Essential oils extracted from the bark of cinnamon; leaves of sweet basil, peppermint, spearmint, kaffir lime, and sweet fennel; peel of kaffir lime; seeds of black pepper were commercially acquired from Tropicalife Company, Ltd. (Bangkok, Thailand). Brain Heart Infusion (BHI) agar and broth were purchased from BBL^TM whereas Rogosa SL and Mueller-Hinton agar were from Difco^TM Becton, Dickinson and Company (Bangkok, Thailand). Artificial saliva was purchased from Ramathibody Hospital (Bangkok, Thailand). Streptococcus mutans KPSK2 and clinical strain Lactobacillus casei were provided by the Department of Oral Microbiology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand. Chlorhexidine mouthwash (0.2% and 0.5%) was prepared by the Faculty of Dentistry, Mahidol University, Bangkok. This experimental in vitro study was carried out during June 2015 to August 2016.

All selected herbal essential oils were screened for their antimicrobial activity by agar disk diffusion method [18]. Briefly, S. mutans KPSK2 and L. casei clinical strains were freshly sub-cultured on BHI agar and incubated at 37°C with 5% CO₂ for 48 hours. Few colonies of each bacterium were collected, cultured in BHI broth and incubated under the condition mentioned for 24 hours. All culinary herb essential oils (200 μL) were primarily diluted with 10% Dimethyl Sulfoxide (DMSO) (10 mL DMSO in 90 mL BHI broth) (800 μL) to be 20% (v/v). A 20 μL of diluted herbal essential oil was added into each sterile 6 mm-diameter paper disk and air dried. The disks were placed on the Mueller Hinton agar that was previously inoculated evenly with bacterial suspension (0.5 McFarland standards, 1-2x10⁸ CFU/mL). The plates were incubated at 37°C with 5% CO₂ for 24 hours. Expression of antimicrobial activity of each herb was analysed from inhibition zone appearance, measured and recorded. The 0.2% chlorhexidine and 10% DMSO were used as positive and negative controls, respectively. The experiment was done in triplicate each with three repeats.

Each herbal essential oil was further examined for its Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) against both cariogenic bacteria by broth microdilution method with some modification [19]. In brief, each herbal essential oil was 2-fold serially diluted with 10% DMSO in Phosphate-Buffered Saline (PBS) to obtain concentrations ranging from 20% to 0.04% (v/v). The cariogenic bacteria were prepared as mentioned previously. Each well of a 96-flat bottom well polystyrene plate (substratum) was filled with 15μL of bacterial suspension in BHI (5x10⁶ CFU/mL). Then, a 135μL of each concentration of herbal essential oil (columns 1-10), 0.2% chlorhexidine in BHI (column 11) and 10% DMSO (column 12) was added and designated as tests, positive and negative controls, respectively. The plate was incubated at 37°C with 5% CO₂ for 24 hours. MIC was the lowest concentration of a certain herb required to impede the growth of cariogenic bacteria (visually clear well). Minimum bactericidal concentration (MBC), defined as the lowest concentration of herb capable of eliminating the tested organisms, was evaluated by transferring 25μL of the solution in each clear well to BHI agar and incubated in the environment described above for 24 hours. The experiment was done in triplicate each with three repeats.

The culinary herb essential oils with potent antimicrobial property, especially against the planktonic S. mutans KPSK2 were finally examined for their anti-plaque effects against S. mutans biofilm which include preventive effect on the formation of bacterial biofilm in vitro and reductive effect on the pre-established biofilm, with minor modification [20].

Preventive effect on the formation of S. mutans biofilm in vitro [20]: A 96-well plate was firstly coated with 50μL of varied concentrations of herbal essential oils ranging from MIC, 2xMIC to 4xMIC, 0.2% chlorhexidine as well as BHI with 3% sucrose (BHI-S) and was designated as tests, positive and negative (untreated substratum) controls, respectively then the plate was incubated at 37°C in 5% CO₂ incubator for different time-intervals (1, 2, 4, and 24 hours). Each well was emptied and washed twice with PBS, pH 7.2. The plate was then applied with 100 μL artificial saliva, incubated at room temperature for three hours and washed with PBS before applied with 200μL of S. mutans suspension containing 1x10⁶ CFU/mL or BHI-S (blank control). This was then incubated aerobically with 5% CO₂ at 37°C for 24 hours. The supernatant was gently decanted and washed with 200μL PBS. The bacterial biofilm was fixed with 100μL absolute methanol for 15 minute then air dried at room temperature. The biofilm was stained with 110 μL of 0.4% crystal violet solution for 30 min before rinsed thoroughly with tap water until the well of blank control appeared colorless. The biofilm mass was quantitated by the addition of 200μL of 95% ethanol, shaken for 30 minute, then 100μL of solution was transferred to a new 96-well plate and absorbance (optical density, OD) was measured at a wavelength of 596 nm [Table/Fig-1]. Preventive effect of the spice essential oils tested was interpolated to be the percentage of inhibition on the formation of bacterial biofilm by using the equation shown below. The experiment was done in triplicate each with three repeats.

