DISSERTATION SYNOPSIS

DR. GARIMA BAJPAI POST GRADUATE STUDENT DEPARTMENT OF PEDODONTICS AND PREVENTIVE DENTISTRY

A.J. INSTITUTE OF DENTAL SCIENCES, KUNTIKANA P.O,

BATCH 2012-15 Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore.

ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION 1 . Name of the Candidate And Address ( in block letters ) DR. GARIMA BAJPAI POST GRADUATE STUDENT, DEPARTMENT OF PEDODONTICS AND PREVENTIVE DENTISTRY, A .J. INSTITUTE OF DENTAL SCIENCES, KUNTIKANA P.O., NH-17, MANGALORE.-575004 2 . 3 . Course of study and subject Name of the institution KARNATAKA. A .J .INSTITUTE OF DENTAL SCIENCES, MANGALORE MASTER OF DENTAL SURGERY-PEDODONTICS AND PREVENTIVE CHILDREN DENTISTRY. BRANCH-VIII. 4 . 5 . Date of admission to course Title of the topic : COMPARATIVE EVALUATION OF ANTIMICROBIAL EFFICACY OF ALOE VERA, GINGER AND NEEM EXTRACT ON STREPTOCOCCUS MUTANS, LACTOBACILLI & CANDIDA SPECIES ISOLATED FROM SALIVA OF PRESCHOOL CHILDREN. 23/5/2012

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Brief resume of the intended work : 6.1 Need for the study: Dental caries is multifactorial and one of the most common disease of childhood, which is neither self limiting nor amenable to short pharmacological management. Endogenous oral bacteria species such as S.mutans, S. sobrinus and L. acidophilus species are the active pathogens involved in the initiation and progression of dental caries {1}. During early childhood period children are more susceptible to opportunistic microbial colonization in the oral cavity especially Candida albicans{2} , due to immature immune system and not fully established micro flora. Effective prevention of these infections can be achieved by mechanical plaque removal sufficiently and thus chemotherapeutic antimicrobial mouth rinses such as chlorhexidiene may be used to limit these infections {3} . These chemical agents used in the form of either dentifrices or mouth rinses may have undesirable side effects such as tooth staining, taste alteration and development of hypersensitivity reactions {4,5}. Although antibiotics are routinely used to prevent the systemic infections, they are not recommended for regular prevention of dental biofilm formation because of risk of bacteria developing resistance to them. For the past few years, extracts or oils of medicinal plants have been used for prevention of various infections {6-9}. Amongst these are; Aloe Vera, Ginger (Zingiber officinale) and Neem (Azadirachta indica). ALOE VERA: Aloe Vera is a well known medicinal plant belonging to the Liliaceae family. The mucilaginous tissue in the centre of the Aloe vera leaf (Aloe vera gel) has traditionally been used for treatment of digestive tract disorders, sunburn and wounds. The gel consists of 98-99% water and the remaining 1-2% contains the active compounds,

including aloesin, aloin, aloe-emodin, aloemannan, aloeride, naftoquinones, methylchromones, flavonoids, saponin, amino acids and vitamins. The pharmacological actions of Aloe vera gel as studied in vitro and in vivo include anti-inflammatory, antibacterial, antioxidant, immune-boosting and hypoglycaemic properties. GINGER (Zingiber officinale): It is a medicinal plant that has been widely used in Chinese and ayurvedic medicines all over the world. Especially the rhizome part of the ginger has been used in traditional medicine. Ginger has antimicrobial and therapeutic effects{9}. NEEM(Azadirachta indica) : Neem leaves extracts and oils have been used since ages for their known antibacterial and antiseptic effect. Presence of fluoride is documented in Neem, which is known to exert an anticariogenic action against Streptococcus species. The aim of this study is to evaluate the efficacy of these medicinal plant extracts in efficient and effective reduction of cariogenic pathogens in oral cavity, thereby incorporating them in mouthwashes or topical preparations as caries prevention measures in children which is an important objective of pediatric dentistry. The purpose of this study is to compare and evaluate antimicrobial efficacy of Aloe Vera, Ginger & Neem extract on S.mutans, Lactobacilli spp. & Candida spp. isolates taken from saliva of pre-school children (age <6 years).

6.2 Review of literature :

Mohammadmehdi Fani and Jamshid Kohanteb (2012){1}

Investigated the inhibitory activities of Aloe vera gel on some cariogenic (Streptococcus mutans), periodontopathic (Aggregatibact actinomycetemcomitans, Porphyromonas gingivalis) and an opportunistic periodontopathogen (Bacteroides fragilis) isolated from patients with dental caries and periodontal diseases. The study concluded that Aloe vera gel at optimum concentration could be used as an antiseptic for prevention of dental caries and periodontal diseases.
Geeta Bhatt, Praveen Kudva & Vidya Dodwad (2011){10}

A total number of 15 subjects were evaluated for clinical parameters like plaque index, gingival index, probing pocket depth at baseline, followed by scaling and root planing (SRP). Test site comprised of SRP followed by intra-pocket placement of Aloe vera gel, which was compared with the control site in which only SRP was done, and clinical parameters were compared between the two sites at one month and three months from baseline. The low plaque index observed in these subjects could be explained by the fact that Aloe vera is a good antibacterial.
Roopal V Patel, Vidhi T Thaker, VK Patel (2011){11}

