The study was conducted at Mahatma Gandhi Dental College and Hospital in Jaipur over a period of one month in the year 2012. Before the start of the study clearance was obtained from the institutional ethical review board of the Dental College Hospital. A lab based invitro experimental study design was employed. Five male subjects aged between 50-52 years, diagnosed with periodontal disease based on clinical examination were selected from the Department of Periodontology and Implantology of the dental college and hospital. Voluntary written informed consent was obtained from all the selected subjects prior to the start of the study. Diagnosis of periodontal disease was made according to the criteria of Community Periodontal Index [9]. All the selected subjects had a score of 3 (pocket depth of 4-5mm) as determined using the Community Periodontal Index probe. All the patients selected were using tooth brush and toothpaste (without fluoride and anti-plaque agents) once daily in the morning as a primary oral self care. None of the patients were using mouthwash, dental floss or inter-dental cleaning aids. Those volunteers agree to refrain from oral hygiene procedures 24 hours prior to plaque collection and subjects having periodontal disease corresponding to score 3 on CPI index were included in the study. The selected subjects did not suffer from any cognitive deficiencies, chronic medical conditions, systemic diseases or infectious diseases. There was no history of drug, alcohol or tobacco addiction. Subjects did not have any other oral diseases other than periodontal disease. They did not have crowns, veneers, dentures or any other prosthesis or oral appliances. The selected subjects were not taking any medications/antibiotics in the recent past.

Buccal surfaces of maxillary and mandibular premolar and molar teeth were selected. The subjects were asked to swish their mouth with plain water to reduce the contamination of plaque with soft debris. The area of plaque collection was isolated with cotton gauze. Sterile stainless steel jaquette scaler was used to collect the supragingival plaque by inserting it slightly into the gingival sulcus and running it over the surface occlusally and a sterile Gracey curette was inserted into the pocket to collect the subgingival plaque sample. Plaque samples thus collected were transported to the laboratory in 200μl peptone water as the transport medium.

Isolation, identification and characterization of Actinobacillus actinomycetemcomitans from dental plaque samples was done using Tryptic Soy Serum Bacitracin Vancomycin agar (TSBV agar). TSBV agar is an enriched selective medium for the isolation and presumptive identification of Actinobacillus actinomycetemcomitans. TSBV agar media was poured in sterile petriplates and inoculated with 05 different dental plaque clinical samples. The plates were incubated at 37^oC for 24 hours.

Actinobacillus actinomycetemcomitans was identified from the incubated plaque samples based on cultural, microscopic, biochemical characterization and multiple drug resistance patterns. Based on the above screening tests, isolate 1 was identified as Actinobacillus actinomycetemcomitans [Table/Fig-1].

Fresh leaves of the green variety of Ocimum sanctum (Linn.) were plucked from courtyards and were dried in sunlight. The dried leaves were then powdered finely. Three hundred grams of finely powdered Ocimum sanctum (Linn.) was then macerated with 100% ethanol. It was then subjected to filtration with Whatman filter paper to obtain a clear filtrate. The filtrate so obtained was reduced at a low temperature of less than 60^oC to obtain a solid residue of Ocimum sanctum (Linn.) extract. From 300 grams of Ocimum sanctum (Linn.) powder dissolved in 1 liter of ethanol, 18 gram of extract (residue) was obtained and thus the yield was 6% w/v.

One gram of cold extract of Ocimum sanctum (Linn.) was dissolved in 10 milliliter (ml) of dimethyl sulfoxide to obtain a 10% concentration of extract. 1ml of the extract was transferred to a sterilized test tube and labelled as 10%. The remaining 9 ml of the extract was then diluted further with dimethylsulfoxide to obtain 09 different concentrations (1%,2%,3%,4%,5%,6%,7%,8%,9%) of Ocimum sanctum (Linn.) extract. A total of 10 concentrations ranging from 1% to 10% were prepared.

Testing for antimicrobial activity of Ocimum sanctum (Linn.) extract:

The antimicrobial activity of Ocimum sanctum (Linn.) extract against Actinobacillus actinomycetemcomitans was performed by agar well diffusion method on Muller Hinton agar media using 50 micro liter (μl) of 0.2% chlorhexidine as the positive control and 50μl dimethyl sulfoxide as the negative control.

The hot media was poured into petriplates and allowed to cool for 20 minutes. Three wells were punched in the plate at a distance of 25 millimeter (mm) from each other, one well for the Ocimum sanctum(Linn.) extract, one for the positive control and one for the negative control. 50μl of the particular concentration of the Ocimum sanctum (Linn.) extract was taken in a micropipette and loaded into the well marked for test sample. Ten such plates were loaded corresponding to ten different concentrations of Ocimum sanctum (Linn.) extract. Similarly 50μl of positive control (0.2% chlorhexidine mouth wash) and 50μl of negative control (dimethyl sulfoxide) were loaded into wells marked for positive and negative control respectively. Ten such plates were prepared for ten different concentrations of Ocimum sanctum (Linn.) extract, each plate having a well for one particular concentration of Ocimum sanctum (Linn.) extract, one for the positive control and one for the negative control. The plates were incubated at 37 degree Celsius for 24 hours. Following incubation, the zone of inhibition was measured with unaided eye using a Vernier caliper measuring the zone sizes to the nearest millimeter, including the diameter of the disk in the measurement.

At the 6% w/v concentration of Ocimum sanctum (Linn.) extract, a zone of inhibition of 22mm was obtained. This was the widest zone of inhibition observed among all the 10 different concentrations tested. Hence, the Minimum Inhibitory Concentration of cold ethanolic extract of Ocimum sanctum (Linn.) against clinically isolated Actinobacillus actinomycetemcomitans was 6% w/v [Table/Fig-2]. The zone of inhibition for positive control was 25mm and no zone of inhibition was observed around the negative control [Table/Fig-3].

