FIELD OF THE INVENTION
The present invention relates to the development of herbal mouthwash or mouthrinse formulation for maintaining oral hygiene. It particularly relates to the development of Mentha arvensis (pudina) extract mouthwash or mouthrinse for reducing the cariogenic flora like Streptococcus mutans, Candida albicans, plaque inhibition .and maintaining gingival health on children. Its more specifically relates to the Mentha arvensis ethanolic extract mouthrinse formulation for reducing the cariogenic flora like Streptococcus mutans, Candida albicans, plaque inhibition and maintaining gingival health on children. The invention also pertains to the development of suitable process for preparation of the Mentha arvensis (pudina) ethanolic extract mouthrinse formulation for inhibition of Streptococcus mutans, Candida albicans, Plaque & maintaining Gingival Health. The present invention herbal pudina extract mouthrinse formulation is helpful for maintaining oral hygiene.
BACKGROUND OF THE INVENTION
Dental caries is a complex, multifactorial and microbial disease which is neither self limiting nor amenable to short-term pharmacological management.1"3 It is an infectious disease involving an interplay between internal defense factors such as; tooth surface morphology, general health, nutritional and hormonal factors, and a number of external factors- like diet, the microbial flora, oral hygiene, fluoride availability, plaque and saliva.4Saliva is a unique biological fluid which is amphi-functional, that baths the ecosystem possessing a large number of components that have been identified as protecting the teeth against caries by diluting and eliminating sugars and other substances, buffering capacity, remineralization and antimicrobial action. A lack of its secretion or alteration in physiochemical properties contributes to development of dental caries.s The main oral innate defense factors are the peroxidase systems, lysozyme, lactoferin and histatins. These proteins are known to limit bacterial or fungal growth, interfere with bacterial glucose uptake or glucose metabolism and promote aggregation and thus elimination of bacteria.The main organic acids produced from fermentable carbohydrates are lactic, formic and acetic acid which coincide with a pH drop in plaque, contributing to tooth demineralization. This creates an environment which is advantageous for further growth of microorganisms contributing to cariogenicity of the dental plaque.6 Carious lesion results primarily from the dissolution of minerals in enamel and dentin due to acid released by fermentation of carbohydrates from oral microorganisms. A group of phenotypically similar but genetically different streptococcal species, known as mutans Streptococci, is considered as the main etiological agent for dental caries in human.22 The changes in the homeostasis of the oral cavity with an overgrowth of Streptococcus mutans, is recognized as the primary cause of the disease. Due to the presence of specific cell-surface adhesin like proteins and bacterial polysaccharide, it is highly acidogenic and aids in its primary attachment to the tooth, producing short-chain carboxylic acids which dissolves enamel and dentine. Dental caries can be prevented by targeting the primary risk factors like micro¬organisms, diet, plaque, etc. Sucrose is the major environmental contributor to dental caries

because many of the oral streptococci, including the mutans Streptococci, possess extra cellular enzyme capable of cleaving the ct| and ot2glycosidic bond of sucrose and harnessing the energy produced to yield a glucose polymer and fructose. This group of enzymes, called the glucosyl transferase, accounts for this special relationship between sucrose and caries.28~33Formation of the glucan-mutans polymer allows the cariogenic bacteria, such as MS to accumulate into biofilms to form critical mass. Like most infectious diseases, adherence to specific receptor followed by the formation of critical mass is a prerequisite for disease. Without formation of critical mass, cariogenic bacteria would colonize the oral cavity but not be massed so as to cause destruction of the enamel surface. Accordingly, the formation of critical mass that is uniquely associated with GTF and sucrose is the biological reason for affliction with caries.7 Plaque control can be achieved by using preventive measures like the combination of mechanical and chemo-therapeutic approaches. The various vehicles for delivery of chemical agents with anti-plaque or anti-gingivitis action are toothpastes, mouthrinses, gels, varnishes, chewing gum, spray, irrigators etc.8These antimicrobial agents may assist in protection by reducing bacterial adhesion to the tooth surface, by reducing the growth of microorganisms and plaque accumulation, by selectively inhibiting only those bacteria directly associated with oral diseases, or by inhibiting the expression of virulence determinants, such as acid production or protease activity. 9 Brushes can reach difficult to clean areas such as inter proximal surfaces and can also reduce the growth of biofilms on soft tissues. Mouthwashes with antimicrobial effects perform this task using three methods, which include apoptosis, inhibition of bacterial growth and/or cell metabolic inhibition; and depending on their concentration their bactericidal and/or bacteriostatic properties vary.10
Mouthwash is defined as a non-sterile aqueous solution with a deodorizing, refreshing or antiseptic effect designed to reduce oral bacteria, remove food particles, temporarily reduce bad breath and provide a pleasant taste. Mouthwash acts by altering integrity of cell membrane of bacteria followed by intercellular coagulation and also has capability of absorbing negatively charged bacterial cell membrane phosphates possibly destroying cell wall and increasing permeability. I1,I2A variety of synthetic anti-microbial mouthwashes are available to prevent dental caries. It was shown to inhibit plaque formation, also reduce gingival inflammation and prevent dental caries. Chlorhexidine (0.2%) is the most common mouthrinse which has shown to effectively reduce the level of S.mutans and has been proved of the most effective anti-plaque agent. However, this mouthwash has been reported to have a number of local side effects on its long term use like brownish discoloration of teeth, some restorative materials and dorsum of tongue; taste perturbation; oral mucosal ulcerations and paresthesia; unilateral/bilateral parotid swelling and enhanced supra-gingival calculus formation.15
Taking into consideration the side effects of chlorhexidine, and the liking or faith of people towards herbal/natural products. Herbal medicine is both promotive and preventive in its approach. It is a comprehensive system which uses various remedies derived from plants and their extracts to treat disorders and to maintain good health. Natural plant extract like Pudina as a

