RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA ANNEXURE- II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. NAME OF THE CANDIDATE AND ADDRESS (IN BLOCK LETTERS) DR. BATHULA VIMALA CHAITANYA POST GRADUATE STUDENT, DEPARTMENT OF CONSERVATIVE DENTISTRY AND ENDODONTICS, SRI RAJIV GANDHI COLLEGE OF DENTAL SCIENCES & HOSPITAL, BANGALORE, KARNATAKA.

2.

NAME OF THE INSTITUTION

SRI RAJIV GANDHI COLLEGE OF DENTAL SCIENCES & HOSPITAL, BANGALORE, KARNATAKA. MASTER OF DENTAL SURGERY

3.

COURSE SUBJECT

OF

STUDY

AND BRANCH IV CONSERVATIVE DENTISTRY & ENDODONTICS

4.

DATE OF ADMISSION TO COURSE

25/05/2012

COMPARISON OF ANTIBACTERIAL EFFICACY OF TURMERIC EXTRACT, MORINDA 5. TITLE OF THE TOPIC CITRIFOLIA AND 3% SODIUM HYPOCHLORITE ON ENTEROCOCCUS FAECALIS AND STAPHYLOCOCCUS AUREUS : AN IN VITRO STUDY

6.

BRIEF RESUME OF THE INTENDED WORK

6.1 Need for the study: One of the primary objectives of endodontic therapy is the microbial reduction or their elimination, to promote normal healing & reestablishment of health of periradicular tissues. Mechanical instrumentation cannot sufficiently disinfect the root canals & hence irrigating solutions & intracanal medicaments are required to eradicate microorganisms.1 Sodium hypochlorite, the commonly used irrigant, has many potential properties; but has a cytotoxic effect when injected into the periapical tissues, also known to produce allergic reactions; a foul smell and taste, tendency to bleach clothes and corrosive potential.1 Currently, most bacteria isolated from endodontically infected teeth are anaerobic, microorganisms such as Enterococcus faecalis, aerobes like Staphylococcus aureus, are the most resistant species, and one of the possible causes of root canal treatment failure.1 The increase in antibiotic resistant strains caused by synthetic drugs led to search for herbal irrigants like Turmeric, Morinda Citrifolia etc. Advantages of using herbal alternatives are easy availability, low toxicity and lack of microbial resistance.2 The present study is intended to compare the antimicrobial properties of different herbal derivatives like Turmeric and Morinda Citrifolia and chemical irrigant i.e Sodium hypochlorite against E.Faecalis and S.aureus

6.2 Review of Literature: Mithra N Hegde,Shishir Shetty,Mahalaxmi Yelapure et al conducted a study to evaluate antimicrobial activity of aqueous and hydro alcoholic curcuma longa extracts against endodontic pathogens. 2.3% NaOCl was used as a positive control. Both extracts showed good antimicrobial properties against endodontic pathogens.1

Prabhakar J, Senthilkumar M, Priya MS et al conducted a study to evaluate the antimicrobial efficacy of Triphala, green tea polyphenols, MTAD and 5% NaOCl against E.Faecalis biofilm formed on tooth substrate. The study concluded that 5% NaOCI showed maximum antibacterial activity against E.Faecalis biofilm followed by Triphala and Green tea polyphenols and MTAD.2

Murray PE, Farber RM, Namerow KN et al conducted a study to compare the in-vitro effectiveness of morinda citrafolia juice with NaOCI and chlordexidine gluconate to remove smear layer from canal walls of endodontically instrumented teeth.The study concluded that the action of morinda citrifolia juice was similar to NaOCI in conjunction with EDTA as an intracanal irrigant.3

