Introduction
Dental caries is a chronic, infectious, transmissible disease that extends throughout the life span. The essential process of this disease involves bacterial adherence to tooth surfaces, dental plaque formation, and localized demineralization of tooth enamel by acids of bacterial origin produced from the fermentation of dietary carbohydrates[1]. Streptococcus mutans has been implicated as a principal etiological agent, although other oral bacterial species like Streptococcus sobrinus, Streptococcus mitis, Streptococcus sanguinis, Streptococcus anginosus and Streptococcus salivarius and non-S. mutans streptococci, Lactobacillus, Propionibacterium, Veillonella, Actinomyces, Bifidobacterium probably contribute to this disease[2].
Research in the field of caries prevention has been focusing on ways for reducing or totally eradicating cariogenic flora from oral cavity. Caries can be prevented by regular tooth brushing and flossing. However, it is seen that these microorganisms are difficult to be eliminated from pits, fissures and proximal surfaces by mechanical means alone. For effective caries control, brushing and flossing can be combined with the chemoprophylactic agents like chlorhexidine (CHX) and antibiotics, which act by lowering the number of microorganisms or inhibiting dental plaque formation. Several undesirable side-effects, including tooth staining and emergence of bacterial resistance have been seen with these chemoprophylactic agents who stimulate the search for alternative agents[1].
Also, with increasing incidence of Dental caries in underdeveloped and developing countries, there is an urgent need to promote traditional preventive measures that are acceptable, easily available, and are cost effective[3].
Ayurveda is the traditional medicinal form, prevalent in India since 2000 BC. The Ayurvedic treatment is entirely based on herbs, which have certain medicinal value or property. In the ancient times, the Indian sages believed that Ayurvedic herbs are one-stop solutions to cure a number of health R09;related problems and diseases. They conducted thorough study about the same, experimented with herbs to arrive at accurate conclusions about the efficacy of different plants and herbs that have medicinal value. Most of the Ayurvedic herbs, thus formulated, are free of side-effects or reactions. This is the reason why Ayurveda is growing in popularity across the globe. The Ayurvedic herbs that have medicinal quality provide rational means for the treatment of many internal diseases, which are otherwise considered incurable in other systems of medicine[4].The World Health Organization reported that 80% of the world’s population rely chiefly on traditional medicine and a major part of the traditional therapies involve the use of plant extracts or their active constituents[1].
Plant extracts constitute rich sources of novel compounds with a variety of pharmacological activities. In many countries, plant extracts have been traditionally used for the treatment of oral mucosal lesions and periodontal diseases without any scientific validation[1].
One such herb which has the medicinal value is the product Azadirachta Indica’, belongs to the family Meliaceae, commonly known as neem. It is used in traditional medicine as a source of many therapeutic agents. The importance of Azadirachta indica has been recognized by U.S. National Academy of Science, which published a report in 1992 entitled “Neem a tree for solving global problems”[5]. It is known to exhibit wide pharmacological activities including antibacterial, antifungal, anti-inflammatory and analgesic action. The biological activities are attributed to the presence of many bioactive compounds including alkaloids, flavonoids, triterpenoids, phenolic compounds, carotenoids, steroids and ketones[6].
“Triphala” is also among the most common formulas used in Traditional Ayurvedic Medicine. It is composed of the fruits of three trees, Indian gooseberry Amalaki (Embilica offi cinalis), Bibhitaki (Terminalia beleria), and Haritaki (Terminalia chebula). Triphala is mentioned throughout the ancient literature of Ayurvedicmedicine as a tonic, highly prized for its ability to regulate the process of digestion and elimination[7]. Formulations of Triphala is claimed to have anti-viral, antifungal and anti-bacterial effect. It has been used extensively as a drug against different diseases[8].
Chlorhexidine is used as a gold standard against which other antimicrobial agents are compared. It has been studied extensively and is currently the most potent chemotherapeutic agent against the bacterial flora involved in dental caries[9]. Therefore, an attempt is made to compare the antimicrobial activity of Neem andtriphala with 0.2% chlorhexidine against Streptococcus mutans and Streptococcus mitis.

Aim and Objectives
•    To evaluate the antibacterial efficacy of Neem and Triphala extract on microorganisms (Streptococcus mutans andStreptococcus mitis) involved in dental caries.
•    To compare the effect of Neem and Triphala extract with commercially available Chlorhexidine mouthwash.

Materials and Method
The following materials were used in this study
1.    Test materials used:
a.    Neem stem bark (Azadirachta indica)
b.    Fresh maturated fruits of Amlaki (Embilica offi cinalis), Bibhitaki (Terminalia beleria), and Haritaki (Terminalia chebula).
c.    0.2% Chlohexidine
d.    Universal solvent- DMSO (Dimethyl Sulfoxide)
2.    Two species of Freeze-dried microorganisms (procured from IMTECH Chandigarh): (Fig.1)
a.    Streptococcus mutans (MTCC 890)
b.    Streptococcus mitis (MTCC 2695)
3.    Nutrient agar plates and blood agar plates
4.    Himedia Zone Scale - C, PW 279 (Himedia Laboratories Pvt. Limited, Mumbai 400086, India).

