Formulation And In-Vitro Evaluation of Herbal Mouthwash for The Management of Halitosis

The microbial load in the oral cavity is decreased by using mouthwashes. Therapeutic on the tooth surface and the mouth is provided by the mouthwashes. Mouthwashes are liquids that show Anti-inflammatory, Antioxidant, Antimicrobial, and Analgesic action. There are two types of Mouthwashes available – chemical and herbal. The main Purposes of using mouthwash are good oral hygiene can be obtained by using the mouthwashes routinely at home, they provide anti-inflammatory, anti-Microbial activity.  Mouthwash is defined as a liquid that is passively held in the mouth by contraction of the oral muscles and movement of the head, and it may be gargled, in which the head is tilted back and the liquid bubbles in the mouth. Herbal mouthwashes are free of alcohol, artificial preservatives, flavours, and colours. Hence Herbal mouthwashes can be used in place of chemical mouthwashes to Maintain oral hygiene, owing to the additional Benefits provided by herbal Preparations.^[1]

Types of Herbal mouthwash: ^[1]

1. Fluoride mouthwash: The cavities in the teeth can be prevented by using the salts of fluoride present in the mouthwashes.
2. Antiseptic mouthwash: It is the common type of mouthwash. It contains alcohol and is typically utilized to stop bacterial growth especially for the people with mouth infections and halitosis or bad breath.
3. Cosmetic mouthwash: It is a mouthwash that are not having any therapeutic effect to the oral care but it masks the bad breath and freshens the bad breath.
4. Natural mouthwash: As the name suggests it contains only natural herbal ingredients obtained from the nature. It is an alcohol-free and chemical lacking mouthwash. The herbal ingredients are safer to use as compared to other types of mouthwashes.

Halitosis is a term derived from the Latin word halitus (breath) and the Greek suffix osis (condition or action); in English it is known as Bad breath. Bad breath, halitosis, is an unpleasant problem that people experience often and dislike it. Halitosis can be a symptom of a serious disease. though it is not a serious Disorder, it affects the overall health of the individual in the society. ^[3] Halitosis refers to the offensive or unpleasant odor which may come from the mouth or cavities of the nose, sinuses and pharynx. Fetor oris, oral malodor, fetor ex oris, foul or bad breath are the various names given to this objectionable condition. Halitosis is  classified as genuine halitosis, pseudo-halitosis or halitophobia. Halitosis whose origin is either physiological or pathological can be termed as Genuine Halitosis whereas pseudo-halitosis may occur where no reasonable cause is known. Almost 80-90% of cases in genuine halitosis, bacterial activities occur in the oral cavity. In the oral microbiota, over 600 species of microorganisms have been detected by researchers. These microorganisms act on sulfur amino acids such as cystine, cysteine and methionine which results in volatile sulfur compounds that include hydrogen sulfide (H2S), methyl mercaptan (CH3SH) and dimethyl sulfide (CH3SCH3).^[4]

Causes of Halitosis- Physiologic causes of halitosis are Lack of flow of saliva during sleep, Food where the metabolism of certain foods and beverages produces volatile fatty acids or other malodorous substances which can be  excreted through the lungs, Smoking, Menstruation where Women have bad breath during periods, which is secondary to hormonal changes.

Advantages of herbal mouthwashes over chemical mouthwashes:

• Herbal mouthwashes are non-irritant and they have non-staining properties.
• They are less harmful and have very few or no side effects.
• Herbal mouthwashes are gentle for even the most sensitive mouth.^[5]

Disadvantages:

• Herbal drugs cannot be found everywhere, they are least availability.
• Pungent in smell.
• Stability problems during the formulation process. ^[6]

Uses: ;

• To enhance oral hygiene.
• To control dental plaque.
• For eradicating bacteria present in oral cavity.^[7]

MATERIALS AND METHODS:

Collection of plant materials:

