in partial fulfillment of the requirements for the degree of

METRONIDAZOLE AND 3% SODIUM HYPOCHLORITE AS ROOT CANAL IRRIGANTS – AN IN VIVO STUDY

A Dissertation submitted

in partial fulfillment of the requirements for the degree of

MASTER OF DENTAL SURGERY

BRANCH – IV

CONSERVATIVE DENTISTRY AND ENDODONTICS

THE TAMILNADU DR. MGR MEDICAL UNIVERSITY CHENNAI – 600 032

2017 – 2020

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation titled "COMPARATIVE EVALUATION OF ANTIMICROBIAL EFFICACY OF TRIPHALA, AZADIRACTA INDICA, METRONIDAZOLE AND 3% SODIUM HYPOCHLORITE AS ROOT CANAL IRRIGANTS – AN IN VIVO STUDY" is a bonafide and genuine research work carried out by me under the guidance of Dr.B.RAMAPRABHA M.D.S., Professor, Department Of Conservative Dentistry and Endodontics, TamilNadu Government Dental College and Hospital, Chennai -600003.

K.VINOTHINI

CERTIFICATE BY GUIDE

This is to certify that Dr K. VINOTHINI, Post Graduate student (2017-2020) in the Department Of Conservative Dentistry and Endodontics, Tamil Nadu Government Dental College and Hospital, Chennai-600003 has done this dissertation titled

" COMPARATIVE EVALUATION OF ANTIMICROBIAL EFFICACY OF

TRIPHALA, AZADIRACTA INDICA, METRONIDAZOLE AND 3% SODIUM HYPOCHLORITE AS ROOT CANAL IRRIGANTS– AN IN VIVO STUDY " under my direct guidance and supervision in partial fulfillment of the regulations laid down by the TamilNadu Dr.M.G.R Medical University Chennai- 600032, for M.D.S., Conservative Dentistry and Endodontics (Branch IV) Degree Examination.

Dr.B.RAMAPRABHA M.D.S., Professor &GUIDE

Department of Conservative Dentistry and Endodontics.

TamilNadu Government Dental College and Hospital Chennai- 600003

HEAD OF THE INSTITUTION

This is to certify that the dissertation titled " COMPARATIVE EVALUATION OF ANTIMICROBIAL EFFICACY OF TRIPHALA, AZADIRACTA INDICA, METRONIDAZOLE AND 3% SODIUM HYPOCHLORITE AS ROOT CANAL IRRIGANTS – AN IN VIVO STUDY” is a bonafide research work done by Dr.K.VINOTHINI, Post Graduate student (2017- 2020) in the Department Of Conservative Dentistry & Endodontics under the guidance of Dr.B. RAMAPRABHA M.D.S., Professor, Department Of Conservative Dentistry & Endodontics, TamilNadu Government Dental College and Hospital, Chennai-600003.

Dr.M.KAVITHA M.D.S., Dr.G.VIMALA M.D.S

Professor & H.O.D., Principal

Department of Conservative Dentistry

& Endodontics

TamilNadu Government Dental College and Hospital.

Chennai- 600003.

I would like to express my heartfelt gratitude to my guide DR.B.RAMAPRABHA M.D.S., Professor for supporting me and guiding throughout my dissertation .Her expertise, wisdom, immense support and motivation during my course of the study has really made me to get the better of myself. It is indeed a great pleasure to describe my gratefulness to my Head of the Department & Professor DR.M. KAVITHA M.D.S, for being an inspiration and for her constant support, guidance and encouragement in my P.G life.

I take this opportunity to convey my gratitude to DR.G.VIMALA M.D.S. Principal, Tamil Nadu Government Dental College and Hospital, Chennai for permitting me to utilize the available facilities in this institution.

My sincere thanks to Dr. K. AMUDHALAKSHMI M.D.S., Dr. D. ARUNA RAJ MDS., Dr. A.NANDHINI M.D.S and Dr. P. SHAKUNTHALA, MDS., Associate Professors, for all the support and guidance, both clinically and academically providing me with their knowledge and experience in this field.

My extended thanks to Dr. M.S. SHARMILA M.D.S., Dr. M. SUDHARSHANA RANJINI M.D.S., Dr.S.VENKATESH M.D.S., Dr. S.JOTHILATHA M.D.S., Dr.S.DHANALAKSHMI M.D.S., Dr. VELAYUDAM M.D.S., Dr.PADMAPRIYA M.D.S.

I thank Dr. PADMAVATHY, Msc, PHD, senior Microbiologist, Sree Balaji Dental College & hospital, Chennai for her guidance in the microbiological analysis. I thank Dr.

SRINIVASAN M.D.S for his statistical guidance and help.

My sincere thanks to staff nurses, male health workers, female health workers for all

my PG life. I would also like to extend my regards to my seniors and juniors for their great support in finishing my thesis and my course of study.

My special thanks to my parents, my sister, my fiancé and my friends for their moral and emotional back up in all my academic pursuits and endeavors.

Above all I pray and thank THE ALMIGHTY GOD for His continuous grace and blessings in every endeavor of my life.

TITLE OF DISSERTATION COMPARATIVE EVALUATION OF OF

ANTIMICROBIAL EFFICACY OF

TRIPHALA, AZADIRACTA INDICA, METRONIDAZOLE AND 3% SODIUM HYPOCHLORITE AS ROOT CANAL IRRIGANTS – AN IN VIVO STUDY

PLACE OF STUDY TAMIL NADU GOVERNMENT

DENTAL COLLEGE AND HOSPITAL DURATION OF COURSE 3 YEARS

NAME OF THE GUIDE DR B. RAMAPRABHA HEAD OF THE DEPARTMENT DR M. KAVITHA

I hereby declare that no part of the dissertation will be utilised for gaining financial assistance or any promotion, without obtaining prior permission of the Principal., TamilNadu Government Dental College and Hospital, Chennai -600 003. In addition, I declare that no part of this work will be published either in print or in electronic media without the guide who has been actively involved in dissertation. The author has the right to preserve for publish of the work solely with the prior permission of The Principal, Tamil Nadu Government Dental College and Hospital, Chennai- 600 003.

HOD GUIDE Signature of the Candidate

This is to certify that this dissertation work titled “COMPARATIVE EVALUATION OF OF ANTIMICROBIAL EFFICACY OF TRIPHALA, AZADIRACTA INDICA, METRONIDAZOLE AND 3% SODIUM HYPOCHLORITE AS ROOT CANAL IRRIGANTS – AN IN VIVO STUDY”

of the candidate Dr.K.VINOTHINI with Registration Number 241717006 for the award of M.D.S in the branch of CONSERVATIVE DENTISTRY AND ENDODONTICS (Branch IV). I personally verified the urkund.com website for the purpose of plagiarism check. I found that the uploaded thesis file contained pages from introduction to the conclusion and the result shows 10 percentage of plagiarism in the dissertation.

