Dental plaque is a diverse population of isolated bacteria in a matrix with a salivary origin that plays an important role in dental caries and gingivitis. ¹ Extracted acid from carbohydrate metabolism causes reduced oral pH and will lead to demineralization of hydroxyapatite and dental caries. ² Streptococcus mutans and lactobacilli are the two most important factors in tooth decay. S. mutans is involved at the beginning of all dental caries. ³ More than half of the bacteria found in gingivitis due to dental plaque are Gram-positive pathogens. Among them, S. mutans, S. sanguis, S. mitis, S. oralis, and S. intermedius are more commonly found, ⁴ which induce dental caries, with possible following cardiovascular complications. ⁵

The use of chemical complementary methods such as oral rinses combined with mechanical tooth cleaning can be effectively used in the control of supragingival dental plaque, gum problems, and improvement of oral ulcers. ⁴ , ⁶ , ⁷ Therefore, the use of mouthwash because of its anti-inflammatory and antiplaque properties and the prevention of the formation or spread of microbial plaque is highly recommended. ⁸ , ⁹ Desirable oral rinses should have broad-spectrum antimicrobial effects, low pharmaceutic resistance, and have no effect on oral normal flora. ⁴ Chlorhexidine is a broad-spectrum antiseptic and mouthrinse that has been approved by the Food and Drug Administration (FDA) and American Dental Association (ADA). ¹⁰ , ¹¹ Due to its cationic properties, it attaches to the cell walls of bacteria and destroys them. It can have various side effects such as discoloration of teeth, taste change, burning of the oral mucosa, dry mouth, and if swallowed negative systemic effects. ¹¹ , ¹² , ¹³

In recent years, the increasing resistance of pathogenic microorganisms to various antibiotics and the high cost of obtaining new drugs has attracted the attention of many researchers around the world to the use of medicinal plants. ¹⁴ , ¹⁵ These include plants such as Aloe vera, ¹⁶ Glycyrrhiza glabra, ¹⁷ Matricaria chamomilla, ¹⁸ Mellisa officinalis, ⁵ and Satureja khuzestania. ¹ Zataria multiflora Boiss is one of the Lamiaceae family species which has been considered the most important medicinal plant in Iran after Foeniculum vulgare. ¹⁹ It has attracted the attention of dentists. ¹ , ²⁰ Its essence contains a high concentration (64%–70%) of the two substances thymol and carvacrol. ¹ , ²¹ The strong anti-bacterial properties of this plant are due to these two phenolic isomers. The effect of Z. multiflora essence on S. mutans, S. sanguinis ²² and the effect of methanolic extract of this plant on S. mutans ²³ , ²⁴ and also the effects of its alkaline extracts on Staphylococcus aureus and Staphylococcus epidermidis ²⁵ , ²⁶ have been studied previously.

According to Jafari et al., the in vitro antibacterial effect of commercially available Z. multiflora extract (Barig Essence Company) against S. mutans colonized on orthodontic elastic rings was compared with chlorhexidine. ²⁰ In another in vitro study, the previous commercial extract was compared with sodium hypochlorite, hydrated calcium hydroxide, and normal saline as a canal irrigating solution against some streptococci in 1, 5, and 15 min. ²² Based on our search, there are limited studies on other Z. multiflora extracts such as the determination of minimal inhibitory and minimal bactericidal concentrations for other aqueous and alcoholic extracts with different time range on other oral streptococci.

Hence, in this in vitro study, the antibacterial effects of aqueous and alcoholic extracts of Z. multiflora on S. mutans and Streptococcus sanguis compared with chlorhexidine mouthwash are reported.

This is an in vitro research aiming assessment of the antibacterial effects of aqueous and alcoholic extracts of Z. multiflora on S. mutans and S. sanguis compared with chlorhexidine mouthwash which was ethically approved by Hormozgan University of Medical Sciences (Approval ID: IR.HUMS.REC.1400.021).

