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. DHEERAJ D. KALRA

DEPARTMENT OF PREVENTIVE

AND COMMUNITY DENTISTRY,

COLLEGE OF DENTAL SCIENCES,

DAVANGERE-577 004,

KARNATAKA.

2 NAME OF THE INSTITUTION COLLEGE OF DENTAL SCIENCES,

DAVANGERE-577 004,

KARNATAKA.

3 COURSE OF STUDY AND

SUBJECT

MASTER OF DENTAL SURGERY

(MDS) IN PREVENTIVE AND

COMMUNITY DENTISTRY.

4 DATE OF ADMISSION TO

COURSE

5 TITLE OF THE TOPIC:

ANTIMICROBIAL, ANTIPLAQUE AND ANTIGINGIVITIS EFFECTS

OF FOUR HERBAL EXTRACTS INCORPORATED IN DENTIFRICES:

AN IN-VITRO AND IN-VIVO STUDY.

6 BRIEF RESUME OF THE INTENDED WORK

6.1 Need for the study:

Dental caries and gingivitis are the most prevalent diseases of

mankind and these diseases are multifactorial in origin. In India, nearly 60–70%

of the child population is affected by dental caries1. Dental plaque plays an

important role in the development of dental caries, which results in both tooth

dysfunction and loss. Streptococcus species have been implicated as primary

causative agents of dental caries in humans and experimental animals.

Especially, Streptococcus mutans have been isolated from human dental plaque

and are known as the cariogenic oral bacteria. Many attempts have been made to

eliminate Streptococcus mutans from the oral flora. Antibiotics such as

ampicillin, chlorhexidine, erythromycin, penicillin, tetracycline and vancomycin

have been very effective in preventing dental caries. However, excessive use of

these chemicals can result in derangements of the oral and intestinal flora and

cause undesirable side effects such as microorganism susceptibility, vomiting,

diarrhea and tooth staining. In this direction the use of natural products in the

prevention and treatment of oral conditions has increased recently and could be

of benefit to low-socioeconomic level urban and rural communities.2

Plaque-induced gingivitis is one of the most frequent periodontal

diseases, affecting more than 90 % of the population, regardless of age, sex or

race.3

Aloe vera, popularly known as ‘babosa’, is a plant commonly found in the

Northeast of Brazil. Its foliage, extract and resin present antimicrobial, anti-

inflammatory and healing properties and are indicated in hepatic and stomach

diseases. The antimicrobial effect of a dentifrice containing Aloe vera has been

demonstrated in an in-vivo study, in which this phytotherapic agent inhibited the

growth of diverse oral microorganisms.4

'Triphala' has been described as a classic ayurveda remedy, probably the

best known among all ayurvedic compounds. It is the combination of ripe,

healthy and dried fruits in equal quantities of Amalaki (Emblica officinalis),

Haritaki (Terminalia Chebula) and Vibhitaki (Terminalia Belerica).5

The antimicrobial activity of Punica granatum Linn has been widely

investigated. The findings of several studies, including some relating to

inhibition of adherence, suggest that the phytotherapeutic use of this plant might

be a viable option in controlling different microbial species.6

Tulsi, scientifically known as Ocimum sanctum, is a time-tested premier

medicinal herb. It is a plant of Indian origin, worshipped by the hindus and used

in ayurvedic medicine since ancient times. It is a herb that is bestowed with

enormous antimicrobial substances and is used to treat a variety of illnesses

ranging from diabetes mellitus, arthritis, bronchitis, skin diseases, etc.7

Hence, this study will be conducted to investigate the antimicrobial,

antiplaque and antigingivitis effects of the above mentioned four herbal extracts

incorporated in dentifrices.

6.2 Review of literature:

A in-vivo study was conducted in Brazil wherin a dentifrice containing

Aloe vera was used on 30 adult subjects to evaluate the reduction of plaque and

gingivitis using Plaque index (PI) and gingival bleeding index (GBI) at day 0

and 30 days which revealed that the toothpaste was efficient in plaque

reduction.4

A randomized controlled clinical trial was conducted in India wherein the

role of triphala was assessed in the management of periodontal disease with

metronidazole as control. It was concluded that the drug triphala or

metronidazole, alone is capable to provide partial relief but when both are used

combinedly, more effective for the treatment of periodontal disease.5

An in-vitro study conducted in Brazil investigated the antimicrobial effect

of a Punica granatum Linn (pomegranate) phytotherapeutic gel and miconazole

(Daktarin® oral gel) against three standard streptococci strains, minimum

inhibitory concentrations of the gels on the adherence of these microorganisms

to glass was assessed. The Punica granatum L. gel demonstrated greater

efficiency in inhibiting microbial adherence than the miconazole.6

An in-vitro study was conducted in India to determine the antimicrobial

activity of Tulsi (Ocimum sanctum) extract against Streptococcus mutans wherin

15 concentrations of tulsi were investigated. Tulsi extract demonstrated an

antimicrobial property against Streptococcus mutans.7

7

6.3 Objectives of the study

1. To determine the Minimum Inhibitory Concentration (MIC) and

Minimum Bactericidal Concentrations (MBC) of all the four herbal

extracts.

