Introduction Endodontic treatment goals at complete disinfection of entire root canal system. The process mainly revolves around chemomechanical preparation, in which a chemical solution is used along with mechanical instrumentation in the root canal space. Various investigations have demonstrated that a thorough and complete debridement of root canal system with all its ramifications and anatomical irregularities is impossible with mechanically driven or hand instrumentation therefore the endodontic preparation should be supported by irrigating solutions. The ideal irrigants should be bactericidal, have the ability to dissolve necrotic tissue, remove the smear layer besides lubricating the canal, and should not be irritating to healthy tissues1 . Most commonly used endodontic irrigants in primary teeth are NaOCl, CHX, EDTA and Citric acid, mixture of doxycycline and citric acid with detergent. None of the irrigants has optimal properties to be termed an ideal irrigant. So it has been recommended to use of two or more solutions in a specific sequence or in combinations for better results. The weaknesses of NaOCl include the unpleasant taste, toxicity, and its inability to remove the smear layer by itself, as it dissolves only organic material. For this reason ethylenediaminetetraacetic acid (EDTA) was used, which effectively demineralises the smear layer but has to be used along with NaOCl to remove the organic part and does not have disinfective ability. Chlorhexidine is another commonly used antimicrobial agent which is being used to irrigate the canals due to its wide spectrum antimicrobial activity, biocompatibility and ability to disinfect the infected root canals. However it does not have 2 tissue dissolving capabilities and also has some undesirable effects as it may discolour the teeth, may lead to dryness of the oral cavity and may even cause burning sensation of the mouth. Constant increase in antibiotic resistant strains, side effects, safety concerns and cytotoxic reactions of synthetic irrigants has encouraged researchers to look for herbal alternatives2 . Herbal medicine is defined by world Health Organization as a plant originated preparation or material that includes processed or raw components from one or more plants that have medicinal properties. The use of herbal alternatives in dentistry is an emerging branch and it has been termed as phytodentistry3 . Over the past decades, interest in drugs derived from medicinal plants has markedly increased. In dentistry herbal medicines has been used as antiinflammatory, antibiotic, analgesic, sedative, and also as endodontic irrigant. Miswak (Salvadora Persica) contains large amounts of salvadorime chloride, trimethylamine and fluoride.It shows significant anti-microbial effect4 . Alcoholic extract is found to have more significant antimicrobial effect than the water extract. Neem (Azadirachta indica) is a commonly seen medicinal tree in India. Use of neem as an endodontic irrigant is advantageous because it is a biocompatible, antioxidant and thus not likely to cause any injuries to patients that is commonly seen with NaOCl. The Neem extract has been shown to have a wide spectrum of antibacterial efficacy against Gram positive, as well as Gram negative microorganisms. These effects have been particularly seen against Streptococcus mutans and Enterococcus feacalis. High levels of antimycotic activity have also been reported with extracts from different parts of neem. Tea Tree Oil (Melaleuca alternifolia) has many properties that makes its use in dentistry. It has antiseptic and antifungal properties. It also has mild solvent action and thus could be used as a potential root canal irrigant for dissolving the necrotic pulp tissue5 . Recent advances and scientific research in endodontics lead to introduction of newer chelating agents for smear layer removal in root canal systems. 3 Phytic acid is a new chelating agent proposed to have superior properties. Plant seeds and bran contains phytic as the major storage form of phosphorus (IP6, inositol hexaphosphate ) that contributes to a wide variety of cellular functions. On the basis of these proposed properties. IP6 has the potential to replace EDTA as a root canal chelating agent. Thus considering these properties of herbal irrigants, objective of this study is to form the sequence of herbal irrigation and compare its antibacterial efficacy with conventional irrigation in primary endodontic therapy

Methodology: 

1)Study design: It is Split mouth Randomized Controlled Trial. 

2)Study setting: Study will be carried out in Department of Pediatric and Preventive dentistry and Department of Microbiology after gaining clearance from institutional ethical committee. 

3)Study population: Maxillary or mandibular primary multirooted teeth of children of age group of 4-8 years reporting to the department of Pediatric and Preventive Dentistry for endodontic treatment after gaining written consent from parent/guardian. 

4)Sample size: The sample size was calculated using Openepi software (v3.0) at 95% confidence interval and 80% power in reference the study conducted by Varghese S et al. The formula for the same is: N = (σ1 2 + σ2 2 / κ) (Z1 – α /2 + Z1 – β )2 Δ 2 The notation for the formulae are: N = sample size σ1 = standard deviation of Group 1 σ2 = standard deviation of Group 2 Δ = difference in group means κ = ratio = 1 Z1 – α /2 = two-sided Z value (eg. Z=1.96 for 95% confidence interval). Z1 – β = power N= ( 4.06 x 4.06) + (3.13 x 3.13) (1.96 +0 .84)2 / (1.26 x 1.26) = 12 The calculated sample size 12 samples per group. So minimum sample size in the study will be 12 in each group.Total sample size is 24. Though the sample size is 24 according to the formula, we will be considering total sample size as 30 (15 samples in each group) 14 5)Sampling technique and blinding: Simple randomized sampling technique to select the right or left quadrant of the maxilla or mandible for treatment with the sequential herbal irrigation with the help of flip coin method will be used. Head will denote right side and tail will denote left side. It will be a double blinded study in which neither the participant nor the operator will know which intervention participants are receiving. 