Materials and bacteria strains

S. mutans PTCC 1683 and S. sanguis PTCC 1449 were purchased as lyophilized from the local center of the Iranian Research Organization for Science and Technology. Chlorhexidine was also prepared in a 0.2% (2 mg/ml) solution purchased from Nazho Pharmaceutical Company (Tehran, Iran).

Extracts preparation

Alcoholic extract preparation

Z. multiflora alcoholic extract was purchased from Soha-Jissa company (Salmanshahr, Mazandaran, Iran) with a primary concentration of 20 mg/ml (Batch No. IEE059.01).

Aqueous extract preparation

To prepare the aqueous extract of Z. multiflora, 30 g of the dried leaves of the plant were soaked, cleaned, and then pulverized by a mill. ²⁷ The dried powder of the leaves was poured into 300 ml of sterile deionized distilled water (ratio 1:10) and stirred for 24 h in a dark chamber at room temperature on a shaker at 100 rpm. The extract was then filtered with Whatman No. 1 filter paper (made in England). The filtered extract was placed in an oven at 40°C for 24 h until complete evaporation of the solvent. The dried extract was collected from the glass surface and stored in a sterile, dark glass container at 4°C for later use. ²⁸

Disk diffusion assay

The prepared bacteria were cultured on blood agar for 24 h at 37°C to achieve the most suitable cultivation. ²⁹ Then samples were taken from the cultured bacteria with a sterile swab, added to 10 ml of physiological saline, and mixed well to obtain a uniform bacterial suspension. This bacterial suspension was set into the standard density of 0.5 McFarland turbidity with a spectrophotometer (2100 Unico, China) at a wavelength of 625 nm resulting in (1.5 × 10 ⁸CFU/ml) bacterial concertation. ³⁰ , ³¹ Then, comparison of growth inhibition zone with disk diffusion method was performed in triplicate by measuring the diameter of inhibition zone in primary concentrations of extracts with an antibiogram ruler after 48 h of incubation in the incubator at 37°C and comparison with Chlorhexidine 0.2% (as the positive control). ³²

Minimal inhibitory concentration and minimal bactericidal concentration

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined by the serial dilution method in triplicate. At this stage, 200 μL of sterile physiological serum was added to 10 sterile microtubes, then 200 microliters of extracts were added to the first microtube. After mixing the extract with physiological serum, 200 μl of this microtube was removed and added to microtube number 2. This operation was continued until the last one when 200 μl of solution was discarded from it. ³³ Therefore, a serial dilution was created from microtube one to microtube ten. Then, 200 μl of the bacterial suspension previously prepared at a turbidity of 0.5 McFarland was added into each microtube. 100 μl was taken from each sample and used for surface culture on Mueller–Hinton agar in a 37°C incubator for 24 h. ³⁴ Chlorhexidine 0.2% was used as the positive control and other dilutions were used as the tests.

The lowest concentrations of the extract with visible colonies and no visible colonies on culture media were defined as MIC and MBC, respectively. The number of colonies was also counted for each sample, and its means were reported. We also note that these tests were performed in triplicate ³⁵ .

Statistical analysis

A one-way analysis of variance (ANOVA) and comparison of the data with Duncan's multiple domain test were performed using SPSS software (version 26.0; SPSS Inc., Chicago, IL, USA). An independent t-test was used to compare the antibacterial effects of each extract on two bacteria. The level of significance of the results in all cases was considered to be 5% (P < 0.05).

Primary PH determination

Results of primary PH determination of Chlorhexidine, alcoholic extract and aqueous extract are shown in Table 1.{Table 1}

Results of disk diffusion assay

After performing the disk diffusion test and ensuring that it has antibacterial properties and comparing the diameter of the growth inhibition zone, it was found that the bacterial growth inhibitory properties for chlorhexidine mouthwash are higher than alcoholic extract and for alcoholic extract are higher than aqueous extract as shown in Table 2.{Table 2}

Minimal inhibitory concentration and minimal bactericidal concentration

The results of the bacterial colony count of S. mutans and S. sanguis culture on Mueller–Hinton agar culture after the effect of compounds are shown in Table 3.{Table 3}

No identical Latin lowercase letters in each column indicate a significant difference in each sample and non-identical Latin uppercase letters indicate significant differences in each row (P < 0.05).