Guide & Supervisor Sign with Seal

IkII Reference No: 7/IRB/20a 7

?1l/hl BER SECRETARY

f£r^l” Dr.M.B.Aswath Narayanan, BSc",MDb., SIEMBERS

Prof. Dr.MaJieaswan Rajendran, MDS., Prof. Dr.S,Jayachandran, MDS., PhD„

Prof. Dr.M.Kavitha, MDS., Prof. Dr.C.Sabarigirinalhan, MDS., Prof. Dr.S.Geetha, MD., tc‹neral Mñicine) Prof. Dr.R, Vanaja, MD., l Microbiology) Prof Dr.K.M.Sudhn MD., (Phanni‹»logy) Prof. Dr.S.Siza, MW., (Biochemist)

Prof. Dr.Bhanthi N Jayantlii,MD„ (Gen.Patsolo yj Prof. Dr.M.Cliandrasekar, MVSc, PhD., Mr,G.K. Muthukumar, B.Com., LLB., Mr.Shantaram

Project Title:

Comparative evaluation of antimicrobial efficacy of "friphaJa.

Azadiracta Indica, Metronidazole and 3% sodium hypochlorim as root canal irrigants — An in vivo study

Principal Investigator: Dr.Vinotbini.K

Revjpw: New/Revised/Expedited Date of revitw: 13.11.17

Date of previous review, if revised application: Not Applicable Decision of the IRB: Approved

Member Secrets ,.. ,

The lnvesñgator / Investigating tram is advised to adbtre t0 the guide lines given below:

• ShoUld bg calTied out under the dirett supervision of the Guide

^intaIn c0nfidentia|lt}'.

without exfra exprndiU re to the InstiMtion

• Inform the IRB in case or any change of St jJdy procedure, site, lnYeStigalor and Guide.

* N0l deviate from the area of work for which

or scrious adverse reactions. Should

‘ ^P!8!8 lh9 W0fk \¥it#!n 5p0CIfiC pefi0d Btd if Bpy gy(Chsign ttmg iS ft9ti ired, sh0tild apply for Remission again to do the work,

• Submit the summary of the work to the IRB : Students-evety 3 months;

Fzculy-evcp6monhs.

• Should not claizt any kind of funds ffora the institution for doing th work or on completion/ or

f0F 88}' tm§ 0f C0Ilt I2flS8fiOflS.

• The members Ofthe IkB have the right la monitor the work gjth yt pit t nt|y /)

^ III - 1fiV#StlgBt0f Blld f*iLl7#B Sft0Ul# 48Ch d€C)Zfg (h8( flq ]jlByj0rj5ly i5 tfly0lygd |q ai, WhOlC $hjd

ané enclose the undertaking in disscxation/ thesis,

TRIPARTITE AGREEMENT

This agreement herein after the “Agreement” is entered into on this day Jan 2020 between the Tamil Nadu Government Dental College and Hospital represented by its Principal having address at Tamil Nadu Government Dental College and Hospital, Chennai - 600 003, (hereafter referred to as, „thecollege„)

And

Mrs. Dr. B. RAMAPRABHA aged 50 years working as Professor in Department of Conservative Dentistry & Endodontics at the college, having residence address at 191/5, Green Fields Apts. R-30A, Ambattur, Thirumangalam High Road, Mugappair,Chennai-3 (herein after

referred to as the Principal Investigator„) And

Ms. Dr. K.VINOTHINI aged 27 years currently studying as Post Graduate student in Department of Conservative Dentistry & Endodontics, Tamil Nadu Government Dental College

and Hospital, Chennai 3 (herein after referred to as the PG student and coinvestigator„).

Whereas the PG student as part of her curriculum undertakes to research on

“Comparative evaluation of of antimicrobial efficacy of triphala, azadiracta indica,metronidazole and 3% sodium hypochlorite – an in vivo study” for whichpurpose the Principal Investigator shall act as principal investigator and the college shall provide the requisite infrastructure based on availability and also provide facility to the PG student as to the extent possible as a Co-investigator.

Whereas the parties, by this agreement have mutually agreed to the various issues including in particular the copyright and confidentiality issues that arise in this regard.

Now this agreement witnesseth as follows

1. The parties agree that all the Research material and ownership therein shall become the vested right of the college, including in particular all the copyright in the literature including the study, research and all other related papers.

2. To the extent that the college has legal right to do so, shall grant to license or assign the copyright so vested with it for medical and/or commercial usage of interested persons/entities subject to a reasonable terms/conditions including royalty as deemed by the college.

3. The royalty so received by the college shall be shared equally by all the threeparties.

4. The PG student and Principal Investigator shall under no circumstances deal with the copyright, Confidential information and know – how - generated during the course of

6. All expenses pertaining to the research shall be decided upon by the Principal Investigator/ Coinvestigator or borne solely by the PG student.(co-investigator)

7. The college shall provide all infrastructure and access facilities within and in other institutes to the extent possible. This includes patient interactions, introductory letters, recommendation letters and such other acts required in thisregard.

8. The Principal Investigator shall suitably guide the Student Research right from selection of the Research Topic and Area till its completion. However the selection and conduct of research, topic an area of research by the student researcher under guidance from the Principal Investigator shall be subject to the prior approval, recommendations and comments of the Ethical Committee of the College constituted for thispurpose.

9. It is agreed that as regards other aspects not covered under this agreement, but which pertain to the research undertaken by the PG student, under guidance from the Principal Investigator, the decision of the college shall be binding and final.

10. If any dispute arises as to the matters related or connected to this agreement herein, it shall be referred to arbitration in accordance with the provisions of the Arbitration and Conciliation Act 1996.

In witness where of the parties herein above mentioned have on this day, month and year herein above mentioned set their hands to this agreement in the presence of the following two witnesses.

College represented by its Principal PG Student

Witnesses Guide

1.

2.

AIM:

To evaluate and compare the antimicrobial efficacy of Triphala, Azadirachta indica, Metronidazole and 3% sodium hypochlorite (NaOCl) as root canal irrigants.

MATERIALS AND METHODS:

Eighty maxillary incisors with asymptomatic apical periodontitis were randomly assigned to one of the four groups corresponding to the irrigant to be tested; Triphala (n = 20),

Azadiracta indica (n = 20), Metronidazole (n=20) and 3% Sodium hypochlorite (n = 20).

After the root canal access opening, a root canal culture sample was taken with a paper point designated as sample 1 and cultured both under aerobic and anaerobic conditions. Cleaning and shaping was completed along with irrigation by 15 mL of the respective irrigants and sample 2 was taken with sterile paper point immediately and cultured under both aerobic and anaerobic conditions. The patients was recalled after 3 days and sample 3 was taken using the paper point and cultured as mentioned before followed by analysis by counting the colony forming units (CFUs).

RESULTS:

Triphala, Azadiracta indica, Metronidazole and 3% NaOCl showed a significant reduction (P

< 0.05) in the mean percentage of CFUs under both aerobic and anaerobic culture .Under anaerobic culture, there is no statistical significant difference between Sodium hypochlorite, Metronidazole, Azadiracta indica and Triphala both immediately and on 3^rd day recall visit.

Under aerobic culture, Sodium hypochlorite shows the highest percentage reduction followed by Metronidazole, Azadiracta indica and the least by Triphala.

CONCLUSION:

Metronidazole, Azadircata indica and Triphala have the necessary antimicrobial efficacy and hence they could be considered as an alternative to the potentially dangerous Sodium

KEYWORDS : Root canal irrigants, Antimicrobial efficacy, Herbal irrigants, maxillary incisors, culture method, colony forming units.

S.NO. TITLE PAGE NO.