The herbal ingredients which are used to formulate the herbal mouthwash such as, Cinnamon, Clove, Fennel, Licorice, rose and rosemary were purchased from the local General stores and were subjected for authentication by “Central Ayurveda Research Institute, Bengaluru”. The herbal materials were identified and authenticated as Cinnamomum verum J. Presl belonging to the family Lauraceae (RRCBI-20516), Syzygium aromaticum (L.) Merr. & L.M. Perry belonging to the family Myrtaceae (RRCBI-mus151), Foeniculum vulgare Mill. Belonging to the family Apiaceae (RRCBI-mus145), Glycyrrhiza glabra L. belonging to the family Fabaceae (RRCBI-mus356), Rosa centifolia L. belonging to the family Rosaceae (RRCBl-mus518), Salvia Rosmarinus Schleid. Belonging to the family Lamiaceae (RRCBI-mus519) respectively. After Authentication the herbal materials  were chopped into small pieces and they were pulverized into a sterile mixer. The powder was sieved using a fine mesh of 44mm. The powder was stored in an airtight container. Chemicals used in the formulation such as Alcohol or food grade ethanol, Glycerol, Sodium lauryl sulphate, Peppermint oil and Distilled water etc. were used from “The Oxford College of pharmacy, Bangalore” ^[4]

Extraction process:

About 5g of all the herbal powders of clove, cinnamon, fennel, licorice and rosemary were transferred into a conical flask which contains 100ml of distilled water. It was kept for maceration at 37°C for about 24 hours in a stoppered flask. After maceration the obtained extract was filtered using the Muslin cloth, then again it was Filtered using a filter paper. After filtration a clear liquid extract of the cinnamon which is brown in color, clove which is reddish brown in color, fennel which is light green in color, licorice which is yellow in color and Rosemary which is dark green in color was obtained. The extracts were boiled, cooled and stored in a closed container.^[5] The rose petals were boiled in a beaker containing 100ml of distilled water for about 30 Minutes. The petals were stirred occasionally by using a spatula or a stirrer. After 30 minutes it was made to cool down and the extract has been filtered in a Muslin Cloth. The red liquid extract with good aroma was obtained and was transferred to an air tight container.^[6]

Pre-Formulation studies:

Compatibility studies: Prior to formulation of our product, a Compatibility study of the herbal drug extractions was carried at “Pooranayu Research Labs, Bengaluru”. To check the interaction of the herbal drugs with each other. Compatibility is the key characteristics to proceed with the formulation of herbal drugs. So, all the herbal extractions of cinnamon, clove, Fennel, liquorice, rose and rosemary 1ml of  each extract were collected separately in Eppendorf tubes. Along with these a mixture of glycerol, alcohol and sodium lauryl sulphate was poured in 1ml Eppendorf tube. The compatibility of the herbal ingredients along with the chemicals was tested and based on the result further formulation of the mouthwash was done.

Interpretation of FT-IR spectra of cinnamon

Interpretation of FT-IR spectra of clove

Interpretation of FT-IR spectra of Fennel

Interpretation of FT-IR spectra of Licorice

Interpretation of FT-IR spectra of Rose

Interpretation of FT-IR spectra of Rosemary

Interpretation of FT-IR spectra of Alcohol, SLS and glycerol

Conclusion of FTIR: FTIR is a technique that is used to analyze the chemical composition of materials by measuring how they absorb infrared rays. Many functional groups are found in the herbs which shows the chemical elements of the herb. These chemical elements are found to be compatible with each other, which means the herbal drugs when used together in the formulation do not show any interactions. From this compatibility study we conclude that Clove, Cinnamon, Licorice, Rosemary, Rose and the chemical ingredients Alcohol, SLS, Glycerol are having good compatibility which are used for the further formulation of the herbal mouth.

Formulation of herbal mouthwash: Mouthwashes were prepared by adding varying proportions of Clove, Cinnamon, Rose, Fennel, Licorice, Rosemary, Peppermint oil, Glycerol, Sodium lauryl sulphate, Alcohol and Distilled water. A total six formulations of mouthwashes F1, F2, F3, F4, F5 and F6 were prepared. ^[7]

