in Dental and Maxillofacial Sciences Evaluation of the Effect of Four Herbal Extracts on Growth of Streptococcus mutans and Lactobacillus

ARTICLE INFO Article Type Original Article


Introduction:
There are about 500 microbial species in the mouth, some of which are responsible for the development of oral infectious diseases.Reduction of oral pathogenic microorganisms is very important in the healing of oral ulcers and infections. (1,2)Dental caries is the most common chronic disease in the world, and its main etiologic agents are Streptococcus mutans (S. mutans) and Lactobacilli.Symptomatic and regenerative treatments will fail if the cause of the disease is disregarded. (3)Mutans streptococci also play a role in the development of other diseases, such as angular cheilitis and parotid glandular inflammation. (4)Also, Lactobacilli are effective in the development of gingival diseases. (5)aily use of mouthwashes in combination with tooth brushing and regular flossing effectively reduces the microbial population of the mouth and ultimately prevents gingival diseases and dental caries and accelerates wound healing. (6)(8) However, this mouthwash has several side effects such as dental discoloration, dysgeusia, burning of oral mucosa, allergies, xerostomia, and adverse systemic effects when swallowed. (7,9)12) The Iranian traditional medicine is one of the old fundaments of medicine and contains valuable information on the use of herbs in the treatment of diseases.Maintenance of oral and dental health has also been addressed in the Iranian traditional medicine, and a chapter has been devoted to oral and dental diseases in the related books. (13)st previous studies have been conducted on specific pathogens that affect the cutaneous, respiratory, digestive, and urinary systems, and few studies have been done on oral mucosal pathogens. (11)Therefore, in this study, the effect of ethanolic extracts of thyme, garlic, clove, and cinnamon on the growth rate of S. mutans and Lactobacilli was assessed, in comparison with 0.2% chlorhexidine, by measuring the inhibition zone diameter at the microbiology laboratory of Shahid Beheshti University of Medical Sciences in 2017.

Materials and Methods:
The research was carried out experimentally.In this study, standard strains of S. mutans (PTCC5027) and Lactobacillus (PTCC1608) were obtained from the Iranian Research Organization for Science and Technology.To confirm the identity of microorganisms, diagnostic tests were used.The bacteria were then stored in Müller-Hinton agar culture medium at -70°C.To prepare bacterial suspensions, 0.5 McFarland concentration was used.Using a sterile swab, we inoculated some of the bacterial suspensions on Müller-Hinton agar culture medium.
The herbs were obtained and then extraction was performed by soaking the herbs.We dried thyme, clove, garlic, and cinnamon plants in the shade at room temperature (25°C) and powdered or chopped the plants using a mill.100 ml of ethanol was mixed with 10 g of each dried plant such that the whole powder was covered and soaked.After 48 to 72 hours, we filtered the solution using a Whatman no.1 filter paper.The filtered solutions were sterilized using a 0.45-μm membrane filter.These solutions were then evaporated in a vacuum evaporator under pressure reduction conditions.The dried extracts were stored at -20°C. (14)fter preparing the herbal extracts, we dissolved 100 mg of the extract in 1 cc of 2% dimethyl sulfoxide (DMSO) to obtain the stock solution.The amount of 10 μl of each extract was poured onto the prepared blank discs.We waited a few minutes so that the extract on the disc was absorbed and then placed the discs on plates containing S. mutans and Lactobacillus bacteria.A disc containing the control groups, including 0.2% chlorhexidine (Iran Daru Pharmaceutical Co., Tehran, Iran) and a blank disc, was also placed on the plates.The plates were placed in a jar for 24 hours at 37°C and then the diameter of the inhibition zone around each disc was measured in millimeters (mm) using a ruler.
In previous studies, the number of samples in each group was at least 1 to a maximum of 10 repetitions.In this research, based on the maximum, 10 replicates and according to 6 groups for each bacterium, 120 samples were examined.In this study, intervening variables such as the concentration of ethanolic extracts, amount of ethanolic extracts, contact time of herbal extracts and microorganisms, microorganism strain, and work environment temperature were matched.Data were analyzed by analysis of variance (ANOVA) and post hoc tests.

