Description:Description of the Invention
FIELD OF THE INVENTION
The present invention relates to an antioxidant, anti-inflammatory, anti-precancerous and anti-plaque mouthwash composition comprising encapsulated Coenzyme Q10 for smokers and tobacco users. The composition further comprises antioxidants, erythritol, xylitol, and water-soluble polymers to soothe the sensitive gums of smokers and tobacco users, without causing xerostomia and a burning sensation.

BACKGROUND OF THE INVENTION
Mouthwashes are aqueous topical medications that are used for rinsing and gargling of the oral cavity so as to enhance oral hygiene. Antiseptic and anti-plaque mouth rinse claims to kill the microbes and fight against dental plaque causing dental caries, gingivitis, and bad breath. Anti-cavity mouth rinse uses fluoride to protect against tooth decay. There are multiple mouthwashes available over the counter and by prescription with a wide range of active ingredients that can be used as an adjunct to aid in the multifactorial management of complex oral conditions.

These days’ consumers prefer a mouthwash product that is free of alcohol content due to the negative effects and risks posed for the development of cancer associated with the regular use of alcohol- containing oral products. Some of the studies also indicate consistent exposure to alcohol may sensitize peripheral nerve endings, leading to diminished response because ethanol destroys nervous tissue by extracting cholesterol and other lipids, further emphasizing the use of alcohol-free mouthwash whenever possible and especially in long-term use. It is also reported that long term use of alcohol based mouthwash leads to laryngitis and difficulty in swallowing.

The PCT application WO0033802 discloses mouth rinse compositions comprising a high concentration of Coenzyme Q10 combined with a solubilizing agent such as a polyoxyethylene sorbitan monostearate, a polyethylene glycol, a non-ionic poloxamer surfactant, a polyethylene glycolparaisooctyphenyl ester, a glycerol ester of fractionated fatty acid having a chain length of 8 to 10 carbons, or a propylene glycol diester of a saturated fatty acid having a chain length of 8 to 10 carbons. The mouth rinse composition disclosed have high alcohol content.

Another PCT application WO2012001337 discloses mouthwash composition comprising Pelargonium, vitamin A, vitamin C, vitamin E, folic acid, niacinamide, zinc, coenzyme Q10, arginine, calcium carbonate, fluoride, strontium chloride, and papain. Mouthwash disclosed contains alcohol. The mouthwash composition comprises alcohol as a carrier for the flavor and as an anti-bacterial.
Smoking leads to the production of reactive oxygen species (free radicals) with each puff of cigarette smoke containing up to 1017 oxidant molecules. Smoking involves the combustion of tobacco, which releases a complex mixture of chemicals leading to inflammation of the oral mucosa and salivary gland openings, causing tissue inflammation, in the initial stages and hence increasing the risk of various oral health diseases, dryness of mouth, precancerous lesions and conditions, in the long run.
While there has been awareness among smokers about the impact of smoking on the lungs but, not many people are aware of the impact smoking has on oral health. People who smoke are prone to damage such as tooth loss, teeth stains, and various oral diseases. Therefore, it is necessary that smokers take additional care of their oral hygiene, including using mouthwash at least twice daily. The inventors of this application did a small survey to identify the reason for the lack of compliance by smokers towards the use of already marketed mouthwashes. One of the common issues faced by the smokers was the burning sensation, strong taste and xerostomia (dry mouth) caused by currently marketed mouthwashes. People who smoke are prone to problems like, smokers' breath or bad breath, tooth staining (nicotine & tar stains), smokers melanosis (brown to black pigmentation of oral tissues i.e the gums, cheeks, palate, etc.), inflammation of oral tissues and burning sensation in the mouth. The inflammation of the salivary gland openings in the mouth can lead to nicotinic stomatitis, decreased flow of saliva leading to conditions like dry mouth (xerostomia), altered taste & smell sensation, increased build-up of plaque and tartar on the teeth, increased risk of dental caries, increased bone loss within the jaw, increased risk of precancerous lesions like leukoplakia (white patches in mouth) and increased risk of developing gum disease (gingivitis, periodontitis), a leading cause of tooth loss.

Hence, the inventors of the present invention have developed a mouthwash composition that does not cause burning or xerostomia in smokers, thereby improving smoker's compliance with the mouthwash product. The inventors identified that the combination of encapsulated Coenzyme Q10, and antioxidants, along with some other anti-inflammatory and antioxidant molecules, provides a mouthwash with the desired mouthfeel that soothes the sensitive gums. In conclusion, the alcohol-free mouthwash composition with encapsulated Coenzyme Q10 is an innovative solution that addresses smokers' oral hygiene needs. The soothing effect of the composition of the present invention on sensitive gums is an excellent alternative to marketed mouthwashes that cause burning and xerostomia. Additionally, the mouthwash composition is free of harsh chemicals such as artificial colorants, dyes, parabens, whiteners, abrasives, and strong surfactants such as sodium lauryl sulphate.