Alper Kustarci, Elif Aybala Oktay, Abdullah Kilic,et al conducted a study to evaluate antimicrobial effect of 2.5% sodium hypochlorite, 12.5% propolis, 25% propolis, OCT and 2% chlorhexidine on microorganisms with different structural characteristics. S.aureus, E. faecalis, E.coli, C.albicans were included in the study. 2.5% NaOCl, 2% chlorhexidine and OCT produced inhibitory zones against all microorganisms tested. NaCl was ineffective against all microorganisms; however, 2.5% NaOCl, 2% chlorhexidine and OCT produced inhibitory zones against all microorganisms tested. 2.5% NaOCl and 2% chlorhexidine showed significantly larger average zones of inhibition compared to other experimental irrigants. While 12.5% propolis extract produced only slight inhibition on S.aureus, 25% propolis was effective on S.aureus, E.faecalis and C.albicans.4

Naoki H, Yoshinori M, Masato I et al carried out a study to test the antimicrobial efficacy of Japanese green tea. Extracts of four kinds of Japanese green tea were tested against 24 bacterial strains isolated from infected root canals. The study concluded that the extracts of all four kinds of tea had antibacterial and bactericidal actions against many of the bacteria.5 Reneta Dornelles-Morgental, Juliane maria Guerreiro Tanomaru et al conducted a study to evaluate the antibacterial efficacy of irrigating solutions (2.5% NaOCl, 2.5% NaOCl plus 10% citric acid, 2.5% NaOCl plus apple cider vinegar, apple cider vinegar, 2% chlorhexidine solution, 1% peracetic acid, saline solution) against E.faecalis. All solutions promoted reduction of E.faecalis after instrumentation, but bacterial counts were higher in the final sample. 2.5% NaOCl, 2% chlorhexidine solution and 1% peracetic acid had lower bacterial counts than other groups.6

Maria Teresa Arias Moliz, Carmen Maria Ferrer Luque et al conducted a study to evaluate the minimal biofilm eradication concentration of NaOCl, chlorhexidine, EDTA and citric and phosphoric acids after 1, 5 and 10 minutes of exposure to biofilms of E.faecalis. They concluded that NaOCl was most effective agent, capable of eradicating the biofilms after 1 minute at a concentration of 0.00625%. chlorhexidine eradicated biofilm after 5 minutes at 2%. EDTA, citric and phosphoric acid solutions were not effective against biofilms at any concentration or time tested.7

6.3 Aims and Objectives of the study: The aim of this study is to evaluate & compare the antibacterial efficacy of two herbal
irrigants i.e Turmeric and Morinda Citrifolia and chemical irrigant i.e.3%sodium hypochlorite (NaOCl) against Enterococcus faecalis and Staphylococcus aureus.

7.

MATERIALS AND METHODS:

7.1 Source of Data:

This in-vitro study will be conducted in department of Conservative Dentistry and Endodontics, Sri Rajiv Gandhi College of Dental Sciences and Hospital with the help of Wingene Biotech Research Labs, Bangalore.

Materials:
50 %Turmeric extract

6% Morinda citrifolia juice 3% Sodium hypochlorite solution Enterococcus faecalis strains(ATCC 21224) Staphylococcus aureus strains(ATCC 1295) Sterile distilled water Nutrient broth Agar media plates Incubator Sterilized swabs McFarland scale

7.2 Method of collection of data (including sampling procedure, if any) materials to be used in the study

50% Turmeric (curcuma longa) extract, 6% Morinda citrifolia juice & 3% Sodium hypochlorite will be used for determination of antibacterial efficacy.

Organisms selected : 1. Enterococcus faecalis 2. Staphylococcus aureus

The density of selected organisms will be adjusted equal to that of the 0.5 McFarland standards (1.5 x 108 CFU/ml) by adding them to nutrient broth for Staphylococcus aureus and Enterococcus faecalis. A 24 hour old culture will be used for the preparation of bacterial suspension. McFarland standards will be used as a reference to adjust the turbidity of microbial suspension so that the number of microorganisms would be within a given range. Agar well diffusion method will be used to conduct the anti microbial susceptibility test. A sterile cotton swab will be dipped into the respective microbial suspensions and surplus removed by rotation of the swab against the sides of the tube above the fluid level. The agar media plates will be inoculated with the respective organisms by even streaking of the swab over the entire surface of the plate three times, rotating the plate approximately 60 degrees after each application to ensure an even distribution of the inoculums. Finally, it will be swabbed all around the edge of the agar surface. Four Wells of 8 mm size will be made with sterile borer into agar plates containing the bacterial inoculums. Named as groups I,II,III,IV.

