Gingivitis associated with dental plaque is the most common form of gingival disease that leads to destruction of periodontal structure or periodontitis if it is not treated. ¹ Formation of dental plaque starts with accumulation of Gram-positive Streptococcus and develops with Gram-negative anaerobic bacterial colonies. ² Oral cavity hosts a significant number of Gram-negative and Gram-positive bacteria. ³ The endogenous bacterial species of oral cavity include Actinobacillus actinomycetemcomitans, Fusobacteria, Porphyromonas gingivalis, Streptococcus mutans, Streptococcus sobrinus, Bacteroides, and Prevotella, whose metabolites play a major role in the inhibition and development of oral cavity infections. ⁴ , ⁵

Effective prevention of oral infections is accomplished by mechanical removal of dental plaque via toothbrushing and dental flossing. ⁶ Despite the significance of mechanical control of dental plaque, gingival inflammation is highly prevalent due to inefficient implementation of these methods. Hence, chemical methods such as use of toothpaste and mouthwashes containing anti-inflammatory and anti-plaque compounds along with mechanical methods are recommended for a proper oral hygiene. ⁷ Among all available mouthwashes, CHX has a high capacity in reducing dental plaque and pathogenic microorganisms like S. mutans. ⁸ Yet, chlorhexidine (CHX) has side effects such as tooth discoloration, taste change, and mouth drying and burning. ⁹ Nowadays, scholars have turned to traditional medicine to find better drugs for the treatment of microbial infections. ¹⁰ Plant extracts may be used as antibacterial and antifungal agents against common antibiotic-resistant pathogens. ¹¹

Aloe vera is a member of Aloaceae family ¹² that is increasingly being used as an herbal drug for dental problems. ¹³ A. vera gel has inhibitory effects on a number of caries-inducing bacteria and periopathogens. ¹⁴ Anthraquinones or phenolic compounds, saponin, tannin, sterol, and organic acids are active agents in A. vera gel. ¹⁵ Flavonoids such as catechin are bioactive compounds in the leaves of green tea, the most important of which is epigallocatechin gallate. ¹⁶ The green tea catechin has bactericidal effects on black pigment bacteria and Gram-negative anaerobic rods, and a combination of mechanical methods and green tea catechin is effective in improving the periodontal condition. ¹⁷

Numerous studies on animal models have shown that green tea has anti-inflammatory, antibacterial, antidiabetic, and most importantly anticancer properties. ¹⁸ Chamomile belongs to the Asteraceae family and its pharmaceutical properties are owing to the flavonoid compounds and breakdown of volatile oils. The extracts of this plant have various pharmacological properties such as anti-itching, anti-inflammatory, antimicrobial, antiviral, and antioxidant properties. ¹⁵ The main compound of Matrika mouthwash is the extract of chamomile, which inhibits the microbial growth. ¹⁹

Several studies have been done on the herbal products. Abdelmonem et al. observed that the number of bacterial colonies of Porphyromonas gingivalis and Prevotella intermedia significantly decreased in the group receiving A. vera mouthwash following SRP treatment. ²⁰ Araghizadeh et al. showed the inhibitory effect of green tea on Actinobacillus actinomycetemcomitans, Prevotella intermedia, P. gingivalis, and S. mutans by agar disc diffusion and broth microdilution methods. ²¹ Hashemipour et al. also investigated the antimicrobial effects of CHX mouthwash (Shahr Darou Co.) and chamomile on Streptococcus sanguis, Candida albicans, Streptococcus viridans, S. mutans, and Lactobacillus casei using serial dilution and reported the higher efficacy of CHX compared to chamomile. ²²

Considering the abovementioned studies and presence of antibacterial agents in A. vera, green tea, and chamomile plant extracts, the current study was conducted to determine and compare the antimicrobial effects of chamomile (Matrika), A. vera-green tea, and CHX mouthwashes on S. sanguis, S. oralis, S. mutans, P. gingivalis, and Eikenella corrodens bacterial species.

This prospective single-blind in vitro study investigated the antibacterial effects of Matrika (Barij Essence, Kashan, Iran), A. vera-green tea (Barij Essence, Kashan, Iran), and CHX 0.2% (Vi-One, Iran) as well as distilled water as control on five bacterial species, including S. oralis, S. sanguis, and S. mutans as primary colonizers and P. gingivalis and E. corrodens as secondary colonizers at the Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, in 2016.