SUMMARY OF THE INVENTION
According to the first aspect of the present invention, there is provided an alcohol-free mouthwash for smokers and tobacco users comprising a synergistic combination of encapsulated Coenzyme Q10 and antioxidants selected from the group consisting of vitamin C, pomegranate peel extract, green tea extract, or a combination thereof.

According to one embodiment of the first aspect of the present invention, the mouthwash has a pH of about 6 to about 7, throughout the shelf life.

According to one embodiment of the first aspect of the present invention, the mouthwash is more effective in reducing inflammatory markers and oxidative stress associated with smoking as compared to the mouthwash comprising non-encapsulated Coenzyme Q10.

According to another embodiment of the first aspect of the present invention, the mouthwash has a viscosity of about 100 to about 300 cps, throughout the shelf life.
According to another embodiment of the first aspect of the present invention, the mouthwash is free of artificial colorants, dyes, fluoride, parabens, whiteners, abrasives, and sodium lauryl sulphate.
According to another embodiment of the first aspect of the present invention, the mouthwash further comprises one or more pharmaceutically acceptable excipients selected from the group consisting of emollients, mild surfactants, natural flavors and preservatives.
According to the second aspect of the present invention, there is provided stable alcohol-free mouthwash for smokers and tobacco users comprising
i. encapsulated Coenzyme Q10,
ii. one or more antioxidant selected from the group consisting of vitamin C, pomegranate peel extract, green tea extract or a combination thereof,
iii. anti-inflammatory agent selected from the group consisting of hyaluronic acid or salt thereof,
iv. one or more water-soluble polymer, and
v. one or more sweetener,
wherein the composition has a pH of about 6 to about 7 and a viscosity of about 100 to about 300 cps.

According to another embodiment of the second aspect of the present invention, the mouthwash comprises sodium carboxymethylcellulose as water-soluble polymer.
According to another embodiment of the second aspect of the present invention, the mouthwash is free of the artificial colorants, dyes, parabens, whiteners, abrasives, and sodium lauryl sulphate. This is done to avoid any sensitivity to the inflamed oral mucosa.

According to the third aspect of the present invention, there is provided a process for the preparation of a stable alcohol-free mouthwash for smokers and tobacco users comprising encapsulated Coenzyme Q10 comprising the steps of:
a) mixing encapsulated Coenzyme Q10 with an antioxidant, an anti-inflammatory agent, a water- soluble polymers, one or more sweeteners till a uniform dispersion is obtained;
b) adding flavoring agent to the above dispersion; and
c) adding emollient to the above dispersion and stirring the dispersion till a uniform dispersion is obtained.

According to another aspect of the invention, there is provided an antioxidant, anti- inflammatory, anti-plaque and anti-fungal alcohol-free mouthwash for improving the oral hygiene of smokers and tobacco users comprising encapsulated Coenzyme Q10 wherein the composition does not cause a burning sensation or xerostomia to the users and is more effective in reduction in inflammatory markers and oxidative stress associated with smoking as compared to the mouthwash comprising non-encapsulated Coenzyme Q10.

BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 graphically represents the mean plaque scores between the groups at baseline, after 15 days, and after 30 days
FIG. 2 graphically represents the mean gingival scores between the groups at baseline, after 15 days, and after 30 days
FIG. 3 graphically represents the median VAS scores (for burning sensation) between the groups at baseline, after 15 days, and after 30 days
FIG. 4A graphically represents the distribution of participants according to pigmentation at baseline between the groups
FIG. 4B graphically represents the distribution of participants according to pigmentation after 15 days between the groups
FIG. 4C graphically represents the distribution of participants according to pigmentation after 30 days between the groups
FIG. 5 graphically represents the mean salivary flow rate (ml/min) between the groups at baseline, 15 days, and 30 days

DETAILED DESCRIPTION OF THE INVENTION

The term stable as used herein means the pH of the composition remains within the critical range of pH about 6 to about 7 throughout the shelf life, when the stability studies are carried out after storing the composition under 40°C/75% RH for a period of 1 year.

For purposes of the present invention, the term alcohol-free shall mean that alcohol is substantially absent, if not completely absent from the formulation. Minute or trace amounts below 1.0% may be found as a result of the flavor oils or any other ingredient used in the present invention, but these amounts are so low that they will not pose any health risk.

Coenzyme Q10 is a potent water-insoluble antioxidant that is involved in electron transport and oxidative phosphorylation. The mouthwash composition of the present invention harnesses the potent natural antioxidant, Coenzyme Q10, to combat oxidative stress and shield against damage caused by free radicals generated in the body due to exposure to nicotine, cigarette smoke, and tobacco. The encapsulation such as microencapsulation provides enhanced bioavailability, absorption, and overall efficiency of Coenzyme Q10 in the mouthwash, ensuring a superior and effective experience to the consumer. The encapsulation process also increases the stability of the enzyme. The encapsulation of the composition may be carried out by any conventional method such as by spraying a solution or suspension of the excipient into a fluidized bed filled with Coenzyme Q10 particles. The amount of Coenzyme Q10 may vary from 0.01 to 10% w/v of the mouthwash composition.