As shown in Table 3, MIC values for the effect of aqueous extract, alcoholic extract, and chlorhexidine on S. mutans were obtained in 1/4, 1/128, and 1/512 dilutions, also MBC values of these samples were evaluated as 1/2, 1/64, and 1/256.

On the other hand, the MIC values for the effects of aqueous extract and alcoholic extract on S. sanguis used in this study were obtained in dilutions of 1/2 and 1/128. This index for chlorhexidine should be seen at concentrations lower than 1/1024, which was not within the range of the samples in this study. Furthermore, the values set for MBC of these samples were evaluated as 1, 1/64, and 1/1024.

The colony count results showed that the difference in the number of colonies grown in all concentrations of the aqueous extract in S. sanguis was significantly higher than in S. mutans (P < 0.05).

About alcoholic extract, it was also found that the ability of this extract to induce growth inhibition against S. sanguis in all dilutions <1/64 is significantly lower than S. mutans (P < 0.05) Table 3.

Comparison of the means of bacterial colony counts counted under different dilutions of chlorhexidine mouthwash showed that this substance had the ability to kill S. sanguis in all dilutions, but this effect against S. mutans was observed up to a dilution of 1/256 (P > 0.05) and lesser dilutions were deficient. Furthermore, in both dilutions of 1/512 and 1/1024, the ability of this substance to kill S. sanguis was significantly higher than that of S. mutans (P < 0.05).

In the present study, the effects of different concentrations of aqueous and alcoholic extracts of Z. multiflora leaves on the growth of S. mutans and S. sanguis were studied, and the culture results were compared with those of 0.2% chlorhexidine mouthwash. Examination of this effect by disc diffusion showed that all three compounds have the ability to limit the growth of both bacteria. The statistical comparison showed that the effect of the alcoholic extract of Z. multiflora on S. mutans and S. sanguis is significantly more effective in inhibiting the growth of both bacteria compared to the aqueous extract of this plant. But what is important is the higher ability of chlorhexidine to inhibit the growth of both bacteria compared to aqueous and alcoholic extracts. The findings of this method indicate that for all three compounds used, there is no significant difference in their effects on two different bacteria. That is, chlorhexidine, alcoholic extract, and aqueous extract have shown the same effect in inhibiting the growth of both bacteria with this method. MIC is the gold standard for measuring the resistance of a variety of bacteria to a variety of antibiotics. MBC, is also the minimum lethal concentration, the lowest concentration of an antibiotic that inhibits the growth of the target bacterium after repeated cultures in antibiotic-free media. ³⁶ The findings of MIC and MBC for applied standardization in the pharmaceutical industry are some of the most important indicators that should be considered in the research of new materials and compounds. In general, the results of the growth inhibition mentioned in this study are consistent with the results of growth inhibition in culture medium. That is, in both methods, 0.2% chlorhexidine showed the greatest inhibitory effect on both bacteria, followed by alcoholic extract and finally aqueous extract Table 3. The ability to inhibit growth in the samples used is determined under aqueous extract < alcoholic extract < chlorhexidine for both bacteria, respectively. This effect increases with increasing concentrations of alcoholic and aqueous extracts on both bacteria Table 3.

