Oral and dental health is an essential and nonintegrated part of general health ¹ and plays an important role in the prevention of chronic diseases such as diabetes as one of the public health hazards. ² Tooth decay, gingivitis, and periodontitis are the most common oral and dental health diseases which can be seen frequently in different age groups and in different parts of the world. ³ In the development of these diseases, various microorganisms, such as Streptococcus mutans(SM), have a significant effect. ⁴ This microorganism plays a key role in carbohydrate fermentation resulting in lactic acid production which leads to demineralization of enamel. This bacterium results in the enhancement of cariogenicity and easy absorption of carbohydrates by production of extracellular polysaccharide that is one of the components of dental plaque. ⁵ , ⁶ SMhas been identified as a microorganism that initiates dental caries due to its adhesion to oral and dental tissues. ⁷ Since plaque-associted diseases tend to be localized, the use of local antimicrobial agents may be more effective than systemic use. ⁸ Brushing with toothpaste is the most common form of dental disease prevention in many countries around the world. ⁹ Today, in toothpastes, a wide range of chemical agents such as fluoride, which are mainly antimicrobial agents, have been added to create a direct inhibitory effect on plaque formation. ⁹ , ¹⁰ , ¹¹ Several clinical studies have shown the inhibitory effect of antimicrobial toothpastes on oral and dental bacteria, ⁹ , ¹¹ , ¹² as well as reduction of toothbrush's microbial contamination. ¹¹ When these agents are added to toothpastes, they eliminate microorganisms by destroying the cell wall and disrupting enzymatic activity. They also prevent bacterial accumulation and reduce the proliferation of microorganisms and inhibit their endotoxin release. ¹³ , ¹⁴ However, in the recent years, the use of herbal products against chemical products in toothpastes has become more popular among people due to fewer side effects and greater impact. ¹⁵ Calendula flowers and salvia are among the medicinal herbs that have been used in ancient medicine in Iran and the rest of the world and their anti-inflammatory and antimicrobial properties have been proven. ¹⁶ , ¹⁷ Calendula extract contains flavonoids, polyphenols, coumarins, essential oils, and fatty acid, which has wound healing, anti-microbial, and anti-inflammatory properties. ¹⁸ , ¹⁹ , ²⁰ Salvia extract has spasmolytic, antiviral, antifungal, antibacterial, and antioxidant properties; its antimicrobial properties are due to the presence of its hydrocarbon, monoterpene, and sesquiterpene. ²¹ , ²² In this study, the antimicrobial effect of one Iranian herbal toothpaste that contains Calendula and sage clay extracts in comparison with its chemical type against SM, Lactobacillus (LB), and Candida albicans (CA) was studied in vitro.

In this experimental study, the effect of two herbal and chemical toothpastes on three microorganisms of LB, SM, and CA was investigated. For better comparison and reduction of possible errors, a non-Iranian toothpaste (Crest cavity protection) was considered as a positive control and sterile distilled water as a negative control. To provide various concentrations of herbal and chemical toothpastes, three tubes were purchased from three different stores in Tehran. However, non-Iranian toothpaste was one type provided from a store. Then, from each of the toothpastes, three different concentrations of full, 1:1, and 1:3 were prepared. In order to obtain full concentration, after calibration of the scale (Sartorius, Germany) with a precision of 0.0001, 150 mg of toothpaste from each tube was transferred to a 6-cm plate and mixed with vortex mixer (Techne, South Korea) for 2 min. To prepare 1:1 concentration, 150 mg of each toothpaste with 150 ml deionized sterilized distilled water and, for 1:3 concentration, 150 mg of each toothpaste with 450 ml deionized sterilized distilled water, in a 6-cm plate, were mixed by vortex mixer for 2 min.

To incubate SM (ATCC35668), LB (ATCC39392), and CA (ATCC10231) microorganisms, brain–heart infusion broth, De Man, Rogosa and Sharpe agar (MRS). All microorganisms were incubated at 37°C ± 0.5°C for 24 h. After incubation, the concentrations of 1.5 × 10 ⁸colony-forming unit/ml (equivalent to the half-McFarland standard) were provided from broths using a spectrophotometer.