1 INTRODUCTION

1

2 AIM AND OBJECTIVES

6

3 REVIEW OF LITERATURE

7

4 MATERIALS AND METHODS

29

5 RESULTS

46

6 DISCUSSION

62

7 SUMMARY

72

8 CONCLUSION

74

9 BIBLIOGRAPHY

i-xx

CFU Colony Forming Units

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

ISM Indian System Of Medicine

MBC Mean Bactericidal Concentration

MIC Mean Inhibitory Concentration

PMN Polymorphonuclear Neutrophils

PRCFU1A Percentage Reduction Of Colony Forming Units Immediately After Irrigation- Aerobic

PRCFU1AN Percentage Reduction Of Colony Forming Units Immediately After Irrigation- Anaerobic

PRCFU2A Percentage Reduction Of Colony Forming Units 3 Days After Irrigation- Aerobic

PRCFU2AN Percentage Reduction Of Colony Forming Units 3 Days After Irrigation- Anaerobic

S.No. TABLE PAGE No.

1. COLONY FORMING UNITS UNDER AEROBIC AND ANAEROBIC CONDITION

46

2. PERCENTAGE REDUCTION CALCULATION OF COLONY FORMING UNITS

48

3. TEST OF NORMALITY:SHAPIRO -WILK TEST 51

4. KRUSKAL WALLIS TEST 52

5. POST HOC DUNN TEST WITH BONFERRONI CORRECTION (PRCFU1A)

53

6. POST HOC DUNN TEST WITH BONFERRONI CORRECTION (PRCFU2A)

54

S.NO TITLE PAGE NO

1 CLUSTER BAR DIAGRAM-MEAN OF

PRCFU1A AND PRCFU2A

55

2 CLUSTER BAR DIAGRAM- MEAN OF

PRCFU1AN AND PRCFU2AN

56

INTRODUCTION

Eradication of micro-organisms and prevention of re-infection is the ultimate goal for success of the root canal treatment. Hand and rotary instruments under irrigation are used to biomechanically prepare the root canal space to remove the inflamed and necrotic tissue, microbial biofilms, and other debris. However proportionally large areas of the main root-canal walls remain untouched by the instruments used for biomechanical preparation as revealed by studies using micro computed tomography,¹⁰⁹ emphasizing the importance of chemical means of disinfection.

Irrigation has a central role in the success of the root canal treatment. During and after instrumentation, the irrigant facilitates removal of the microorganisms, dentin chips and tissue remnants from the root canal through a flushing mechanism and also helps to prevent packing of the hard and soft tissue debris in the apical area of the root canal thereby preventing extrusion of infected material into the periapical area. Irrigating solutions primarily exert its action by dissolving organic and inorganic tissues in the root canal. In addition, several irrigating solutions have desirable antimicrobial activity when introduced in direct contact with the microbes.

However to exert its action these irrigating solutions should face few challenges present in the root canals like smear layer, dental erosion, cleaning of an uninstrumented part of the root canal, biofilm, safety versus effectiveness in the apical root canal.⁵⁰ An optimal irrigant should have all or most of the positive characteristics, but it should not possess any of the negative or harmful properties.

Sodium hypochlorite (NaOCl), a halogenated compound, is routinely used by the dentists for irrigation in varying concentrations between 0.5% to 6% during the root canal treatment. It is used either as an unbuffered solution at pH 11 in various concentrations, or buffered with bicarbonate buffer (pH 9.0), usually as a 0.5% (Dakin solution) or 1% solution exerting its antimicrobial action.^32,118,124 Sodium hypochlorite ionizes to sodium and oxy-chloride in water.

Between pH values 4 and 7, chlorine ion exists as hypochlorous acid whereas, at pH above 9, oxychloride predominates. In addition to its antibacterial action, sodium hypochlorite also has a nonspecific proteolytic action by which it dissolves the pulpal remnants⁶ and organic components of the dentin. In addition, it partially neutralizes the necrotic tissues or any antigenic component left in the root canal space²⁹ and removes all the pulpal remnants and predentin on the uninstrumented canal surfaces.¹ Regardless of its significant effect on the organic components of the dentin, it has no effect on dentin's inorganic component which is a major drawback for its clinical applications.

On the contrary Sodium hypochlorite has several undesirable disadvantages in clinical usage.¹⁰⁸ Ideally, irrigants should be always confined to the root canal system during biomechanical preparation, which is not always possible as extrusion of irrigants beyond the apical foramen may occur during over-instrumentation, in teeth with open apices,¹²³ and through the sites of perforation or external resorption.¹⁵⁷ Binding of the irrigation needle’s tip inside the root canal and usage of too much irrigation pressure also extrudes the irrigant into the periradicular tissues, leading to necrosis and tissue destruction.¹⁴⁰ Thus when used injudiciously, NaOCl is very destructive to intraoral soft tissues, the periradicular vasculature and cancellous bone eliciting severe inflammatory responses and degradation of the organic components leading to tissue necrosis. In addition to this the other drawbacks of NaOCl includes the unpleasant taste, and its inability to remove the smear layer.⁴⁹ Added to this, NaOCl has limited antimicrobial effectiveness in vivo, which could be due to the problems in penetration to the most peripheral parts of the root-canal system and inactivation from exudates from the periapical area, pulp tissues, dentin collagen, and microbial biomass.¹³³ Hence this finding raises the question of whether hypochlorite may elevate the risk of iatrogenic vertical root fracture. Thus the search is on for an irrigant that can match or better this gold standard irrigant without undesirable side effects.

Metronidazole is a nitroimidazole group of drugs with broad antibiotic spectrum. It is effective

against obligate anaerobic bacteria than on facultative anaerobic and aerobic bacteria. Anaerobic bacteria contain electron transport components such as ferrodoxin that have a negative redox potential donating electrons to metronidazole. This single electron transfer reaction forms a reactive nitro radical action that targets DNA of bacteria by inhibiting the nucleic acid synthesis.⁹¹ The advantage of this drug is it does not develop resistance among anaerobes as suggested by the consensus of clinical and experimental evidence, which is due to decreased uptake of drug or reduced drug efficiency or prevention of entry of the drug or efflux. There is no problem with re- administration of the metronidazole which has made the drug as ‘priority drug’.⁹¹Also it is bactericidal for anaerobic organism at very low concentrations of 0.5%.

Thus the advantage of using metronidazole in dentistry as an irrigant includes ready availability, rapid bactericidal action, good penetration, cost effectiveness, acceptable pharmacokinetics and pharmacodynamics, undiminished antimicrobial activity and inability of susceptible organisms to develop resistance.⁹¹

Herbal products have been used in medical ⁷⁸ and dental practice ^62,64, since ancient times, and in the recent times the trend is growing again towards it due to their high antimicrobial activity, excellent biocompatibility, anti-inflammatory and anti-oxidant properties. Various herbal extracts, such as neem and tulsi extracts, curcuma longa, and turmeric, Aloe vera, Morinda citrifolia, have antimicrobial, antiinflammatory, and therapeutic effects and hence are promising to be used as endodontic irrigants.

Triphala is an established powdered combination in the Indian system of medicine (ISM), being used in Ayurveda since ancient times. Triphala consists of equal parts of the Emblica officinalis, Terminalia chebula, and Terminalia belerica.⁶¹ It consists of Tannins, Quinones, Flavones, flavonoids, and flavonols, Gallic acid and Vitamin C as active constituents.⁶¹

Triphala has been used in dentistry in the treatment of carious teeth ¹¹³ ; as root canal irrigant ^35,

132; in periodontal diseases ^2,88; and also as a mouth rinse⁶⁰ due to its potent anti-microbial, anti-

oxidant and anti-collagenase activity. Triphala in an invitro study has shown significant anti- bacterial activity against three and six week biofilms.¹¹³ Triphala seems to fulfill most of the requirements of irrigant without any adverse effect on oral tissues and also at very minimal cost as compared to commercially available products today. Hence the use of triphala as a root canal irrigant might prove to be advantageous considering the several undesirable characteristics of NaOCl.

