Evaluation of antifungal activity of Miswak (Salvadora persica) and toothpaste against oral cavity Candida species

Abstract:

Around the world, several oral health measures have been implemented. The most popular method is to use a toothpaste. However, chewing sticks are still used in many cultures around the world in a conventional manner. Chewing sticks have a mechanical cleansing action similar to a toothbrush in addition to their antimicrobial effect. The purpose of this study is to evaluate the effect of Miswak on the growth of Candida species and to compare it to that of toothpaste. A cross-sectional study was carried out on 120 selected randomly volunteers from January to April, 2022. Two groups of participants were used for two weeks. The participants were separated into a group used Miswak and a group use ordinary toothpaste. Samples were collected before and after two weeks in these two groups. Samples were immediately processed for microbiological phenotypic conventional methods and in vitro susceptibility testing of the microbial isolates to antimicrobial. The findings show that a significant effect of Miswak against an oral cavity candida species. Total candida count was 27.5 ± 18.48, P = 0.001 (Mean ± SD) from Miswak group and 247.0 ± 90.14, P = 0.979 (Mean ± SD) in toothpaste group. The most frequent Candida isolates organisms were C. albicans: 22 (36.67%) in the Miswak group and 24 (40.0%) in the toothpaste group, the second isolate was C. dubliniensis: 04 (6.69%) in the Miswak group and 12 (20.0%) in toothpaste group followed by C. glabrata: 02 (3.33%) in Miswak group. In conclusion, Miswak has a considerable antifungal impact immediately and after two weeks of use.

Introduction

The oral cavity is a habitat for a large number of microorganisms' species that coexist with one to another as a normal microbiota [1]. There are more than 20 species of candida, the most common opportunistic oral fungus associated with oral candidal infection, which colonize from 20.0% to 80.0% of adults without an evidence of infection [2, 3]. Candida albicans are the predominant oral cavity species in medically compromised and otherwise healthy individuals [4]. Oral candidiasis is a major condition that arises due to some predisposing factors such as poor oral hygiene, immune-suppression, nutritional deficiencies, long-term of use antibiotic/radiation therapy, dental prostheses, diabetes mellitus and a high carbohydrate diet or cigarette smoking [5]. Oral health has been linked to a number of chronic and systemic disorders and good oral hygiene is vital for disease prevention [6]. Modern dental care tools are designed to provide mechanical and chemical means through regular removal of plaque and food residues from the surface and spaces among the teeth. One of the most popular ways to maintain oral hygiene is by using a toothbrush and dentifrices. Other notable methods include chewing gum, mouthwash and dental floss [7]. In many countries, chewing sticks made from the twigs, stems or roots of various plant species are chosen and prepared as natural methods of brushing teeth [8]. Salvadora persica is the most commonly used plant for this practice [9]. This plant is commonly known as the Miswak tree. Due to its broad association with the practice, the Arabic word Miswak, which means tooth cleaning stick, becomes a common name for S. persica [10]. Due to their ability to defend against some oral infections and their potential to advance oral health, chewing sticks of S. persica have been recommended for use in oral hygiene by the World Health Organization [11]. S. persica has been comprising a wide variety of organic and inorganic compounds within its extract. Among the organic compounds were glycoside, saponins, flavonoids, alkaloids, tannins, benzyl derivatives, phenol compounds and organic acids [12]. For inorganic compounds, i.e. anionic compounds such as fluoride, chloride, sulphate, thiocyanate and nitrate have been identified [13]. Long fibers and the specific complexity of phytochemicals and minerals offer it an advantage as a tool for oral and dental health care by supplying all of the necessary means of mechanical and chemical cleaning [14]. Miswak at the time of usage stimulates saliva production and buffer sits pH [15]. These ingredients have the ability to remineralize dental hard tissue, stimulate the gingiva and treat inflammatory gums. Chewing sticks also contain volatile oils, tannic acid, sulphur and sterols which contribute to anti-septic, astringent and bactericidal properties. It reduces plaque formation, has anti-carious properties, eliminates bad odors and improves taste sensation [16]. It contains a strong antimicrobial activity [17] as well as protecting from pathogens that enter the body through the mouth [18]. Thus. The present study aimed to assess the effect of Miswak on the growth of Candida species and to compare with toothpaste use.

