INTRODUCTION:

Oral diseases are common now a days, it is to be a very big issue worldwide^1,2. Oral health is affected the quality of life ³. Since the modernization, advertisement changes the life style; dental caries have been observed to be more frequent. This is related with the alteration in human diet and techniques of food preparation, meals become more starchy, rich sugar, sticky and adherable ⁴. The more refined food items do not stimulate saliva flow efficiently and also do not ensure self cleaning as their unrefined counter parts, desperate buffering and re mineralization of enamel and cause dental plaque accumulation ⁵.

Modernization of healthcare and access to food varieties per periodontal diseases (due to Vitamin C deficiency) are decreases but the ineffective removal of dental plaque in association with chronic stress, inherent smoking, dyslipidemia, diabitiesetc may result in periodontitis affects a large number of peoples worldwide ⁶. It also generates other problems like tissue degeneration as well as neoplasms and their treatment may result in hypo salivation, immune deficiency, malnutrition with other oral diseases ⁷. Oral hygiene is must for healthy oral cavity. Brushing and flossing are necessary for it ⁸.

Mouthwashes play a vital role in oral care, offering therapeutic benefits through gargling and rinsing the mouth. Various oral issues, spanning from bad breath to periodontal diseases, can necessitate the use of mouth rinses⁹. They are especially important for managing secondary infections like oral mucositis. Now varieties of mouthwashes are available in the market. They are dominated by the use of many synthetic additives, some of the long-term health effects of these relatively new substances such as sodium lauryl sulfate (SLS) and triclosanetc.

Looking at the importance and significance of the work, the present paper is designed on the concerned topic. A green mouth was his consisted with pure essential oils which is distilled liquids extracted from flowers, leaves, bark, stems, roots, shrubs and trees and botanicals ingredients that have been known for their medicinal benefits for thousands of years ¹⁰. It has antibacterial, antimicrobial, and anti-inflammatory properties.

EXPERIMENTAL:

Collection of Plant materials:

Leaves of Ocimum tenuiflorum (tulsi), Azadirachta indica (neem), Psidium guajava (guava), rhizome of Zingiber officinale (ginger), rhizome of Curcuma longa (turmeric) were randomly collected from matured plants from various areas of Bhopal on the basis of geographical availability, cleaned, shadedried for 5 days. After five days leaves were taken and pulverized into moderately coarse powder and stored in airtight container for further use.

Extraction with single solvent by maceration method:

Powdered plant leaves and oils were weighed (20 gm) and packed in citrus juice were added as extraction solvent in the bottles and labeled. Further it is kept for 24 hrs and then filtration was performed to separate the solvent and powdered plant material. The solvent was evaporated to gain pure extract.

The mouthwash is prepared by adding a natural product with essential oils in the appropriate amount of solvents. Green Sample Dissolve the Tulsie xtract 2.0 ml, Neem extract ml, Ginger extract ml, Turmeric extract and Gauva extract 1.0 ml in a separate container and add Clove oil and mix it properly then slowly add distilled water to make a volume up to 100ml and obtaina clear solution and well shake it.

Phytochemical screening:

Phytochemical examinations were carried out for green sample of the extracts as per the standard methods and results suggested the presence of Phenols, Flavonoids and Alkalides.

Organoleptic properties of prepared formulations:

Physical Appearance:

Physical parameter such as colour, odour, taste and consistency were examined by visual examination. The colour of the sample was light brown and taste was good.

pH –The pH of sample was 7.23.

Stability Study:

Two sets of formulation are taken to check their stability, put it first in the normal temperature for several days and after that in the refrigerator for some interval of days and after that conclude that all these formulations are stable at room temperature as well as cool temperature.

Palatability Determination:

It is the property of being acceptable to the mouth. The mouthwash is tested separately for that criterion by three members in a blind style. The test of green mouthwash is good.