Groups : Group I: 50% Aqueous turmeric extract Group II: 50% Hydro alcoholic turmeric extract Group III: 6%Morinda Citrifolia juice Group IV: 3% Sodium hypochlorite 100l volume of each of the irrigants will be dispensed into the wells of inoculated plates. Sterilized distilled water will be used as a negative control. 3% sodium hypochlorite itself will be the positive control since it is the commonly used irrigant

and has excellent antimicrobial activity against the above mentioned organisms. The plates thus prepared will be refrigerated for 60 minutes allowing the diffusion of the irrigant into the agar. After incubation for h at the late w ll e observed.If

antibacterial activity is present on the plates, it will be indicated by an inhibition zone surrounding the well containing the irrigant. The zone of inhibition will be measured and expressed in millimeters. Antibacterial activity will be recorded if the zone of inhibition is greater than 8 mm. The antibacterial activity results will be expressed in terms of the diameter of zone of inhibition and <9mm zone will be considered as inactive; 9-12mm as partially active; while13-18mm as active and >18mm as very active. Experiment will be performed 12 times & the mean and standard deviation of the diameter of inhibition zones will be calculated.

PLAN FOR DATA ANALYSIS:

Mean comparison between the groups will be carried out by ANOVA test. If ANOVA is significant, further multiple comparisons will be done using Tukey test. P <0.05 will be considered as statistically significant.

7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If so, please describe briefly. No. 7.4 Has ethical clearance been obtained from your institution in case of above? yes

8 .LIST OF REFERENCES

1. Dr. Mithra N Hegde, Dr. Shishir Shetty, Dr Mahalaxmi Yelapure, Amit Patil. Evaluation of antimicrobial activity of aqueous and hydroalcoholic curcuma longa extracts against endodontic pathogens. IOSR Journal of Pharmacy; Mar Apr 2012; Vol 2(2) pp: 192-198.

2. Prabhakar J, Senthilkumar M, Priya MS, Mahalakshmi K, Sehgal PK, Sukumaran VG. Evaluation of antimicrobial efficacy of herbal alternatives (Triphala and green tea polyphenols), MTAD and 5% sodium hypochlorite against Enterococcus faecalis biofilm formed on tooth substrate: An in-vitro study. J Endod 2010; 36(1):83-86.

3. Murray PE, Farber RM, Namerow KN, Kuttler S, Garcia-Godoy F. Evaluation of Morinda citrifolia as an endodontic irrigant. J Endod 2008;34(1):66-70

4. Alper Kustarci, Elif Aybala Oktay, Abdullah Kilic, Ulku Ozan, Deme Altunbas. Evaluation of antimicrobial efficacy of sodium hypochlorite, propolis, octenidine dihydrochloride and chlorhexidine on microorganisms. Cumhuriyet Dent J 2011;14(3):183-190

5. Naoki H, Yoshinori M, Masato I, Toru M, Hiroshi N. A pilot study of Japanese green tea as a medicament:Antibacterial and bactericidal effects. J Endod 1991; 17(3):122-24.

6. Reneta Dornelles-Morgental, Juliane maria Guerreiro Tanomaru, Norberto Batista de Faria-Junior, Marco Antonio Hungaro- Duarte, Milton Carlos Kuga, Mario Tanomaru Filho,et al.Antibacterial efficacy of endodontic irrigating solutions and their combinations in root canals contaminated with Enterococcus faecalis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011; 112:396400.

7. Maria Teresa Arias Moliz, Carmen Maria Ferrer Luque, Miguel Espigares-

Garcia, Pilar Baca. Enterococcus faecalis Biofilms Eradication by Root Canal Irrigants. J Endod 2009;35:711-714