2. To compare the MIC and MBC of all the four herbal extracts.

3. To evaluate the clinical efficacy of four herbal extracts in reducing

plaque and gingival scores.

4. To investigate the effect of four dentifrices containing herbal extracts

and two control dentifrices on Mutans streptococci and Candida

species.

5. To compare effect of four dentifrices containing herbal extracts on

Mutans streptococci and Candida species and also their efficacy in

reducing plaque and gingival scores.

MATERIAL AND METHODS

7.1 Source of data:

A total of 120 adults of the age group of 35 – 44 years will be included

in the study. School teachers fulfilling the eligibility criteria will be selected

randomly from schools in Davangere city, Karnataka.

7.2 Method of collection of data (including sample size, inclusion criteria,

exclusion criteria if any):

Study design:

Randomized controlled doubled blinded (observer and subject) clinical

trial with parallel group study design.

Sample size and design:

Sample size is determined using the formula

2

n= 2 Zα (σ)

d where Zα is determined from table values,

σ = Standard Deviation.

d= x1-x2

= 2.78-2.65

= 0.13

2 n = Sample size

n= 2 2(0.2)

0.13

n= 18.9

So the sample size is approximately 20.

A total of 120 adult subjects in the age group 35-44 years will be considered as

the study population. They will be randomly divided into 6 groups with 20

participants in each group.

The six study groups are,

Group I - Punica granatum Linn (Pomegranate) based dentifrice.

Group II - Aloe vera based dentifrice.

Group III - Triphala based dentifrice

Group IV - Ocimum sanctum (Tulsi) based dentifrice.

Group V - A commercially available dentifrice with one of the active

ingredient as fluoride as positive control.

Group VI - A placebo dentifrice as negative control.

Preparation of the extracts:

The herbal ingredients will be obtained from local market. The extracts

will be prepared by macerating 20 grams of dry powder of each herb with 100

milliliters of 70% w/v ethyl alcohol for a week. The extracts will be then filtered.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum

Bactericidal Concentration (MBC):

The extracts obtained will be used to determine MIC & MBC against

Mutans streptococci  and Candida species by standard susceptibility broth

dilution technique.8

Preparation of dentifrices:

All the test dentifrices will contain sodium lauryl sulfate as detergent,

silicon dioxide as abrasive, carboxymethylcellulose as binder, flavoring agent,

methyl-p-oxybenzoate as preservative, saccharin, sodium metaphosphate and de-

ionized water along with the respective herbal extract in concentrations as

determined by the in-vitro anti-microbial analysis.

STUDY POPULATION:

Inclusion criteria:

1) Subject with informed consent.

2) Subjects having Gingival Bleeding Index (GBI) score >40%.

3) Presence of at least 20 natural teeth.

Exclusion criteria:

1) Subjects with medical disorders or probing depth >3 mm.

2) Individuals under antimicrobial therapy at least 1 month prior to the

study and using mouthrinses or dentifrices containing substances with

anti-inflammatory properties.

3) Smokers and pregnant women.

Method of Examination:

Purpose of the study will be explained to the study subjects and

demographic details will be taken in a proforma along with the written consent

from all the subjects.

The clinical examination will be conducted in the Department of

Preventive and Community Dentistry, College of Dental Sciences, Davangere.

All the subjects will be provided with their assigned products and

instructed to use it twice daily. All the subjects will be instructed to follow their

routine oral hygiene practices along with the assigned products.

The volunteers will be examined for plaque and gingivitis at baseline

and after 30 days and 60 days by a single, previously calibrated examiner using

Gingival Bleeding Index (GBI) (Harold G. Carter and George P. Barnes) and

Plaque Index (PI) (Silness J. and Loe H.).

Plaque sample collection:

Before assigning the respective dentifrices to the groups baseline Mutans

streptococci and candidial counts will be estimated from all the subjects.

Samples will be collected at baseline from the buccal surface of one quadrant

using a swab. After sample collection, Mutans streptococci and candida colonies

will be cultured on Mitis Salivarius Bacitracin agar and Sabouraud’s Dextrose

agar respectively. Then again Mutans streptococci and candidial counts will be

estimated on 30th day and 60th day from the commencement of use of dentifrice.