Identification of extracted sample through instrumental Methods:

Voltametric and Polarographic Methods:

Apparatus:

An Elico (Hydrabad) pulse polarograph model CL-362 coupled with an x-y polarocard (recorder) model LR-108 and an electrode assembly consisting of three electrodes, a dropping mercury electrode used as an indicator electrode, a SCE as reference electrode and a coiled platinum electrode used as an auxiliary electrode. A known concentration (10ml) of sample of mouthwash formulation was taken in 0.01M Ammonium Tartrate buffer solution as supporting electrolyte and gelatin as maximum suppressor at рН= 9.0±0.1. The рН was adjusted using HCl/NaOH solution. This solution was taken in a polarographic cell. The nitrogen gas was bubbled through the solution for 5 minutes than polarograms were recorded with the help of printer.

RESULTS AND DISCUSSION:

Figure 1 are Direct Current Polarogram (DCP)& Differential pulse Polarogram (DPP)green mouthwash sample. It shows well defined polarographic wave/ peak with E_1/2/Ep - 0.05/-0.05 V and -0.22 / 0.24 V vs SCE in DCP and DPP mode. In DPASV the peak potential Ep are -0.36 and -0.16 V vs SCE using carbon fiber electrode as indicator electrode.

Figure 1. DCP, DPP and DPASV of Green Sample of Mouth Wash in 0.01M Ammonium Tartrate and gelatine at рН= 9.0±0.1

To confirm the presence of above mentioned species in the sample, a known quantity of standard solution was added to the analyte and resulting polarograms were recorded, which increased the observed wave / peak height of added sample without any change in E_1/2 /Ep values.Thus the results confirming the presence of all species the extracted sample and also confirm the enabling, developed procedure for an accurate qualitative as well as quantitative analysis of natural origin extracts obtained from plants. The standard deviation is about in the range 0.02 confirm the reliability of the analysis.

Antimicrobial Study of green mouth wash:

Marketed Chlorhexidine, sample and green sample were tested against various pathogens viz. Actinobacillus, Actinomycetem comitans, Prophromonas gingivalis and Prevotella intermedia respectively by disk diffusion method (11). On the basis of results obtained it could be concluded that green extracted sample was found to be effective antibacterial activity against gram positive and gram negative bacteria and compare this with the standard antibiotic Penicillin. The range of concentrations of the samples used for microbial activity against various micro-organisms is in between 100-500 mg/ml and the 37⁰C temperature was selected and time duration was 36 hours. A good number of reproducible results have been observed.

The result of antibacterial study of standard antibiotic Chlorohexidine and extracted green mouth wash with gram-positive and gram-negative bacteria of marketed (Actinobacillus, Actinomycetemc omitans, Prophromonas gingivalis and Prevotella intermedia) at a concentration of 0.5 mg/ml and 1mg/ml. The results of which are reported in Table 1.

On the basis of the obtained results by antimicrobial activity against mentioned various bacteria it could be concluded that green sample shows very good results like against Actinobacillus at 1mg/ml concentration it show 50% inhibition over control antibiotic and 72% over chlorohexidine. It shows 41% inhibition over control antibiotic and 47% over control chlorohexidine against Prevotella intermedia pathogen. These are remarkable results but it not shows effective results with other two microbes i.e. Actinomycetemc omitans and Prophromonas gingivalis

Pharmacological Analysis:

Plaque samples were collected and sent for microbiological analysis to estimate colony-forming units. Prior to this analysis, all subjects received a complete oral prophylaxis treatment. Marginal plaque samples were collected along the gingival margin of all teeth. To prevent contamination from saliva, the site was isolated with cotton rolls and gently dried with an airway syringe. Marginal plaque samples were collected using a sterile jaquette scaler and were immediately transported to the laboratory in brain heart infusion broth as the transport medium. Subsequent microbial analysis was conducted ^6,7.

The participants were randomly divided into two groups. Each group comprised of 12 participants. In each sample, participants were instructed to rinse with 10 ml of the assigned mouthwash sample for 30 seconds, twice daily, without revealing the specific type of mouthwash. In addition to the mouthwash usage, all participants were provided with an orthodontic tooth brush for use during the study.

Follow-Up:

After the 10th and 23rd days, the participants were recalled for further assessments. During these follow-up visits, we recorded the gingival and bleeding indices. Furthermore, we collected supra gingival plaque samples using sterile jaquette scalers for subsequent microbiological analysis.

