Comparative clinical data for gingivitis treatment using gels from Ocimum sanctum (Tulsi) and chlorhexidine (CHX)

Ocimum sanctum (Tulsi) has various properties like anti bacterial, anti inflammatory, anti oxidant for curing diseases. It is a plant with known medicinal value in Indian system of medicine. Therefore, it is of interest to evaluate the effectiveness of Ocimum sanctum with Chlorhexidine (CHX) which is a standard material for the treatment of gingivitis. We used 30 gingivitis subjects divided into 2 groups. Group I used Tulsi gel (n= 15) and Group II used CHX gel (n = 15) for treatment. Tulsi and CHX gel use was advised for 1 month. The Clinical parameters assessed were gingival Index (GI), plaque Index (PI), probing depth (PD) and clinical attachment loss (CAL) assessed at a time interval of 30 days. Statistical analysis was completed using the SPSS software 23.0. Data showed that GI and PD for Tulsi and CHX in pre and post groups are not significant with p > 0.05. Moreover, PI is not significant with p>0.05 among pre Tulsi, pre CHX and post CHX. However, data is significant with p<0.05 for Tulsi group. CAL is significant with p<0.05 among pre/post Tulsi groups. However, this is not significant with p>0.05 among pre/post CHX groups. Data shows that 2% of Tulsi is effective in reducing gingival bleeding and inflammation. Thus, clinical data shows that Tulsi gel is promising for the treatment of gingivitis.


Background:
Plaque is the main factor for gingivitis (gingival inflammation). Oral hygiene failure leads to plaque formation [1]. The mechanical supra gingival plaque control measures includes toothbrush, floss, wood sticks, and interdental brushes. Oral hygiene maintenance is in plaque control measures [2]. There is increasing inclination towards biofilm properties and microbial colonies [3]. Different stages of dental biofilm formation are (a) absorption of bacterial molecules to host tooth surface [4-6]; (b) transport of bacteria to tooth surface [7-9]; (c) adherence of late colonies to early colonies [10-12]; (d) multiplication of the attached organisms [13][14][15][16][17][18] and (e) active detachment [19,20]. Gingivitis is the inflammation of gingiva without apical migration of junction epithelium [21]. Prolonged use of Chlorhexidine leads to brown colour stains on the teeth and change in pattern of taste. It also leads to formation of calculus and swelling of the parotid gland [22]. Plant extracts are used as the main component in mouthwash to reduce gingival inflammation and gel forms are mostly used to reduce periodontitis. These products have low side effects because of their herbal nature [23]. Some Indian plant species that contain antimicrobial activities are as follows they are Aloe vera, Piper betel, Piper nigrum, Syzygium aromaticum, Coriandrum sativum, Eucalyptus globules, Allium sativum, Curcuma longa, Camellia sinensis, Alliumcepa, Carica papaya, Solanum tuberosum and Ocimum sanctum [24]. Ocimum sanctum (Tulsi) belongs to the basil family Lamiaceae. Tulsi is an aromatic shrub [25]. Tulsi has both medicinal properties and spiritual properties [26]. The 2 types of Tulsi are Sri Tulsi and Krishna Tulsi. Sri Tulsi has green coloured leaves and Krishna Tulsi has purple coloured leaves [27]. Tulsi is available in various forms like dried leaves powder; fresh leaves and herbal tea [28]. Tulsi was used as a mouthwash to control plaque and gingival conditions [29]. A 4% of prepared Tulsi plant extract showed highest antibacterial activity against Streptococcus mutans [30]. Thus, Tulsi is considered as "The master of the herbs'' for its magical healing properties and antiinflammatory properties [31]. Therefore, it is of interest to document the comparative clinical data for gingivitis treatment using gels from Ocimum sanctum (Tulsi) and chlorhexidine.

Materials and Method:
Preparation of 2% Ocimum sanctum gel: Data for the preparation is shown in Table 1.
Preparation of supercritical fluid: 250 g of Tulsi powder is taken and soaked in 1000 mL of Ethyl alcohol for 48 hours. Filter with Whartman's filter. Filter liquid evaporated -Supercritical Fluid and stored in the fridge is shown in Figure 1 and Figure 2.

Preparation of Ocimum sanctum gel:
Carbopol 940 soaks it in distilled water containing 0.2% sodium benzoate throughout the night. HPMC solution was added. Propylene glycol was added to it. 2ml of SCF (Homogenized) was added. Tri ethanol amine was added in drops. Check for pH 6-6.5.The gel was stored at ambient temperature. The prepared gel is firm for the time interval of six months. Alteration in pH changes were noted and adjusted according to the protocol ( Figure 3).

