Antifungal and antibacterial activities of polyherbal toothpaste against oral pathogens, in vitro

Background and Purpose: Herbal toothpastes are more secure and efficacious and less poisonous due to containing natural chemicals as compared with the synthetic toothpastes. The present study aimed to formulate a polyherbal toothpaste using accessible medicinal plants in Iran and evaluate its efficiency in the protection of oral hygiene and prevention of dental caries. Materials and Methods: The developed toothpaste was made of the leaf extracts of Artemisia dracunculus, Satureja khuzestanica (Jamzad), and Myrtus communis (Linn), combined at four different dilutions, namely 1:4 (25%), 1:1 (50%), 3:4 (75%), and (100%), with sterile distilled water. The product was tested against five microorganisms, including Streptococcus mutans, Lactobaccilus caseie, S. sanguis, S. salivarius, and Candida albicans, using agar well diffusion method. Results: After 24 h of incubation, the maximum mean diameters of inhibition zone against L. caseie and C. albicans were obtained as 17-30 and 10-25 mm, respectively. Furthermore, the minimum mean diameter of inhibition zone against S. salivarious was estimated as 15-20 mm. Conclusion: The formulated toothpaste showed potent inhibitory activities against Gram-positive bacteria and C. albicans. Therefore, more studies are required to accurately investigate the efficacy of the formulated toothpaste.


Introduction
ne of the most common chronic oral infections in the world is dental caries [1]. Oral pathogenic microorganisms have been the cause of dental plaques, dental caries, as well as gingival and periodontal diseases [2]. Streptococcus mutans is one of the main opportunistic pathogens of dental caries, which is responsible for the formation of dental plaque and caries [3]. Other microorganisms associated with this oral condition include Escherichia coli, S. aureus [4], and Candida species.
C. albicans is the most frequent yeast isolated from the oral cavities with poor oral hygiene [2]. The formulation ingredients of commercial toothpastes are mostly chemical substances, such as fluoride and whitening agents [2]. The literature contains evidence regarding the adverse effects of fluoride and bleaching agents (e.g., peroxide-based agents) used in the commercial toothpastes [5].
With this background in mind, the present study aimed to formulate a polyherbal toothpaste without any fluoride or whitening agents and evaluate its antimicrobial properties. The main ingredients of this toothpaste included the leaf extracts of Artemisia dracunculus L. (ADL), Satureja khuzestanica (Jamzad; SKJ), and Myrtus communis (Linn; MCL). The MCL or myrtle, belonging to the Myrtaceae (Lamiaceae) family, is an aromatic evergreen small tree with small foliage and numerous branches [6]. In ancient medicinal herbs, myrtle leaves and flowers were used for the treatment of respiratory problems, dysentery, urinary tract infections, and candidiasis as a mouthwash [7].
According to the literature, the essential oil of MCL has pharmacological activities, including antioxidant [8], antimicrobial [9,10], and antifungal activities [11,12]. There is evidence regarding the inhibitory activity of the essential oil of MCL against clinically isolated oral pathogenic microorganisms [13]. Artemisia is a small and continual aromatic shrub from the Asteraceae family [14], which is called "Tarkhon" in Iran. In traditional medicine, this plant is used for the remedy of stomach pains, fever, and diabetes and is known to have anti-inflammatory, anti-parasitic, antioxidant, and antimicrobial activities [15]. On the other hand, SKJ, belonging to the Lamiaceae family, is extensively grown in the northern Khuzestan and southern Lorestan provinces of Iran [16]. This plant has traditionally been used for relieving tooth pain, strengthening the gum, and healing the wound in the southern part of Iran [17]. Moreover, this herb has been applied for antimicrobial [18] and antifungal [19] purposes, as well as the treatment of infectious diseases [20][21]. With regard to the previous studies reporting the antimicrobial properties of polyherbal toothpaste [22,23], the current study was conducted to formulate a new polyherbal toothpaste containing the aqueous herbal extracts available in Iran and evaluate its antimicrobial potency against oral pathogens.

