INTRODUCTION:

Our oral cavity is the home for several kinds of microorganisms like bacteria, fungi, viruses and some types of protozoa. Oral cavity provides an unique environment to these microorganisms in the form of secretions, tissues and consumed food^1,2. In some cases, these organisms proliferate in case of disease conditions such systemic diseases, AIDS, immunosuppression, oncological treatment, drug misuse, antibiotic treatment, lifestyle etc.^3,4. Oral thrush was reported in infants of women’s with lack of overall health standard awareness⁵.

Like bacteria, fungi can be part of the natural oral micro flora and it can shift from the balanced microflora to infection and finally disease.

Candidiasis commonly called yeast infection, oral thrush, oral candidosis, candidal stomatitis, sometimes it is also referred as biofilm disease⁶. In the initial stages of infection patches are inside the cheek, under tongue and throat but as the organism grows it may progress into esophagus³ causing sore throat, problems in swallowing, loss of appetite and chest pain. If left unchecked esophageal candidiasis can cause severe respiratory complication⁷.

There are various treatments for oral thrush including mouthwashes, gargles, tablets, mouth paints and lozenges⁸. Advanced thrush treatment includes nystatin, clotrimazole, fluconazole and ketoconazole. Furthermore, a long-term treatment with the commonly used antifungal agent, such as amphotericin B or ketoconazole alone or combined, have toxic effect and finally leading to drug resistance^9,10. These antibiotics have various side effects like nystatin causes nausea and bad taste in mouth, clotrimazole causes burning sensation and local irritation, ketoconazole cause nausea, vomiting, loss of appetite, headache, and menstrual irregularities due to suppression of estradiol synthesis. However, toxicity and resistance to synthetic antifungal drugs is a major problem¹⁰. Thus there is need to search new treatments to encounter these problems^12,13,14. Researchers are focusing on certain natural origin sources like endophytic fungi for treating oral infections¹².

Saliva has antibacterial and antifungal properties that help to protect the healthy patient from infection. Proteins such as secretary IgA, lysozyme, mucin and lactoferrin have inhibitory effects on Candida that stop adhesion and multiplication on the mucosal surface¹⁵. Decrease in salivary flow or alteration of salivary composition gives an opportunity for Candida infection.

Lysozyme has been studied against different genotypes of Candida and it has shown very good inhibitory effect against many Candida genotypes^16,17 has suggested that lysozyme has bimodal action on C. albicans killing the organism at higher concentrations and modulating aspartyl proteinase (sap) metabolism at lower concentrations. The main functions of sap are probably to provide nutrition, aid penetration and invasion, and evade immune responses.

Coccinia indica belongs to the family Cucurbitaceae. It is a rapidly growing, perennial climber or trailing vine¹⁸. Traditionally different parts of this plant namely the roots, leaves and fruits are used in folklore medicine for several purposes like jaundice, diabetes, wound healing, ulcers, stomach ache, skin disease, fever, asthma, cough^19,. Mistry et al. demonstrated the hepatoprotective action of C. indica. The chemical constituents of fruits are taraxerone, taraxerol, B-carotene, lycopene, cryptoxanthin²⁰.

Mouth paints are viscous liquid preparations used for mouth and throat infection. Viscous nature of mouth paints allows the drug to remain in contact or adhered with mucous membrane for sufficiently long time to give desired inhibitory action. Glycerin is commonly used as a base because of its viscous nature and appreciable taste. Also, glycerin is an excellent solvent for various compounds²¹.

This study was designed to prepare mucoadhesive mouth paint with prolonged transit time in oral cavity by using natural antifungal lysozyme and C. indica fruit extract. The prepared formulation is homogeneous in nature, it has an agreeable taste and colour and dose of it can be easily adjusted according to the need of a patient. This is a novel and first time attempt for controlling oral thrush.