Result:
In the present study, the effect of ethanolic extracts of thyme, cinnamon, clove, and garlic on the growth rate of S. mutans and Lactobacillus microorganisms, in comparison with 0.2% chlorhexidine, was assessed by measuring the inhibition zone diameter with 10 samples per group and a total of 120 samples.The diameters of the inhibition zones according to the type of microorganisms and categorized A) S. mutans: The data showed that the largest inhibition zone diameter was observed with chlorhexidine (22±1 mm), followed by clove (17.3±1.3 mm), thyme (15.4±1 mm), garlic (13.1±0.7 mm), and finally cinnamon (12.2±0.75 mm; Figure 1).ANOVA showed that the difference in the inhibition zone diameter in the five groups is statistically significant (P<0.0001).Pairwise comparisons by post hoc tests also showed significant differences (P<0.001).The data showed that the largest inhibition zone diameter was observed with chlorhexidine (19.6±2 mm), followed by clove (16±1.2mm), thyme (14.7±0.6 mm), garlic (13.8±0.9 mm), and finally cinnamon (12.7±0.5 mm; Figure 2).ANOVA showed that the difference in the inhibition zone diameter in the five groups is statistically significant (P<0.0001).Pairwise comparisons by post hoc tests also showed significant differences (P<0.001).

Discussion:
In the present study, the effect of ethanolic extracts of clove, thyme, garlic, and cinnamon on the growth rate of S. mutans (PTCC5027) and Lactobacillus (PTCC1608) microorganisms was assessed, in comparison with 0.2% chlorhexidine, by measuring the inhibition zone around discs containing the herbal extracts; we tested 10 samples per group and a total of 120 specimens.The data showed that all four ethanolic herbal extracts were able to inhibit the growth of S. mutans and Lactobacillus.For both bacteria, the largest inhibition zone diameter was observed with chlorhexidine, followed by clove, thyme, garlic, and finally cinnamon.ANOVA and post hoc tests also showed that there is a significant difference in the In a study by Hiregoudar et al, the antibacterial activity of ethanolic extracts of clove, turmeric, and ginger was evaluated using zone of inhibition (Z.O.I) and minimum inhibitory concentration (MIC) measurements against S. mutans under laboratory conditions. (15)Their results showed that all the extracts exhibited antimicrobial activity against this bacterium.In both methods, ethanolic extract of clove exhibited the most prominent antibacterial property, followed by turmeric and ginger (P<0.001).(15) These results were similar to those of the present study.
In a study by Mirpour et al, the antibacterial effects of ethanolic and methanolic extracts of clove and gallnut on S. mutans and S. salivarius were assessed. (16)The antimicrobial activity of the extracts was evaluated by measuring the diameter of the Z.O.I and by measuring MIC and minimum bactericidal concentration (MBC).In the Z.O.I method, methanolic and ethanolic extracts of clove showed more significant antibacterial effects than ethanolic and methanolic extracts of gallnut; (16) these results are similar to ours.In the MIC and MBC methods, the antibacterial activity of methanolic extract of clove was higher than that of its ethanolic extract, and the antibacterial activity of ethanolic extract of gallnut was higher than that of its methanolic extract.Methanolic extract of clove showed the best rates of MIC and MBC against both bacteria. (16)n a study by Soltan Dallal et al, the antimicrobial effect of Shirazi thyme (Zataria multiflora) essential oil on antibiotic-resistant Staphylococcus aureus (S. aureus) strains was evaluated using the MIC and MBC methods. (17)The results showed that the essential oil of Shirazi thyme has good effects on tetracycline-, erythromycin-, trimethoprim-sulfamethoxazole-, and methicillin-resistant S. aureus (MRSA). (17)ani et al assessed the inhibitory effect of aqueous garlic extract on multidrug-resistant S. mutans species using disc sensitivity and broth dilution methods.(18) Of the 92 groups of S. mutans, 28 groups (30.4%) showed multidrug resistance, that is, they were resistant to four antibiotics or more.The MIC of chlorhexidine for multidrug-resistant and drug-sensitive S. mutans was 2-16 μg/ml and 0.5-1 μg/ ml, respectively.All S. mutans groups showed sensitivity to garlic extract with a MIC of 4-32 μg/ml. (18)oleimani et al evaluated the antibacterial effect of Cinnamomum verum and Ferula gummosa essential oils on some gram-positive and gram-negative bacteria. (19)To evaluate the antimicrobial activity of the essential oils, the well-diffusion method was used and the MIC was determined on several standard bacterial strains.In both methods, cinnamon essential oil showed higher antibacterial effects than Ferula gummosa.Based on the results of agar welldiffusion method, the largest inhibition zone diameter by cinnamon was observed against Staphylococcus saprophyticus (47 mm), and the largest inhibition zone diameter by Ferula gummosa was detected against Staphylococcus epidermidis (33 mm).The MIC results showed that cinnamon essential oil has the highest inhibitory effect against Staphylococcus saprophyticus. (19)n studies by Soltan Dallal et al (17) , Fani et al (18) , and Soleimani et al (19) , the antibacterial effects of thyme, garlic, and cinnamon have been shown, which have also been confirmed extracts on Lactobacillus, which has been limitedly taken into consideration by other studies and is one of the most important cariogenic bacteria, can be mentioned.
In this study, standard strains of bacteria were used in vitro; nevertheless, these herbal extracts may show different antibacterial effects in the oral cavity due to the association of S. mutans and Lactobacillus with other microorganisms.Therefore, it is suggested that future studies examine the antimicrobial effect of these herbal extracts in the oral environment.
Research on clove suggests that its chemical compounds include eugenol and eugenol acetate, flavonoids, saponins, tannins, triterpenoids, and steroids such as phytosterol.This plant has antinociceptive, antifungal, and antibacterial properties; eugenol is its effective ingredient.Aqueous and ethanolic extracts of clove have antimicrobial effects against S. mutans and S. salivarius and antifungal effects against C. albicans. (14,20)hyme also has antibacterial properties due to its phenolic compounds.These phenolic substances include carvacrol, thymol, and eugenol.Thymol, which has a fragrant smell and spicy taste, is an antibacterial and antifungal agent.The analgesic, anti-inflammatory, antibacterial, and antifungal properties of these two herbs have been of practical value. (21)