[Table/Fig-1]:

Methodology to determine preventive effect of essential oil on the formation of S. mutans biofilm in vitro.

Reductive effect against the S. mutans biofilm established in vitro [20]: A 96-well plate was first coated with 100μL of artificial saliva and incubated at room temperature for three hours. The plate was washed twice with PBS before 20 μL of S. mutans inoculum (1x10⁶ CFU/mL) was applied into each well containing BHI-S that was 200μL in total volume. The bacterial biofilm was allowed to form in 37°C incubator with 5% CO₂ for 24 hours. As mentioned in the section of preventive effect, various concentrations of herbal essential oils, chlorhexidine and BHI-S were added to the pre-established biofilm for different time-interval ranging from 1, 2, 4 to 24 hours and designated as tests, positive, and negative controls, respectively. After being washed, the biofilm mass was finally determined by crystal violet staining as described above [Table/Fig-2]. Reductive effect of the tested herbal essential oils against the pre-established S. mutans biofilm was interpolated to be the percentage of bacterial biofilm being disrupted by using the equation described below. The experiment was performed in triplicate each with three repeats.

[Table/Fig-2]:

Methodology to determine reductive effect of essential oil against the S. mutans biofilm established in vitro.

Anti-cariogenic plaque efficacy (preventive and reductive effects) was described as the percentage of retardation of biofilm formation and reduction of the pre-established biofilm, respectively. Influence of the culinary herb essential oil concentration and exposure time on the anti-plaque efficacy was further analysed by two way analysis of variances and Tukey’s multiple comparison tests at significance level of 0.05 using SPSS program version 18.0.

All tested herbal essential oils at concentration of 20% (v/v), except black pepper displayed different degrees of inhibitory activity against both S. mutans KPSK2 and L. casei through the agar disk diffusion method. No inhibitory activity against L. casei was observed from black pepper essential oil even though the concentration of the oil was increased to 50% (v/v). Cinnamon bark essential oil expressed the most potent antimicrobial activity against both cariogenic bacteria whereas, 10% DMSO used as a diluent did not show such activity. Inhibitory efficacy of all tested essential oils against the cariogenic microorganisms was summarized in [Table/Fig-3,4].

[Table/Fig-4]:

Inhibitory effect of 20% (v/v) of culinary herb essential oils including cinnamon (Cin), sweet fennel (Fen), spearmint (Spe), kaffir lime (Kaf) and 10% (v/v) dimethyl sulfoxide (DMSO) (negative control) against S. mutans KPSK2, determined by agar disk diffusion method.

The essential oils with antimicrobial activity against both tested microorganisms were further examined for their MIC and MBC values by broth microdilution method. The growth of planktonic S. mutans KPSK2 and L. casei was strongly inhibited by the essential oil extracted from the cinnamon bark with MIC and MBC values of 0.08% and 0.16% (v/v), respectively. Essential oils extracted from either peel or leaf of kaffir lime expressed the weakest antimicrobial activity with MICs and MBCs of 2.5% and 5.0% (v/v) against S. mutans KPSK2 and L. casei, respectively. The MIC and MBC values of the selected herbal essential oils were illustrated in [Table/Fig-5].

Preventive effect on the formation of S. mutans biofilm in vitro: After the substratum was primed with 0.25% chlorhexidine mouthwash, the formation of S. mutans biofilm was affected differently ranging from >60% inhibition (24-h priming) down to >30% inhibition (1-h priming). Inhibition of S. mutans biofilm development was also observed on the substratum being primed with each tested herbal essential oil. Similar to antimicrobial effect against the planktonic culture, the formation of S. mutans biofilm was retarded by >80% on the substratum that was primed with cinnamon bark essential oil at its MIC for 4 or 24 hours, compared to the negative control (untreated substratum) [Table/Fig-6]. In contrast, the formation of bacterial biofilm was inhibited by approximately 60% on the substratum primed with this herb for only one or two hour. Additionally, high concentration {4xMIC; 0.32% (v/v)} of the cinnamon bark oil with 1-h priming on the substratum inhibited the formation of S. mutans biofilm by >80%. Sweet basil essential oil was the second best among the tested herbs in restraining the formation of such bacterial biofilm (>70% inhibition). Other essential oils illustrated lower degree of preventive effect (not statistically significant, p>0.05) against the formation of S. mutans biofilm, compared to those of cinnamon bark and sweet basil oils. Preventive effect of all tested herbal essential oils against the formation of S. mutans biofilm in vitro was summarized in [Table/Fig-6,7].