Ahmadi et al., by investigating the effect of ethanolic extract of Z. multiflora on inhibiting the growth of S. aureus and E. coli by the plate propagation method, reported a growth inhibition zone of 22 mm and 16 mm, respectively. ³³ In the present study, the index for the effect of alcoholic extract on the growth inhibition of S. mutans and S. sanguis was 35.8 and 33.2 mm, respectively. In the study by Zomorodian et al., it was reported that the MIC of Z. multiflora essential oil for both S. mutans and S. sanguis was 1/4. ¹ In the present study, this index was the same for S. mutans in the aqueous extract, but the alkaline extract used in this study showed a MIC of 1/128 for both bacteria. This indicates that the alcoholic extract has more antibiotic ability than the essential oil used in the study by Zomorodian et al.

The higher efficiency of alcoholic extract than aqueous extract should be found in the nature of extraction of phenolic materials with these two solvents. The presence of large amounts of thymol and carvacrol in Shirazi thyme and other thyme species has been confirmed. ³⁷ Various studies on the antibacterial properties of thymol and carvacrol have been shown to be due to their ability to alter the structure of bacterial cell membranes. According to the findings, the ability of these materials to bind to the fat membrane of the cell wall increases the curvature of this membrane. The hydrophilic portion of these molecules attaches to the polar part of the membrane, while the hydrophobic part of the benzene ring of these molecules sinks into the inner part of the membrane. This causes instability of the fat layers and changes in the structure of the membrane, leading to a decrease in elasticity and an increase in membrane fluidity. This process increases the permeability of potassium and hydrogen ions. ²² These compounds also lower the pH by passing through the membrane and thus act as proton exchangers. These compounds, which contain a hydroxyl radical, are released from the membrane into the cytoplasm, where they release their protons. It then returns to the cell membrane to remove a potassium ion from the cytoplasm. This cation is released and thymol or carvacrol can trap another proton again and this cycle will be repeated. This process is associated with the depletion of cellular ATP stores and causes the loss of large amounts of cellular energy and ultimately the death of bacteria. ²⁴ The entry of these compounds into the bacterial cell also affects the activity of inner membrane proteins such as enzymes and receptors.

Thymol and carvacrol, by binding to membrane proteins, cause their deformation and consequently, their inefficiency. Therefore, the two factors of changing the cell membrane elasticity and changing the function of membrane proteins, and membrane depolarization ³⁶ are the main factors affecting the effects of these molecules on bacterial cells. The unique nature of thymol and carvacrol, i.e., the hydrophobic property of the benzene ring together with the hydrophilic property of the hydroxyl agent (OH), causes the mentioned processes and creates a special ability for them to destroy different bacteria. ³⁶

The solubility of thymol and its isomer (carvacrol) in alcoholic solvents is much higher than in water. ³⁸ , ³⁹ The solubility of thymol in ethanol is reported to be 90%, ³⁹ and the solubility of this substance in water is reported to be 0.1%. ³⁹ On the other hand, the study by Ultee et al. showed that the solubility of carvacrol in octanol is 10,000 times higher than water. ⁴⁰ This difference in the extraction of thymol and carvacrol, which are the most important antibacterial substances of the extracts studied in this study, could be the main reason for the greater ability of the alcoholic extract to inhibit the growth of both S. mutans and S. sanguis. The study by Chen et al. showed that by increasing the concentration of alcohol from 2% to 5%, the solubility of thymol increased from 0.52 to 0.62 mg/ml and the solubility of carvacrol at the same concentrations. It also increased from 0.46 to 0.57 mg/ml. A similar increase has been observed in other compounds such as eugenol and trans-cinnamaldehyde. ⁴¹ Hydroalcoholic extraction of thymol from Z. multiflora with different percentages of alcohol (26%, 37%, and 72%) also showed that the amount of thymol extracted was equal to 2.7, 3.7, and 6 mg/g, respectively. That is, the content of extracted thymol depends entirely on the percentage of ethanol used, and with increasing alcohol concentration, more thymol is extracted. ⁴² The concentration-dependent effect of the alcoholic extract of Z. multiflora on the growth inhibition of methicillin-resistant S. aureus has also been confirmed by Yadegar et al. ²⁶ The effect of Z. multiflora essential oil on cultured samples of Staphylococcus aureus, Bacillus subtilis, Proteus mirabilis, Klebsiella pneumoniae, Escherichia coli, Candida albicans, and Aspergillus niger, showed that in all cases, with increasing essential oil concentration, halo diameter growth has also increased. ⁴³