The mouthwash composition further comprises antioxidants selected from the group consisting of vitamin C or derivative thereof, pomegranate peel extract, green tea extract or a combination thereof. The amount of antioxidants may vary from about 0.01 to about 30% w/v of the mouthwash composition. These components possess several beneficial properties other than antioxidant properties.

Vitamin C is a powerful antioxidant that aids in preventing gum diseases and supporting teeth through collagen synthesis. The Vitamin C derivatives may be selected from the group consisting of 3-O-ethyl ascorbic acid, L-ascorbic acid, calcium ascorbate, calcium 1-ascorbate dihydrate, magnesium ascorbate, potassium ascorbate, magnesium L-ascorbyl phosphate L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, (+) sodium L-ascorbate, dehydro-1-(+)-ascorbic acid dimer, sodium ascorbyl phosphate, ascorbic acid-2-glucoside, ascorbyl dipalmitate, ascorbyl methylsilanol pectinate, ascorbyl stearate, disodium ascorbyl sulfate, ascorbyl 6-palmitate and combinations thereof. In a preferred embodiment, 3-O-ethyl ascorbic acid may be included as a stable and potent form of vitamin C.

Similarly, pomegranate peel extract supports periodontal health with antioxidant and antifungal properties, efficiently curbing harmful oral bacteria while preserving beneficial probiotics. The pomegranate peel extract is also known to have anticancer effect. The green tea extract poses antiviral and antimicrobial properties. The extract also prevents formation of sulphur compounds thereby combats bad breath.

The mouthwash further comprises anti-inflammatory agents such as hyaluronic acid or pharmaceutically acceptable salt thereof. Hyaluronic acid is known to remove plaque, moisturize oral tissues and exhibits anti-inflammatory properties. The amount of anti-inflammatory agent may vary from 0.01 to 10% w/v of the mouthwash composition.

The mouthwash may comprise mild surfactants such as the alkylamidopropyl betaine used in the present invention may be selected from one or more of cocamidopropyl betaine, lauramidopropyl betaine, myristamidopropyl betaine, oleamidopropyl betaine, and mixtures or any two or more thereof. In particular the surfactant is cocamidopropyl betaine.
The mouthwash of the present invention promotes the healing of oral soft tissues & maintains periodontal health. It also helps reduce detrimental diseases associated with smoking and tobacco use like gum disease (gingivitis, periodontitis, etc.), and oral fungal disease. It also helps in reducing post-smoking burns due to cigarette smoke, and its compounds. The mouthwash prevents the build-up of plaque & tartar. It also combats the bad odour, also called smoker’s breath. It also relieves dryness of the mouth by improving the flow of saliva and moisturizing the oral tissues. Due to the presence of the above-mentioned ingredients, the composition exhibits holistic support to oral hygiene and health of the consumer as antioxidant, anti-inflammatory, anti-plaque and anti-fungal.

The viscosity of the mouthwash composition is from about 100 to about 300 cps at 25?C. This viscosity provides a better mouthfeel for the product to the user along with higher efficacy of antioxidants and anti-inflammatory agents present in the composition. The composition is in the form of solution, suspension, colloid or nanosuspension, however, the suspended particles do not settle or separate throughout the shelf life of the product. The mouthwash of the present invention is free of particulate matter.

Sweeteners such as sorbitol, aspartame, sodium saccharin, erythitol, and xylitol may be individually or in combination added for better taste in amounts of from about 0.001% w/v to about 50.00% w/v and preferably in an amount of approximately 0.01% w/v to 30.00% w/v. In particular, the sweetener is xylitol, erythitol, or sorbitol. It is preferred that the sweetener is xylitol as it starves harmful bacteria of their food source and reduces their ability to produce the acids that lead to cavities. It also helps prevent dry mouth in consumers due to its smoothening effect. Similarly, erythritol effectively decreases the weight of dental plaque and adherence of common streptococcal oral bacteria to tooth surfaces. It also inhibits the growth and activity of associated bacteria like S. mutans.

Many people use mouthwash as part of their oral health care regimen. However, the low pH of oral care products may cause problems for the users. Many mouthwashes are too acidic (pH below 5) or somewhere near the critical pH of our mouth (around 5.5) and are unable to balance the overall pH of saliva, weakening the tooth enamel by aiding in demineralisation and dental caries. Therefore, the pH of the present invention is maintained around pH about 6 to about 7. By keeping the pH above the critical pH, the composition also facilitates remineralization of the tooth. This is accomplished generally by adding an acid, a base or a buffer comprising a weak acid and its salt or a weak base and its salt to the final composition.