Azadiracta indica (Neem) has been extensively used in Ayurveda, Unani and Homoeopathic medicine and has become a wonder tree of modern medicine.¹⁴²It has been used traditionally for many years as an therapeutic agent to cure inflammation, infections, fever, skin diseases and dental problems. The active constituents of neem are Azadirachtin, Nimbin, Nimbidin and Nimbidene, and the important phytoconstituent is azadirachtin, a limonoid. However, in the year 1942, three active constituents of neem were identified namely: nimbin, nimbidin and nimbidene. Nimbin is a constituent of triterpenoid assumed to be credited for majority of the properties of neem. Nimbidin is isolated from the oil of seeds which potentially can sedate central nervous system and nimbinene is the active constituent isolated from the leaves and bark of neem (Kraus and cramer in 1981).⁹⁷

Neem has potent antioxidant, antibacterial, antifungal, antiviral, anti-inflammatory antimutagenic and anticarcinogenic properties.¹²¹Aqueous and ethanolic extract of neem leaf inhibits S. mutans and E. faecalis.¹⁵ Literature suggested that the Neem (Azadirachta indica) leaf extract has significant antimicrobial effect against E. faecalis derived from the infected root canal samples compared with 2% sodium hypochlorite.¹²¹ Use of neem as an endodontic irrigant might be advantageous because it is a biocompatible antioxidant and hence not likely to cause the severe toxic reactions to patients that occurs when NaOCl is used.

Culture methods are considered as gold standard for bacterial identification as it exclusively detects viable cells, has good sensitivity and enumeration, and is reliable.⁵⁷ Culture methods rely on the growth of micro-organisms in one or more nutrient media, and are designed in such

a way that any growth causes a change in the medium’s property, which can be measured (e.g. by visual assessment of turbidity or colour). Both quantitative and qualitative testing of microorganisms is possible with culture.¹²⁰ Traditional techniques has been evolving towards the detection of bacteria with increased sensitivity and specificity.⁷² Culture is still an irreplaceable key for studying bacterial diseases, even if routine diagnosis can be efficiently achieved by using other (although generally culture-derived) tools, including genetic amplification.⁷¹ In the world of emerging infectious organisms, fulfillment of Koch’s postulates, which requires culture, remains a very necessary model of rigorous proof and scientific thinking.¹⁰¹

Inspite of knowing the advantages of herbal medicine in various in vitro studies, there is a lack of sufficient clinical studies reporting various herbal root canal irrigants and their antibacterial actions. Hence, the present study was performed to evaluate antimicrobial effect of herbal root canal irrigants (triphala, Azadirachta indica), metronidazole and to compare it with 3% sodium hypochlorite by assessing the mean colony forming units for both aerobic and anaerobic bacteria separately.

AIMS AND OBJECTIVES

Aim

The aim of this study is to compare and evaluate the antimicrobial efficacy of Triphala, Azadiracta indica, Metronidazole and 3% Sodium hypochlorite as root canal irrigants.

Objectives

1. To evaluate the potential of Triphala, Azadiracta indica, Metronidazole and 3% Sodium hypochlorite to be used as an intracanal irrigant clinically by assessing the mean CFU counts for aerobic and anaerobic bacteria preoperatively, postoperatively and after a 3 day recall visit following irrigation.

2. To Compare the antibacterial activity of these herbal irrigants and metronidazole with 3% sodium hypochlorite.

dilutions of sodium hypochlorite was evaluated using three independent biological models.

Dilutions as low as 1:1000 caused complete hemolysis of RBC's in vitro. Undiluted and 1:10 dilutions produced moderate to severe irritation to rabbit eyes which healed after 24 to 48 h.

Intradermal injections of undiluted, 1:1, 1:2, and 1:4 dilutions of NaOCl produced skin

of 5% NaOCl for biomechanical preparation of root canals is a clinically acceptable and highly effective procedure, it must be used judiciously and with great caution to prevent it

REVIEW OF LITERATURE SODIUM HYPOCHLORITE

Pashley et al (1985) ¹⁰⁵ reported that intradermal injection of 5.25% NaOCl produced immediate hemorrhage and edema lasting hours after injection. The cytotoxicity of various

ulcerations following intradermal injections.They concluded by saying that although the use

from reaching the periapex where it can elicit severe inflammatory reactions.

Siqueira JF et al (1997) ¹³⁶ evaluated the effectiveness of 4% NaOCl against Enterococcus faecalis in vitro reporting that it was significantly more effective than saline solution (control group) in disinfecting the root canal.

Vissers M et al (1998) ¹⁵⁵ compared the human red cell lysis by hypochlorous and hypobromous acids and reported that NaOCl breaks down into water and hypochlorous acid immediately when it is mixed with plasma. Hypochlorous acid then induces hemolysis almost immediately through membrane protein modification.

Siqueira J F et al (2000) ¹³⁷ evaluated the in vitro intracanal bacterial reduction produced by instrumentation and irrigation with 1%, 2.5%, and 5.25% sodium hypochlorite (NaOCl) or saline solution.They concluded that regular exchange and the use of large amounts of irrigant should maintain the antibacterial effectiveness of the NaOCl solution, compensating for the effects of concentration

M. Hülsmann & W. Hahn (2000) ⁸¹ discussed that inadvertent injection of 1% sodium hypochlorite beyond the apex produced symptoms like pain, burning sensation, ecchymosis and profuse haemorrhage from the root canal.

Gomes BP et al (2001) ⁴³ assessed the in vitro, the effectiveness of several concentrations of NaOCl (0.5%, 1%, 2.5%, 4% and 5.25%) and two forms of chlorhexidine gluconate (gel and liquid) in three concentrations (0.2%, 1% and 2%) in the elimination of E. faecalis. They found that all irrigants were effective in killing E. faecalis, but at different times. CHX in the liquid form at all concentrations tested (0.2%, 1% and 2%) and NaOCI (5.25%) were the most effective irrigants. However, the time required by 0.2% chlorhexidine liquid and 2%

chlorhexidine gel to promote negative cultures was only 30s and 1min, respectively.

Zwischenberger et al (2002)¹⁵⁹ discussed that long-term complications of chemical ingestion of sodium hypochlorite can include esophageal stricture formation, which typically develops between 3 and 8 weeks after ingestion-related burns

Vianna M E et al (2004)¹⁵⁴ investigated in vitro the antimicrobial activity of 0.2%, 1%, and 2% chlorhexidine gluconate (CHX gel and CHX liquid), against endodontic pathogens and compare the results with the ones achieved by 0.5%, 1%, 2.5%, 4%, and 5.25% sodium hypochlorite (NaOCl). All tested irrigants eliminated Porphyromonas endodontalis, Porphyromonas gingivalis, and Prevotella intermedia in 15s. The timing required for 1.0% and 2.0% CHX liquid to eliminate all microorganisms was the same required for 5.25% NaOCl

Charles H et al (2006) ²³ discussed that Enterococcus faecalis is the primary organism in the persistent endodontic infections because of virulence and survival in the root

canal in nutrient depleted states and the current method to eradicate the microorganism was to irrigate with 2% CHX combined with NaOCl.