whole or in combination have been scientifically proven to be safe and effective against various oral health problems especially in preventing tooth decay.
PUDINA: Pudina {Mentha arvensis) a perennial aromatic herb belong to the family Labitae and genus Mentha is an important culinary plant with immense medicinal use. The entire plant is antibacterial, antifebrile. It yields an essential oil and menthol which exert, through their rapid evaporation, lightly anesthetic and anodyne local effect. Pudina contains menthol as anti¬oxidants as a form of biologically active component. 17Peppermint leaves contains about 0.5-4% volatile oil that is composed of 50-78 % free menthol, monoterpene, menthofurane and traces of jasmine (0.15 %).The antimicrobial activity of terepenoids on microorganisms is mainly by membrane disruption.I6,l4K.umar et.al.(201 l)found a significant antifungal activity by both aqueous, ethonolic and methonolic mint leave extracts activity on C.albicans.14
There are reports which reported the herbal mouthwash or mouthrinse, the important ones include Ranjan Malhotra et al 2011 [Ranjan Malhotra, Vishakha Grover, Anoop Kapoor, Divya Saxena, Journal p/"Indian Society of Periodontology - Vol 15, Issue 4, Oct-Dec 2011] which discloses the Comparison of the effectiveness of a commercially available herbal mouthrinse with Chlorhexidine gluconate at the clinical and patient level. Ratika Sharma et. al, 2014 [Ratika Sharma (Dr.), Mamata Hebbal, Anil V.Ankola, Vikneshan Murugaboopathy, Sindhu Jayasimha Shetty, Journal of Traditional and Complementary Medicine, Volume 4, Issue 4, October-December 2014, Pages 272-278] which discloses the effect of two Herbal Mouthwashes on Gingival Health of School Children]. MM Pathan et al, 2017 [Multazim Muradkhan Pathan, Kishore Gajanan Bhat and Vinayak Mahableshwar Joshi, J Indian Soc.Periodontol. 2017 Jul-Aug; 21(4): 270-275] which discloses the comparative evaluation of the efficacy of a herbal mouthwash and chlorhexidine mouthwash on select periodontal pathogens: An in vitro and ex vivo study.
Though there are publications in the literature on the herbal mouthwash or mouthrinse but none of the prior art in the literature discloses the pudina mouthrinse formulation/composition which is efficient for reducing the cariogenic flora like Streptococcus /nutans, Candida albicans, plaque inhibition and maintaining gingival health on children. Thus, there exists several drawbacks or limitations with the prior art herbal mouthwash. Therefore, the present inventors have developed pudina ethanolic extract herbal mouthrinse formulation which is efficient for reducing the cariogenic flora like Streptococcus mutans, Candida albicans, plaque inhibition and maintaining gingival health on children and also a suitable process for preparation of herbal mouthrinse. The aim of this invention is to evaluate the efficacy of the medicinal plant extract is efficient and effective reduction of cariogenic pathogens in oral cavity, thereby incorporating them in mouthwashes as caries preventive measures in children which is important objective of pediatric dentistry. In vitro studies indicate that pudina ethanolic extracts are inhibitory to oral streptococci which are responsible for various oral diseases. Literature review revealed very few in vivo

studies worldwide assessing the effects of pudina ethanolic aqueous extracts on plaque, gingival, . S. mutans and Candida albicans.
OBJECTIVES OF THE INVENTION
The primary object of the present invention is the development of herbal mouthwash for inhibition of bacterial growth and maintaining oral hygiene.
The particular object of the present invention is the development of herbal mouthrinse formulation for reducing the cariogenic flora like Streptococcus mutans, Candida albicans, plaque inhibition and maintaining gingival health in children.
The specific other object of the present invention is the development of Mentha arvensis ethanolic extract mouthwash formulation for reducing the cariogenic flora like Streptococcus mutans, Candida albicans, plaque inhibition and maintaining gingival health on children.
The specific other object of the present invention is the development of Mentha arvensis ethanolic extract mouthwash formulation for inhibition of Streptococcus mutans, Candida albicans, Plaque & maintaining Gingival Health.
The other object of the present invention is the development of Mentha arvensis ethanolic extract mouthwash formulation for reducing the cariogenic flora like Streptococcus mutans, Candida albicans, plaque inhibition and maintaining gingival health on children which employs the naturally existing ingredients.
The other object of the present invention is the development of Mentha arvensis ethanolic extract mouthwash formulation for reducing the cariogenic flora like Streptococcus mutans, Candida albicans, plaque inhibition and maintaining gingival health on children which successfully maintains the oral hygiene.
The other object of the present invention is the development.of Mentha arvensis ethanolic extract mouthwash formulation for reducing the cariogenic flora like Streptococcus mutans, Candida albicans, plaque inhibition and maintaining gingival health on children which is cost-effective and eco-friendly.