[Table/Fig-7]:

In vitro formation of S. mutans biofilm on the untreated substratum and those substrata being primed with various concentrations (at MIC, 2xMIC and 4xMIC) of cinnamon bark essential oil (Cin) or 0.2% chlorhexidine (CHX) for 24 h.

Reductive effect against the in vitro established S. mutans biofilm: A 24 hours in vitro-established S. mutans biofilm mass was reduced by ≤35% and ≤19% after the bacterial biofilm was treated with 0.25% chlorhexidine for 24 hours and one hour, respectively. Cinnamon bark essential oil exhibited the strongest reductive effect but not statistically significant (p>0.05) against the 24 hours pre-established S. mutans biofilm among the tested herbal essential oils. Approximately, 80% of biofilm mass was reduced after the pre-established bacterial biofilm was exposed to the cinnamon essential oil at its MIC, 2xMIC {0.16% (v/v)}, and 4xMIC {0.32% (v/v)} for 24 hours. Within the shortest exposure time (1 h), the oil was able to reduce the biofilm mass by 60% approximately. The reductive effect of all tested essential oils against the 24 hours pre-established S. mutans biofilm was demonstrated in [Table/Fig-8].

Culinary herbs or spices used in Thai cuisine have been well-known not only as food flavoring but also as herbal medicine in the traditional remedy. These edible herbs contain many medicinal properties including antimicrobial (especially against medically important pathogens) and anti-inflammatory activities [11-16]. Presently, the safety and side effects of the medicinal herbs used worldwide have not been extensively analysed yet. However, these criteria are required before the herbs will be used as pharmaceutical drugs but not as dietary supplements [21]. Dental plaque control by lowering the levels of dental caries related microorganisms (mutans streptococci, lactobacilli, etc.,) and restraining their biofilm formation has been recommended as an effective strategy for oral health promotion [22]. The study clearly illustrates that the essential oils extracted from cinnamon, sweet basil, peppermint, spearmint, sweet fennel, black pepper, and kaffir lime not only were antimicrobials against cariogenic bacteria including S. mutans and L. casei, but also were anti-plaque agents to retard the formation of S. mutans biofilm in vitro.

Different degrees of antimicrobial activity of those culinary herb essential oils against the growth of both cariogenic bacteria were observed and categorized into 3 groups according to the MIC values: strong {MIC ≤ 0.1% (v/v)}, moderate {MIC ≤ 1.0% (v/v)}, and weak [MIC > 1.0% (v/v)] activities, respectively. Cinnamon bark essential oil demonstrated the most potent inhibitory activity against the growth of planktonic S. mutans KPSK2 and clinical strain L. casei (strong activity group). This finding was associated with the previous study by Zainal-Abidin et al., but it was significantly different from those studied by Fani and Freires [23-25]. Sweet basil leaf essential oil moderately inhibited the growth of such cariogenic Streptococcus (moderate activity group) then followed by peppermint leaf, spearmint leaf, black pepper seed, sweet fennel seed, kaffir lime peel and leaf essential oils (weak activity group). Antimicrobial potential of sweet basil examined in this study was slightly weaker than those reported previously [26]. Dissimilar to the report by Wongsariya et al., a much stronger antimicrobial activity of kaffir lime essential oil against S. mutans KPSK2 was observed [27]. Intriguingly, both peppermint and spearmint oils did express inhibitory activity against the tested streptococci with moderate degree. This was completely different from the previous study showing that antimicrobial activity against such oral Streptococcus was observed from neither peppermint nor spearmint essential oils [11]. These discrepancies may be the consequences of active component variation in herb origin due to geographic location, seasons, harvesting time, extraction procedure, etc., strains of the tested microorganisms as well as testing methods [28].