Preparation of extracts
The ADL was purchased from the local market, and MCL and SKJ were prepared with the aids of the Agricultural and Natural Resources Center, Ahvaz, Iran. The collected plants were air dried in shade. About 10 g of each powdered air dried plant was added to100 ml sterile distilled water (1:1 W/V) in a glass beaker for maceration, and then incubated on a rotary shaker for 72 h [24]. In the next step, the filtration of suspension was accomplished using Whatman filter paper No.1. The filtrated aqueous extracts were evaporated and dried at the room temperature. The extracts were stored in air-tight containers at -20˚C until future use.

Microorganisms and inoculum preparation
The antimicrobial activity of four anaerobic bacteria (Gram-positive) isolated from clinical isolates and Candida species was assessed. A total of twelve anaerobic bacteria, including S. sanguis, S. salivarius, S. mutans, and L. casei, were prepared from the frozen stock cultures obtained from the Department of Medical Microbiology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. In addition, five C. albicans strains were isolated from patients with periodontitis and gingivitis referring to the Educational Clinics of Dentistry School, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
These samples were subcultured, and then diluted in a sterile normal saline solution (0.9%) to obtain a concentration of 5×10 5 CFU/ml for fungal strains and a colony forming unit of 10 6 (CFU/ml) for bacterial strains, adjusted with the turbidity of 0.5 McFarland [25,26].

Formulation of polyherbal toothpaste
The ingredients of the fluoride-free polyherbal toothpaste was prepared according to the procedure adopted by Sekar and Zulhilmi Abdullah [22] with some modifications (Table 1).

Screening polyherbal toothpaste for antimicrobial activity
The formulation of the toothpaste was accomplished using three plants, namely ADL, MDL, and SKL, which were previously confirmed to have antimicrobial activities by in vitro assays. The dentifrice solution was prepared according to the previously reported procedures [2,27].
The solution was tested against five microorganisms, including S. mutans, L. caseie, S. sanguis, S. salivarius, and C. albicans, by using agar well diffusion method following the previous studies [28,29]. The agar plates inoculated with bacteria were kept in an anaerobic cabinet supplied with CO2 at 37°C for 24, 48, and 72 h. On the other hand, those agar plates inoculated with C. albicans were incubated at 30°C for 48 h [30].

Statistical analysis
Statistical analysis was performed in SPSS software (version 20.0). The mean diameters of the inhibition zones were calculated. P-value less than 0.05 was considered statistically significant.

Quality parameters of formulated polyherbal toothpaste
The organoleptic investigation of polyherbal toothpaste, including color, taste, odor, and texture, were carried out by sensational and visual surveys according to the modified procedure of Sekar and Zulhilmi Abdullah [22]. Results Table 2 summarized the inhibition zones produced by four polyherbal toothpaste dilutions of full strength, 3:4, 1:1, and 1:4 against S. mutans, S. salivarius, S. sanguis, L. casei, and C. albicans. The mean values of the microbial inhibition zones are shown in Table 2. The results demonstrated that L. casei showed the highest sensitivity against the dilutions of polyherbal toothpaste ranging from 5-30 mm in 24 h, followed by C. albicans (10-25 mm), S. mutans (5-25 mm), S. salivarius (10-25 mm), and S. sanguis (5-20 mm) at different dilutions of the toothpaste (figures 1 and 2; Table 2). All dilutions of polyherbal toothpaste were effective in inhibiting the growth of the tested bacterium and fungus; however, they had no inhibitory effect on L. caseie, S. sanguis, and S. mutans at the dilution of 1:4.
The polyherbal toothpaste showed a significant antimicrobial activity against all tested bacterium and yeast (P<0.001). Table 3 tabulates the results of onesample t-test for each dilution of toothpaste against all the tested strains. The comparison of antimicrobial activities against tested microorganism in different concentrations is demonstrated in Table 4. Table 5 exhibits the results of the physicochemical parameters of the formulated toothpaste.