Mohammadi Z (2008) ⁹² discussed that despite NaOCl’s very safe profile when used topically, it can sometimes be injected beyond the dermis and into soft tissue causing iatrogenic injury.

This most often occurs in endodontics, where sodium hypochlorite is used as an irrigant of the root canal system. When this occurs, it can lead to severe tissue reactions characterized by pain, swelling, hemorrhage and potentially secondary infection or lasting paresthesia

Bosch X et al (2009) ¹⁶ reported that local injection of a large amount of concentrated NaOCl into soft tissue could cause significant muscle breakdown, releasing muscle contents that can lead to acute kidney injury (AKI) via rhabdomyolysis.

Peck et al (2011) ¹⁰⁷ examined the toxicity associated with NaOCl exposure and analyze potential mechanisms of injury, placing special emphasis on the potential for renal toxicity.

They concluded by saying that Reactive oxygen species generated by hypochlorous acid can lead to both AKI in the form of ATN and also contribute to existing renal disease. This may be an additional mechanism by which exposure to sodium hypochlorite can lead to AKI, especially in circumstances of accidental injection, which can strike blood vessels.

Peck et al (2014) ¹⁰⁶ described the first case of accidental sodium hypochlorite injection into the infraorbital tissue during a dental procedure. He speculated that oxidative species induced by sodium hypochlorite caused AKI secondary to the renal tubular injury, causing mild acute tubular necrosis.

Elakanti et al (2015) ³⁹ compared the antimicrobial efficacy of QMix™ 2 in 1, sodium hypochlorite (NaOCl), and chlorhexidine (CHX) against Enterococcus faecalis and

Candida albicans and the results showed no statistical significant difference was found between 5.25% NaOCl and 2% CHX (P > 0.001) against E. faecalis and C. albicans.

Al-Sebaei M O et al (2015) ⁴ described a serious and life-threatening complication of the use of sodium hypochlorite as an irrigation solution in root canal therapy and elaborated that extrusion of the NaOCl beyond the root apex during root canal treatment is a rare complication, but if it occurs, it causes chemical burns and tissue necrosis.

Abuhaimed T S & Abou Neel E A (2017) ³ discussed that regardless of the conflict seen in literature about the action of sodium hypochlorite on dentin bond strength, some natural antioxidants could also increase the resistance of dentin to biodegradation and hence stabilize the resin adhesive-dentin interface by attacking any residual bacteria or inhibiting the degradation of the hybrid layer.

Divia A R et al (2018) ³⁵ compared the antimicrobial efficacy of Morinda citrifolia (MC), green tea polyphenols and Triphala was compared with 5% NaOCl against Enterococcus faecalis. Sixty extracted human premolar teeth were infected with E. faecalis, a Group D Streptococci for 48 h. At the end of 48 h, the vital bacterial population was assessed by counting the number of colony-forming units (CFUs) on blood agar plate. Samples were divided into five groups; Group I (distilled water), Group II (NaOCl), Group III (MC), Group IV (Triphala), and Group V (green tea polyphenols). The samples were irrigated with individual test agents and CFUs were recorded. The results obtained were sodium hypochlorite‘s antibacterial effect was comparable to Triphala and among the experimental groups, Morinda citrifolia showed the minimum antibacterial effect.

Browne et al (2019) ¹⁸ determined the collagen change within 0.5 mm of canal or root surfaces, with or without mature roots or periodontal involvement and concluded that EDTA surface treatment of polished dentine surfaces enhanced FTIR detection of NaOCl‐induced collagen changes. Both root maturity and irrigation protocol influenced the ability of NaOCl to alter dentinal collagen up to 0.5 mm from the canal lumen.

METRONIDAZOLE

Ingham et al (1977) ⁵⁹ carried out a controlled clinical trial comparing metronidazole and parenteral penicillin in the treatment of 16 patients presenting with acute apical infections has been carried out; all patients receiving metronidazole are said to have responded satisfactorily.

Hoshino E et al (1996) ⁵⁴ evaluated the antibacterial efficacy of mixture (Metronidazole, Ciprofloxacin and Minocycline, with and without Rifampicin) on the bacteria from infected dentin of root canal walls, and authors reported that this drug combination is sufficiently potent in eradicating bacteria from infected dentin of root canals. Although, metronidazole can be used as intra-canal medicament causing inhibition of growth of all obligate anaerobes and more effective than other intra-canal medicaments in eliminating obligate anaerobes present within the root canal.

Siqueira JF & de Uzeda M (1997) ¹³⁹ evaluated the antibacterial activity of 0.12%

chlorhexidine gel, 10% metronidazole gel, calcium hydroxide plus distilled water, calcium hydroxide along with camphorated paramonochlorophenol (CPMC) and calcium hydroxide along with glycerine using an agar diffusion test. Authors found that, metronidazole caused inhibition of the growth of all obligate anaerobes tested and showed better effectiveness than calcium

hydroxide/ CMPC paste against two of the strains, whereas, calcium hydroxide/ CMPC paste and chlorhexidine were effective against all bacterial strains tested.

Krithikadatta J et al (2007) ⁷³ evaluated the disinfection of dentinal tubules using 2%

chlorhexidine gel, 2% metronidazole gel, bioactive glass (S53P4) in comparison with calcium hydroxide and concluded from the present study that 2% chlorhexidine gel alone was most effective against E. faecalis when compared to other medicaments tested.

Kargul B & Kadir T (2010) ⁶⁷ conducted a clinical trial, where metronidazole has been used as intra-canal medicament combined with pulpectomy in infected primary molars. When freshly mixed metronidazole cream was applied as intra-canal medicament for one week, it has shown success rate of 85% after 24 months, which proves metronidazole’s effectiveness against endodontic bacteria (obligate anaerobes).They concluded by saying that hese results suggest that main factors responsible for failure may be associated with uncertain mixing proportions of the metronidazole paste and inadequate maxillary restorations. But some modifications in preparing the paste could increase its efficacy.

Bhangdia et al (2014) ¹⁴ evaluated the efficacy of metronidazole gel versus metronidazole solution against Enterococcus faecalis in abscessed primary molars. Twenty pulpally involved non-vital carious human primary mandibular second molars with furcal abscess were randomly allocated into two groups to evaluate the efficacy of metronidazole gel (3 % w/v) and metronidazole solution (0.5 % w/v) against E. faecalis. Subjects in the first experimental group were subjected to treatment with metronidazole gel (3 % w/v) and subjects in the second experimental group were treated with metronidazole solution (0.5 % w/v). Two microbial samples (pre-operative and post-operative samples) were obtained from the root canals of each subject from both the groups; sub cultured and efficacy of both the

groups were evaluated they concluded that Metronidazole gel (3 % w/v) was more effective than metronidazole solution (0.5 % w/v) against E. faecalis.

Tan E E et al (2019) ¹⁴⁷ evaluated the the immune response of macrophages to bacterial infection in response to the combination of ciprofloxacin or amoxicillin and metronidazole and concluded that apart from disinfection of the root canal system, the combination of ciprofloxacin and metronidazole also exerts an immunomodulatory effect, which may aid in periapical healing.

TRIPHALA

Maurya D K et al (1997) ⁸⁸ evaluated 60 cases of either sex and different age with different stages of inflammatory periodontal diseases with metronidazole and triphala and they concluded by saying that the efficacy of the drug, triphala in relieving the signs and symptoms in patients of periodontal disease was proved as identical in comparison to the modern drug and the drug is effective against many signs and symptoms of the periodontal disease.