The other object of the present invention is the development of Mentha arvensis ethanolic extract mouthwash formulation for reducing the cariogenic flora like Streptococcus /nutans, Candida albicans, plaque inhibition and maintaining gingival health on children which is having good shelf life and stability.
The other object of the present invention is the development of Mentha arvensis ethanolic extract mouthwash formulation for reducing the cariogenic flora like Streptococcus mutans, Candida albicans, plaque inhibition and maintaining gingival health on children which is easy to use with little technical expertise.
The other object of the present invention is the development of Mentha arvensis ethanolic extract mouthwash formulation for reducing the cariogenic flora like Streptococcus mutans, Candida albicans, plaque inhibition and maintaining gingival health on children which is not having any adverse or side effects, safe and practical to use.
Yet another object of the present invention is the development of suitable process for preparation of Mentha arvensis ethanolic extract mouthwash formulation for reducing the cariogenic flora like Streptococcus mutans, Candida albicans, plaque inhibition and maintaining gingival health on children.
SUMMARY OF THE INVENTION
Mouth rinses are widely used as an adjunct to mechanical oral hygiene measures due to their anti-microbial, anti-inflammatory and anti-oxidant properties. Of the commercially available mouth rinses, Chlorhexidine mouth rinse is considered to be the 'gold standard' for routine home oral hygiene measure. However, these mouth rinses are not safe for a routine use in children due to various side-effects; and are not only expensive but also not easily available in the remote rural areas in our country. In order to overcome these problems, currently mouth rinse formulations containing herbal products like Neem, Tulsi, Garlic, Mango, Pudina, Amlaetc are being proposed.
The aim of this invention is to evaluate comparative effect of Pudina extract and Chlorhexidine (0.2%) mouthwash on Streptococcus mutans, Candida albicans, plaque inhibition & gingival health on children. The study protocol was reviewed and approved by the ethical committee of the institution; and permission from the school authorities was obtained. An informed consent was obtained from the parents of the study subjects before the onset of the study. The present study, conducted on 45 healthy children between age group of 9 to 12 years of both the sexes were selected randomly from the Residential school. They were divided into 2 groups, 15 in each

group:Group A:ChIorhexidine mouthwash(0.2%)-control group, Group B:Pudina mouth rinse. The dental health assessment of the children was recorded using simplified oral hygiene index, gingival index, plaque index, DMFT index and deft index. Children having DMFT/deft scores of 4 were selected for the study. The children of each group performed an oral rinse by swishing 10 ml (undiluted) of the solution in the mouth for 60sec once daily. The samples were then subjected to microbiological analysis to get the colony forming units of streptococcus mutans per ml of saliva by using Mitis Salivarius bacitracin agar and Candida albicans using CHROM agar respectively. The data was statistically analyzed using SPSS vl7.0 software with one-way ANOVA and Turkey's POSTHOC HSD, Kruskal Wallis, Bonferroni statistical test. This study found a significant fall in the colony counts of Streptococcus mutans in all the three mouth rinse groups. A statistical significant fall in Candida albicans colony counts was found in Pudina group. Gingival index scores also showed the same results. Plaque index scores and oral hygiene index scores was significantly lower in all 2 groups at baseline, 14lh day and 21st day. Against Candida albicans, Pudina mouth rinses were found to be better. Pudina mouthwash has almost equivocal effect on S.mutans and C.albicans when compared to Chlorhexidine. The findings of this study indicate that Pudina mouth rinses could be the economical safer alternatives to the commercial products especially in low income groups and remote areas where modern health care facilities are not accessible.
STATEMENT OF THE INVENTION
Herbal mouthwash formulation for maintaining oral hygiene comprising a plant extract of Mentha arvensis (pudina) with other suitable ingredients. The plant extract is of Mentha arvensis leaves. The plant extract is ethanolic extract of Mentha arvensis leaves. Mentha arvensis extract is 40 to 60% (w/w) of the formulation preferably 50% (w/w) of the formulation. The other ingredients are selected from group of Pepperment oil, Sodium saccharine, Methyl paraben and distilled water. The amount of other ingredients is 1 g/L of Pepperment oil, 1 g/L of Sodium saccharine, 0.05% (w/w) Methyl paraben.
Process for preparation of herbal mouthwash formulation for maintaining oral hygiene comprising the extracting of Mentha arvensis and mixing of the effective amount of plant extract with other suitable ingredients selected from group of Pepperment oil, Sodium saccharine, Methyl paraben and distilled water to obtain the herbal mouth rinse composition.
BRIEF DESCRIPTION OF THE INVENTION
Fig. 1: Investigation design
Fig. 2:Gender distribution
Fig. 3: Mean Age Distribution Among Groups
Fig. 4: Mean and standard deviation of DMFT/deft among three groups
Fig. 5: Inter-group comparison of S.mutans counts at baseline, 14th and 21st days
Fig. 6:Inter-group comparison of Candida albicans counts at baseline, 14th and 21st days