The study aimed to evaluate the in vitro effects of ginger and honey on micro-organisms on carious teeth by employing antibiotic sensitivity test in two hundred & fifty (250) extracted carious teeth. Results indicate a considerable antibacterial activity of ginger & honey. sThe combined extracts were most effective against Staphylococcus aureus (30.0.0 1.5) mm but least effective against L. acidophilus (21.0 0.7) mm.
Meenal N Gulve, Nitin D Gulve (2010){12}

This study compared the effectiveness of ginger extract and 2% sodium hypochlorite against Enterococcus Faecalis by using the agar diffusion test. Ginger extract showed better antibacterial efficacy against Enterococcus Faecalis .
Manohararan K Pillai, Md. Zaini Asmawi, Tan Soo Choon, S. Sasidharan,

Subramanion J Lachumy, Rusli Ismail (2010){9} Ethanolic and water extracts obtained from Malaysian and Thailand gingers were evaluated for their inhibitory effect against eight bacterial isolates viz. Escherichia coli, Salmonella typhi, Bacillus subtilis, Bacillus thuringiensis, Micrococcus luteus, Pseudomonas aeroginosa, Staphylococcus aureus and Pseudomonas merobilis and two fungal isolates viz. Aspergillus niger and Candida spp.
Atai. Z, Atapour. M, Mohseni. M (2009){13}

The study investigated the antifungal activity of Zingiber officinale (Ginger) on Candida albicans. Ethanolic extract was prepared. The results showed that the ethanolic extract was effective on Candida albicans (2 mg mL-1).
Marco Antonio Botelho1, Rinaldo Araujo dos Santosh, Jose Galberto et al,

(2008){14}: The aim of this study was to compare the short-term efficacy and safety of a Azadirachta indica mouthrinse on gingival inflammation and microbial plaque, compared to 0.12% chlorhexidine. Additionally, the count of cariogenic bacteria (Streptococcus mutans) in the saliva was assessed before and after treatment. All clinical index scores were reduced in both groups seven and 30 days after treatment. The S. mutans count in the A. indica group was reduced from a median of 9550 CFU/ml to 1200 CFU/ml (P<0.001).

 Prashant GM, Chandu GN, Murulikrishna KS, Shafiulla MD (2007){15}

The study was conducted to evaluate the antimicrobial effects of mango and neem chewing sticks on the microorganisms Streptococcus mutans , Streptococcus salivarius , Streptococcus mitis, and Streptococcus sanguis which are involved in the development of dental caries.Mango extract, at 50% concentration, showed maximum zone of inhibition on Streptococcus mitis. Neem extract produced the maximum zone of inhibition on Streptococcus mutans at 50% concentration. Even at 5% concentration neem extract showed some inhibition of growth for all the four species of organisms. 6.3 Objectives of the study: 1.To evaluate the antimicrobial efficacy of Aloe vera extract on S.mutans, Lactobacilli spp. & Candida spp. 2. To evaluate the antimicrobial efficacy of Ginger extract on S.mutans, Lactobacilli spp. & Candida spp. 3. To evaluate the antimicrobial efficacy of Neem extract on S.mutans, Lactobacilli spp. & Candida spp. 4. To compare antimicrobial efficacy of herbal extracts (Aloe vera, Ginger and Neem) with saline (as control)

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Material and methods: 7.1 Source of data : 80 healthy pre-school children (age <6 years) of both sexes, who visit OPD at AJ institute of dental sciences, Mangalore, will be selected by Conventional/Universal sampling technique. Inclusion criteria: 1. Children whose parents give the consent for the examination. 2. Children with dmft/dmfs score >3. Exclusion criteria: 1. Children who have undergone any dental restoration,especially ZOE or GIC in past six months. 2. Children using Chlorhexidiene mouthwash or under antifungal therapy. 3. Using any herbal dentrifices. 4. Children under medication like antibiotics & steroid therapy in past one month prior to the study. 5. Children whose guardians do not agree for the consent.

7.2 Methods of collection of data: 1. The caries experience (DMFT/defs) of the children will be recorded using visible light, mouth mirror and CPI probe. 2. 2 ml of unstimulated saliva will be collected by asking the patient to drool passively into a sterile plastic bottle for 5 minutes. Subjects will be informed in advance not to eat or drink(except water) one hour before saliva collection to minimize possible food debris and stimulation of saliva. The samples will be then transferred to the microbiological lab where it will be prepared for the isolation of strains of, Streptococcus mutans, Lactobacilli spp and Candida spp. using mitis salivaris agar media with bacitracin, rogusa agar media and sobhourds agar media respectively, under sterile environment. Twenty isolates of each species will be derived. 7.3 METHODOLOGY: The obtained isolates of S.mutans, Lactobacilli spp. and Candida spp. each, will be then transferred into blood agar media separately to test the antimicrobial activity of the extract. Inoculation will be performed by using sterile swab brushes across the media. Each strain will be further cultured in 4 different plates A,B,C,D. A- 50% concentration of Aloe vera extract. B- 50% concentration of ginger extract. C- 50% concentration of neem extract D- Normal saline 0.9% weight by volume.

