2) PLANT PROFILE:

Table 1: Plant profile of selected herbs^8-11

Sr.No.	Common Name	Scientific name	Family	Plant part used	Chemical Constituents	Uses
1.	Meswak	Salvadorapersica	Salvadoraceae	Twig	Salvadorine, flavonoids	Antiplaque
2.	Myrrh	Commiphoramyrrha	Burseraceae	Resin	α-, β- and γ- Commiphoric acids	Wound Healing
3.	Clove	Syzygiumaromaticum	Myrtaceae	Flower bud	β-caryophyllene	Antibacterial
4.	Thyme	Thymus vulgaris	Lamiaceae	Leaf, stem	Thymol	Anti-inflammatory
5.	Stevia	Stevia rebaudiana	Asteraceae	Leaf	Stevioside, rebaudioside	Sweetening agent
6.	Lemon	Citrus limon	Rutaceae	Fruit	Phenolic acid	Antioxidant

3) MATERIALS AND METHODS:

3.1 Collection of Plant Powders:

Roots, twigs, and stem of Salvadora persica (Meswak), buds of Eugenia caryophyllus (clove), reddish-brown dried sap a thorny tree Commiphora Abyssinia (myrrh), Thymus vulgaris (thyme), leaves of Rebaudiana Bertoni (stevia) and Citrus limon (lemon oil) purchased from online and local market of Nashik.

3.2 Formulation of Herbal Mouthwash:

The selection of herbs was made, keeping in mind the anti-microbial efficacy along with their efficacy along with their excipient’s properties namely, preservative, sweetening, and flavoring effects, which are required to develop an ideal mouthwash.

Table 2: Master formula of Polyherbal Mouthwash

Sr. No	Ingredients	Botanical names	Role	Quantity (ml)
1.	Meswak	Salvador persica	Antiplaque	30
2.	Myrrh	Commiphoramyrrha	Wound Healing	20
3.	Clove	Syzgiumaromaticum	Antibacterial	20
4.	Thyme	Thymus vulgaris	Anti-microbial	15
5.	Stevia	Rebaudiana Bertoni	Sweetening agent	10
	Pomegranate
6.	Lemon oil	Citrus limon	Flavoring agent,	q.s.

Formulation Table:

We had prepared four different formulations with different quantities of herbs in each formulation. The quantities of the herbs were decided on the basis of a literature review and also the minimum concentration of herbs at which they exhibit their desired properties.

Table 2: Four different formulations of polyherbal mouthwash

Sr. No	Ingredients	F1 (ml)	F2 (ml)	F3 (ml)	F4 (ml)
1.	Meswak	25	30	25	30
2.	Myrrh	25	20	25	20
3.	Clove	20	20	20	20
4.	Thyme	10	15	10	15
5.	Stevia	10	10	10	10
6.	Pomegranate peel	10	5	-	-
7.	Lemon oil	-	-	10	5

1. A 20gm weighed quantity of each ingredient was taken.

2. The powdered herb was taken mixed with a 200ml quantity of water.

3. Then it is kept for incubation for 72 hours at 37°C.

4. After 72hours the mixture is removed and mixed properly in an appropriate proportion desired for our formulation.

5. Finally preservative added and the product will be packed in an attractive, well-closed container^14-16.

Development of formulation and evaluation was the continuous process. Several characteristic issues like fungal growth on storage, strong aromatic odourwere observed and to rectify the same several combinations were tried in formulation.

3.3 Preparation of extract:

The Aqueous extract of each plant material was prepared using incubation method^12,13 where the powder sample is soaked in sterile distilled water and maintained in Incubator at 37°C for 72hours. The herbal extracts were filtered using Whatman filter paper, washed with 10ml of sterile distilled water, and pressed.

4) EVALUATION:

4.1 Organoleptic Evaluation:

Table 3: Organoleptic evaluation for Herbal Mouthwash

Evaluation parameter	Observation
Colour	Brown
Odour	Strong, spicy, citrus odor

4.2 pH:

With the help of calibrated pH meter, the differences and variability in pH of formulation was calculated. The pH of formulation was found to be 6.7 which is suitable for use in oral cavity.

4.3 Viscosity:

Brookfield viscometer was used to measure the viscosity of formulation. Viscosity was calculated using Spindle no. 61 at 50rpm and it was found to be 1.92 CP.

4.3 Stability testing:

We have formulated the same formulation after a time period of 3 months for checking its efficacy over a period of time and have done all physicohemical tests on it and was in the desired range of the parameters. Both the samples has shown same pH, viscosity and potent anti-microbial activity.

So, we can conclude from here that our formulation is effective over a 3 month period of time.

Furthermore, more evalaution can be done after a period of 6 or 9 or 12 months to check its stability.

4.4 Anti-microbial testing:

The Polyherbal Mouthwash we have developed has been evaluated for checking its effectiveness of anti-microbial activity. The use of natural antimicrobials may contribute incontrolling the disordered growth of oral microbiota, thus overcoming problems caused by species resistant to conventional antimicrobials.

4.4.1 Method:

i. Pure strains of E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcusaureus ATCC 25923, Candida sp. were used.

ii. The medium which was used to study was nutrient agar. The Agar well diffusion technique was used for determining the Minimum inhibitory concentrations (MIC).

iii. The minimum inhibitory concentration (MIC) was used to evaluate the inhibitory effects of herbal mouthwash.

iv. The test standard used were Gentamycin and Nystatin against herbal mouthwash.

v. The plates were incubated at 37°C overnight and the zone of inhibition of growth was measured in millimetres.

vi. The antimicrobial activity of the mouthwash with different test standard was screened by Agar well diffusion technique and the zone of inhibition was measured in mm diameter. The images of the zone of inhibition are given in the figure no 1.