Fig. 7:Inter group comparison of OHI scores at baseline, 141 and 21st days of the 2 groups Fig. 8:Inter-group comparison of Plaque index scores at baseline, 14th and 21st days
DETAILED DESCRIPTION OF INVENTION
Materials and Methods: Following ethical clearance from the Institutional Ethical committee,45 healthy children between the age group of 9-12years from the schools will be divided randomly into 2 groups of 15 each based on the mouth rinse prescribed: -Chlorhexidine mouth rinse 0.2% (Group A) as a control -50% concentration of Pudina mouth rinse (Group B)
INCLUSION CRITERIA:
1. Children whose parents give the consent for the examination.
2. Systemicaliy healthy subjects.
3. Atleast four restored/decayed and/or missing teeth (deft/DMFT)
EXCLUSION CRITERIA:
1. Children who cannot expectorate completely.
2. Children who could not brush their teeth on their own.
3. Children with a history of taking antibiotics 3 months prior to and during the study period.
4. Children undergoing orthodontic treatment or with an intraoral prosthesis.
5. Presence of any intra oral pathology.
6. Medically compromised children.
MATERIALS AND EQUIPMENTS:
Plane mouth mirrors, Probes(WHO probe), Tweezers, Kidney trays, Sterilizing solution (Korsolex), Gloves and mouth masks, Mouth rinses (Chlorhexidine and Pudina mouth rinses), Sterile plastic containers, Disposable cups Mouth rinses Used:Mouthrinses with an acceptable mild flavor.
Chlorhexidine(0.2%)mouth rinse as commercially available (CLOHEX,DR. Reddy's). Pudina mouth rinse: Pudina leaves were obtained from market and were dried in sunlight. The dried leaves were then powdered finely. lOOgm of finely powdered Pudina was then macerated with 500m! of 100% ethanol. It was then subjected to Soxlet filtration then with Whatman filter paper to obtain a clear filtrate. The filtrate so obtained was reduced at a low temperature of less than 60 degree Celsius to obtain a solid residue of Pudina extract.50 gram of extract was dissolved in 10ml of dimethyl formamide to obtain 50% concentration was made.
The ethanolic extract obtained was stored in a refrigerator for preparation of the mouthwash solution. Authorized Sweetening agent -Pepperment oil(lgm/l)and sodium saccharine(lgm/l) ,preservative-Methyl Paraben (0.05%) were added to it .The final concentrate was then diluted with 1000ml of sterile distilled water to make a mouthwash and it was dispensed in sterile air tight containers.

METHODOLOGY:
1. The study and treatment protocol will be explained in detail to the parents and the child and informed consent will be obtained for the same.
2. 30 healthy children in the age group of 9-12years were selected, 30 children will be selected based on the above mentioned selection criteria and divided into 2 groups. The study will be conducted for 21days.The children were instructed to brush twice daily. A base line non stimulated saliva sample will be collected only after a week of following the same.
A base-line non-stimulated whole salivary sample (2 ml) will be collected in the morning 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 collected in sterile bottles will be carried in the ice box containing ice (used as transport media).The samples will be tested for the number of colony forming units for Streptococcus mutans and Candida albicans by using Mitis Salivarius Bacitracin agar, and CHROM agar respectively.
These children will be randomly divided into 2 groups of 15 each and they will be prescribed Chlorhexidine (0.2%) and Pudina mouthrinse. They will be advised to rinse the mouth for lmin using 5ml of the mouth rinse daily after breakfast. The second sample will be taken only after 14 days after using mouthrinses and counted for colonies. The same procedure is advised to continue for one more week. At 21s1 day the children salivary sample is taken using same procedure and colonies are counted for S.mutans and C.albicans.
CLINICAL ASSESSMENTS:
1. Simplified oral hygiene index, 2. Gingival index, 3. Plaque index, 4. DMFT/deft index.
SIMPLIFIED ORAL HYGIENE INDEX: Simplified (OHI-S index) Index was described by John C Greene and Jack R Vermillion in 1964. Instruments: mouth mirror and explorer. Teeth selected: 16, 11, 26, 36, 31, 46.Surface examined: 4 posterior and 2 anterior teeth, buccal surface of selected upper molar and lingual surface of selected lower molar.
Criteria for classifying debris:
Score 0- No debris or stains.
Score 1- Soft debris covering not more than l/3r of the tooth surface, or presence of extrinsic
stains without other debris regardless of surface area covered.
Score 2- soft debris covering more than l/3r, but not more than 2/3r of exposed tooth surface.
Score 3- soft debris covering more than 2/3r of exposed tooth surface.

Criteria for classifying calculus.
Score 0-No calculus present.
Score 1-SupragingivaI calculus covering not more than l/3rd of the exposed tooth surface.
Score 2- Supragingival calculus covering more than l/3rd but not more than 2/3rd of the exposed
tooth surface or the presence of individual flecks of subgingival calculus around the cervical
portion of the tooth or both.
Score 3- Supragingival calculus covering more than 2/3rd of the exposed tooth surface or the
continuous heavy band of subgingival calculus around the cervical portion of the tooth or both.
OHI-S score for each individual is calculated by, the debris and calculus scores are totalled and
divided by the number of tooth surfaced scored.
Average of each individual debris and calculus i.e simplified debris index (DI-S) and simplified
calculus index (CI-S) will be scored from range of 0-3.
Good- 0.0 - 0.6, Fair- 0.7-1.8; Poor- 1.9-3.0
Some of the DI-S and CI-S will give the OHI-S values ranging from 0-6.
Good- 0.0- 1.2; Fair- 1.3-3.0; Poor- 3.1-6.0
GINGIVAL INDEX: Index was described by Loe H and Sillness J in I96341
Instruments: mouth mirror and blunt instrument, such as periodontal probe
Teeth selected: 16, 12, 24, 36, 32 and 44.
Scoring units: Disto-facial papilla, facial margin, mesial-facial papilla and the entire lingual
gingival margin.
Scoring criteria:
Score 0-absence of inflammation/normal gingiva.
Score 1- Mild inflammation, slight change in colour, slight edema, no bleeding on probing.
Score 2-Moderate inflammation; moderating glazing, redness, edema and hypertrophy, bleeding
on probing.
Score 3-severe inflammation; marked redness and hypertrophy, ulceration,tendency to
spontaneous bleeding.
Gingival score for each tooth is calculated by totaling the scores around each tooth. If the scores
around each tooth are totalled and divided by four, gingival index score for the tooth is obtained.
0.1- 1.0- mild gingivitis.
1.1-2.0- moderate gingivitis.
2.1- 3.0- severe gingivitis.
PLAQUE INDEX: Described by Sillness P and Loe H in 196449
Instruments used: mouth mirror and explorer/dye.
Index can be used both as a simplified index or full mouth index.
Teeth selected: 16, 12, 24, 36, 32, 44.
Surface examined: labial or buccal surface are divided into mesio-facial, facial and disto-facial,
Palatal or lingual surface is considered as single surface.
Scoring criteria: Score 0-No visible plaque. When probe is passed along the cervical portion of the tooth surface no plaque adheres to the tip of the explorer.
Score I- No visible plaque. When probe is passed along the cervical portion of the tooth surface a film of plaque adheres to the tip of the explorer.