7.4 DETERMINING ANTIMICROBIAL EFFICACY: Antimicrobial efficacy will be evaluated by Ditch plate method. Ditch will prepared on the agar plates and will be filled with 50l extract. Sterile normal saline will be taken as control. The plates will then be incubated at 37C for 48 hrs after which they will be examined for the size of the inhibition zones. The inhibition zones will be measured with vernier calliper. STATISTICAL ANALYSIS: Statistical analysis will be done by chi square test and Kruskall-Wallis test.

7.5 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If so, please describe briefly. Yes.

7.6 Has ethical clearance been obtained from your institution in case of 7.5? Yes.

INVESTIGATION DESIGN

Saliva samples from 80 healthy preschool children will be collected and cultured to isolate..

N= 20 strains of S.mutans.

N= 20 strains of Lactobacilli spp.

N= 20 strains of Candida spp.

Microbiological analysis by culturing in Aloe vera, Ginger and neem extract in comparison with normal saline as control.

Determination antimicrobial efficacy by zone of inhibition.

Statistical analysis will be done by chi square test and Kruskall-Wallis test.

8.

LIST OF REFERENCES: 1. Mohammadmehdi Fani and Jamshid Kohanteb (2011) Inhibitory activity of Aloe vera gel on some clinically isolated cariogenic and periodontopathic bacteria. Journal of Oral Science, 2012, Vol. 54, No. 1, 15-21. 2. De Carvalho et al,(2007): review articles on presence of candida spp. in infants oral cavity and its association with early childhood caries. Brazillian journal of oral sciences 2007,vol 6(20),pg no:1249-1253. 3. Neeti Bajaj, Shobha Tondon (2011). The effect of triphala and chlorhexidiene mouthwash on dental plaque, gingival inflammation and microbial growth. Intr journal of ayurveda research2011. vol 2(1). 4. Chang YC, Huang FM, Tai KW, Chou MY (2001) The effect of sodium hypochlorite and chlorhexidine on cultured human periodontal ligament cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001. 92, 446-450. 5. Beaudouin E, Kanny G, Morisset M, Renaudin JM, Mertes M, Laxenarie MC, Mouton C, Jacson F, Moneret-Vautrin DA (2004) Immediate hypersensitivity to chlorhexidine: literature review. Eur Ann Allergy Clin Immunol 36, 123-126. 6.Pai MR, Acharya LD, Udupa N (2004) Evaluation of antiplaque activity of Azadirachta indica leaf extract gel a 6 week clinical study. J Ethnopharmacol 90, 99-103 7. Fine DH, Furgang D, Barnett ML, Drew C, Steinberg L, Charles CH, Vincent JW (2000) Effect of an essential oil-containing antiseptic mouthrinse on plaque and salivary Streptococcus mutans levels. J Clin Periodontol 27, 157-161. 8. Fani MM, Kohanteb J, Dayaghi M (2007) Inhibitory activity of garlic (Allium sativum) extract on multidrug-resistant Streptococcus mutans. J Indian Soc Pedod Prev Dent 25, 164-168.

9. Manoharan Karuppiah Pillai, Mohd. Zaini Asmawi,Tan Soo Choon et al Pharmacologyonline 3: 951-958 (2010). 10. Geeta Bhatt, Praveen Kudva & Vidya Dodwad (2011) Aloe vera: Nature's soothing healer to periodontal disease. J Indian Soc Periodontol. 2011 Jul-Sep; 15(3): 205209. 11. Roopal V Patel, Vidhi T Thaker, VK Patel (2011) Antimicrobial activity of ginger and honey on isolates of extracted carious teeth during orthodontic treatment. Asia pacific Journal of tropical Biomedicine, 2011,S58-s61. 12. Meenal N Gulve, Nitin D Gulve (2010) Comparison of antimicrobial efficacy of ginger extract and 2% sodium hypochlorite against Enterococcus Faecalis using agar diffusion method. JIDA,vol4 no.10 October 2010. 13. Atai. Z, Atapour. M, Mohseni. M (2009) The inhibitory effect of ginger on Candida albicans. American journal of applied sciences 2009:vol 6: no.6:1067-69. 14. Marco Antonio Botelho1, Rinaldo Araujo dos Santosh, Jose Galberto et al, (2008) Efficacy of a mouthrinse based on leaves of the Neem tree (Azadirachta Indica) in the treatment of patients with chronic gingivitis: A double-blind, randomized, controlled trial. Journal of Medicinal Plants Research Vol. 2(11), pp. 341-346, November, 2008. 15. Prashant GM, Chandu GN, Murulikrishna KS, Shafiulla MD. The effect of mango and Neem extract on four organisms causing dental caries: Streptococcus mutans, Streptococcus salivavius, Streptococcus mitis, and Streptococcus sanguis: An in vitro study. Indian J Dent Res 2007;18:148-51.