One or more flavors may be used in the practice of the present invention. The flavor could be from peppermint extract, lemon oil or blend of flavor oils. Other flavors such as citrus oils, vanillin, cardamom oil, clove oil, menthol and the like may be incorporated to provide further taste variations. This unique blend of flavor oils not only provides for a pleasant tasting mouthwash but also serves to taste mask the bitter- tasting ingredients in the mouthwash. The amount of flavor may comprise from 0.01% w/v to 1.5% w/v.
The mouthwash composition comprises water soluble polymer such as hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and polyvinyl pyrrolidone. The water soluble polymer is present in the range of 0.01 to 30%, preferably 0.1 to 15%, more preferably 1 to 5%, by weight of the composition.

The mouthwash composition may comprise humectants such as sorbitol, propylene glycol or polyethylene glycol, or mixtures thereof; which may be present in the range from 5 to 70%, preferably 5 to 40%, more preferably 10 to 30% by weight of the composition.
The mouthwash composition may comprise emollients such as PEGylated oils for example, PEG-40 hydrogenated castor oil. PEGylated oils are derivatives of polyethylene glycol (PEG)—products of the etherification and esterification of glycerides and fatty acids with ethylene oxide. PEGylated oils function as a surfactant (emulsifying or solubilizing agents). PEG-40 hydrogenated castor oil (trade name Cremophor RH 40) is often used in mouthwashes. It is a polyethylene glycol derivative of hydrogenated castor oil where an average PEG chain is 40. PEG-40 hydrogenated castor oil is the most used PEGylated oil in mouthwashes. PEG-40 hydrogenated castor oil functions as an emulsion/suspension stabiliser and surfactant, thereby helps solubilise fragrances and oils.

Because of their high-water content, mouthwashes are challenging to preserve, this is particularly seen in mouthwash that are free of alcohol. The mouthwash composition also comprises preservative selected from the group consisting of sodium benzoate, potassium sorbate, benzyl alcohol, and the combinations thereof.

The composition may further comprise pH adjusting agents such as acidifying agents, basifying agents or buffers, to adjust the pH of the composition within the desired range of pH about 6 to about 7.

Mouthwash compositions according to the present invention may be prepared by admixing the ingredients in the appropriate relative amounts in any order, if necessary adjusting the pH to give the final critical pH value.

EXAMPLE 1
Ingredients Percentage of Ingredients
1. Sorbitol 30.00
2. Pomegranate peel extract 2.00
3. Green Tea Extract 2.00
4. Cocamidopropyl betaine 1.00
5. Sodium benzoate 0.50
6. Encapsulated Coenzyme Q10 0.10
7. PEG-40 hydrogenated castor oil
(Cremophor RH 40) 0.50
8. Vitamin C 0.10
9. Sodium Hyaluronate 0.10
10. Sodium Carboxymethyl Cellulose 0.10
11. Menthol 0.10
12. Xylitol 0.10
13. Erythitol 0.10
14. Cardamom spicy flavor 0.10
15. Sodium Hydroxide Q.S.
16. Demineralized Water Q.S. to 100.00

BRIEF MANUFACTURING PROCESS:
1. Encapsulated Coenzyme Q10 was mixed with sorbitol, erythritol, pomegranate peel extract, Green Tea Extract, cocamido propyl betaine, sodium benzoate, vitamin C, sodium hyaluronate, sodium carboxymethyl cellulose and xylitol in water and stirred for 30 minutes at 20 rpm to obtain a uniform dispersion.
2. PEG-40 hydrogenated castor oil and menthol were added to the dispersion of step 1 and were stirred again.
3. The flavor was added to the above dispersion.
4. The pH of the above dispersion was checked and pH was adjusted to 6-7 using sodium hydroxide.

The pH of the final mouthwash composition was found to be 6.15.
The above composition was subjected to stability studies at 40?C and 75% RH and were tested for specification parameters. The composition was found to be stable for 6 months under specified conditions.

Table 1: Stability data for mouthwash composition of Example 1
PARAMETERS Specification 24 hrs. 15 days 1 Month 3 month 6 month
RESULT RESULT RESULT RESULT RESULT RESULT
pH 6.00 - 7.00 6.29 6.14 6.12 6.02 6.45
Foam Height NLT 50 ml 50 ml 50 ml 50 ml 50 ml 50 ml
Colour Intensity Pale yellow, transparent
liquid. Complies Complies Complies Complies Complies
Homogeneity No visible
particles Complies Complies Complies Complies Complies
Odour Minty Complies Complies Complies Complies Complies

EXAMPLE 2
Ingredients Percentage of Ingredients
1. Sorbitol 30.00
2. Pomegranate peel extract 2.00
3. Green Tea Extract 2.00
4. Cocamidopropyl betaine 1.00
5. Sodium benzoate 0.50
6. Encapsulated Coenzyme Q10 0.10
7. PEG-40 hydrogenated castor oil
(Cremophor RH 40) 0.50
8. 3-O-ethyl ascorbic acid 0.10
9. Sodium Hyaluronate 0.10
10. Sodium Carboxymethyl Cellulose 0.10
11. Menthol 0.10
12. Xylitol 0.10
13. Erythitol 0.10
14. Vanilla flavor 0.10
15. Sodium Hydroxide Q.S.
16. Demineralized Water Q.S. to 100.00

The composition of Example 2 was prepared according to the process given in Example 1.