Jagtap AG & Karkera SG (1999) ⁶¹ tested the aqueous extract from Terminalia chebula for its ability to inhibit the growth and some physiological functions of Streptococcus mutans.

Their results proved that the extract strongly inhibited the growth, sucrose induced adherence and glucan induced aggregation of S. mutans. Mouthrinsing with a 10% solution of the extract inhibited the salivary bacterial count and salivary glycolysis. Mouthrinsing with the extract significantly reduced total bacterial counts and the total streptococcal counts in the saliva samples obtained up to and including 3 h after rinsing, compared with the counts obtained prerinsing or after placebo rinsing. They concluded by saying that the extract of T. chebula may be an effective agent in the treatment of carious teeth, owing to its ability to inhibit the growth and accumulation of S. mutans on the surface of the tooth. This would

prevent the accumulation of acids on the surface of the tooth, and thus the further demineralization and the breakdown of the tooth enamel.

Abraham S et al (2005) ² evaluated the inhibitory activity of triphala on PMN-type matrix metalloproteinase (MMP-9) expressed in adult periodontitis patients and compared its activity with another ayurvedic drug, kamillosan, and doxycycline, which has known inhibitory activity. Matrix metalloproteinases (MMPs) were extracted from gingival tissue samples from 10 patients (six males, four females) with chronic periodontitis. Triphala showed a 76.6%

reduction of MMP-9 activity, whereas kamillosan showed a 46.36% reduction at a concentration of 1,500 microg/ml (crude extract) and doxycycline showed a 58.7% reduction at a concentration of 300 microg/ml (pure drug) and they stated that the present study had showed the strong inhibitory activity of triphala on PMN-type MMPs involved in the extracellular matrix (ECM) degradation during periodontitis.

Kumar et al (2009) ⁷⁴ prepared anointment from the Triphala extract (10% w/w) and assessed for in vivo wound healing on infected rat model by rate of healing, bacterial count, biochemical analysis, and expression of matrix metalloproteinases. Assessment of granulation tissue on every fourth day showed significant reduction in bacterial count with significant level of collagen, hexosamine, uronic acid, and superoxide dismutase in the treated group (P < 0.01).

Reduction of matrix metalloproteinase expression observed in the treated group by gelatin zymography and immunoblotting confirms our in vivo assessment. The above results showed the antibacterial, wound healing, and antioxidant activities of Triphala ointment, necessary for the management of infected wounds. Active principles of the Triphala may be further evaluated and used as an excellent therapeutic formulation for infected wounds.

Loganathan et al (2009) ⁸⁰ evaluated the ethanolic extracts of the triphala formulation for its total antioxidant activity using improved ABTS radical cation decolorizing assay and antibiotic assay against Streptococcus mutans (predominantly involved in bio-film formation on human teeth). An effort was also made to correlate its antiplaque activity using an in-vitro assay (conditions were kept similar to oral cavity) with Triphala and two commercial toothpastes (Product 1 and product 2). Their results proved that the herbal extract effectively inhibited the bio-film formation and the better antioxidant activity exhibited by the extract might protect the gum cells effectively from free radicals than the commercial toothpastes.

Tandon S et al (2010) ¹⁴⁸ conducted a study on 1501 students in the age group of 8- 12 years with the aim of determining the effect of Triphala mouthwash on prevention of dental caries (manifest caries) as well as incipient carious lesions, and also comparing the effect of Triphala and chlorhexidine mouthwashes. The incipient caries was recorded at 3, 6, 9 months intervals and manifest caries at 9 months interval. No significant increase in the DMFS scores was found at the end of 9 months. Also, there was no significant increase in the incipient caries score towards the conclusion of the study. It was concluded that there was no significant difference between the Triphala and the chlorhexidine mouthwashes and with the use of Triphala, although there was an increase in the DMFS scores, it was not significant. The trend in the caries status was comparable to that of chlorhexidine. dmfs scores could not indicate the efficacy of mouthwash as this was a long-term study of nine months and a few of the carious teeth recorded at baseline were exfoliated at the conclusion of the study. However, no new manifested carious lesions were observed.

Prabhakar et al (2010) ¹¹³ evaluated the antimicrobial efficacy of Triphala, green tea polyphenols (GTP), MTAD, and 5% sodium hypochlorite against E. faecalis biofilm formed on tooth substrate. 5% sodium hypochlorite showed maximum antibacterial activity against

E. Faecalis biofilm formed on tooth substrate. Triphala, green tea polyphenols and MTAD showed statistically significant antibacterial activity. They stated that the use of herbal alternatives as a root canal irrigant might prove to be advantageous considering the several undesirable characteristics of NaOCl.

Pujar M et al (2011) ¹¹⁷ evaluated the antimicrobial efficacy of Triphala, Green tea polyphenols (GTP) and 3% of sodium hypochlorite against E. faecalis biofilm formed on tooth substrate. Human extracted teeth were biomechanically prepared, vertically sectioned and placed in wells containing E. faecalis to form a biofilm. After 2 weeks all groups were treated for 10 minutes with test solutions (Triphala, GTP, 3% of sodium hypochlorite and saline) and were analysed quantitatively. Sodium hypochlorite has shown maximum antibacterial activity against E. faecalis biofilm formed in tooth substrate. Triphala and GTP have shown significantly better antibacterial activity. Thus Herbal alternatives can be used as root canal irrigants, considering the undesirable effects of sodium hypochlorite.

Velmurugan A et al (2013) ¹⁵³ compared the effect of 20% aqueous extract of Terminalia chebula and Emblica officinallis with chlorhexidine as an anticaries mouthwash in vivo . Forty five high caries risk patients were divided into group 1,2,3 [ n=15]and asked to rinse with 20%

aqueous extract of Terminalia chebula and Emblica officinalis, 0.2% Chlorhexidine for 40 seconds respectively. Salivary samples were collected for pH, buffering capacity before and after rinsing at repeated intervals of 10, 30,60 and 90 minutes and except for microbial analysis for which it was collected before and 90 minutes after rinsing. The pH and the buffering capacity analysis was done using chairside kit and percentage of reduction of Streptococcus mutans and Lactobacillus were tabulated and statistically analysed. For salivary pH, Emblica officinalis showed the highest peak value followed by Terminalia chebula. For salivary buffering capacity, there was no significant difference between any of the groups. For microbial count, highest percentage of reduction was seen in Emblica

officinalis followed by Terminalia chebula and 0.2% Chlorhexidine . However 0.2%

Chlorhexidine was more efficient at 90 minutes compared to other groups. Thus the aqueous extract of Embilica officinalis extract and Terminalia chebula was more effective anticaries mouthwash but with less time of action than chlorhexidine.

Prasad P S et al (2014) ¹¹⁴ used Atomic Force Microscope and Energy Dispersive X-ray Spectroscopy to evaluate the effect of 5% NaOCl, 17% EDTA and triphala on ProTaper and iRaCe rotary Ni-Ti instrument. A total of eight Ni-Ti rotary files, four files each of ProTaper - S2 (Dentsply) and iRaCe - R3 (FKG DENTAIRE) were used. Three out of four files each from ProTaper and iRaCe were immersed in 5% NaOCl, 17% EDTA and Triphala separately for five minutes. The Roughness average (Ra), Root Mean Square (RMS) and Mean Height of Roughness Profile Elements (Rc) of the scanned profiles were then recorded using AFM and the elemental composition was evaluated with EDS. Data were analyzed by Student's t test, One Way ANOVA and Duncan's Multiple Range Test. Topographic irregularities at the nanometric scale were observed for all files. Files immersed in EDTA and NaOCl showed highly significant surface roughness than untreated files. Short- term contact with 17% EDTA and 5% NaOCl can cause significant surface deterioration of ProTaper and iRaCe rotary NiTi files. AFM proves to be a suitable method for evaluating the instrument surface.