A similar effect has been previously reported by Faraji et al. (2018) in increasing the growth inhibitory property of zinc and oxycinose by increasing the annual concentration of methane (methanolic and ethanolic) in melissa (Melissa officinalis). ⁵ The extraction percentage of each of these materials is very diverse according to the efficiency of the extraction method ⁴⁴ and, as a result, will be very effective in the subsequent use of these extracts.

On the other hand, Haghighati et al. on the effect of extracts of 10 medicinal plants on growth inhibition of Candida albicans, S. mutans, and Actinobacillus showed that discs containing alcoholic extract of Z. multiflora were able to create a growth inhibition zone of 11.6 mm. ²³ This barrier diameter is smaller than the findings of the present study (35.8 mm). This discrepancy can most likely be related to the discrepancy between the materials used in these two studies. The alcoholic solvent used in the research of Haghighat et al. was methanol alcohol, while the solvent used in this research was ethanol. The group also noted that by increasing the purity of methanol from 80% to 100%, the inhibitory capacity of Z. multiflora extract on all three pathogens increases.

Thymol of three species of thyme (Thymus vulgaris, Thymus zygis, and Thymus citriodorus) was extracted using 3 different solvents (ethanol, limolin, and ethyl lactate) and it was found that the ability to extract thymol in the ethanol solvent was significant (1/1). Higher by 12% than limolin solvent (9.4%) and ethyl lactate (9.2%). ⁴⁴ Furthermore, Gas Chromatography Mass Spectrometry (GC/MS) studies have shown great diversity in 32 different compounds in the extracts of these three species, which can have a different effect on the performance of each of the same pathogens. ⁴⁵

In different studies, significant differences in the ability to inhibit the growth of different essential oils and alcoholic extracts (ethanolic or methanolic) compared to the aqueous extract of the same plant on different pathogens have been reported in different medicinal plants. Goudarzi et al., by examining the effects of aqueous and alcoholic (ethanolic) extracts of Z. multiflora on hemorrhagic E. coli by the well-drying method, stated that the MIC of alcoholic extract for this bacterium was 1/64 in dilution and aqueous extract, even with zero dilution, had no effect on the lack of growth of this bacterium. ⁴⁶ This group showed that with increasing the concentration of the extract in the well, the diameter of the growth inhibition zone of this extract also increases. In another study, Kamkar found that ethanolic dill extract has more antioxidant capacity than its essential oil. ⁴⁷ A comparison of the properties of different essential oils and extracts of ethanol, acetone, and aqueous on the inhibitory effect of the growth of 50 medicinal plants on the fungus Candida albicans showed that the effect of essential oils of Thymus kotschyanus and Z. multiflora is more effective than ethanolic and acetoic extracts of these two plants. ⁴⁷ A noteworthy point in this study was the three-fold effect of Shirazi thyme ethanolic extract compared to mountain thyme. The effect of solvent on the extraction of plant compounds has already been reported in the case of phenolic substances extracted from potato peel. ⁴⁸ By extracting the extract with five different solvents, including water, methanol, ethanol, hexane, and acetone, this group showed that the highest amount of phenolic substances is present in methanolic extract. By preparing fenugreek extract by two methods of extraction, with pure methanol and with a methanol/water mixture (ratio of 1: 1), it was shown that the extract prepared with pure methanol has more phenolic compounds and its antioxidation effect is also higher. ⁴⁹ A comparison of the effects of aqueous and alcoholic extracts prepared from turbid (Daphne oleoides) plants also showed that the ability to inhibit the growth of alcoholic extract of this plant (with a diameter of no growth equal to 20.55 mm) on S. mutans is greater than that of aqueous extract. ⁵⁰