[Image 1]

Procurement of plant extracts
Crude plant extracts were obtained from HSK College of Pharmacy Bagalkot, Karnataka after their standardized taxonomic identification was performed at BVVS Ayurvedic College Bagalkot, Karnataka and extraction was done using 70% ethanol by soxhlet extraction method[10]. The extracts were then diluted to 5%, 10%, 50% concentrations for antimicrobial evaluation.

Procurement of microorganisms
Freeze-dried forms of microorganisms, Streptococcus mutans, Streptococcus mitis, were obtained fromInstitute of Microbial Technology (IMTECH), Chandigarh.

Preparation of Culture Media
The ampoules containing freeze - dried forms of the microorganisms were added to the nutrient broth, which was incubated at 37°C for 24 hours. A sterile cotton swab was dipped into the nutrient broth and then inoculated onto the blood agar plate which was incubated at 37°C overnight. The growth obtained on the blood agar plate was transferred onto agar plate to test the antimicrobial activity of the herbal extracts.

Agar well diffusion method
Agar well diffusion method prescribed by National Committee for Clinical laboratory Standards (NCCLS 2000) was employed in antimicrobial susceptibility testing[11]. Wells of 6mm diameter were made on the agar surfaces using standard borerat five individual quadrants streaked. All the test compounds, positive control 0.2% CHX, negative control DMSO, Neem andtriphala extracts with different concentrations were added to the respective labelled wells (Fig. 2) and the plates were incubated at 370C for 48 hours which were evaluated for the zones of inhibition (Table 1). The procedure was repeated five times. The inhibition zones were measured on the underside of the plate by using Himedia Antibiotic Scale (Fig. 3, 4)

[Image 2]

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Statistical Analysis
The collected data was analysed using the following statistical tests:
1.    Student t- test
2.    One way analysis of variance (ANOVA)
(SPSS version 17.0, Inc. 233 South Wacker Drive, 11th Floor, Chicago, II, 60606-6412).

Results
Mean zones of inhibition of 11.0, 11.0 and 11.9 mm were observed at 5%, 10% and 50% concentration of neem against Streptococcus mutans and 10.3, 11.3 and 11.8 mm were observed against Streptococcus mitis, while mean zone of inhibition of 0.2% Chlorhexidine against Steptococcus mutans was observed to be 18.6 and against S. mitis was 15.6 respectively (Table 2 and Graph 1).
Similarly, mean zones of inhibition of 8.7, 9.7 and 10.4 mm were observed at 5%, 10% and 50% concentration against S. mutans and 8.9, 9.9 and 11.4 mm were observed against S. mitis with Triphala while mean zone of inhibition of 0.2% Chlorhexidine against Steptococcus mutans was 17.4 and against S. mitis was observed to be 16.2 mm respectively (Table 3 and Graph 2). Both the species were resistant to the action of dimethyl sulfoxide (negative control) (Table 2 & 3, Graph 1 & 2).
Tables 4 and 5 are showing the results of intra-group and inter-group comparison of mean zones of inhibition of Streptococcus mutans and mitis using different concentrations of Neem and Triphala.
On comparison of antimicrobial activity of 0.2% Chlorhexidine with negative control (DMSO) and with different concentrations of neem and triphala against S. mutans and S. mitis, results were found to be highly significant (p<0.05).

[Image 6]

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[Image 10]

[Image 11]