Score 2- A thin to moderate accumulation of plaque is seen on the tooth surface of the cervical
portion of the crown.
Score 3- abundance of plaque is seen on the tooth surface at the cervical potion of the crown.
Plaque score for each tooth is calculated by adding the plaque scores of the mesio-facial, facial,
disto-facial, palatal surface and divided it by 4. The individual scores of all the teeth are added
and divided by the total number of teeth examined.
Excellent- 0; Good- 0.1 to 0.9; Fair- 1.0 to 1.9; Poor- 2.0 to 3.0.
DMFT/deft INDEX: DMFT index was developed by Henry T.Klein, Carrole E. Palmer and
Knutson J.W in 1938 and deft index was modification by WHO of def index given by Grubbel
A.O in 1944.1nstruments used: mouth mirror and explorer.
Surface examined: 5 surface- posterior teeth (facial, lingual, mesial, distal and occlusal) and 4
surface-anterior teeth(facial, lingual, mesial, distal)
Teeth examined: All erupted teeth except unerupted and supernumerary teeth.
D/d- decayed teeth.M/e- teeth lost due to dental caries. F/f- filled teeth.
DMFT/def score for individual is calculated by total of each component i.e., D/d, M/e, F/f
separately, then , total D/d+M/e+F/f=D/dM/eF/f
MICROBIOLOGICAL ASSESSMENTS
Streptococcus mutans: 0.1 ml saliva sample would be spread on Mitis Salivarius agar supplemented with 0.2U/ml bacitracin and colony forming units (CFU) were identified by morphology, size and color, and were counted using a stereomicroscope
Candida albicans: 0.1ml saliva sample would be spread on CHROM agar and colony forming units(CFU) were identified by morphology, size and color, and were counted using a stereomicroscope.
STATISTICAL ANALYSIS: The data was statistically analyzed by using Mean, Standard Deviation, One-Way variance ANOVA, Tukey's POSTHOC HSD test, Bonferroni, Kruskal Wallis test and Chi-Square Test in SPSS software 17.0. The mean and Standard deviations were used for age distribution, defs, and colony count observations for all three mouth rinse groups. The results were considered statistically significant at 0.05 probability level. One-Way variance ANOVA test was used to compare the mean of differential colony counts in all the three mouth rinse groups. Tukey's POSTHOC HSD, Bonferroni, Kruskal Wallis Analysis test was used for comparative analysis of three mouth rinse groups at different intervals.

Table 1 shows gender distribution. Out of total forty five samples, our study consisted of 16 males and females 14 . In Chlorhexidine group, there were 6 males (40%) and 9 females (60%). In Pudina group there were 10 males (66.7%) and 5 females (33.3%).
Table 2 shows mean age. The mean age of Chlorhexidine group was found to be 10.40 in 15 samples and in Pudina group it was 9.95 in 15 samples.
Table 3 shows mean DMFT/deft. The mean DMFT/deft of Chlorhexidine was found to be 5.2667 and in Pudina group it was 4.8667.

Table 4 and Table 5 shows Inter group comparison of S.mutans counts at baseline, 14th and 21st days of the 2 groups.
The mean salivary S. mutans counts in Chlorhexidine group is found to be 7.6099(S.D=0.23776) at baseline, 5.7117 (S.D= 0.16401) at 14th day and 4.5533 (S.D=0.23911) at 21st day. In Pudina group it was 7.4712 (S.D=0.17836) at baseline, 4.2830 (S.D=0.14451) at 14th day and 4.0029 (S.D=0.18010) at 21st day. This result was found to be statistically significant 03=0.001*).
Table 6 shows mean difference of S. mutans counts at different time intervals.
The result shows the mean difference was found to be 1.898l7(p<0.001 ) in Chlorhexidine group
and 0.18821 (p=0.012) in Pudina group between baseline and 14lh days.
Mean difference was 3.05663(p<0.00f),0.34499(p=0.056) and 0.26829 (p<0.001*) for
Chlorhexidine and Pudina respectively between Baseline and 21s' day
The result shows the mean difference was found to be 1.15846(p<0.001 ) in Chlorhexidine group
and 0.08009 (p=0.600) in Pudina group between 14th day and 21st days.

Table 7 shows inter group comparison S. mutans counts in Chlorhexidine and Pudina groups. The mean difference -0.81642 between Chlorhexidine and Pudina (p<0.001 ) at baseline. The mean difference -2.37196 between Chlorhexidine and Pudina groups (pO.001*) at 14th Day. The mean difference -3.52805 between Chlorhexidine and Pudina groups was found to be -3.64964(p<0.001*) at 21st day.
Table 8 and Table 9 shows Inter group comparison of Candida albicans counts at baseline, 14th and 21sl days of the 2 groups. The mean salivary Candida albicans counts in Chlorhexidine group is found to be 4.723 (S.D=0.270) at baseline, 3358 (S.D= 0.308) at 14th day and 3.075 (S.D=0.361) at 21st day. In Pudina group it was 4.853 (S.D=0.094) at baseline, 3.300 (S.D=0.I02) at 14Ih day and 3.246 (S.D=0.112) at 21st day. This result was found to be statistically significant (p=0.001 ) in Chlorhexidine and Pudina group.