Shakouie S et al (2014) ¹³² compared the antimicrobial activity of Triphala (a plant- derived solution) with 0.5, 1, 2.5 and 5% concentrations of sodium hypochlorite (NaOCl), against Enterococcus faecalis (E. faecalis). wo hundred plates of cultured E. faecalis, were divided into 5 experimental groups (n=38) and two positive and negative control groups. The antimicrobial activity of the test solutions was determined by measuring the zone of inhibition in the culture media. The antimicrobial activity of the test solutions was determined by measuring the zone of inhibition in the culture media. Triphala exhibited better

antimicrobial activity against E. faecalis compared to 0.5 and 1% NaOCl (P<0.05). Thus under the circumstances of this in vitro study, Triphala was more effective on cultures of E. faecalis compared to 0.5 and 1% NaOCl.

Thomas S et al (2017) ¹⁴⁹ evaluated the antimicrobial efficacy of diode laser, triphala, and sodium hypochlorite (NaOCl) against Enterococc-cus faecalis contaminated primary root canals. Forty-nine single-rooted human primary teeth were reduced up to cemento-enamel junction and biomechanically prepared. After sterilization, five teeth were selected as negative controls and remaining teeth were inoculated with E. faecalis. The teeth were then randomly divided into four groups. The first group was irradiated with diode laser, the second group was irrigated with sodium hypochlorite, and the third group with triphala solution. The fourth group served as the positive control. The antimicrobial efficacy was tested by collecting transfer fluid saline from the canals and counting the colony forming units (CFUs) of viable E. faecalis on agar plates. Laser group showed significant reduction in the colony count compared to the other groups. Triphala group showed better antibacterial activity than NaOCl, but the difference was not statistically significant.

Susan A C et al (2019) ¹⁴⁶ compared the smear layer removal ability of different solutions of Triphala (TA) when used in specific irrigant protocols in curved canals. Seventy- four mandibular first molars with 25–35 degrees of curvature of mesial roots were selected and standardized, and canals were prepared. Seventy-four mandibular first molars with 25–35 degrees of curvature of mesial roots were selected and standardized, and canals were prepared.

As the initial rinse solution (8 mL), 5% sodium hypochlorite was used. Samples were divided into control (Group I—normal saline, Group II—17% ethylenediaminetetraacetic acid) and experimental (Group III, IV, V, VI, VII, VIII, and IX) groups based on the type of final rinse solution (5 mL) used, that is, TA-premixed (P), TA- (P)-sonic, TA-(P)-ultrasonic, 3% TA solution, 5% TA solution, 10% TA solution, and 10% citric acid . The samples were

dehydrated, split buccolingually, splutter coated, and examined in field emission scanning electron microscope. Among the experimental groups, Triphala-(P)- ultrasonic presented the least amounts of smear and debris in all thirds of the root canal.Thus the use of TA as a final rinse solution during biomechanical preparation seems promising.

Manoraj A et al (2019) ⁸⁴ determined the effect of Triphala on antibiotic properties of gentamicin and oxacillin against multi-drug resistant organisms. The checkerboard method was used to determine the synergy of Triphala with gentamicin and oxacillin against multi-drug resistant (MDR) Gram negative bacilli and methicillin-resistant Staphylococcus aureus (MRSA) using 2,3,5-triphenyltetrazolium chloride (TTC) assay. Fractional inhibitory concentration (FIC) index was calculated. The results showed that Triphala has synergistic activity with gentamicin against the selected MDR Gram negative bacilli and with oxacillin against MRSA isolates warranting further studies on the possibility of clinical use.

AZADIRACTA INDICA

Van Der Nat JM et al (1987) ¹⁵² investigated the aqueous extract of the stem bark of Azadirachta indica with different parts of the human immune system. The extract showed strong anticomplementary effects which were dose-and time-dependent and most pronounced in the classical complement pathway assay. Moreover, a dose-dependent decrease in the chemiluminescence of polymorphonuclear leukocytes was observed and a dose-dependent increase in the production of migration inhibition factor by lymphocytes.

Nayak A et al (2011) ⁹⁹ evaluated the antimicrobial potential of Neem leaf aqueous and alcohol extracts. To determine the inhibitory effect of Azadirachta indica (aqueous and alcoholic extract of neem) on Streptococcus mutans, Enterococcus faecalis and Candida albicans. The activity of Azadirachta indica against Candida albicans, Streptococcus mutans and

Enterococcus faecalis was tested by serial broth dilution method and was expressed by minimum inhibitory concentration (MIC). The minimum inhibitory concentration (MIC) of the aqueous neem extract to all the organisms was 7.5%. The MIC of the alcoholic neem extract for E. faecalis, S.mutans, C. albicans were 1.88%, 7.5%, and 3.75% respectively. Aqueous and alcoholic extract of neem leaf showed significant antibacterial activity against S.mutans and E.faecalis and significant antifungal activity against C albicans. There was no statistical difference between the efficacies of alcoholic over aqueous neem extract.

Chatterjee A et al. (2011) ²⁴ assessed the efficacy of neem based mouth rinse regarding its antigingivitis effect. Forty five subjects with plaque induced gingivitis were selected for the study. They were equally divided into three groups. Group I patients were asked to rinse with 15 ml of neem mouthwash twice daily, group II with 15 ml of chlorhexidine mouthwash twice daily, and group III with 15 ml of saline twice daily. The three groups were asked to perform the routine oral hygiene procedures thought out the study period. Bleeding on probing and gingivitis were evaluated by Muhlemann and Son's Sulcus bleeding index (1971) and Loe and Sillness gingival index (1963), respectively, at base line, after every week till one month. Our result showed that an A. indica mouthrinse is equally effective in reducing periodontal indices as Chlorhexidine. The results demonstrated a significant reduction of gingival, bleeding, and plaque indices in both groups over a period of 21 days as compared to placebo. A. indica-based mouth rinse is equally efficacious with fewer side effects as compared to chlorhexidine and may be used as an adjunct therapy in treating plaque induced gingivitis.

Dutta A & Kundabala M (2013) ³⁷ analyzed the antimicrobial effect of five irrigants formulated from different parts of the tree Azadirachta indica (Neem) and compared with 2.5%

sodium hypochlorite and 0.2% chlorhexidine gluconate through an agar diffusion test. A clinical isolate of Candida albicans was innoculated on Sabourad Dextrose Agar and

Enterococcus faecalis (ATCC 29212) on Sheep Blood Agar. Wells with 6 mm diameter were created in agar and 100 μL aliquiots of each irrigant were introduced to five different wells.

After incubation, the largest uniform diameter of the inhibition zone was recorded. The leaf extract of the tree and a mixture of the seed-bark powder dissolved in dimethyl sulfoxide were active against both organisms. The other neem-based irrigants, a leaf powder dissolved in dimethyl sulfoxide, aqueous bark decoction and neem oil, did not possess any antimicrobial efficacy. Sodium hypochlorite completely inhibited growth of C. albicans and the leaf extract had larger inhibition zones than chlorhexidine (p = 0.011) or the seed-bark irrigant (p = 0.008).