6)Method of selection of study subject: 

I) Inclusion criteria: 1.Primary molars with irreversible pulpitis. 2.Adequate coronal structure to support rubber dam. 3.Teeth having at least 2/3rd of root length. 4.Patients who had not received antibiotics 4 weeks prior to microbiology sampling. 

II) Exclusion Criteria: 1.Teeth with unrestorable crowns. 2.Teeth with abnormal anatomy and calcified canals. 3.Teeth with evidence of extensive internal/external pathological root resorption and having excessive mobility. 4.Teeth requiring pulp therapy due to periodontal problems. 5.Any systemic disorder.

STUDY MATERIAL 1. Ethanolic extract of Miswak (Salvadora persica) 2. Ethanolic extract of Neem (Azadirachta indica) 3. Tea tree oil (Melaleuca alternifolia) 4. Phytic acid 5. NaOCl (Prime Dental Products Pvt.Ltd) 6. Normal Saline (Infutec Healthcare Ltd.Mumbai,India) 7. EDTA (Prime Dental Products Pvt.Ltd) 8. Chlorhexidine (Dentochlor, Ammdent)

 ARMAMENTARIUM: 1. Rubber dam 2. Barbed broach (Mani.inc, Tochigi, Japan) 3. Paper points 4. Tweezer 5. 5 ml syringe with 24gauge needle 6. K files (Mani.inc, Tochigi, Japan) 7. Sterile vials (Himedia Laboratories Pvt. Ltd, Mumbai, India) 8. Nutrient broth 9. Petri dish (Himedia Laboratories Pvt. Ltd, Mumbai,India) 10. Selective agar media 

STUDY GROUPS: Group 1 (Control group) NaOCl followed by Normal Saline followed by EDTA followed by Chlorhexidine 15 

Group 2 (Test group) Ethanolic extract of Miswak and Neem followed by Tea tree oil and phytic acid

EXPERIMENTAL DESIGN: 

Collection of samples:

 Conventional irrigation : Pre irrigation Sample (C1) Post irrigation Sample (C2) 

  Herbal irrigation: Pre irrigation Sample (H1) Post irrigation Sample (H2) 

Samples will be collected using sterile paper point which will be introduced into the root canal and moved circumferentially along the walls of the canal without being lifted till the apical foramen, it was left for 1 minute in the canal, then with sterile tweezers the paper points will be removed and transferred to vials containing 5ml nutrient broth and it will be transported to microbiological laboratory for culturing procedure. 

 ENDODONTIC PROCEDURE: Patient will be instructed to rinse his/her oral cavity for 30 sec with 5ml povidine iodine. Local anesthesia will be administered and rubber dam application will be done. After rubber dam application, the access opening will be done with sterile diamond round bur (#330). On completing access cavity, the pulp will be extirpated using barbed broach and the pre irrigation sample will be taken as mentioned. The biomechanical preparation will be performed. Irrigation will be done with use of the irrigating solution in the respective group. after this the canals will be dried with sterile paper points and post irrigation samples of the root canal contents will be taken in the same manner as mentioned before. Both the sample will be transferred to the microbiological laboratory for culturing procedure.

 â€¢ Procedure for Conventional irrigation21: (Group 1) NaOCl During instrumentation canal should be irrigated copiously with NaOCl to dissolve pulp remnants Normal saline (An intermediate irrigating solution will be used to prevent interaction between irrigating solutions like NaOCl and EDTA) EDTA (EDTA irrigating solution will be used to remove the smear layer) Normal saline Chlorohexidine (The canals are dried and irrigated with a final irrigating solution which is chlorhexidine to disinfect the canals) 

 â€¢ Procedure for herbal irrigation: (Group 2) Ethanolic extract of miswak followed by ethanolic extract of neem Tea tree oil (Dissolves necrotic tissue) Phytic acid as chelating agent (To remove smear layer) Final rinse with Ethanolic extract of miswak followed by ethanolic extract of neem will be done 

PROCESSING AND EVALUATION OF SAMPLE: The samples will be immediately transferred to the Department of Microbiology, where processing will be done within 2 h of collection of the sample. Samples will be inoculated in separate agar plates and incubated aerobically and anaerobically for 24 to 48 h at 37° C for identification of bacteria in primary teeth root canal such as enterococcus faecalis, prevotella intermedia by using selective agar media. Quantification and identification of microbial growth will be done. 

STATISTICAL ANALYSIS: Statistical analysis will be done with Statistical Package for Social Sciences (IBM SPSS Statistic for window, version 21.0. Armonk, NY: IBM Corp.) at 95% CI and 80% power to the study. Intergroup comparison will be done using quantitative analysis test. Descriptive statistics was performed in terms of mean, standard deviation. Statistical significance was calculated at p<0.05 19 8. 

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