Table 10 shows Mean difference of C. albicans counts at different time intervals (baseline, 14lh and 21st day). The result shows the mean difference was found to be 0.23510 (pO.OOl ) in Chlorhexidine group and 0.25348 (pO.OOl*) in Pudina group between baseline and 14lh days. Mean difference was 0.35165 (p<0.001*)and 0.30760 (p=0.001*) for Chlorhexidine and Pudina respectively between Baseline and 21st day. The result shows the mean difference was found to be 0.11654 (p=0.351) in Chlorhexidine group and 0.05412 (p=0.475) in Pudina group between 14th day and 21st days.
Table 11 and 12 shows intergroup comparison OHI scores at different time intervals (baseline,
14lh day and 21 sldays) of the 2 groups.
The mean OHI scores in Chlorhexidine group is found to be 1.4400 (S.D=0.07368) at baseline,
0.9333 (S.D=0.8997) at 14th day and 0.4533 (S.D=0.07432) at 21stday. In Pudina group it was
1.4600(S.D=0.24142) at baseline, 1.0133 (S.D=0.26690) at 14th day and 0.6467 (S.D=0.23563)
at 21s1 day.
This result was found to be statistically significant (p=0.001) in intergroup comparison.
Statistically significant (p<0.001 ) in both groups at time wise comparison of OHI scores at
baseline, 14lh day and 21st days.

Table 13 shows inter group comparison of OHI scores in Chlorhexidine and Pudina groups. The
mean difference -0.367 between Chlorhexidine and Pudina (p=0.977NS) at baseline.
The mean difference -0.500 between Chlorhexidine and Pudina groups (p=0.535 NS) at 14th day.
The mean difference -0.560 between Chlorhexidine and Pudina groups was found to be -0.193
(p=0.011*)at21slday.
Table 14 shows mean difference of OHI scores at different time intervals.
The result shows the mean difference was found to be 0.50667 (pO.OOl*) in Chlorhexidine
group and 0.44667 (pO.OOl ) in Pudina group between baseline and 14' days.
Mean difference was 0.98667 (p<0.00r)and 0.81333(p=0.00r) for Chlorhexidine and Pudina
respectively between Baseline and 21sl day
The result shows the mean difference was found to be 0.4800 (pO.OOl ) in Chlorhexidine group
and 0.36667 (pO.OOl*) in Pudina group between 14th day and 21sl days.

Table 15 and 16 shows intergroup comparison plaque index scores at different time intervals
(baseline, 14th day and 21st days) of the 2 groups.
The mean plaque index scores in Chlorhexidine group is found to be 1.2933 (S.D= 0.07037) at
baseline, 0.7467 (S.D= 0.05164) at 14th day and 0.5467 (S.D=0.05164) at 21st day. In Pudina
group it was 1.3067 (S.D=0.08837) at baseline, 1.0267 (S.DO.10328) at 14th day and 0.8333
(S.D=0.8333)at21s,day.
This result was found to be statistically significant (pO.OOl) in all of the groups at intergroup
comparison.
Table 17 shows mean difference of plaque index scores at different time intervals.
The result shows the mean difference was found to be 0.54667 (pO.OOl ) in Chlorhexidine
group and 0.28000 (p<0.001*) in Pudina group between baseline and 14lh days.
The result shows, the mean difference was found to be 0.74667 (PO.OOl ) in Chlorhexidine
group and 0.47333 (PO.001*) in Pudina group between baseline and 21s1 days.
The result shows the mean difference was found to be 0.2000 (pO.OOl ) in Chlorhexidine group
and 0.19333 (pO.OOf) in Pudina group between 14lh day and 21st days.
Table 18 shows inter group comparison of plaque index in Chlorhexidine and Pudina groups, The mean difference -0.047 between Chlorhexidine and Pudina groups (p-0.867 NS) at baseline. The mean difference -0.367 between Chlorhexidine and Pudina groups (pO.001*) at 14th day. The mean difference -0.360 (pO.001 ) between Chlorhexidine and Pudina groups was found to be -0.287 (pO.001*) at 21st day.

Table 19 and 20 shows intergroup comparison gingival index scores at different time intervals
(baseline, 14th day and 21st days) of the 2 groups.
The mean gingival index scores in Chlorhexidine group is found to be 1.3333(SD=0.08165) at
baseline, 0.8400 (S.D= 0.10556) at 14,h day and 0.6133(S.D=0.07432) at 21st day. In Pudina
group it was 1.3667 (S.D=0.07237) at baseline, 0.9467 (S.D=0.12459) at 15th day and 0.9400
(S.D=0.05071)at21s,day.
This result was found to be statistically highly significant (p<0.001 ) at intergroup comparison
during 14th day and 21s'day.
Statistically significant (pO.001) in all the groups at time wise comparison of gingival index
scores at baseline, 14lh day and 21s1 days.
Table 21 shows inter group comparison of gingival index in Chlorhexidine and Pudina groups. The mean difference,-0.033 between Chlorhexidine and Pudina groups (p=0.455 NS) at baseline. The mean difference -0.107 between chlorhexidine and pudina groups (p=0.021 NS) at 14lh day. The mean difference between Chlorhexidine and Pudina groups was found to be -0.327 (P<0.001*)at21s,day.