Plants are one of the important sources of medicine [10]. It is estimated that 25% of modern medicines of the 21^st century have plant derivatives as their active ingredients or principles [11]. Among a wide array of medicinal herbs, Ocimum sanctum (Linn.) or the Indian Holy Basil stands out as a time tested premiere medicinal herb.

The chemical composition of Ocimum sanctum (Linn.) is highly complex containing many biologically active compounds and nutrients. Ocimum sanctum (Linn.) leaves have antibacterial agents mainly in the form of essential oils. The five major constituents found in the essential oils of Ocimum sanctum (Linn.) leaves are eugenol, caryophyllene, germarene-A, clemene and caryophylline oxide. [11]. They also contain many biologically active compounds in the form of phytochemicals like ursolic acid, rosmarinic acid and oleanolic acid [6]. The essential oils and biologically active compounds have antibacterial properties and are effective against gram positive and gram negative bacteria [10–13].

Studies have shown that the essential oils and biologically active compounds in fresh leaves of Ocimum sanctum (Linn.) are effective against bacteria like E coli, Shigella species, Salmonellatyphi, B.anthracis and P.aeruginosa [10,13]. The oils are known to inhibit the invitro growth of M. tuberculosis bacteria [1,4]. A study conducted in Lahore, Pakistan found that essential oils from fresh Ocimum sanctum (Linn.) leaves displayed marked antibacterial efficacy against Pseudomonasaeuroginosa, E.-coli, Klebsiella, Proteusmirabulus and Staphylococcus aureus [14].

The anti microbial action of essential oils in Ocimum sanctum (Linn.) is attributed to monotrepene components which are mostly phenolic in nature. They exert membrane damaging effects to microbial strains and stimulate leakage of cellular potassium which is responsible for a lethal action related to cytoplasmic membrane damage [14].

Based on this mechanism it may be supposed that the essential oils and phytochemicals in Ocimum sanctum (Linn.) effective against systemic disease causing bacteria might have also acted against the periodontal pathogen Actinobacillus actinomycetemcomitans in human dental plaque.

In this study, leaves of green variety of Ocimum sanctum (Linn.) were used (green leaves and white to purplish flowers) over other varieties for no specific reason other than convenience and ease of availability. Studies have shown that the green and black varieties of Ocimum sanctum (Linn.) have similar chemical constituents and common medicinal properties [12].

Ethanol was used as a solvent because the essential oils in Ocimum sanctum (Linn.) are more soluble in alcohol when compared to distilled water [15]. Dimethyl sulfoxide, an inert solvent, was used to dilute the Ocimum sanctum (Linn.) extract to neutralize the effect of alcohol, which itself is an antiseptic, attributing the result solely to Ocimum sanctum (Linn.).

In the present study, 0.2% chlorhexidine was found to be more effective against Actinobacillus actinomyctemcomitans when compared to Tulsi extract. However, the well known side effects of chlorhexidine, i.e., staining of teeth and restorations, alteration of taste sensation and development of resistant organisms, may limit its long term use. In comparison, Ocimum sanctum (Linn.) has a high safety margin with exceptionally low toxicity and no human drug interaction data is currently available [6,16]. Hence, it may be recommended for long term use. Moreover, herbal medicine like Ocimum sanctum (Linn.) is abundantly available, easily accessible, economically feasible and culturally acceptable. Hence, using herbs like Ocimum sanctum (Linn.) in the management of oral diseases can overcome many barriers that exist for the utilization of dental services like affordability, accessibility, availability and acceptability.

Literature search revealed very few studies that have tested the antimicrobial action of Ocimum sanctum (Linn.). on oral disease causing organisms. A triple blind randomised clinical trial which tested 4% w/v of Ocimum sanctum (Linn.) mouthrinse against 0.12% chlorhexidine mouthwash and saline water as placebo found that Ocimum sanctum mouthrinse was equally effective in reducing plaque and gingivitis as chlorhexidine [17]. An in vitro study found that the methanolic extracts of A. indica (neem), O.sanctum (Tulsi), M.elengi (bakul), T.cardifolia (Giloy) and chlorhexidine gluconate had antimicrobial activity against common endodontic pathogens like S. mutans, E.faecalis and S. aureus. At 3mg concentration, O.sanctum was the most effective against S. mutans and chlorhexidine was the most effective against S.aureus [18].

Further, the laboratory results obtained in this study need reconfirmation in real life situations. If confirmed so, the active ingredients of Ocimum sanctum (Linn.) could be incorporated into oral hygiene products such as toothpastes and mouthwashes as an agent to fight the periodontal disease causing organisms.

One of the limitations of the present study was that it employed an invitro lab based study design. Hence, the laboratory results need reconfirmation in real life situation. Further, the study tested the anti-microbial effect of Ocimum sanctum (Linn.) against only one periodontal pathogen i.e. Actinobacillus actinomyctemcomitans. However, many other microorganisms have been implicated in the causation of periodontal disease including P.gingivalis and Prevotella intermedia. Hence, it is also recommended to determine the anti-microbial effect of Ocimum sanctum (Linn.) extract against other common periodontal pathogens like P.gingivalis and Prevotella intermedia to find out the spectrum of its activity.

Given the high prevalence of periodontal disease in developing countries like India, the undesirable effects due to prolonged use of currently used antibacterial agents and financial considerations there is a need for alternate preventive and treatment strategies that are safe, effective and economical when compared to existing treatment methods. In this direction, natural phytochemicals isolated from traditional medicinal plants like Tulsi serve as a good alternative.