaOCl and ethanolic A. indica leaf extract combination as an endodontic irrigant would prove beneficial. Sebatni and Kumar[40] found A. indica leaf extract in distilled water to have significant smear layer removal followed by NaOCl. The biocompatibility, antioxidant, antimicrobial properties, effective removal of smear layer and E. faecalis biofilm make it a potential agent for root canal irrigation as an alternative to NaOCl.[23]

Triphala is an ayurvedic polyherbal medicine containing Amalaki (E. officinalis Gaertn.), Bibhitaki (T. chebula [Gaertn.] Roxb.) and Haritaki (T. belerica Retz.). In various classic Ayurvedic medicine literature Triphala is considered as multi-therapeutic and with cure-all properties, it is even labeled as panacea historically.[5455]

Triphala has been used conventionally in Ayurvedic medicine as an antimicrobial agent.[56] The citric acids from the fruits aid in the removal of the smear layer and act as chelating agents. Different solvents have been used in the preparation of the Triphala extract such as distilled water, ethanol, DMSO; the latter being the most commonly used. Various in-vitro studies found Triphala (ethanolic extract) to be more effective than 0.5% and 1% NaOCl solution and also Triphala (10% DMSO) was marked equally effective as compared to 2.5% and 3% NaOCl solutions at eliminating E. faecalis.[3639] Satti et al.[32] demonstrated higher antimicrobial efficacy of aqueous extract Triphala as compared 3% NaOCl whereas a study by Saxena et al.[38] revealed 2.5% NaOCl to be more effective than ethanolic extract of Triphala.

Significant removal of smear layer was observed by Thomas et al. when Triphala was used in combination with 17% EDTA and 5% NaOCl or 5% NaOCl and ultrasonic activation.[39] Another study also showed effective removal of smear layer by 17% NaOCl along with Triphala. It was concluded that its use as an adjunctive herbal root canal irrigating solution might prove to be advantageous. Triphala thus represents a potential therapy to eliminate E. faecalis as more side-effects and risks being associated with NaOCl although further clinical trials have been recommended to evaluate its potential use as a root canal irrigant.

MCJ has been relatively new to endodontics and recommended as an alternative to NaOCl because of its chelating ability to remove the smear layer and antimicrobial activities, especially against anaerobic bacteria such as E. faecalis and C. albicans. Murray et al., investigated the antimicrobial effectiveness of MCJ against E. faecalis and found its MIC to be 6%.[43] Ex-vivo studies evaluating the efficacy of M. citrifolia as root canal irrigant showed that MCJ to be effective against E. faecalis biofilms and also in the removal of smear layer, especially when used in combination with EDTA but not to the same extent as NaOCl.[4445] Authors however have recommended the use of MCJ as an irrigant because of it is biocompatibility, antioxidant property, and safety profile as it is not likely to cause severe injuries to patients which might occur with the use of NaOCl accidents or if extruded out.

Herbal formulations are advantageous in many ways. They are safe, easily available, better shelf life, economical, and absence of microbial resistance so far. They are most effective when used with proper knowledge. Considering the low toxicity and antibacterial effectiveness, these herbs could be used as an adjunct to NaOCl thereby lowering its toxicity and also providing the added antioxidant advantage. Due to their high safety profile, these irrigants have the potential to replace chemical irrigants in patients with a history of allergic reactions to NaOCl and also in pediatric patients where open apices limit the use of chemical irrigants. Many studies have concluded that herbal extracts produce promising results when used as endodontic irrigants. Further studies like quantitative real-time-polymerase chain reaction and confocal microscopic examination of the biofilms could provide better insight into the herbal irrigants. Combination of these herbal irrigants can also be tested for their synergistic action and also the possible interactions between the ingredients. This review has revealed that there is a paucity of comparative clinical trials utilizing these herbal endodontic irrigants despite having good baseline data showing promising results from in-vitro and ex-vivo studies.

Randomized clinical controlled trials can be carried out to further substantiate the clinical efficacy of these herbal irrigants. However, this scoping review could not quantitatively assess and combine the research data and could only provide an overview of the literature pertaining to the use of A. indica, M. citrifolia and Triphala as herbal root canal irrigants.

Conclusion

The article reviewed studies utilizing A. indica A. juss, Morinda Citrofolia, and Triphala as endodontic irrigants. Various in vitro studies of herbal products have yielded promising results, but more clinical trials are needed to substantiate the results from in-vitro studies and evaluate the biocompatibility before they can conclusively be recommended as intracanal irrigating solutions. Clinical studies investigating synergistic action utilizing various combinations of herbal irrigants at different concentrations and solvents are recommended to bridge the gaps in preclinical and clinical research activities and promote green dentistry in clinical practice in the true sense.

Financial support and sponsorship

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Conflicts of interest

There are no conflicts of interest.