Biofilm form, a favored mode of growth, of oral streptococci is found to be a major cause of dental caries development and hard to control apart from its planktonic counterpart. So, it is necessary to assess the anti-plaque effects of the selected spice essential oils in terms of preventing bacterial biofilm formation and reducing or disrupting the pre-established biofilm. For preventive effect, the formation of S. mutans biofilm was inhibited by 80% at least on substratum being primed with cinnamon bark essential oil as low as its MIC or four times the MIC for four or one hour, respectively. The present finding has restated the previous report that this oil contained preventive effect against the formation of S. mutans biofilm [29]. Relatively weaker degree of preventive effect against the formation of S. mutans biofilm was found in other essential oils extracted from sweet basil, peppermint, spearmint, sweet fennel, black pepper and kaffir lime, compared to the one of cinnamon bark oil. Only 25-40% of S. mutans biofilm was allowed to form on the substratum primed with these essential oils at the MIC for 24 hours or at four times the MIC for one to four hours. Interestingly, the study clearly shows that the preventive effect of all selected culinary herb essential oils against the formation of S. mutans biofilm was in dose- and exposure time-dependent manners. Different degrees of preventive effect among these culinary herb essential oils observed may result from a variety of phytochemicals or bioactive components with distinct properties, especially the adhesion inhibition property, and quantities comprised in individual plant. Previous report has demonstrated that cinnamon extract contained glucosyltransferase adherence-inhibition effect on S. mutans [30].

All tested essential oils demonstrated reductive effect on the S. mutans biofilm pre-established in vitro, apart from their preventive effect against the formation of such cariogenic biofilm. Cinnamon bark essential oil also showed the strongest efficacy in disrupting the mass of pre-established bacterial biofilm. Approximately 80% of biofilm mass was reduced after the pre-established biofilm exposed to the cinnamon bark oil with the concentration at 4 times the MIC for 24 hours, whereas other oils were able to reduce the biofilm mass by at least 50%. This finding was associated with the previous study [31]. The reductive effect of each essential oil against the pre-established S. mutans biofilm clearly depended on the exposure times rather than the doses. This experiment has reiterated that the complex infrastructure of bacterial biofilm acts as a shelter to protect the embedded microorganisms from any antimicrobial agents. In other words, the biofilm form is more tolerant to any harmful substances, especially antimicrobials than its planktonic counterpart because several mechanisms have been proposed to contribute to phenotypic resistance of biofilm. These mechanisms include decrease in antimicrobial penetration, different growth rates and nutrient ingredients within the biofilm, persister phenomenon, induction of resistance mechanisms, and mutational resistance [32].

Additionally, all tested essential oils, except black pepper seed oil, demonstrated different degrees of antimicrobial activity against another cariogenic bacterium, L. casei (clinical strain). Cinnamon bark essential oil was still the best culinary herbal oil that strongly inhibited the growth of planktonic L. casei, as shown against S. mutans KPSK2. This study is probably the first report demonstrating MIC values of cinnamon bark oil against both cariogenic microorganisms. Interestingly, floating L. casei seems to be less susceptible to all tested spice essential oils because high concentrations of the oils were required to restrain the growth of the bacterium. Dissimilar to previous study, sweet basil leaf essential oil illustrated potent inhibitory effect on the tested Lactobacillus [25]. The peppermint leaf oil inhibited the growth of L. casei whereas the black pepper seed oil did not affect adversely on such bacterium as reported previously [33,34].

The primary bacterial colonizers such as streptococci and Gram-positive rods (Actinomyces species) interact with receptor molecules in the pellicle through specific stereo-chemical molecular interactions, alter microenvironment on the tooth surfaces and allow the secondary and tertiary colonizers to adhere and to form biofilm at last [35,36]. The impediment in primary colonization of oral streptococci should directly impact on the dental biofilm development. Therefore, anti-L casei plaque effect of the selected herbal essential oils was not studied.

S. mutans biofilm was developed as single-species plaque under the static environment. This in vitro pre-established bacterial biofilm may not represent the actual properties of multi-species dental plaque with structural complexity and dynamic condition developed on hard and soft tissues of oral cavity. Different responses of dental plaque to those culinary herb essential oils may be observed which could not be done in the present study.

This study has indicated that cinnamon, sweet basil, peppermint, spearmint, sweet fennel, kaffir lime and black pepper are excellent not only as culinary but also as anti-caries herbs. The essential oils extracted from these herbs, especially cinnamon bark illustrated potent medicinal properties including antimicrobial (against planktonic cariogenic bacteria) and anti-plaque (retardation of S. mutans biofilm formation and reduction of the established biofilm) effects. Further investigations for other pharmacological properties, toxicity, etc., of these herbal essential oils are required prior to being qualified and applied as clinically effective, inexpensive and safe plaque control supplements to promote oral health condition.