A study was conducted to compare the efficacy of the mouthwash of the present invention with a marketed mouthwash.

Materials and Methods:
Total sample of 40 smokers, aged 25 to 55 years, were randomly divided into 2 groups, Group 1 and Group 2 with Mouthwash I and II respectively, during phase 1 and 2 of the study which was of 15 days each.

Data was recorded - At baseline, after 15 days and after 30 days.

Mouthwash according to the present invention was labelled as Mouthwash I had encapsulated Coenzyme Q10, Green Tea extract, Pomegranate peel extract, Vitamin C (3-O-Ethyl Ascorbic Acid), Sodium Hyaluronate, Xylitol, Erythritol and Carboxymethyl cellulose.

Mouthwash II had purified water, Hyaluronic acid, Sodium Monofluoro Phosphate, Coenzyme Q10 (non-encapsulated), Citric Acid, Sodium Citrate, Menthol, PEG 40 hydrogenated castor oil, Linalool, sodium benzoate, Tea tree oil, Aloevera, Myrrh, Menthol, Color Brilliant Blue and Xylitol. The Mouthwash II is a suspension with particulate matter. The pH of the comparative mouthwash was found to be 5.2 to 5.8.

Total sample size - 40
Group 1 - 20 (3 females, 17 males)
Group 2 - 20 (2 females, 18 males)

No. Of Cigarettes smoked by the individuals - 3-15 cigs/day

Following Parameters were monitored in both the groups
Plaque index - (Silness and Loe)
Gingival index - (Loe and Silness)
Burning sensation - (VAS)
Gingival pigmentation - (HMI score)
Salivary Flow Rate - (Unstimulated Saliva - ml/min) - (The Spit Technique)
Salivary pH - Hydrion pH paper

Selection criteria -
(1) Patient should have been smoking for at atleast three years or more.
(2) Patient should not have had any known systemic conditions that could influence periodontal health.
(3) Patient should not have been subjected to periodontal therapy or any antibiotic medication during the last 6 months.

The data was analyzed using SPSS for Windows [ver. 26.0, IBM Corp., Armonk, NY]. The data was assessed for normal distribution using the Shapiro-Wilk test. Continuous data was compared between the groups using the unpaired t-test and Mann-Whitney U test. Categorical data was compared between the groups using the Chi-Square test. Results were presented using graphs and tables. The level of significance was set at P = 0.05.
The Silness-Loe Plaque Index (PI) is a tool used to evaluate the level and rate of plaque formation on teeth. It's used to assess dental caries and periodontal disease. The PI is based on a four-point scale that measures the amount of plaque on the tooth surface:
Score 0: The tooth surface is clean
Score 1: The tooth surface appears clean, but plaque can be removed with a sharp explorer
Score 2: Plaque is visible along the gingival margin
Score 3: The tooth surface is covered with abundant plaque
To calculate the PI:
1. The plaque score was measured in four areas of each tooth: distal facial, mesial-facial, facial, and lingual
2. The plaque score was recorded for each area of each tooth and total score was recorded
3. The total score was divided by 4 and recorded for both the groups at baseline, day 15 and day 30, as given in Table 2 and Figure 1.
The higher PI indicates increased deposition of plaque.
Table 2: Comparison of mean plaque scores
Number Mean SD t P value
Baseline Group I 20 1.105 0.55 -1.27 P = 0.19
Group II 20 1.085 0.48 NS
After 15 days Group I 20 0.895 0.51 -1.27 P = 0.904
Group II 20 0.955 0.47 NS
After 30 days Group I 20 0.705 0.38 -1.27 P = 0.703
Group II 20 0.85 0.4 NS
SD-standard deviation; NS-not significant using unpaired t-test

Observation for PI
Baseline
It was found that mean plaque scores at baseline were almost similar among participants in both groups. The difference in mean plaque scores between the groups was not statistically significant (P = 0.19).

After 15 days
It was found that mean plaque scores after 15 days were almost similar among participants in both groups. The difference in mean plaque scores between the groups was not statistically significant (P = 0.9).

After 30 days
It was found that mean plaque scores after 30 days were marginally higher among participants in Group II compared to Group I. The difference in mean plaque scores between the groups was not statistically significant (P = 0.703). The data related to Plaque Index for all the subjects has been summarized in Table 2 and Figure 1.