Table 22 shows mean difference of gingival index scores at different time intervals. The result
shows the mean difference was found to be 0.49333 (pO.OOl ) in Chlorhexidine group and
0.42000 (pO.OOl*) in Pudina group between baseline and 14th days.
The result shows the mean difference was found to be 0.72000(P<0.001 ) in Chlorhexidine
group and 0.42667 (PO.OOl ) in Pudina group between baseline and 21st day.
The result shows the mean difference was found to be 0.22667 (p<0.001 ) in Chlorhexidine
group and 0.00667 (p=l .000 NS) in Pudina group between 14lh day and 21st days
Many children have inadequate oral and general health because of active and uncontrolled caries. It is the single most common chronic childhood disease. Owing to its non-life-threatening nature and ubiquitousness has minimized its significance in over all human health. Initiation of dental caries and the microbial composition of plaque have generally involved either S. mutans and also a fungi, recently being implicated in the etiology of dental caries namely Candida albicans. Children with high dmft have increased S. mutans count. As a result variety of anti-plaque agents has been examined for their ability to control S. mutans.2 Plaque control can be achieved with the combination of mechanical and chemo-therapeutic approaches. Chemical antimicrobial agents can reach difficult to clean areas such as inter proximal surfaces and can also reduce the growth of biofilms on soft tissues and as a local drug delivery agent.
Among the chemotherapeutic agents used in mouthwashes, chlorhexidine is the positive control for comparison with other substances due to its proven efficiency.2 ' J4 Chlorhexidine is regarded as the 'gold standard' for ahtiplaque, antigingivitis treatment. However, it can cause a number of side effects including extrinsic tooth and tongue staining, taste alteration, supragingival calculus formation, desquamation of the oral mucosa restricting its usage in children.8'l2-25'26,27Long term use of mouthrinses can also lead to microbial drug resistance. Formulation of mouth rinses with widely available condiments in the home could address these problems. In the recent years there has also been an increase in acceptability and use of herbal products; a survey revealed that 9.3% of adults all over the world have found herbal products to be more beneficial than conventional medicine without any side effects. Various studies have shown a high caries and periodontal disease prevalence in India.40"48
In this study, two widely available and used herbal products in Indian homes namely. Pudina leaves as mouth rinses were studied by comparing their anti-microbial efficacy to CHX, the golden standard antibacterial and antiplaque mouth rinse in children. The anti-microbial efficacy was evaluated against the primary etiologic bacteria namely S.mutans and fungi-Candida albicans. Present study also evaluated on the gingival health and plaque control in vivo. Since,

the main effect of mouthwash is for plaque control, indices were used to evaluate the same, using a disclosing agent.
Age is the critical factor in subject selection, of which the most important is the number of tooth surface at risk. Subject with age group 9-12 years were chosen because they were entering a period of high caries activity in the mixed dentition, with increase in accumulation of S.mutans.
The study was conducted in a residential school, as all the subjects consumed the same diet during the period of investigation. Acessibility and convenience to the investigator for daily supervision was also a factor. Dental caries is a dietary carbohydrate modified infectious disease, and the frequency of exposure to a cariogenic diet and the form of intake of cariogenic food substances appear to be important factors in the development of dental caries. Hence, diet as a causative factor was controlled in our study because all children were consuming the same type of diet.34 JS
In the present study, stimulated saliva was collected because unstimulated saliva is less reliable. Chewing helps to wash out bacteria from the tooth surface and mix them with secretion. The stimulation of flow rate also minimizes intra individual variation of secretion flow and increases the shedding of bacteria from the teeth.
Our study consisted of 57.8% males and 42.2% females. The mean age of the population was 10.40years in Chlorhexidine group and 9.93 years in Pudina group.
Effect on salivary streptococcus mutans count
In this study we found that salivary Streptococcus mutans count was less among the Pudina group. In the present study, the mean value of S. mutans counts index scores in Chlorhexidine group was found to be 7.6099*0.23776 at baseline, 5.7117±0.16401 at 14lh day and 4.5533±0.23911 at 21SI day. Statistically significant reduction in salivary mutans streptococci counts was found in all groups (p=0.001) in which Chlorhexidine was the most followed by Pudina.
The mean value of S.mutans counts in Pudina group was found to be 7.4712±0.17836 at baseline,4.2830±0.14451 at 14,h day and 4.0029±0.18010 at 21st day. Significant reduction in salivary mutans streptococci counts was found between three different time intervals that isbetween baseline and 14th day (p=0.012), between 141 and 2 Is1 day (p=0.600) and between baseline and 21sl day (p=0.001*) in Pudina group.
L.G.Vijayaalakshmi et al in her invitro study it was noted that herbal mouth wash was checked for its antibacterial activity in reducing Streptococcus Mutans in comparison with Chlorhexidine mouth wash and results showed that herbal mouthwash was as equivalent to conventional mouth wash21. According to many studies that have been conducted on the effects of mouthwashes on