Instruments and materials:

Mouth mirror, Explorer, Tweezers, Kidney tray, Enamel bowl, Gloves,

Mouth mask, Cotton, Korsolex disinfectant solution, Culture media and

Petridish.

Statistical analysis:

For MIC and MBC, means and standard deviations will be calculated.

One-way Analysis of Variance (ANOVA) followed by the Tukey’s post hoc test

will be performed for comparison of MIC and MBC of the different herbal

extracts. Kruskal-Wallis Analysis of Variance will be used for multiple group

comparison. Mann-Whitney U test for group wise comparison and Wilcoxons-

signed rank test for comparing microbial parameter with baseline. For

comparison of GBI and PI scores at 0, 30, and 60 days One-way Analysis of

Variance (ANOVA) followed by the Tukey’s post hoc test will be used. p value

<0.05 will be considered statistically significant.

Duration of study: 6 months

7.3 Does the study require any investigation (or) intervention to be

conducted on patients (or) other humans (or) animals? If so please describe

briefly :

Yes.

Baseline examinations will be done by a single examiner and scores of

Gingival Bleeding index and Plaque index will be recorded. All the subjects will

be provided with their assigned products and instructed to use them twice daily.

All the subjects will be instructed to follow their routine oral hygiene practices

along with the assigned products. At 30 days and 60 days, assessment will be

made by the same examiner using the same indices and plaque sampling will be

done followed by analysis of microbial counts.

7.4 Has ethical clearance been obtained from your institution?

Yes.

8. REFERENCES

1. Damle SG. Pediatric Dentistry-scope and rationale 1st edition. Arya

publisher, 2006.

2. Botelho MA, Nogueira NAP, Bastos GM, Fonseca SGC, Lemos TLG,

Matos FJA, et al. Antimicrobial activity of the essential oil from Lippia

Sidoides, carvacrol and thymol against oral pathogens. Braz J Med Biol

Res. 2007;40(3):349-56.

3. Villalobos OJ, Salazar CR, Sánchez GR. Efecto de un enjuague bucal

compuesto de aloe vera en la placa bacteriana e inflamación

gingival. Acta Odontol Venez. 2001;39(2):16-24.

4. Oliveira SMA, Torres TC, Pereira SLS, Mota OML, Carlos MX.

Effect of a dentifrice containing aloe vera on plaque and gingivitis control.

A double-blind clinical study in humans J Appl Oral Sci. 2008;16(4):293-6

5. Maurya DK, Mittal N, Sharma NK, Nath G. Role of triphala in the

management of Periodontal disease. Ancient Science of Life 1997:17

(2);1-6.

6. Pereira JV, Pereira MSV, Sampaio FC, Sampaio MCC, Alves PM,

Araújo CRF, et al. In vitro antibacterial and antiadherence effect of

Punica granatum Linn extract upon dental biofilm microorganisms. Braz J

Pharmacogn 2006; 16:88-93.

7. Agrawal P, Nagesh L, Muralikrishnan. Evaluation of the antimicrobial

activity of various concentrations of Tulsi (Ocimum sanctum) extract

against Streptococcus mutans: an in-vitro study. Indian J Dent Res

2010;21:357-9.

8. Ibrahim M. Determination of Minimum Inhibitory Concentration (MIC)

of the isolated pure compounds. http:/www.jesbd.info/MIC.pdf. Accessed

on 17.1.2011.

9. SIGNATURE OF THECANDIDATE ( Dr. DHEERAJ D. KALRA )

10. REMARKS OF THE GUIDE

11. NAME AND DESIGNATION OF

(IN BLOCK LETTERS)

11.1 GUIDE

Dr. PRASHANT G.M., M.D.S., READER ,DEPARTMENT OF PREVENTIVE AND COMMUNITY DENTISTRY, COLLEGE OF DENTAL SCIENCES, DAVANGERE – 577004.

11.2 SIGNATURE

11.3 Co-GUIDE (IF ANY)

11.4 SIGNATURE

11.5 HEAD OF THE DEPARTMENT

Dr. CHANDU G.N., M.D.S., PROFESSOR AND HEAD OF THE DEPARTMENT, DEPARTMENT OF PREVENTIVE AND COMMUNITY DENTISTRY, COLLEGE OF DENTAL SCIENCES, DAVANGERE – 577004.

11.6 SIGNATURE

12.12.1 REMARKS OF THE CHAIRMAN AND THE PRINCIPAL

12.2 SIGNATURE