Gingival Index developed by Loe and Silness is a dental assessment tool used to measure the severity of gingivitis by evaluating the appearance and bleeding tendency of the gums on a scale from 0 (healthy gums) to 3 (severe inflammation), with scores typically assessed on the facial, lingual, and mesial surfaces of each tooth; essentially, it is a method to clinically grade the level of gum inflammation based on visual observations and probing bleeding.
Scoring system:
0: Normal gingiva, no inflammation or bleeding
1: Mild inflammation, slight color change, no bleeding on probing
2: Moderate inflammation, redness, bleeding on probing
3: Severe inflammation, spontaneous bleeding, marked redness and swelling
Examination method: A dental probe was used to assess the gingival tissue around each tooth, noting the color, texture, and bleeding response. The mean gingival scores between the groups at baseline, after 15 days, and after 30 days was calculated for group I and II, as given in Table 3 and Figure 2.

Table 3: Comparison of mean gingival scores
Number Mean SD t P value
Baseline Group I 20 0.825 0.29 -0.37 P = 0.709
Group II 20 0.86 0.29 NS
After 15 days Group I 20 0.635 0.26 -1.29 P = 0.21
Group II 20 0.745 0.28 NS
After 30 days Group I 20 0.46 0.19 -2.54 P = 0.015*
Group II 20 0.65 0.27
SD - standard deviation; NS - not significant and *statistically significant at P<0.05 using unpaired t-test

Observation for Gingival Score
Baseline
It was found that mean gingival scores at baseline were almost similar among participants in both groups. The difference in mean gingival scores between the groups was not statistically significant (P = 0.19).

After 15 days
It was found that mean gingival scores after 15 days were marginally higher among participants in Group II compared to participants in Group I. The difference in mean gingival scores between the groups was not statistically significant (P = 0.21).

After 30 days
It was found that mean gingival scores after 30 days were higher among participants in Group II compared to participants in Group I. The difference in mean gingival scores between the groups was statistically significant (P = 0.015). The data for all the subjects has been summarized in Table 3 and Figure 2.

Burning Sensation - A Visual Analog Scale (VAS) score for a burning sensation ranges from 0 to 10, where 0 represents "no burning sensation" and 10 represents "the worst burning sensation imaginable," allowing patients to mark a point on the scale that best reflects their current level of burning discomfort.
Interpreting VAS scores for burning sensation:
0-2: Minimal or no burning sensation
3-5: Mild burning sensation
6-8: Moderate burning sensation
9-10: Severe burning sensation

The VAS scores (for burning sensation) was recorded for both the groups at baseline, after 15 days, and after 30 days and median was calculated.

Observation for Burning Sensation
Baseline
It was found that participants in Group I had higher median VAS scores (for burning sensation) when compared to participants in Group I. The difference in median VAS scores between the groups was not statistically significant (P = 0.17).

Table 4: Comparison of median VAS scores (for burning sensation)
Number Median IQR Mann Whitney U P value
Baseline Group I 20 2 0.0 - 3.0 152.5 P = 0.17
Group II 20 0.5 0.0 - 2.0 NS
After 15 days Group I 20 0.5 0.0 - 1.0 174 P = 0.44
Group II 20 1 0.0 - 1.75 NS
After 30 days Group I 20 0 0 90 P = 0.001**
Group II 20 1 0.0 - 2.0
IQR-interquartile range; NS - not significant and *statistically significant at P < 0.05 using the Mann-Whitney U test

After 15 days
It was found that participants in Group II had higher median VAS scores (for burning sensation) when compared to participants in Group I. The difference in median VAS scores between the groups was not statistically significant (P = 0.44).

After 30 days
It was found that participants in Group II had higher median VAS scores (for burning sensation) when compared to participants in Group I who did not experience any burning sensation. The difference in median VAS scores between the groups was statistically significant (P = 0.001). The data for all the subjects has been summarized in Table 4 and Figure 3.

Gingival Pigmentation - Hedin Melanin Index, a scoring system used to assess the extent of gingival pigmentation, where a score of 0 indicates no pigmentation and a score of 4 represents a continuous ribbon of pigmentation across the entire area between the canines; essentially, it measures how much pigmented area is present on the gums based on its distribution and severity.
Scoring system:
Score 0: No pigmentation;
Score 1: One or two solitary units of pigmentation in the papillary gingiva;
Score 2: More than three units of pigmentation in the papillary gingiva without forming a continuous ribbon;
Score 3: One or more short continuous ribbons of pigmentation;
Score 4: One continuous ribbon, including the entire area between the canines.

Table 5.1: Distribution of participants according to pigmentation at baseline between the groups
Group I Group II Total Chi value P value
N (%) N (%) N
Score 0 6 (30) 1 (5) 7 6.038 P = 0.16
NS
Score 1 2 (10) 4 (20) 6
Score 2 6 (30) 6 (30) 12
Score 3 5 (25) 5 (25) 10
Score 4 1 (5) 4 (20) 5
Total 20 (100) 20 (100) 40
N-number; %-percentage; NS-not significant using the Chi-square test;

At the baseline, it was found that 5% of participants in Group I had a score of 4 compared to 20% of participants in Group II. About 25% and 30% of participants in both groups had a score of 3 and a score of 2 respectively. In addition, about 30% of participants in Group I had a score of 0 when compared to 5% of participants in Group II. The distribution of participants according to pigmentation at baseline between the groups was not statistically significant (P = 0.16). The same is summarized in Table 5.1 and Figure 4.1.