oral microorganisms, the Chlorhexidine mouthwash was the most superior amongst all mouthwashes.36*37
Effect on Candida albicans
On comparing the antifungal activity of CHX and Pudina against Candida albicans, Pudina
mouth rinse was found to be as potent as CHX mouth rinse. Statistically significant reduction
was seen in the both CHX and Pudina between baseline and 21st day intervals (p<0.001*Plaque status
In the present study, the mean value of plaque index scores in Chlorhexidine group was found to
be 1.2933±0.07037 at baseline, 0.7467*0.05164 at 14,h day and 0.5467±0.05164 at 21st day.
Same while, in Pudina group the mean plaque index scores was
1.3067±0.08837,1.0267±0.10328 and 0.8333±0.09759 at baseline,14,h day and 21stday,and result
was statistically significant (p=0.000).
After rinsing with the respective mouthwashes, statistically significant differences were found
between Chlorhexidine and Pudina at 14th day and 21sl day time intervals.
Gingival status: In the present study, the mean value of gingival index scores in Chlorhexidine
group was found to be 1.3333*0.08165 at baseline, 0.8400±.10556 at 14th day and
0.6133±0.07432 at 21s1 day. Meanwhile, the Pudina group showed the mean gingival scores of
1.3667±0.7237, 0.9467±0.12459 and 0.9400±0.5071 at baseline, 14th day and 21st day
respectively. It was statistically significant (p=0.001).After using the mouthwashes, significant
differences in gingival indices were found at 14thday and 21slday intervals. Pudina and
chlorhexidine mouthwashes had equivalent effect on gingival scores at 14 days and at 21 days
evaluation.
Oral health status: In present study we found that the all 2 groups (Chlorhexidine and Pudina)
significantly reduced the oral hygiene index scores at baseline, 14th day and 21sl day (PO.001),
and a statistically significant (p=0.001) in mean difference of all 2 groups (Chlorhexidine and
Pudina) between baseline and 14lh day and between baseline and 21s1 day. In contrast to our
study Shreya Shetty et al showed that there was no statistically significant (p>0.5) changes
between the groups (Herbal Hiora and Chlorhexidine mouthwash) in oral hygiene index scores at
the end of 5 days.38
Inference: No adverse effects on the oral hard and soft tissues or abnormal symptoms/ sensation
was detected after using the Pudina mouthrinse and was well accepted by the children for a
period of 21 days. Mentha piperita L. (peppermint) is a medicinally important plant, Peppermint
leaves contains about 0.5-4 % volatile oil that is composed of 50-78 % free menthol,
monoterpene, menthofurane and traces of jasmine (0.15 %). Anti-microbial property is due to the
presence of Peppermint oil and menthol against both gram-positive and gram negative bacteria
(Saeedetal.2006).l9J9
Pudina mouthwash found to be safe with regular use, Since, the plaque inhibition and gingival
health was evaluated using indices, this could show a variation in subjects since mouthrihsing

was used only as an adjunct. Pudina mouthwash was evaluated in our study for its potential as novel antimicrobial agent. However, the study was of short duration and on a small sample size. So more invivo studies, on a larger sample need to be conducted to prove its efficacy as an effective antimicrobial components in mouthwashes, that can be prescribed for children. From the present study, the following conclusions were drawn. Our invention demonstrated, Pudina mouth wash (Group B) showed statistically significant reduction in salivary mutans Streptococci levels and gingival index scores at baseline, 14Ih day and 21sl day whereas Pudina containing mouthwash was more effective against Candida albicans. Pudina containing oral rinse were effective in reducing salivary Streptococcus mutans counts, plaque, gingivitis and improvement in oral hygiene status. Hence, it may serve as a natural antimicrobial mouthrinse alternative to conventional mouthwash. The final conclusion of this study is that Pudina oral rinse, causes significant reduction in salivary S. mutans levels and minimal effect on Candida albicans.
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We Claim,
1. Herbal mouthwash formulation for maintaining oral hygiene comprising a plant extract of Mentha arvensis (Pudina) with other suitable ingredients.
2. Herbal mouthwash formulation for maintaining oral hygiene as claimed in claim 1 wherein the plant extract is of Mentha arvensis leaves.
3. Herbal mouthwash formulation for maintaining oral hygiene as claimed in claim 1 or 2 wherein the plant extract is ethanolic extract of Mentha arvensis leaves.
4. Herbal mouthwash formulation for maintaining oral hygiene as claimed in claim 1 wherein the Mentha arvensis extract is 40 to 60% (w/w) of the formulation.
5. Herbal mouthwash formulation for maintaining oral hygiene as claimed in claim 1 or 4 wherein the Mentha arvensis extract is 50% (w/w) of the formulation.
6. Herbal mouthwash formulation for maintaining oral hygiene as claimed in claim 1 wherein other ingredients are selected from group of Pepperment oil, Sodium saccharine, Methyl paraben and distilled water.
7. Herbal mouthwash formulation for maintaining oral hygiene as claimed in claim 6 wherein the amount of other ingredients is 1 g/L of Pepperment'oil, 1 g/L of Sodium saccharine, 0.05% (w/w) Methyl paraben.
8. Process for preparation of herbal mouthwash formulation for maintaining oral hygiene comprising the extracting of Mentha arvensis (Pudina) and mixing of the effective amount of plant extract with other suitable ingredients selected from group of Pepperment oil, Sodium saccharine, Methyl paraben and distilled water to obtain the herbal mouth rinse composition.
9. Process for preparation of herbal mouthwash formulation for maintaining oral hygiene as claimed in claim 8 wherein the extract is ethanolic extract of Mentha arvensis at an effective amount of 40 to 60% (w/w) of the formulation.
10. Process for preparation of herbal mouthwash formulation for maintaining oral hygiene as claimed in claim 8 or 9 wherein the amount of Mentha arvensis extract is 50% (w/w) of the formulation.