Materials and methods

Study design: This study was conducted at University of Tripoli, Faculty of Medical Technology, Department of Medical Laboratory Sciences, from January to April, 2022, and was approved by the Research Ethics Committee of the University (2021). It was performed on 120 volunteers who divided into two main groups. Group Miswak uses, whereas, the other group uses toothpaste for two weeks, three times daily.

Study population: 120 subjects aged 15 - 65 years (78 females and 42 males) consented to participate and were enrolled in the study. During the two weeks, neither antibiotic nor antiseptic mouthwash had been utilized by the chosen participants. The study excluded smokers and none of the individuals had ever utilized Miswak. There were 60 volunteers split between the two groups. Data were collected by questionnaire including age, sex and oral condition health.

Sample collection culture and identifications: Sampling was carried out two times:

Miswak group: The first sample was before using Miswak and after utilizing Miswak for two weeks, the second sample was taken. Volunteers were instructed about the Miswak use, advised to use it three times daily and shown how to keep it fresh by cutting off the edge of the Miswak every day and storing it in the refrigerator at night.

Toothpaste group: The first sample was taken before using toothpaste and the second sample was taken after using toothpaste for two weeks as directed. By using an oral concentrated rinse, samples were acquired by washing the mouth for 30 seconds with 10 ml of sterile water. The mouthwash liquid is then placed in conical tubes and placed in an insulated container until it is microbiologically processed. The sample suspension was washed three times in sterilized 0.10 M phosphate-buffered saline pH 7.4 (PBS) by centrifugation at 3 500 rpm .The pellet was then resuspended in 01.0 mL of PBS and 100 μl aliquots were inoculated by spiral plating system onto the surface of Sabouraud’s dextrose agar with an antibiotic. The growth of the colonies on the plates was counted and expressed as the number of Candida colony forming units per milliliter (CFU per ml) of rinse after 24 - 72 hours of incubation at 30 ^oC [19]. Yeasts species identified by germ-tube production, micromorphology and chlamydospores production on corn meal agar plus 01.0% Tween 80. On corn meal agar, all isolates tested positive for germ tube test and chlamydospores and Tween 80 was identified as C. albicans or C. dubliniensis, Sunflower seed agar. Candida species were identified by chromogenic media [20].

Statistical analysis: The raw data were entered into spreadsheets in Excel and then imported into SPSS software version 26 (IBM Corp, Armonk, N.Y, USA). All data were expressed as Mean and standard deviation (Mean ± SD). Descriptive statistics were used to calculate the frequency distribution, mean, standard deviation and median. For the total number of microorganisms in the two groups, t-test was used. The level of significant difference was set at ^*p < 0.05, ^**p < 0.01 as highly significant and ^***p < 0.05 as very high significant.

Results

In this study, of the 120 adult participants, 78 were females (65.0%) and 42 were males (35.0%). The mean age of the participants was 34.2 ± 17.42 years old (range: 15 - 65 years). In the two groups, 60 participants were Miswak users and 60 participants were toothpaste users. The present findings of the biochemical tests used in this study to identify isolated strains were given in Table 1. The results show that there is statistically significant difference between the collected samples after two weeks of using Miswak compare to toothpaste according to the microbial growth on Sabouraud’s dextrose agar. Where that Miswak is more effective than toothpaste, showed a significant decrease in the total number of colonies for each sample with a correlation coefficient. This shows that there is a positive correlation between the Miswak and the overall number of candida species with p < 0.001. Tables 2 and 3 show the effect of Miswak on the candida. Thus, the total candida count was 27.50 ± 18.48, IQR = 30, p = 0.001 from Miswak group and 247.0 ± 90.14, IQR =110, p = 0.979 in toothpaste group. Table 4 depicts the distribution of all the organisms from the Miswak group and toothpaste group that has been isolated. The most frequent Candida isolated organisms were C. albicans 22 (36.67%) in the Miswak group and 24 (40.00%) in the toothpaste group, the second isolate was C. dubliniensis 04 (6.687%) Miswak group and 12 (20.0%) in toothpaste group followed by C. glabrata 02 (03.33%) in Miswak group.

Table 1: Phenotypic and microscopic characteristics of isolated candida species

Table 2: Candida count in the different brushing groups

IQR = Interquartile Range

Table 3: Comparison between Miswak and toothpaste through cultural growth before and after use

Table 4: Microorganisms isolated from the mouth of the test group