Table 5.2: Distribution of participants according to pigmentation after 15 days between the groups
Group I Group II Total Chi value P value
N (%) N (%) N
Score 0 6 (30) 1 (5) 7
Score 1 6 (30) 4 (20) 10 8.48 P = 0.03*
Score 2 4 (20) 6 (30) 10
Score 3 4 (20) 5 (25) 9
Score 4 0 4 (20) 4
Total 20 (100) 20 (100) 40
N-number; %-percentage; NS-not significant using the Chi-square test.

As given in Table 5.2 and Figure 4.2, after 15 days, it was found that none of the participants in Group I had a score of 4 compared to 20% of participants in Group II. About 20% of participants in Group I had a score of 2 & 3 when compared to 30% and 25% of participants in Group II. In addition, about 30% of participants in Group I had a score of 1 when compared to 20% of participants in Group II. The distribution of participants according to pigmentation at baseline between the groups was statistically significant (P = 0.03).

Table 5.3: Distribution of participants according to pigmentation after 30 days between the groups
Group I Group II Total Chi value P value
N (%) N (%) N
Score 0 7 (35) 1 (5) 8
Score 1 8 (40) 4 (20) 12 14.16 P = 0.007**
Score 2 5 (25) 7 (35) 12
Score 3 0 5 (25) 5
Score 4 0 3 (15) 3
Total 20 (100) 20 (100) 40
N-number; %-percentage; NS-not significant using the Chi-square test

As given in Table 5.3 and Figure 4.3, after 30 days, it was found that none of the participants in Group I had a score of 4 or 3 compared to 25% and 15% of participants in Group II. About 25% of participants in Group I had a score of 2 when compared to 35% of participants in Group II. In addition, about 40% of participants in Group I had a score of 1 when compared to 20% of participants in Group II. About 35% of participants in Group I had a score of 0. The distribution of participants according to pigmentation at baseline between the groups was statistically significant (P = 0.007).

Mean salivary flow rate - To measure unstimulated salivary flow rate using the spit technique, a person would simply spit saliva into a graduated container over a set period of time (in mins), and the volume collected would be divided by the time to calculate the flow rate.

Table 5: Comparison of mean salivary flow rate (ml/min) between the groups at baseline, 15 days, and 30 days
Number Mean SD t P value
Baseline Group I 20 0.471 0.09 0.332 P = 0.74
Group II 20 0.46 0.11 NS
After 15 days Group I 20 0.657 0.12 5.2 P = 0.001**
Group II 20 0.458 0.11
After 30 days Group I 20 0.714 0.09 7.5 P = 0.001**
Group II 20 0.461 0.11
SD-standard deviation; NS-not significant and *statistically significant at P < 0.05 using unpaired t-test

Baseline
It was found that the mean salivary flow rate at baseline was almost similar among participants in both groups. The difference in mean salivary rate between the groups was not statistically significant (P = 0.74).

After 15 days
It was found that the mean salivary flow rate after 15 days was lower among participants in Group II compared to participants in Group I. The difference in mean salivary flow rate between the groups was statistically significant (P = 0.001).

After 30 days
It was found that the mean salivary flow rate after 30 days was lower among participants in Group II compared to participants in Group I. The difference in mean salivary flow rate between the groups was statistically significant (P = 0.001).

The mouthwash of the present invention (Mouthwash I) was compared with the already marketed mouth wash, the mouthwash was found to be more effective in reducing the plaque, gingivitis and gingival pigmentation along with reduced burning and an improved salivary flow rate. The specific combination of ingredients lead to an improved efficacy with reduced side effects.