Conclusion:
Ethanolic extracts of clove, thyme, garlic, and cinnamon showed antibacterial effects against standard strains of S. mutans (PTCC5027) and Lactobacillus (PTCC1608).Ethanolic extract of clove has more prominent antibacterial effects than ethanolic extracts of thyme, garlic, and cinnamon.

Figure 1 :
Figure 1:Inhibition zone diameter (mm) of Streptococcus mutans (S. mutans) divided by the studied groups

Figure 2 :
Figure 2: Inhibition zone diameter (mm) of Lactobacillus divided by the studied groups diameter of the inhibition zone formed by these extracts (P<0.001).In a study by Hiregoudar et al, the antibacterial activity of ethanolic extracts of clove, turmeric, and ginger was evaluated using zone of inhibition (Z.O.I) and minimum inhibitory concentration (MIC) measurements against S. mutans under laboratory conditions.(15)Their results showed that all the extracts exhibited antimicrobial activity against this bacterium.In both methods, ethanolic extract of clove exhibited the most prominent antibacterial property, followed by turmeric and ginger (P<0.001).(15) These results were similar to those of the present study.In a study by Mirpour et al, the antibacterial effects of ethanolic and methanolic extracts of clove and gallnut on S. mutans and S. salivarius were assessed.(16)The antimicrobial activity of the extracts was evaluated by measuring the diameter of the Z.O.I and by measuring MIC and minimum bactericidal concentration (MBC).In the Z.O.I method, methanolic and ethanolic extracts of clove showed more significant antibacterial effects than ethanolic and methanolic extracts of gallnut;(16)  these results are similar to ours.In the MIC and MBC methods, the antibacterial activity of methanolic extract of clove was higher than that of its ethanolic extract, and the antibacterial activity of ethanolic extract of