MATERIALS AND METHODS:

Materials:

Glycerin and Lysozyme were obtained Sigma Aldrich, India, and pure culture of Candida albicans (MCC-1151) obtained from the National Center for Cell Science (NCCS), Pune. Sabouraud dextrose broth was obtained from HiMedia, India.

Methods:

Extraction of plant material:

The fruits of Coccinia indica were collected from the area of Latur, Maharashtra. The fruits were surface cleaned by washing repeatedly, then dried under the shade and pulverized. There are various methods of available for herbal extraction²² here, the coarse powder of fruits (100 g) was extracted with methanol using soxhlet apparatus²³. The extract was dried in hot air oven at 40⁰C and stored in desiccator for further use.

Anticandidal activity:

The anticandidal activity of C. indica fruit extract, lysozyme and combination of both were studied by preparing 500ppm dilutions of each.

Tube dilution susceptibility test:

The sabouraud dextrose broth was prepared, distributed in the test tubes and sterilized at 121^˚C and 15 lbs for 15-20 min. These sterilized broth test tubes were inoculated with 0.1ml suspension of C. albicans having approximate population 5x10⁶ CFU/ml. Then measured amount of dilutions containing fruit extract, lysozyme and combination were added into respective labeled test tubes as mentioned in the table 1.

These tubes were kept for incubation at 37^˚C for 48 hrs. After incubation, the tubes were observed for C. albicans growth. The growth was measured in terms of turbidy at 540nm by colorimeter.

Table 1: Dilutions added in the tubes during susceptibility test

Solution	Tube-1	Tube-2	Tube-3	Tube-4 (+Ve Control)	Tube-5 (-Ve Control)
Nutrient broth	9.8	9.8	9.8	9.9	10
Lysozyme	0.1	-	-	-	-
Fruit extract	-	0.1	-
Fruit extract and Lysozyme combination	-	-	0.1
Inoculum Suspension	0.1	0.1	0.1	0.1
Total Volume	10 ML	10 ML	10 ML	10 ML	10 ML

Preparation of formulation:

The mouth paints are thick viscous preparations, which are prepared by dissolving drug in thick viscous vehicles. In this study, glycerin was used as a vehicle for preparing mouth paint because it is thick and viscous in nature with agreeable taste. The formulation of 500ppm was prepared by dissolving 2.5mg of lysozyme and 2.5 mg of fruit extract in 10ml of glycerin. The prepared formulation was characterized and MIC was determined against C. albicans.

Characterization of formulation:

Determination of pH of formulation:

The pH of formulation was determined using digital pH meter which was calibrated using standard buffer solutions of pH 4 and 7.

Macroscopic and microscopic tests:

Gel formulation was inspected macroscopically and microscopically for clarity, homogeneity, consistency and presence of particles. Homogeneity of formulation was examined by microscopic observation. The consistency of the formulation was noticed by placing a small quantity of gel between the thumb and the index fingers²⁴.

Viscosity test: Sample formulation was settled for 30 min at room temperature before checking viscosity. Viscosity of the formulation was measured at 25°C by using Brookfield (DV-III) viscometer at 100rpm.

Determination of MIC of Formulation:

The formulation was prepared in 1000ppm concentration. This formulation was used as stock and different concentrations were prepared using glycerin as diluents. The resulting dilutions were subjected for determining MIC. The broth was prepared and distributed in test tubes such that it gives final volume 10 ml. The tubes were sterilized and then inoculated with 0.1ml of C. albicans inoculums. The measured amount formulation was added to each test tube. The tubes were incubated at 37^oC for 48 hrs. Growth was measured in terms of turbidity. To determine growth the absorbance was measured at 540nm on colorimeter. MIC is the lowest concentration of the formulation that inhibits the growth.

In above procedure the inhibition of candida growth was observed at 200µg/ml. To determine the accurate MIC dilution range was narrowed from 100µg to 200µg/ml, and MIC was determined by following the same protocol as above.

Statistical analysis:

All the studied experiments were performed in triplicate and results are expressed as ± standard deviation.