Participants using the mouthwash of the present invention (Mouthwash I), which contained synergistic combination of encapsulated Coenzyme Q10, Green Tea Extract, Pomegranate Peel Extract, Vitamin C, demonstrated superior efficacy in reducing oral oxidative stress and inflammation. The combination of Coenzyme Q10 and other antioxidants, including Green Tea Extract and Vitamin C, resulted in a more pronounced reduction in inflammatory markers and oxidative stress associated with smoking compared to Mouthwash II, which included non-encapsulated Coenzyme Q10 and Tea Tree Oil.
In a separate small study, it was observed that antibacterial and antifungal effects were notably better with Mouthwash I. The presence of Pomegranate Peel Extract and Green Tea Extract in Mouthwash I significantly reduced the levels of Streptococcus mutans and Candida albicans in the oral cavity, as well as the overall microbial load, compared to Mouthwash II. Additionally, Mouthwash I showed greater efficacy in preventing the formation of dental plaque and reducing plaque accumulation.
Mouthwash I, due to the presence of sodium hyaluronate, carboxymethyl cellulose and xylitol, outperformed Mouthwash II in terms of alleviating dry mouth and improving oral tissue hydration. This combination provided moisturising properties to the mouthwash that effectively soothed and protected the oral mucosa, providing better relief from dryness and discomfort often associated with smoking.
Mouthwash I also proved more effective in controlling bad breath. The combined action of Green Tea Extract and other ingredients in Mouthwash I led to a notable reduction in volatile sulfur compounds and overall oral malodor compared to Mouthwash II.
The elevated pH of Mouthwash I helps to neutralize the acidic environment often created by smoking. This pH balance reduces the risk of enamel erosion and maintains oral health.
The inventors are also planning further studies on patients with pre-cancerous white lesions, recurrent ulcers, radiation-induced xerostomia, oral candidiasis, and Oral submucous fibrosis (OSMF). The formulation contains micro encapsulated CoQ10 and pomegranate peel extract, which are important in potential anti-cancer effects.
, Claims:1. A stable alcohol-free mouthwash for smokers and tobacco users comprising a synergistic combination of encapsulated Coenzyme Q10,
wherein the composition has a pH of about 6 to about 7, throughout the shelf life and does not cause a burning sensation or xerostomia.
2. The mouthwash according to claim 1, wherein the mouthwash further comprises antioxidants selected from the group consisting of vitamin C or derivative thereof, pomegranate peel extract, green tea extract, or a combination thereof.
3. The mouthwash according to claim 1, wherein the mouthwash is effective in reducing inflammatory markers and oxidative stress associated with smoking compared to the mouthwash comprising non-encapsulated Coenzyme Q10.
4. The mouthwash according to claim 1, wherein the mouthwash has a viscosity of about 100 to about 300 cps.
5. The mouthwash according to claim 1, wherein the mouthwash is free of artificial colorants, dyes, fluoride, parabens, whiteners, abrasives, and sodium lauryl sulphate.
6. A stable alcohol-free mouthwash for smokers and tobacco users comprising
i. encapsulated Coenzyme Q10,
ii. one or more antioxidant selected from the group consisting of vitamin C or derivative thereof, pomegranate peel extract, green tea extract or a combination thereof,
iii. anti-inflammatory agent selected from the group consisting of hyaluronic acid or salt thereof,
iv. one or more water-soluble polymer, and
v. one or more sweetener,

wherein the composition has a pH of about 6 to about 7 and a viscosity of about 100 to about 300 cps.
7. The mouthwash according to claim 6, wherein the mouthwash does not cause a burning sensation or xerostomia.
8. The mouthwash according to claim 6, wherein the mouthwash is more effective in reduction in inflammatory markers and oxidative stress associated with smoking as compared to the mouthwash comprising non-encapsulated Coenzyme Q10.
9. The mouthwash according to claim 6, wherein the Coenzyme Q10 is present in an amount from about 0.01 to about 10% w/v of the mouthwash composition.
10. The mouthwash according to claim 6, wherein antioxidant is present in an amount from about 0.01 to about 30% w/v of the mouthwash composition.
11. The mouthwash according to claim 6, wherein the anti-inflammatory agent is present in an amount from about 0.01 to about 10% w/v of the mouthwash composition.
12. The mouthwash according to claim 6, wherein the sweetener is present in an amount from about 0.01 to about 50% w/v of the mouthwash composition.
13. The mouthwash according to claim 6, wherein the composition further comprises one or more pharmaceutically acceptable excipients selected from the group consisting of emollients, mild surfactants, natural flavors and preservatives.
14. The mouthwash according to claim 6, wherein water-soluble polymer is sodium carboxymethyl cellulose.
15. The mouthwash according to claim 6, wherein the mouthwash is free of artificial colorants, dyes, parabens, fluorides, whiteners, abrasives, and sodium lauryl sulphate.
16. A process for the preparation of a stable alcohol-free mouthwash for smokers and tobacco users comprising encapsulated Coenzyme Q10 comprising the steps of:
a) mixing encapsulated Coenzyme Q10 with an antioxidant, an anti-inflammatory agent, a water-soluble polymer, one or more sweeteners till a uniform dispersion is obtained;
b) adding a flavor to the above dispersion; and
c) adding emollient to the above dispersion and stirring the dispersion till a uniform dispersion is obtained.
17. An antioxidant, anti- inflammatory, anti-plaque and anti-fungal alcohol-free mouthwash for improving the oral hygiene of smokers and tobacco users comprising encapsulated Coenzyme Q10 wherein the composition does not cause a burning sensation or xerostomia to the users.
18. The mouthwash according to claim 17, wherein the mouthwash is more effective in reduction in inflammatory markers and oxidative stress associated with smoking compared to the mouthwash comprising non-encapsulated Coenzyme Q10.