1. Visual Examination: Through visible examination, physical characteristics including odor and color have been checked for the formulated herbal mouthwash. Physical parameters such as color, odor and Consistency are examined by visual inspection. ^[8]
2. Determination of pH: The digital meter was used to measure the pH of the prepared herbal mouthwash. Standard buffer was used to calibrate the pH meter. About 1ml of mouthwash was taken and its pH was determined by pH meter. To check pH of the formulations, the pH meter was connected to the formulations. Readings were taken three times to get an average value.^[9]
3.  Centrifugation test: Using 6 different centrifugation tubes, the herbal mouthwash preparation was centrifuged in the Centrifugation machine at 2800 rpm for 5minutes to examine the phase separation of the Formulated preparation. First ensure that the Centrifugation machine is well calibrated and ready to use.  Transfer 10ml of herbal mouthwash preparation to 6 different Centrifugation tubes. Place the tubes in centrifuge machine and set the following parameters; Speed-2800 rpm (revolutions per minute), Time-5 minutes, Temperature-Room temperature (20-25°C) Start the centrifuge machine and run the sample for the set time. After Centrifugation observe the Sample for any visible sediment or phase separation.^[10]
4. Microscopic test: The transparency of the mouthwash formulation was assessed by using a compound microscope with magnification powers of 10X and 40X.The result is concluded as transparent or non-transparent. ^[11]
5. Viscosity:  Viscosity of the formulation is determined using Ostwald viscometer. Viscometer is mounted on a suitable burette stand. Mouthwash was filled in a viscometer in wider opening side. The formulation was sucked until the mark A. The time was noted in a stopwatch to determine the time take by the mouthwash to flow from mark A to mark B. All the 6 formulation’s viscosity was determined in triplicate to get an average value in seconds. ^[12]
6. Foam height: 1ml of mouthwash is mixed in 50ml of distilled water. The mixture is poured in a measuring cylinder. Distilled water was added in the measuring cylinder to make the volume up to 100ml. The mixture received 25 strokes, after which it was kept aside. The height of the foam above the aqueous volume is observed and noted in all 6 formulations. ^[13]
7. Homogeneity: The mouthwash preparations were packed in the container and tested for homogeneity by visual inspection. It shows whether there is a presence of aggregate in the formulation. ^[14]
8. In-vitro Antibacterial activity:  In-Vitro Antibacterial activity test is performed on isolated colonies of streptococcus mutans. The agar well diffusion method was used for determining the Zone of Inhibition (ZOI) and Minimum Inhibitory Concentration (MIC).

Table 1.2. Equipment’s for antibacterial activity

Test organisms: Streptococcus mutans

Test compound as standard: Tetracycline Inoculum: Streptococcus mutans cell suspension was prepared and grown on media and cultures were incubated for 24hrs at 37°C. The cell suspensions of all the cultures were adjusted to 1-2x 106cells/ml.

Test compound: Sample – 250 microgram, Std-Standard: Tetracycline 25 µg/ml

Procedure:

Determination of Antimicrobial activity: Streptococcus mutans were inoculated on media (90 mm), Test compounds: Sample (25µl), Standard Tetracycline (25µl) Streptococcus mutans were added to the 5mm well on agar plates. The treated plates with Streptococcus mutans were incubated in aerobic chamber at 37°C for 24hrs. The treated plates were observed for zone of inhibition around the wells. ^[15]

RESULTS AND DISCUSSION:

1. Visual Examination:

Table 1.3. Visual recordings

F4 mouthwash looks clear and transparent when compared to other formulations among the all 6 formulated herbal preparations.

2. Determination of pH:

Table 1.4. pH readings

As the skin is having the acidic pH around 5.5, this pH range of the formulated herbal mouthwash is suitable for the oral disorders such as halitosis. Oral problems are appropriate for this pH range of formulation.

Table 1.5. Phase separation

       
           
       
 Fig.1.4 observation of phase separation

4.Viscosity:

       
           
       
Fig.1.5. viscosity observation

Foam height:

Table.1.7. Height of foam

Fig.1.6. Foam height

Homogeneity:

Table.1.8. Homogeneity

In-vitro Antibacterial activity:  Inhibitory activity of test compounds against test organisms:

Conclusion: The zone of inhibition observed against test compound and standard are summarized in Table. Sample shows inhibitory activity against Streptococcus mutans in well diffusion. The zone of inhibition is the circular spot of the sample in which the bacterial colonies do not grow. ZOI between 5-10 mm indicates moderate to high Antibacterial activity. ZOI of 7 mm indicates that the antimicrobial agent has inhibited the growth of streptococcus mutans in a circular area with a diameter of 7 mm around the agent.

Table.1.9 Zone of inhibition measurement

       
           
       
Fig.1.7. Zone of Inhibition