Discussion
Medicinal plants continue to be an important therapeutic aid for alleviating the ailments of humankind. In India, the use of different parts of several medicinal plants to cure specific ailments has been in vogue from ancient times. Neem and triphala is being used since time immemorial in traditional medicine as a source of many therapeutic agents. The present study intended to evaluate the antibacterial efficacy of neem and triphala extracts against Streptococcus mutansand Streptococcus mitis as these are one of the main culprit microorganisms responsible for dental caries.
The extracts of neem and triphala in the concentrations of 5%, 10%, and 50% were tested for antimicrobial activity and agar well diffusion method for antimicrobial susceptibility testing was preferred over other methods as the selection of methods depends on the nature of compounds such as solubility. Furthermore, this method gives both qualitative and quantitative results[12]. More than thousand years ago, neem has been used in South Asia for teeth cleaning and maintaining gum health[13]. Neem contains the alkaloid margosine, resins, gum, chloride, fluoride, silica, sulphur, tannins, oils, saponins, flavenoids, sterols and calcium[14]. In the present study, neem extract showed the maximum zone of inhibition of 11.9 and 11.8mm at 50% concentration against Streptococcus mutans and Streptococcus mitis respectively and this study is in co-relation with the study done by Khalid et al[15]. Neem has shown the maximal antimicrobial property even in minimal concentration of 5%. This data of present study correlates with the study of Prashant et al in 2007[3].
High antibacterial property of neem can be explained on the basis of presence of fluoride, which is known to exert an anti- cariogenic action. Its other constituents like silica acts as an abrasive and prevents accumulation of plaque. Alkaloids are known to exert an analgesic action, oils have carminative, antiseptic and analgesic effects, tannins exert an astringent effect and form a coat over the enamel, thus protecting against tooth decay[3]. Fibrous nature of neem sticks may act as antiplaque agents, as this may cause mechanical removal of plaque[16]. Calcium which is present in neem, acts as an abrasive agent which is responsible for tooth polishing[17]. Shiela et al stated that neem have antiadhesive effects due to the presence of photochemicals which minimizes the plaque formation[18]. Vanka et al concluded the antimicrobial effect of neem mouthwash against the salivary levels of Streptococcus mutans and reversing the incipient carious lesions[19]. Another study conducted by Wolinsky et al stated that pre-treatment of saliva conditioned hydroxyapatite with neem stick extract prior to exposure to bacteria, yielded significant reduction in bacterial adhesion. This result suggests that neem stick extract can reduce the ability of some streptococci to colonize tooth surfaces[20]. EI-Siad et al, described the lower incidence of dental caries among users of chewing sticks compare to nonusers. This attributes to the superior mechanical cleansing action on the teeth and to the antimicrobial properties of neem sticks[21].
Triphala is a key ingredient used in Ayurveda since time immemorial. Triphala has been proven to have antibacterial, antiviral, and antifungal actions. It is also said to possess antihistaminic, anti-inflammatory, antioxidant, antitumor, blood pressure lowering, cholesterol lowering, digestive, diuretic, and laxative properties[22]. In the present study both the neem and triphala showed antimicrobial activity, but in comparison to neem, triphala showed less antimicrobial activity. At 50% concentration the maximum zone of inhibition of Triphala for S. mutans was 10.4mm and for S. mitis was 9.9mm, as compared to zone of inhibition of 11.9mm and 11.8mm of neem against S. mutans and S. mitis respectively. Shrinagesh J et al, Bajaj N et al and Velmurugan et al showed that 0.6% Triphala and 0.1% Chlorhexidine had been shown to have an inhibitory effect on plaque, gingivitis and growth of S. mutans and Lactobacillus[22], [7], [23].
The possible mechanism behind this antibacterial action is not clear right now. A close association between total phenolic contents and antibacterial activity of Triphala components has been observed. Tannins are a group of polymeric phenolic substances which are the active compounds present in Terminalia Chebula, are well recognized for its antimicrobial activity due to their action on membranes of microorganisms or may be due to non specific interaction with the proteins and astringent property[24]. Hence, it can be presumed that antibacterial potential of Triphala is due to the presence of high content of phenolics in triphala components. A study conducted by Shuchi Shah et al, in 2014, concluded that extracts of neem and tulsi demonstrated an antimicrobial activity against S Mutans and L Acidophilus up to certain extent, while extract of triphala has failed to show an antimicrobial activity[14].
Dimethyl Sulfoxide, an inert solvent was used to dilute the extract to different concentrations.
CHXwas considered as a positive control in this study. Chlorhexidine gluconate, which is charged positively, shows high affinity for negative ions found in cell membranes of microorganisms. It indirectly affects the enzymatic function of dehydrogenase and adenosine triphosphatase present in the cell wall of bacteria resulting in the disruption of cell membrane[23]. It abolishes the activity of the phosphoenolpyruvate - phosphotransferase sugar transport system, and thereby markedly inhibits acid production, in oral streptococci cariogenic bacteria in subjects with a high risk of developing caries. CHX, which has been studied extensively for over 25 years, is currently the most potent anti-microbial agent against microorganisms involved in dental caries[25].

In the present study Chlorhexidine was found to be more effective when compared to both the herbal extracts and the results were statistically significant (p<0.05). Another study conducted by Nisarg J Chaudhary et al showed similar results[26], however the well-known side effect of Chlorhexidine i.e. staining of teeth and restoration, alteration of taste sensation, development of resistant microorganisms, may limit the long term use of Chlorhexidine. In comparison herbal products like neem and triphala are abundantly available, easily accessible, economically feasible, and culturally acceptable and may possess minimal side effects and hence can be recommended for long term use.

Conclusion
Ayurveda-based regimens are likely to replace Chlorhexidine soon as intense antimicrobial, palatable, and cost-effective preventive strategies. In the present study, both neem and triphala extracts demonstrated antimicrobial activity against Streptococcus mutans and Streptococcus mitis and the antimicrobial potential increases with increasing concentrations, showing maximum zone of inhibition at 50% concentration level. Neem extract showed slightly more antimicrobial activity than triphala extract. Since, thepresent study was conducted in vitro using herbal extracts; the duration of the contact of the extract with microorganisms in the oral cavity in vivo is not established. Therefore, more scientific work needs to be carried out to prove the efficacy, especially in this era when holistic integrated medicine approach is the need of the hour.

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