Statistical Analysis:

The data collected for this study will be presented in terms of mean values and standard deviations to describe the central tendency and variability of the measurements⁸.

Inter-group Comparison:

To assess differences between the groups, conduct a statistical analysis and employ analysis of variance (ANOVA) with post hoc tests or the Kruskal-Wallis test, depending on the distribution of the data. These methods will be used to examine group variations and make detailed group comparisons as necessary.

Table: 1. Antimicrobial study of Formulation at 1mg/ml concentration

S. N	Test Organism	Inhibition zone (mm) Con. of complex 2mM/10ml (B)	Control antibiotic 1.0 mM/10ml (A)	% Change (A-B/A) ×100	Control Chlorohexidine mM/ml (Y)	% Change (Y-B/Y) ×100
1	Actinobacillus	09	18	50	33	72
2	Actinomycetemcomitans	29	34	14	27	-7.4
3	Prophromonasgingivalis	---	25	--	17	--
4	Prevotella intermedia	10	17	41	19	47

Table 2: Comparison of Gingival Index Associated with the Usage of Mouthwashes in Patients Undergoing Orthodontic Treatment in standard Chlorohexidine and prepared Formulation

Para-meter	Name of Sample	First day		After 14 days		After 21 days			P value
		Mean	S.D.	Mean	S.D.	Mean		S.D.
Gingival Index	Chlorohexidine	1.98	0.46	1.32	0.217	1.11		1.87	<0.001
	Green Mouthwash	1.57	0.482	1.18	0.264	1.03		1.62	0.007
Bleeding Index	Name of Sample	First day		10th day		23rd day				P value
		Mean	S.D	Mean	S.D	Mean	S.D
	Chlorohexidine	2.40	0.520	1.80	0.450	1.10	0.300			0.004
	Formulation	2.45	0.530	1.95	0.570	1.45	1.62			<0.001
Bacterial Colony Counts	Name of Sample	First day		After 14 days		After 21 days				P value
		Mean	S.D	Mean	S.D	Mean	S.D
	Chlorohexidine	87200	8200	60100	7315	51300	8190			<0.001
	Formulation	89250	8110	79550	7325	61750	8170			0.105

Intra-group Comparison:

To evaluate changes within each group over time, perform pair-wise comparisons between different time points and use paired t-tests or the Wilcoxon signed-rank test, depending on the distribution of the data. These tests will help us identify statistically significant differences within each group at various time intervals ^9-11.

A significance level of p<0.05 will be used to determine the statistical significance of the results. Any p-value below 0.05 will be considered indicative of a statistically significant finding in this study.

Table 2 represents a comparison of the Gingival Index (GI) among four samples on the first day, 10th day, and 23rd day of the study. Notably, we observed asignificant difference in mean GI between the first day and the 10th day (p<0.05), while no significant difference was found after the 23rd day. Further more, within each sample, a significant decrease in GI was observed from the first day to the 23rd day, indicating a substantial improvement in gingival health over time. Additionally, we found a significant difference in mean GI within each sample across all three time points. The post hoc test results further confirmed these significant differences.

When bacterial colony counts are compared between the time points within green Formulation and standard marketed Chlorohexidine, it was observed that green Formulation is almost similar effective in decrease in bacterial count as Chlorohexidine ^12-13.

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11. Rupali Y. Gatfane, Rajashri A. Ware, Kalpana S. Denge. Pharmaceutical Analysis of a Herbo-mineral Formulation-Pippalyadi Agad from Yogaratnakar. Research Journal of Pharmacy and Technology. 2024; 17(5): 1927-2. doi: 10.52711/0974-360X.2024.00305

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13. Rupali Y. Gatfane, Rajashri A. Ware, Kalpana S. Denge. Pharmaceutical Analysis of a Herbo-mineral Formulation-PippalyadiAgad from Yogaratnakar. Research Journal of Pharmacy and Technology. 2024; 17(5): 1927-2. doi: 10.52711/0974-360X.2024.00305

Received on 14.12.2023 Revised on 13.05.2024

Accepted on 27.08.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):6032-6035.

DOI: 10.52711/0974-360X.2024.00915