First, some of the suspension containing each bacterium was cultured on the specific culture medium of each species. After incubation, several colonies of each species were separately dissolved in the Trypticase Soy Broth (TSB, BD™, Germany), and a suspension with specific McFarland turbidity was prepared. ²³

In colony-forming unit, 1 cc of each mouthwash and distilled water was separately mixed with 1 cc of the McFarland bacterial suspension and was cultured in a specific culture medium after incubation. Further, the McFarland suspension of each bacterium was cultured on the specific culture medium prior to adjacency. Finally, the number of colonies was counted before and after adjacency. ²³

To analyze the zone of inhibition by disc diffusion method, the McFarland suspension of each bacterial species was cultured on the specific culture medium plate by spread plate technique. Then, the paper discs impregnated with mouthwash or distilled water (20 μL) were placed on the plate and incubated. Finally, the zone of inhibition of each disc was measured. ²³

As for the analysis of zone of inhibition by well diffusion method, the McFarland suspension of each bacterial species was cultured on the specific culture medium plate by spread plate technique. Then, the wells were created on the culture medium and 60 μL of each mouthwash or distilled water was added to the wells and incubated. Finally, the zone of inhibition of each well was measured. ²³ In all methods, anaerobic conditions were considered for P. gingivalis.

To increase the accuracy of each sample in disc diffusion and well diffusion methods, 15 tests were carried out on each bacterial species. Moreover, five tests were performed for each sample in each bacterial species in colony-forming unit. The obtained data were analyzed by SPSS (version 22, SPSS Inc., Chicago, IL, USA) using descriptive statistics and ANOVA, Tukey, Kruskal–Wallis, and Mann–Whitney tests (α = 0.05).

The results of ANOVA showed a significant difference among all the four groups in well diffusion and disc diffusion methods with respect to the mean diameter of zone of inhibition (P < 0.001). Further, the mean diameter of zone of inhibition was significantly different among different bacteria in all three mouthwashes (P < 0.001) Table 1and Table 2. Moreover, the mean diameter of zone of inhibition was equal to 0 in all bacterial species in the control group in both methods.{Table 1}{Table 2}

The findings of Kruskal–Wallis test indicated a significant difference among the four groups in the mean number of the remaining bacterial colonies (P < 0.001). Moreover, there was a significant difference among different bacteria in the number of bacterial colonies in both A. vera-green tea and chamomile (Matrika) mouthwashes (P < 0.001). However, there were no colonies in any of the bacteria in CHX mouthwash Table 3. There was also no significant difference between the mean number of the remaining colonies and the primary colonies in all bacterial species in the control group.{Table 3}

The results of this study showed that CHX (which is known as the gold standard antibacterial mouthwash) had a significantly higher effect on all five bacterial species than the other two mouthwashes. Further, each mouthwash had a different effect on various bacterial species.

Regarding the comparison of zone of inhibition by disc diffusion and well diffusion methods, the findings indicated no significant difference for the effect of CHX in both methods for each bacterial species except E. corrodens. As for herbal mouthwashes in disc diffusion method, no zone of inhibition was observed in most bacterial species and the diameter of zone of inhibition was small. Moreover, the diameter of the zone of inhibition induced by the herbal mouthwashes was higher in well diffusion method than disc diffusion method, and a greater number of bacterial species had no zone of inhibition. The amount of the mouthwash used was higher in well diffusion method than disc diffusion method, but given the similar diameters of the zone of inhibition in each bacterial species in both methods, the difference in CHX mouthwash might probably be due to the difference in the inherent diffusion properties of mouthwashes. It seems that the herbal mouthwashes have not been able to diffuse well from the disc to the culture medium after impregnation of discs in disc diffusion method. In well diffusion method, considering the absence of disc and higher amount of the mouthwash used, the zone of inhibition was formed in a greater number of bacterial species. Moreover, the diameter of the zone of inhibition was larger. Saliem investigated the antibacterial effect of the green tea extract, obtained by alcohol and water, and CHX on P. gingivalis using well diffusion and serial microdilution methods and measured the minimum inhibitory concentration and minimum bactericidal concentration. The researcher concluded that ethanolic green tea was more effective than CHX and water-based green tea. ²⁴ It seems that the interaction of mouthwash and bacteria is an important factor in evaluating the effect of herbal mouthwashes on bacterial species. It can be concluded that the findings of colony-forming unit in which the mouthwash and bacteria blend are closer to reality.

The present study showed that CHX mouthwash had the lowest effect on S. mutans in both disc diffusion and well diffusion methods, while its effect was equal on all bacterial species in colony-forming unit, and the number of colonies after adjacency with the mouthwash was equal to 0. Research has shown that CHX has a high capacity in reducing the dental plaque and pathogenic microorganisms like S. mutans among all mouthwashes available, ⁸ which is in line with the results of the present study with respect to the colony-forming unit.

