Formulation And Evaluation of Herbal Mouthwash

Mouthwash can be suggested to address infections, decrease inflammation, alleviate pain, and combat bad breath, or to administer fluoride directly for the prevention of cavities. The purpose of using mouthwash is mainly to control dental caries, and its therapeutic use is to inhibit or reduce plaque-associated bacteria. The International Standards Organization (ISO) recommends and considers general aspects of mouth rinse safety, such as toothpaste. Possible risks and side effects associated with mouth rinses arise from three factors: the physicochemical characteristics of the rinse, the active components in the products, and additional ingredients. The mouth harbors different microorganisms that come through food intake, warm temperatures, and moisture, which provide a suitable environment for these microorganisms to grow easily and produce dental plaque^1,2. A significant portion of the population is afflicted by plaque-associated oral illness, which is one of the main causes of tooth loss. Herbal products are highly valued compared with chemical products. Because of their antibacterial and antifungal capabilities against human infections, medicinal plants have been used to cure illnesses for a long time^3,4. Throughout history, plants have served to prevent or address a variety of illnesses globally, starting from the emergence of contemporary pharmaceutical medications. Plants contain substances that can be used for therapeutic purposes or as precursors for synthesizing useful drugs. Thus, over 50% of these modern drugs are directly or indirectly derived from natural products. Consequently, these natural substances are essential in the drug development process within the pharmaceutical sector.^5,6. It is easier to prevent oral diseases than to treat them. The widespread use of mouthwashes as an aid to oral hygiene is a relatively recent phenomenon in developing countries. Herbal mouthwashes are in high demand because they act on oral pathogens, relieve pain instantly, and are also less side-effective^7,8,9. The mouthwashes are concentrated aqueous antibacterial solutions that are used against oral microbes to counter oral infection, cleansing to remove bad breath refreshing, and antiseptic. Mouthwash plays a prominent role in oral hygiene, helping to relieve the symptoms of inflamed gum gingivitis¹⁰.   Teeth's supporting tissues are impacted by periodontal disorders. Gingivitis, which is the least severe type of periodontal disease, is typically a result of inadequate oral hygiene. Gum swelling and bleeding are signs of gingivitis. The primary factor contributing to gingivitis is the plaque that develops on the teeth and gum surfaces. As a mainstay for maintaining oral hygiene, mechanical plaque control measures are used. Methods for controlling plaque mechanically are often time-intensive and necessitate a certain level of motivation and skill to execute effectively; therefore, antimicrobial agents have been widely used as supplements to mechanical cleaning^11,12. Several herbal mouthwashes and herbal extracts have been tested in vitro and in vivo in search of a suitable adjunct to mechanical therapy for long-term use. In this study, one such herbal mouthwash was selected, which has proven to be a promising antimicrobial agent in past studies and has shown anti-plaque properties¹³.Jamun leaves have antibacterial properties and are believed to decrease bad odor. Using natural mouthwash alongside brushing and flossing is an excellent approach for minimizing oral bacteria and maintaining optimal oral health and hygiene. Clove extracts are used to treat. periodontitis and stop Staphylococcus aureus from growing. It also has anti-inflammatory, antioxidant, and antimicrobial properties^14,15.  An herbal mouthwash preparation with antibacterial properties was created using extracts of jamun leaves, mint leaves, clove buds, and licorice root under sterilized conditions.

1. MATERIALS AND METHODS

2.1 Collection of plants

Collection of jamun leaves (Syzygium cumini), mint leaves (Lamiaceae), clove buds (Eugenia caryophyllus), and licorice root (Glycyrrhiza glabra) from mature plants.

2.2 Extraction Process^16,17

The leaves of mature plants were collected and washed 2-3 times with sterile water to remove dust and dirt. The leaves underwent a shadow drying technique in which the leaves were spread in container trays and maintained at room temperature for 6-7 days. After 7 days, dried leaves were taken and powdered using a sterile mixer under aseptic conditions. Pulverized leaves were transferred to airtight sterile container jars. A total of 100 ml of sterile distilled water was gathered from four 250 ml conical flasks, and the powdered leaves were measured and placed in distilled water while maintaining sterile conditions. The preparation was heat sterilized at 40°C for 5-10 min and incubated at 37°C for 72 h. After incubation, the extracts were filtered using a sterile Whattmann filter paper no. 1 and a funnel under laboratory conditions. The filtered extracts are boiled vigorously again to kill the bacterial spores, which prevents contamination. After heating, the extract is ready to be used in the formulation of mouthwash.

Table. 1: Formulation Of the Herbal Mouthwash.

2.3 Procedure for the preparation of mouthwash containing the extract^18,19

A weighed quantity of each ingredient is taken. Extracts were taken and mixed thoroughly in a mortar and pestle with a small quantity of water. Mix all ingredients properly, taking care to avoid lump formation. Finally, water is added to increase the volume and preservatives are added, and the product is packed in an attractive, well-closed container.

3. Evaluation of mouthwash^19,20

3.1 Colour and Odour

Physical parameters, such as odour and color, were examined by visual examination.

3.2 Measurement of pH

The pH of the prepared herbal mouthwash was measured using a digital pH meter (kncp-ELICOLI- 120). The pH meter was calibrated using standard buffer solutions of pH 7, 9.2; approximately 1 ml of mouthwash was weighed and dissolved in 50 ml of distilled water, and its pH was measured.

3.3 Homogeneity

All mouthwash formulations were placed on a platform and tested for homogeneity by visual inspection. They were tested for their appearance and the presence of any lump flocculates, or aggregates.

3.4 Test for microbial growth in developed mouthwash.

The developed mouthwash was inoculated into plates of agar media using the streak-the-plate method, and a control was prepared. The plates were placed in an incubator and incubated at 37°C for 24 h. After the incubation period, the plates were removed, and microbial growth was checked by comparing them with the control.

3.5 Stability studies

The formulation and preparation of any pharmaceutical product is incomplete without proper stability studies of the prepared product. A stability study is done to determine the physical and chemical stability of the prepared product and thus determine the safety of the product. A general method for predicting the stability of any product is accelerated stability studies, in which the product is subjected to elevated temperatures as per the ICH guidelines. A short-term accelerated stability study was conducted for 3 months for the prepared formulation. The samples were stored under the following conditions at 35°C. Finally, the samples kept under the accelerated study were withdrawn at monthly intervals and analyzed.

3.6 In vitro antibacterial activity

In vitro, antibacterial activity was assessed in isolated colonies of Streptococcus aureus. The agar well diffusion technique was used to determine the zone of inhibition and minimum inhibitory concentrations (MIC). The strains of S. Aureus were inoculated into a prefabricated agar plate. Plates were dried, and four wells were made with the help of a 6-mm agar well cutter. 1 ml, 2 ml, and 3 ml of prepared mouthwash were loaded into the wells. The agar plates were kept undisturbed to allow the passive diffusion of herbal mouthwash into the agar culture medium. The plates were then incubated at 37°C for 24 h. The commercial antibiotic ampicillin (50mg/ml) was used as a positive control for S. aureus. The zone of inhibition (mm) was recorded for each plate and compared with the control.

4. RESULTS AND DISCUSSION

Table 2: Evaluation parameters of the herbal mouthwash

Table 3: Results of the agar well diffusion antibacterial assay

Fig. 9: Agar diffusion method for assessing antibacterial activity.