5) RESULT AND DISCUSSION:

5.1 Evaluation parameter results:

Based on the marked characterization it has been observed that the Pomegranate content in formulation 1 and 2 has led to fungal growth in the formulation whereas replacement of pomegranate content by the lemon oil has resolved the issue. The lemon oil incorporated in the formulation has developed strong aromatic odor to the formulation which is then rectified by managing the proportions of Meswak and lemon oil. On the basis of all evaluation test so performed it is concluded that the formulation 4 is effective, stable and safe.

Sr No.	Parameters	Evaluation
1.	Color	Brown
2.	Odor	Strong, spicy, citrus odor
3.	pH	6.30
4.	Viscosity	1.92cp
5.	Foam Index	250
6.	Antimicrobial activity	No Zone of inhibition

5.2 Antimicrobial activity report:

The sample F4 were continued to the anti-microbial test where the comparative assessment was also made amongst the formulation which is freshly prepared, the formulation stored over the period of 4 months. The formulation is assessed against the standard antimicrobials like Gentamycin and Nystatin. The observations so made are tabulated below.

Figure 2: Polyherbal Mouthwash

6) CONCLUSION:

The findings of tests are evident to conclude that the combination of herbs is accurate and suitable to use as mouthwash. The antiplaque, wound healing, antibacterial, anti-inflammatory and analgesic properties in formulation helps to solve the oral health issues. The aqueous extract helps to sustain the formulation as alcohol free.

7) REFERENCES:

1. Nigam D, Verma P, Chhajed M. Formulation and Evaluation of Herbal Mouthwash against Oral Infections Disease. International Journal of Pharmacy & Life Sciences. 2020 Jul 1; 11(7):6746.

2. Chitali SC, Dubewar A. “Formulation and Evaluation of Ayurvedic Herbal Mouth Wash against Oral Disorders”.

3. Problems and Proposals for Recording Gingivitis and Plaque. International Dental Journal, 25, 229-235.

4. Shambharkar SB, Thakare VM. Formulation and Evaluation of Herbal Mouthwash. World Journal of Pharmacy Research. 2021 May 28; 10(9):775-91.

5. Lone ZA, Jain NK. Phytochemical Analysis of Clove (Syzygiumaromaticum) Dried Flower Buds Extract and its Therapeutic Importance. Journal of Drug Delivery and Therapeutics. 2022 Aug 15; 12(4-S):87-92.

6. Da Silva NB, Alexandria AKF, De Lima AL, Claudino LV, De Oliveira Carneiro TF, Da Costa AC, et al. In vitro antimicrobial activity of mouth washes and herbal products against dental biofilm-forming bacteria. Contemp Clin Dent. 2012; 3(3):302–5.

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8. Haque MM, Alsareii SA. A review of the therapeutic effects of using miswak (Salvadora Persica) on oral health. Saudi Medical Journal. 2015 May; 36(5): 530.

9. Batiha GE, Wasef L, Teibo JO, Shaheen HM, Zakariya AM, Akinfe OA, Teibo TK, Al-Kuraishy HM, Al-Garbee AI, Alexiou A, Papadakis M. Commiphora myrrh: a phytochemical and pharmacological update. Naunyn-Schmiedeberg's Archives of Pharmacology. 2023 Mar; 396(3):405-20.

10. Kong M, Hwang DS, Yoon SW, Kim J. The effect of clove-based herbal mouthwash on radiation-induced oral mucositis in patients with head and neck cancer: a single-blind randomized preliminary study. Onco Targets and Therapy. 2016 Jul 22:4533-8.

11. Sharma, S., Mali, A. M., and Dixit, S. (2017). Formulation and evaluation of a polyherbal mouthwash for oral health maintenance. Indian Journal of Dental Research, 28(2), 140-145. doi: 10.4103/ijdr.IJDR_162_15

12. Lohar, D. R., Mali, R. G., and Vhyawahare, N. S. Development and evaluation of polyherbal mouthwash for the management of oral diseases. Journal of Ayurveda and Integrative Medicine, 2016; 7(2): 85-90. doi: 10.1016/j.jaim.2016.05.005

13. Ghogre, P. V., Kakade, S., and Deshmukh, V. Formulation and evaluation of herbal mouthwash containing Triphala for improving oral health. Journal of Oral Research and Review, 2018; 10(1): 25-30. doi: 10.4103/jorr.jorr_19_17

14. Mahajan, R., Bansal, K., and Mahajan, A. Comparative evaluation of a polyherbal mouthwash with chlorhexidine on gingival health and microbial plaque. Journal of Clinical and Diagnostic Research. 2017; 11(9): ZC17-ZC21. doi: 10.7860/JCDR/2017/27623.10677.

15. Jeddy N, Ravi S, Radhika T, Lakshmi LS. Comparison of the efficacy of herbal mouth rinse with commercially available mouth rinses: A clinical trial. Journal of Oral and Maxillofacial Pathology. 2018 Sep; 22(3):332.

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Received on 30.09.2023 Modified on 27.03.2024

Res. J. Pharma. Dosage Forms and Tech.2024; 16(3):229-232.

DOI: 10.52711/0975-4377.2024.00036

Sample	*E. coli* ATCC 25922	*Pseudomonas aeruginosa* ATCC 27853	*Staphylococcus aureus* ATCC 25923	*Candida sp.*
F4 stored for 4 months	No zone	No zone	No zone	No zone
Freshly preparedF4	No zone	No zone	No zone	No zone
Gentamycin	28 mm.	29 mm.	28 mm.	–
Nystatin	–	–	–	21 mm.