In this study, the effect of A. vera-green tea mouthwash on S. mutans, P. gingivalis, and E. corrodens was higher than that of Matrika mouthwash in all three methods. However, its effect on S. sanguis was lower than that of Matrika in all three methods, but it showed a significant difference compared to the control group (distilled water). Further, the effect of this mouthwash on Streptococcus oralis was higher than that of Matrika mouthwash in colony-forming unit and well diffusion method.

Sakanaka et al. showed that green tea catechin had inhibitory effects on S. mutans. However, the efficacy of green tea and CHX was not compared in this study. ²⁵ Araghizadeh et al. reported a higher inhibitory effect of green tea on S. mutans than periopathogenic bacteria. It was confirmed that the sensitivity of Gram-positive bacteria might be due to the difference in the cell wall and bacterial polysaccharide charge. ²¹

Hirasawa et al. indicated that green tea catechin had bactericidal effects on the black pigment bacteria and Gram-negative anaerobic rods, and a combination of mechanical methods and green tea catechin improved the periodontal conditions. ¹⁷ In the current study, A. vera-green tea had the highest effect on P. gingivalis and the greatest diameter of zone of inhibition was 17 mm. The results of the present study also showed that the effect of A. vera-green tea on S. sanguis was much lower than those of other bacterial species in colony-firming unit. Since this bacterium is similar to S. mutans and Gram-positive S. oralis cocci, this difference may be due to the intrinsic properties of each species.

Using microdilution and disc diffusion methods, Fani and Kohantalab found that A. vera gel had inhibitory effects on some caries-inducing and periopathogenic bacteria. They concluded that a proper concentration of A. vera gel could be used in toothpaste or mouthwash to prevent caries and periodontal diseases. ¹⁴ The findings of Lee et al. showed that A. vera toothpaste had inhibitory effects on the growth of different oral microorganisms such as S. mutans and S. sanguis, ²⁶ which is in agreement with the results of the present study. Bertolini et al. also found no significant difference between the antimicrobial effects of A. vera-beeswax and CHX gluconate 0.12% mouthwashes on S. mutans in colony-forming unit. ²⁷ However, our study showed that CHX had a significantly higher effect on the studied bacterial species than A. vera-green tea mouthwash in all methods.

Sadeghi reported a significantly higher effect of CHX on S. mutans, S. sanguis, and E. corrodens than chamomile mouthwash using disc diffusion method. ¹⁹ The findings of the study by Salehi et al. on orthodontic patients indicated a significant reduction of microorganisms during the use of Matrika and Persica mouthwashes. ²⁸

In the present study, CHX mouthwash exhibited a significantly higher effect than Matrika on all five bacterial species in all methods. Moreover, Matrika mouthwash showed a significant effect on S. mutans, S. sanguis, and P. gingivalis in colony-forming unit but had no effect on E. corrodens and S. oralis. In addition, Matrika had the highest effect on S. sanguis. Since this bacterium is similar to S. mutans and Gram-positive S. oralis cocci, this difference might be due to the inherent properties of the bacterial species.

The findings of Hashemipour et al. also showed that CHX had a higher effect than chamomile on Lactobacillus casei, C. albicans, S. sanguis, S. viridans, and S. mutans in serial dilution method, ²² which is in line with the results of the current study.

A limitation of this study was lack of access to other species like periopathogens. Moreover, the mouthwashes used in this study were made beforehand and it was not possible to change their concentration. In addition, given the different growth conditions of oral bacterial species compared to in vitro conditions, further clinical studies are suggested to explore the effect of herbal mouthwashes. Furthermore, the adjacency of plant compounds and microorganisms is suggested to be taken into account in analyzing the antimicrobial effects of plant compounds.

The findings of the present study showed that A. vera-green tea and Matrika mouthwashes had potentially antibacterial effects, but these effects were lower than that of CHX mouthwash. Within the limitations of this study, it seems that these herbal mouthwashes cannot be an appropriate substitute to CHX mouthwash. However, more clinical studies are needed to prove the present findings.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

We would like to thank all of participants who help us to conducting this study. Based on a thesis submitted to the school of Dentistry, Isfahan University of Medical Sciences, in partial fulfillment of the requirement for the DDS/MSC degree, this study was supported by Isfahan University of Medical Sciences Research (Grant No. 396279).

Financial support and sponsorship

This study has been supported by the Isfahan University of Medical Sciences.

Conflicts of interest

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