RESULTS AND DISCUSSION:

Susceptibility Test:

The susceptibility were determined by tube dilution method and expressed in terms of optical density (Fig 1). C. albicans has shown sensitivity to all as the tube containing test samples were shown the lesser optical density than control tube. The fruit extract have shown very less activity against C. albicans and lysozyme shown better sensitivity than fruit extract but lesser than combination of both (Fig 2). So from this study, it is clear that the synergistic activity of lysozyme and fruit extract was more prominent than when they are used alone. So formulation containing combination of lysozyme and fruit extract was used for further study. Rukayadi et al., (2009) have been studied the synergistic effect of xanthorrhizol obtained from Curcuma xanthorrhiza and ketoconazole and amphotericine B against C. albicans²⁵. Samaranayake et al., (2009) have shown the synergistic activity lysozyme and antifungal agents against C. albicans²⁶. But both these studies have used chemical antibiotics for synergism while present study shows the synergistic antifungal activity of two natural compounds plant extract and lysozyme. Thus the present formulation is safer than other synergism studies.

Fig.1: Susceptibility test F1-fruit extract, F2-lysozyme, F3-combination of fruit extract and lysozyme.

Fig.2: Susceptibilities of fruit extract, lysozyme combination of both against C. albicans

Characterization of formulation:

Determination of pH:

The pH of prepared formulations was 7.4 ±0.2. Salivary pH varies from 6.5 to 7.7 ^27,28(Goodarzi, Hendi, Abdolsamadi, & Rafieian, 2013; Rehan et al., 2016) according to eating habits and life style. While oral mucosal pH at various site is present between 6.2 to 7.4 ²⁹. The pH of formulation matches with oral cavity pH and suitable for further studies.

Macroscopic and microscopic tests:

The prepared formulations were uniform homogeneous in nature with no bubbles and no lumps when touched and observed under the microscope³⁰.

Viscosity test:

The viscosity of formulation was 2450 cps ±20 at 25^oC. This viscous nature increases transit time of drug in oral cavity so that drug remains in affected area for longer time.

Determination of MIC:

The MIC of the formulation was determined by tube dilution method using sabouard dextrose broth. The tubes were serially diluted with formulation and observed after 48 hrs for C. albicans growth (Fig 3). The inhibition of growth was observed after 200µg/ml concentration of lysozyme and fruit extract combination together (Fig 4). So, for getting exact MIC the dilutions were prepared between 100 to 200µg/ml and the dilution range was narrowed (Fig 5). The lowest concentration of formulation at which no growth was recorded as MIC and it was observed at 160µg/ml (Fig 6) of lysozyme and fruit extract. The global crises of antibiotic resistance³¹ may resolved by developing the natural formulations.

Fig.3: MIC of formulation (12.5 to 200µg/ml)

Fig.4: MIC of formulation (12.5 to 200 µg/ml)

Fig.5: MIC of formulation (100-160 µg/ml)

Fig. 6: MIC of formulation (100-160 µg/ml)

CONCLUSION:

Candiasis of oral cavity treated with conventional antimicrobial agents causes the problem such as toxicity and resistance. To overcome these problems natural agents were used in the formulation lysozyme and fruit extract, which showed good synergetic action by reducing their individual doses. Both lysozyme and fruit extract are obtained from natural sources hence these are safer to use. The glycerin was used as a base that gives a pleasant taste to the formulation by providing required thickness. Thus constituent remains at the site of infection by resisting to flushing action of saliva thus give protection to affected area. This formulation provides effectiveness, safety, dosage flexibility. Finally, it can be concluded that this formulation certainly holds great promise in treatment of oral thrush. However, further studies like drug release study, stability testing and in vivo efficacy are required to evaluate for further clinical application of this formulation.

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Received on 10.11.2022 Modified on 30.11.2022

Asian J. Res. Pharm. Sci. 2023; 13(1):1-5.

DOI: 10.52711/2231-5659.2023.00001