Saoudi et al. investigated that Thymus capitatus essence has more antiacanthamoeba (Acanthamoeba) effects than its alcoholic extract. ⁵¹ This finding is not consistent with the results of the present study, and the reason is the difference in the studied pathogens. Other studies on the effects of some medicinal plants on various pathogens have reported a greater effect of aqueous extracts than alcoholic extracts. As mentioned, the variety of compounds present in the organs of different plants and the species of the pathogen under study make the mode of unique action of each compound against a particular pathogen. ⁵¹

Due to the presence of different compounds in Z. multiflora, especially thymol and carvacrol, the inefficiency of the aqueous extract used in this study compared to the alcoholic extract can be attributed to the difference in the solubility of these two substances in ethanol compared to water.

The ability of chlorhexidine to inhibit the growth of pathogens in oral diseases has been confirmed in many studies. ¹³ , ²⁰ , ²³ However, it has been reported that it also has a great ability to destroy the natural flora of the mouth. ⁴ According to the obtained findings, it can be stated that the ability of chlorhexidine to inhibit the growth of S. mutans and S. sanguis is significantly higher than alcoholic and aqueous extracts. It was proved by both the plate propagation method and the culture method and by counting colonies in the culture medium. The aqueous and alcoholic extracts used in this study had a greater inhibitory effect on S. mutans than S. sanguis. The interpretation of this issue should be related to the characteristic of more acid production by S. mutans than by S. sanguis. ⁶ Under the conditions of multiplication of these two bacteria, the environment is made more acidic by S. mutans. A comparison of the antibacterial effects of thyme extract at different pH showed that its effect at pH 5.5 is greater than pH 6.5. This property is due to the interaction of the cytoplasmic membrane, aqueous medium, and phenolic content of the extract. ⁵²

It should be noted that the use of plant extracts in prophylaxis should not necessarily be due to their bactericidal properties, but rather to the ability of the substance used to prevent the growth of the desired bacteria, which can be used in pre-medicine. Such effects may include changes in the pH of the bacterial cytoplasm, increased permeability of the bacterial membrane to ions and metabolites, inhibition of intracellular or extracellular enzymes in bacteria, and destruction of bacterial metabolic pathways. They can reduce the uptake of other bacteria onto the biofilm, destroy plaque, and prevent the biofilm from spreading to the teeth. ³⁷ Furthermore, nontherapeutic approaches such as eliminating the burning sensation in the mouth and unpleasant odor previously used in the use of peppermint mouthwash to reduce the unpleasant effects of chlorhexidine ⁵² as well as reducing the indicators of gingivitis and plaque, using mouthwash made from the essential oils of three plants (balsam herb, peppermint, and thyme) ⁵³ is another use of plant extracts.

In different compounds tested in thyme extracts, high levels of other compounds such as p-Cymene, a precursor to carvacrol, have also been reported. ⁵¹ , ⁵⁴ , ⁵⁵ , ⁵⁶ These compounds are hydrophobic substances that cause water retention and swelling of the bacterial membrane. It also changes the structure of membranes due to its lipophilic properties and increases their permeability to thymol and carvacrol. ⁵⁶ This is why the use of essential oils or extracts of any of the medicinal plants shows their antibacterial effects in much higher amounts than when each of their constituents is separately. ¹

In this study, it was found that aqueous and alcoholic extracts of Z. multiflora have antibacterial properties against two bacteria, S. mutans and S. sanguis. Therefore, we suggest conducting other controlled studies in in vivo conditions as a mouthwash for investigating streptococcal bacteria reduction.

Acknowledgments

We would like to thank the staff of this university. We also sincerely thank Kaveh Madhoush, director of ViroMed medical laboratory, and his crews (Rasht), for their kind help on bacterial purchasing and cultures.

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.