Assessment of physical properties of 2% Ocimum sanctum gel:
The formulations were assessed for different evaluation tests. They evaluate the physical appearance, homogenous nature, spreadability, grittiness, ability to extrude, and pH evaluation. Estimation: The Physical examinations are Hue (color) and Aspect (look) was assessed. Lubricity: Lubricity is estimated by a wooden block apparatus. The apparatus has a pulley at one of its ends. Basic methodology is to slide and draw the properties of Ocimum sanctum (Tulsi) gel. Two grams of the prepared gel was taken and kept on the bottom slide. The Ocimum sanctum (Tulsi) gel was fitted into the bottom slide; another slide with the same measurements was taken and a hook was attached to it. One kilogram weighing scale was kept on the top of 2 slides for a period of 5 minutes. Hence air bubbles were eliminated to produce a constant form of gel film among slides. Excess gel was removed from the edges. The top plate was put through a jerk of 80 grams with the help of a string which is joined with a hook. The time period taken to envelope a distance of 7.5 centimeters by the top plate was noted in seconds. A shorter interval means better spreadability nature.
Spreadability was evaluated using the approved formula: S = M × L/T Where, S = Spreadability, M = Weight applied the pan (tied to the upper portion of slide), L = to what length moved by the glass slide and T = Time in seconds taken to separate the slides from each other.

Homogeneity:
Once the formulation is set in a container Homogeneity was examined by visible monitoring. It was evaluated for visible and tangible aggregates. It is categorized as good, fair, poor. For good it triple plus scale, fair means double plus, poor means single plus.

Extrudability:
The formulation was stored in a clean aluminium collapsible one ounce tube and it has a nasal tip of 5 mm opening slot. It was assessed by calculating the quantity of the gel expelled through nib when applied with a load of one kilogram was kept. The expelled gel was collected in a container and weighed for the measurement. The expelled gel was measured in percentages, and categories were assigned.

Determination of viscosity:
Viscosity of the formulation was evaluated at 25°C using Brookfield digital viscometer. The dimensions were taken in the established speed settings. The settings range from 10 to 100 rotations per minute. A time interval of 30 seconds among 2 consecutive speeds in a declining manner is used.
pH Measurement: 2.5 grams of the Ocimum sanctum (Tulsi) gel was taken and measured exactly and then poured into 25 ml of water. It was stored for 2 hours. The pH was measured using a standard pH meter.

Study design:
The study design includes a parallelized controlled clinical trial for the subjects from outpatient department of periodontics, Saveetha dental college and hospitals for the eligibility criteria for the study population are as follows: With the above inclusion criteria and exclusion criteria a total of 30 subjects were included in the study. The subjects were asked to massage the gingiva with Tulasi gel and CHX gel 2 times a day for 1 month.

Study group:
Group I: Tulasi gel Group II: CHX gel The assessment criteria included Gingival Index (GI) score, Plaque Index (PI) score, Probing Depth (PD) and Attachment Loss (AL) score that were measured as pre and post Osmium sanctum (Tulasi) gel and Chlorhexidine (CHX) gel.

Statistical analysis:
Differences between the study groups were statistically analyzed by SPSS Software 23.0 version; Paired t-test was used to analyse the difference between the groups. Mean and Standard Deviation were assessed for statistical analysis and the results are tabulated.        Singh et al. discussed the uses of Ocimum sanctum seeds. Oil was extracted from seeds. Oil has anti histaminic activity and anti inflammatory activity because of dual suppression of arachidonate metabolism. The oil has antipyretic activity due to the inhibition of prostaglandin. It also has antiulcer activity. The oil has proven to be efficient against formaldehyde and in addition to induced arthritis. The oil also has immuno modulatory, hypotensive and anticoagulant properties [38]. Ramamurthy et al. showed Ocimum Sanctum (Tulsi) gel have possible anti-oxidant and antiinflammatory effects. It is less toxic than brine shrimp nauplii. Ocimum Sanctum (Tulsi) proved to be the most favourable agent for the therapy of periodontal conditions [39].
Ocimum Sanctum (Tulsi) is equally effective against pathogenic gram +ve and gram -ve bacteria. Use of Tulsi in oral and systemic diseases, anti diabetic, wound recuperating movement, radiodefensive impact, cancer prevention agent, antimicrobial, gastroprotective, inflammatory, eye issue, renal issue, mental issue, skin issue, Tulsi use in oral health tooth ache, periodontal issue, anticariogenic impact, candidiasis, oral submucous fibrosis and ulcer. Therefore, it is of interest to evaluate the effectiveness of Ocimum sanctum with Chlorhexidine (CHX) which is a standard material for the treatment of gingivitis. The Clinical parameters assessed were gingival Index (GI), plaque Index (PI), probing depth (PD) and clinical attachment loss (CAL) assessed at a time interval of 30 days. Statistical analysis was completed using the SPSS software 23.0. Data showed that GI and PD for Tulsi and CHX in pre and post groups are not significant with p > 0.05. Moreover, PI is not significant with p>0.05 among pre Tulsi, pre CHX and post CHX. However, data is significant with p<0.05 for Tulsi group. CAL is significant with p<0.05 among pre/post Tulsi groups. However, this is not significant with p>0.05 among pre/post CHX groups. Data shows that 2% of Tulsi is effective in reducing gingival bleeding and inflammation. Thus, clinical data shows that Tulsi gel is promising for the treatment of gingivitis.

Conclusion:
We report that 2% of Tulsi is effective in reducing gingival bleeding and inflammation. Thus, clinical data shows that Tulsi gel is promising for the treatment of gingivitis.