To determine the antimicrobial effect of the samples, a disc diffusion (agar) method was used. ²³ Sterilized swabs were used for transferring LB, to MRS agar, SM, to the brain–heart infusion agar, and CA to the Sabouraud dextrose agar medium. The microorganisms were cultivated in their special medium near a flame. Then, in all media, the plates were divided into four equal parts, and the paper discs were stacked at the same distance. Each plate contained five discs, three discs containing 150 mg of three different concentrations of a toothpaste, a disc containing 150 ml of deionized distilled water (as a negative control), and an empty disc. Similarly, 21 plates were prepared for each microorganism. In order to grow lactobacilli and SM, the plates were incubated at 37°C ± 0.5°C for 24 h and CA plates were incubated at 37°C ± 0.5°C for 48 h. After 24 h, the LB and SM plates and, after 48 h, CA plates were removed from the jar, and the diameter of the inhibition zone (if present) around the discs was measured by a ruler with a precision of 1 ml by a microbiologist. Results were reported as average. Data were analyzed by SPSS software version 16, two-way ANOVA, one-way ANOVA tests, and regarding the difference variances, Tamhane supplementary tests were used at the significance level of P < 0.05.

The mean and standard deviation of zone of inhibition for the microorganisms studied for the Iranian herbal and chemical toothpastes and positive control are summarized in Table 1. According to the results of this study, Iranian chemical and herbal toothpastes at full concentration had the highest mean diameter of inhibition zone for LB and CA microorganisms, while for SM, the highest mean diameter of the inhibition zone for herbal toothpaste was at full concentration and for chemical toothpaste it was at a concentration of 1:3.{Table 1}

Based on the results, the antimicrobial effect of full-concentration herbal toothpaste on all microorganisms was significantly higher than the concentrations of 1:1 and 1:3 (P < 0.05). However, in the positive control toothpaste, antimicrobial effect of all the three concentrations was the same in all the three microorganisms. The antimicrobial effects of all the three concentrations of Iranian chemical toothpaste on SM and CA were similar, but the effect of full-concentration chemical toothpaste on the LB was higher than that of the other concentrations Table 2. As shown in Table 3, at full concentration, the herbal and chemical toothpastes have the same antimicrobial effect on SM, LB, and CA microorganisms (P < 0.05), while in 1:1 concentration, the chemical toothpaste in comparison with the herbal toothpaste against SM (P < 0.028) and CA (P < 0.001) microorganisms has higher antimicrobial effect. Chemical toothpaste also has higher antimicrobial effect at 1:3 concentrations on SM and CA microorganisms compared with the herbal toothpaste. Based on the results, at full concentration, the antimicrobial effect of the herbal toothpaste was higher than positive control on SM (P < 0.005), LB (P < 0.005), and CA (P < 0.005) microorganisms and, at 1:3 concentration, the antimicrobial effect of positive control toothpaste was higher than that of the herbal one on SM and CA microorganisms. Herbal and positive control toothpastes at the concentration of 1:1 have the same antimicrobial effect on SM and LB microorganisms. The findings also indicate that the antimicrobial effect of the full concentration of chemical toothpaste on SM (P < 0.027), LB (P < 0.001), and CA (P < 0.012) microorganisms is higher than the positive control. At 1:1 and 1:3 concentrations, the antimicrobial effect of the chemical toothpaste was significantly higher than the positive control on SM microorganisms (P < 0.05) Table 3.{Table 2}{Table 3}