Discussion
A good oral hygiene maintenance is the key to prevent oral diseases. The novelty of the herbal toothpaste developed in the current study owes to its natural compounds as aqueous extract. Therefore, it can be claimed that this toothpaste can be used as a completely natural product without the complications of the commercial products. The results revealed that our developed toothpaste had different degrees of effectiveness against the five tested microorganisms. In this regard, the formulated toothpaste exerted a highly significant effect against all tested oral pathogens.
As the results indicated, L. caseie and S. sanguis had the highest and lowest sensitivities to the formulated polyherbal toothpastes, exhibiting inhibition zones of 17-30 and 15-20 mm, respectively. In a clinical study, a commercially available toothpaste was reported to have inhibitory effects on some pathogenic oral microorganisms, such as S. mutans, Micrococcus species, Proteus vulgaris, S. aureus, and C. albicans [29].
There are a number of polyherbal toothpastes playing an important role in dental prophylaxis and improvement of oral health [31]. In a study conducted by Sekar and Zulhilmi Abdullah, S. aureus was reported as the most sensitive species to polyherbal toothpaste with an inhibition zone of 10-15 mm, followed by E. coli (9-12 mm), Bacillus cereus (7-12 mm), and Pseudomonas aeruginosa (9-11 mm) [22]. In another study, the polyherbal dentifrices containing Neem, Pudina, Long, Babul, Turmeric, and Vajradanti showed significant antimicrobial activities against E. coli, S. aureus, S. mutans, and C. albicans [23].
In a study conducted by Sharma et al. (2014), the methanolic extract of polyherbal formulation showed the highest and lowest activities against S. mutans and C. albicans, respectively [32]. In the present study, the formulated toothpaste demonstrated a good activity against C. albicans in various concentrations (i.e., 100%, 75%, 50%, and 25%) and exhibited a strong activity against L. caseie, S. mutans, S. sanguis, and S. salivarius in 100%, 75%, 50% concentrations.
Moreover, the presence of carvacrol (2-methyl-5-[1-methylethyl]-phenol) (96.9%) as the main component of S. khuzestanica essential oils (SKEO) may be responsible for the antimicrobial properties of the present toothpaste. It was also reported that the SKEO has remarkable antibacterial effects, particularly against the resistant S. aureus [33]. In another study, S. khuzestanica leaf extract was reported to show a significant antibacterial activity against S. mutans in the range of 300-500 µg/ml [34].
Zomorodian et al. investigated the antimicrobial activity of seven essential oils against the common species accounting for oral infections, such as S. mutants, S. sanguis, S. salivarius, E. faecalis, S. aureus, C. albicans C. glabrata, C. dubliniensis, and C. krusei. The results of the mentioned study revealed that SKJ and A. sieberi were active against all tested oral pathogens. In addition, in the mentioned study, SKJ showed the highest antimicrobial activities, while A. sieberi exhibited the lowest antimicrobial activity [35].
The consistency between the results of the aforementioned study and those of our research can be due to the utilization two plants used in the mentioned research for the formulation of our polyherbal toothpaste. Moreover, there is evidence confirming the antimicrobial effects of the essential oil of MCL on clinically isolated oral pathogens. All isolates were sensitive to MCL at 125-1000 μg/ml by agar disk diffusion with inhibition zones of 8.1-41.25 mm in diameter. Furthermore, all S. pyogenes, S. mutans, and C. albicans strains showed sensitivity at 62.5 μg/ml.
The S. mutans, as the main etiological agent of dental caries, has been recorded as the second most sensitive pathogen to MCL (21). Previous studies have also reported that the main compounds of the essential oil of MCL are terpenoid compounds (i.e., 1,8-cineole, α-pinene, myrtenyl acetate, limonene, linalool, and αterpinolene), flavonoids and tannin that are responsible for the inhibitory effects of the MCL in the formulated toothpaste [36].
In addition, Sharafati Chaleshtori demonstrated that the essential oil of ADL has an antibacterial activity against S. aureus, Alcaligenes faecalis, Providencia rettgeri, Serratia marcescens, Shigella dysenteriae, Listeria monocytogenes, and Klebsiella oxytoca [37]. Based on these observations, the efficiency of polyherbal toothpaste can be ascribed to the presence of variant phytochemical compounds in plant extracts.
The novelty of the present study is the use of the aqueous extracts of selected plants instead of chemical solvents, such as acetone, ethanol, and methanol. Moreover, the extracts utilized in our polyherbal toothpaste had no side effects as confirmed by an in vivo study previously performed by the authors of the current study.

Conclusion
Further studies are recommended to make it as one of the commercial herbal toothpaste for the treatment of oral microbial infections.