Against E. faecalis, inhibition zones with chlorhexidine were the largest and differed significantly from sodium hypochlorite (p = 0.039), leaf extract (p = 0.008) and seed-bark irrigant (p = 0.011). Two neem irrigants displayed antimicrobial properties. The efficacy of the standard endodontic irrigants varied depending on the organisms tested.

Dutta A & Kundabala M (2014) ³⁸ compared the anti-microbial efficacy of 2.5% sodium hypochlorite (SHC) and 0.2% chlorhexidine gluconate with an experimental irrigant formulated from the Neem tree, Azadirachta indica A. Juss. A sample of 36 single rooted anterior teeth with periapical radiolucency and absence of response to vitality tests that required root canal treatment were selected for this study. The test irrigants and their combinations were assigned to five different groups and saline served as the control. Access cavities were prepared using an aseptic technique and samples collected for both anaerobic culture and Gram stained smears, followed by irrigation and sample collection again. The number of organisms were expressed in colony forming units/ml after 72 h of incubation; the smears were analyzed for their microbial loads and tissue clearance and assessed as per defined criteria. Their results found the maximum reduction in microbial loads, when analyzed by culture method, with a combination of SHC and the experimental neem irrigant.

Maximum tissue clearance on the Gram Stained smears was also found with the same combination.

Mistry K S et al (2014) ⁹⁰ checked the antimicrobial activity of Azadirachta indica (Neem), Ocimum sanctum (Tulsi), Mimusops elelngi (Bakul), Tinospora cardifolia (Giloy) and Chlorhexidine Gluconate (CHX) on common endodontic pathogens like Streptococcus mutans, Enterococcus faecalis and staphylococcus aureus. The agar diffusion test was used to check the antimicrobial activity of the Methanolic extracts of the medicinal plants along with CHX.

Six different concentrations of the tested agents were used for the study. The values of Zone of Inhibition were tabulated according to the concentration of the tested agent and data was statistically analyzed using ANOVA and Bonferroni post- hoc tests. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentrations (MBC) values were also recorded. The Methanolic extract of A.Indica, O.sanctum, M. Elengi, T.cardifolia and Chlorhexidine Gluconate has considerable antimicrobial activity against S. mutans, E. faecalis and S. aureus.

Mistry K S et al (2015) ⁸⁹ determined the antimicrobial activity of Azadirachta indica (Neem), Mimusops elengi (Bakul), and Chlorhexidine gluconate (CHX) on multispecies biofilm of common endodontic pathogens such as Streptococcus mutans, Enterococcus faecalis, Staphylococcus aureus and Candida albicans. The in vitro dentin disinfection model was used to check the antimicrobial activity of the methanolic extracts of the medicinal plants along with Chlorhexidine gluconate. The polymicrobial biofilm was allowed to grow on extracted teeth sections for a period of 21 days. Remaining microbial load in the form of CFU/ml after the antimicrobial treatment was tabulated, and data were statistically analyzed using ANOVA and Bonferroni post-hoc tests. Both the plant extracts showed considerable antimicrobial efficacy as compared to negative control. 2% CHX was the most effective antimicrobial agent having statistically significant difference against plant extracts and negative control (saline).

Joy Sinha et al (2015) ⁶³ evaluated the disinfection of dentinal tubules using Propolis, Azadirachta indica (alcoholic and aqueous extracts), 2% chlorhexidine gel and calcium hydroxide against Candida albicans biofilm formed on tooth substrate. One hundred and five human teeth were infected with Candida albicans for 2 days. Samples were divided into 7 groups. Group I- Propolis, Group II- Alcoholic extract of Azadirachta indica, Group III- Aqueous extract of Azadirachta indica, Group IV- 2% Chlorhexidine, Group V- Calcium hydroxide, Group VI- Ethanol and Group VII- Saline (negative control). At the end of 1,3 and 5 days, the antimicrobial efficacy of medicaments against Candida albicans was assessed at the depths of 200 µm and 400 µm. The overall percentage inhibition of fungal growth (at 200 µm and 400 µm depth) was 99.2% with 2% chlorhexidine gel. There was no statistical difference between propolis, alcoholic extract of Azadirachta indica (neem) and 2%

chlorhexidine. Propolis and alcoholic extract of Azadirachta indica performed equally well as that of 2% Chlorhexidine.

Chandrappa P M et al (2015) ²² assessed the antimicrobial activity of herbal medicines (neem extract, tulsi extract) and chlorhexidine against Enterococcus faecalis in Endodontics. Agar diffusion method was used to evaluate the antimicrobial action of different medicines. Sixty samples were segregated into four groups with 15 samples in each: Group I: chlorhexidine 2%, Group II: neem extract, Group III: tulsi extract, and Group IV: distilled water. The inhibition zones against E. faecalis were recorded and statistically assessed using one-way analysis of variance (ANOVA) test (P < 0.05). Significant antibacterial effect against E. faecalis was observed with chlorhexidine followed by neem extract and tulsi extract. They concluded by saying that Herbal medicines seemed to be effective against E. faecalis compared to 2%

chlorhexidine gluconate.

Sundaram D et al (2016) ¹⁴³ evaluated the efficacy of 40% honey, 100% neem leaf extract and 5.25% sodium hypochlorite as an intracanal irrigant against the isolated microorganisms from infected root canal. The samples were collected from infected root canals of 60 primary molar teeth indicated for pulpectomy. Alpha hemolytic Streptococci, gram negative bacilli, Candida, Staphylococci, Lactobacilli, Enterococci, Spore bearing gram positive bacilli and Micrococci were the microorganisms isolated from the samples. The zone of inhibition against the microbial growth was measured by agar well diffusion method. Statistical analysis was done by Repeated Analysis of Variance (ANOVA) and Bonferroni method. The results indicated that 5.25% sodium hypochlorite is more effective as root canal irrigant when compared with 100% neem leaf extract and 40% honey. It was also observed that 100% neem leaf extract has greater antimicrobial effect than 40% honey.

Anand S et al (2016) ⁵ compared the antibacterial efficacy of Azadirachta indica (Neem), Commiphora myrrha (Myrrh), Glycyrrhiza glabra (Liquorice) with 2% Chlorhexidine (CHX) against E. faecalis by using Real Time PCR. A total of fifty teeth specimens (n=50) were inoculated with E. faecalis for 21 days. Specimens were divided into five groups (Group 1:

Myrrh, Group 2: Neem, Group 3: Liquorice, Group 4: 2% CHX and Group 5: Saline (negative control)). The intracanal medicaments were packed inside the tooth. After 5 days, the remaining microbial load was determined by using real time PCR. Threshold cycle (Ct) values of Myrrh extract, Neem extract, Liquorice Extract, 2% CHX and saline were found to be 30.94, 23.85, 21.38, 30.93 and 17.8 respectively. Myrrh extract showed inhibition of E.faecalis equal to that of 2% CHX followed by Neem, Liquorice and Saline.

Babaji & Prashant et al (2016) ⁷ evaluated the antimicrobial effect of herbal root canal irrigants (Morinda citrifolia, Azadirachta indica extract, Aloe vera) with sodium hypochlorite (NaOCl).In their study highest inhibitory zone against E. faecalis was seen in NaOCl