In the present study, the antimicrobial effect of Iranian herbal, chemical, and Crest cavity protection toothpastes was compared. The results showed different responses at different concentrations against SM, LB, and CA microorganisms. Based on the results of this study, at different concentrations of herbal toothpaste, the full concentration has more antimicrobial effect on three microorganisms at concentrations of 1:1 and 1:3 (P < 0.05). Iranian chemical toothpaste showed the same antimicrobial effect at all concentrations against SM and CA microorganisms, while for LB, its full concentration was more effective. Positive control toothpaste also showed the same antimicrobial effect on all the three microorganisms at all the three concentrations (P > 0.05). According to the results of this study, at full concentration, the antimicrobial effect of the herbal and chemical Iranian toothpastes is similar on SM, LB, and CA microorganisms, but by decreasing the concentration, their antimicrobial effect decreases as well. Similar to this finding, Prasanth has reported that reducing toothpaste concentrations may reduce the antimicrobial effects of toothpastes on CA and SM. ²⁴ In addition, in the study conducted by Sadeghi and Assar, the antimicrobial effect of toothpastes decreased significantly in fluoride-containing toothpastes while the concentration decreased. ²⁵ However, in the study by Shaheen et al., the reduction in toothpaste concentration did not affect the antimicrobial properties of toothpastes. ²⁶ The findings of this study showed that at full concentration, the antimicrobial effect of herbal and chemical toothpastes on SM, LB, and CA microorganisms is similar. While Gibraiel et al. showed that natural agents-containing toothpastes have higher antimicrobial effect than triclosan chemical toothpastes against E. coli and CA, ³ this may be due to the herbs used. Furthermore, in a study by Bhat et al. on CA and S. sanguis microorganisms, it has been reported that herbal toothpastes have antimicrobial activity only against SM microorganisms. ²⁷ Teh JY et al. also reported that there is no significant difference in antimicrobial effect between herbal mouthwashes on SM, Streptococcus sobrinus, Lactobacillus salivarius, Staphylococcus aureus, CA, and Pseudomonas aeruginosa (P < 0.05). ²⁸ In this study, agar diffusion method (disc) has been used because it is the most common method to study the antimicrobial properties of oral and dental products before in vivo studies. In studies by Leyster, ²⁹ Sadeghi and Assar, ²⁵ Bhat et al., ²⁷ de Camargo Smolarek et al., ³⁰ and Shaheen et al., ²⁶ the same method was used to study the antimicrobial properties of toothpastes and mouthwashes.In vivo studies, oral and dental products are diluted by saliva, which may dilute their antimicrobial agents so that the concentrations of 1:1 and 1:3 in this study are similar to the oral state, in which toothpaste is diluted by saliva. Several studies have also used different concentrations of toothpastes. ²⁵ , ²⁶ In this study, by reducing the concentration of the Iranian herbal toothpaste, its antimicrobial effects are reduced as well, while in Crest toothpaste; this is not the case, which is probably due to the specific formulation of Crest toothpaste In such a way, the minimum inhibitory concentration and minimum bactericidal concentration are lower than the concentrations studied in this experiment, so it might be better to make changes in the formulation of the herbal toothpaste, which, despite its dilution, keeps its antibacterial properties. Different results from different studies indicate the effectiveness of herbal toothpastes in preventing dental decay. ²⁶ , ²⁷ In the same field, Shaheen et al., after studying the effect of antimicrobial activity of 12 herbal toothpastes on CA, SM, Streptococcus sanguinis, Actinomyces viscosus, Streptococcus pyogenes, and S. aureus microorganisms, suggested that herbal toothpastes can be effective in improving oral and dental health, similar to other toothpastes. ²⁶

Limitations

One of the limitations of this study is its implementation in laboratory conditions. Therefore, the results cannot be generalized to clinical conditions (in vivo).

According to the results of this study, all the three herbal, chemical, and Crest cavity protection toothpastes have antimicrobial activity against all the three SM, LB, and CA microorganisms. In addition, the findings show that at full concentration herbal toothpastes have the most antimicrobial effect, while decreasing the concentration decreases its antimicrobial effect when compared with chemical toothpastes. This study shows that the mere presence of these toothpastes inhibits the growth of SM, lactobacilli, and CA, which leads to the reduction of biofilm formation.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.