Inhibitory effects of propolis and essential oils on oral bacteria

Introduction: Propolis is a natural composite balsam. In the past decade, propolis has been extensively investigated as an adjuvant for the treatment of periodontitis. This study aimed to investigate antimicrobial activities of propolis solutions and plant essential oils against some oral cariogenic (Streptococcus mutans, Streptococcus mitis, Streptococcus sanguis, Lactobacillus acidophilus) and periodontopathic bacteria (Actinomyces odontolyticus, Eikenella corrodens, Fusobacterium nucleatum). Methodology: Determination of the minimum inhibitory concentration (MIC): The antimicrobial activity of propolis and essential oils was investigated by the agar dilution method. Serial dilutions of essential oils were prepared in plates, and the assay plates were estimated to contain 100, 50, 25 and 12.5 μg/mL of active essential oils. Dilutions for propolis were 50, 25, 12.5 and 6.3 μg/mL of active propolis solutions. Results: Propolis solutions dissolved in benzene, diethyl ether and methyl chloride, demonstrated equal effectiveness against all investigated oral bacteria (MIC=12.5 μg/mL). Propolis solution dissolved in acetone displayed MIC of 6.3 μg/mL only for Lactobacillus acidophilus. At the MIC of 12.5 μg/mL, essential oils of Salvia officinalis and Satureja kitaibelii were effective against Streptococcus mutans and Porphyromonas gingivalis, respectively. For the latter, the MIC value of Salvia officinalis was twice higher. Conclusions: The results indicate that propolis and plant essential oils appear to be a promising source of antimicrobial agents that may prevent dental caries and other oral infectious diseases.


Introduction
Extracellular polysaccharides, mainly glucans, which are synthesized from dietary sucrose by streptococcal glucosyltransferases (GTFs), play a key role in the pathogenesis of dental caries and in plaque formation and accumulation as well [1]. During the last several decades, a remarkable increase has been observed in the use of natural products, especially propolis and essential oils [2,3]. The two mechanisms by which propolis exerts anti-caries properties, such as antimicrobial activity against cariogenic bacteria and inhibition of glucosyltransferase enzymes (GTFs) activity, have been described in earlier studies [4,5]. Among many active compounds present in propolis, flavonoids and terpens display distinct biological properties as effective GTF inhibitors and antibacterial agents, respectively [6]. The main components of the Salvia officinalis and Satureja kitaibelii essential oils are ketones and monoterpene hydrocarbons [7,8]. The products of these plant are lipophilic and capable of penetrating through the cell wall and cellular membranes. They can also increase cell permeability, affect the proton-pump mechanism and deactivate cellular enzymes after denaturing the plasma proteins to cause cellular death [9].
Since propolis and plant essential oils appear to be a promising source of antimicrobial agents, the aim of this work was to evaluate in vitro antibacterial effects of these natural products on the development of caries and strains that cause periodontopathy.
The minimal inhibitory concentrations (MIC) of essential oils (Salvia officinalis and Satureja kitabelii) and of propolis solutions (dissolved in benzene, diethyl ether, acetone and methyl chloride) have been determined for the following strains:

Study design
This prospective study was conducted at the Faculty of Stomatology, Pančevo, from 2010 to 2014.

Collection of the plant material
Salvia officinalis and Satureja kitaibelii, two freegrowing and also cultivated medicinal plant species, were collected in northern Serbia for the purpose of the study.

Bacterial strains
The

Extraction of essential oils
The essential oils of Salvia officinalis and Satureja kitaibelii were obtained by distillation in a Clevengertype apparatus. With respect to the preparation of propolis solutions in the study, propolis of the same species and origin was used -from one apiary near the mountain of Kopaonik (southern part of Serbia), and collected during only one time section (autumn), to ensure the highest homogeneity of the basic raw material. Extraction, as a chemical method, was performed as follows, regardless of the type of solvent. A mixture of solvent and water in a volume ratio of 60:40 to 96:4 was placed in a double-pot mixer (for necessary cooling). Four non-polar solvents (ether, acetone, methyl chloride and benzene) were used to dissolve propolis as well as ethanol, the solvent most commonly used for these purposes. When non-polar solvents, such as ether, acetone, methyl chloride and benzene were used as solvents during the extraction process, 500 mL of each solvent was added to 150 g of propolis. The extraction process took 48 hours, after which 360 mL, 450 mL, 486 mL and 500 mL of filtrate was obtained, respectively. The weight of the propolis extract was 80 g, 80 g, 40 g and 150 g, respectively. The containers with propolis extracts were cooled from 5 °C to 15 °C for thirty minutes and the contents were stirred at a rate of 20 m/s. After that, the suspension was centrifuged three times. The resulting extract was filtered through a Watman filter No. 4. The resulting propolis filtrate was a clear, dark brown liquid, which was further subjected to a vaporization process and stored in a dark flask at 4 °C until use. In this way, solvents that may have toxic effects were eliminated and at the same time the active components of propolis were preserved.

Antimicrobial activity
The essential oils and propolis solutions were individually tested against specific bacteria. The bacteria were cultured overnight at 37 °C in Mueller Hinton broth (HiMedia, Mumbai, India.), pH = 7.4.

Determination of the minimum inhibitory concentration (MIC)
The antimicrobial activity of propolis and essential oils was investigated by the agar dilution method [10]. Serial dilutions of essential oils were prepared in plates, and the assay plates were estimated to contain 100, 50, 25 and 12.5 µg/mL of active essential oils. Dilutions for propolis were 50, 25, 12.5 and 6.3 µg/mL of active propolis solutions. Inoculates were applied to blood agar surfaces (Liofilchem Roseto degli Abruzzi, Italy), producing approximately 10 6 µg/mL of bacteria. All plates were incubated for 48-72 hours under anaerobic conditions. MIC was taken as the lowest concentration of essential oil and propolis that produced no visible bacterial growth as compared to the control growth. The oils and propolis were tested in triplicates.

Results
The MIC values of propolis solutions ranged from 6.3 to 12.5 µg/mL. Solutions I, II and IV, dissolved in benzene, diethyl ether and methyl chloride, respectively, displayed for all tested bacteria the MICs of 12.5 µg/mL. In contrast, fraction III dissolved in acetone displayed the MIC of 6.3 µg/mL only for L. acidophilus ( Table 1).
The antibacterial activities of Salvia officinalis and Satureja kitaibelii essential oils were tested against S. mutans and P. gingivalis. At the MIC of 12.5 µg/mL, essential oils of Salvia officinalis and Satureja kitaibelii were effective against S. mutans and P. gingivalis, respectively. For the latter, the MIC of Salvia officinalis essential oil was twice higher (25 µg/mL) ( Table 2).

Discussion
Because of its complex chemical composition, many biological activities have been attributed to the ethanolic extract of propolis, while some of propolis flavonoids are considered to be antimicrobial agents [11,12]. In the present study, MIC values of propolis solutions I (dissolved in benzene), II (dissolved in diethyl ether) and IV (dissolved in methyl chloride) were 12.5 µg/mL for all investigated strains. In contrast, MIC values of propolis III (dissolved in acetone) were the same for most of the bacteria, except for L. acidophilus (6.3 µg/mL). Confirming the findings of the previous study, about anti-caries properties of propolis type-3 and type-12 [5], Hayacibara et al. demonstrated that chloroform fraction, and especially hexane fraction of both propolis types, were the most effective extracts [13]. In general, the hexane fraction from both propolis types rich in flavonoids, showed the most potent antibacterial and anti-GTFs activity in vitro [13]. Further, it was interesting to note from earlier works that propolis ethanolic extract RS2 with the highest concentrations of flavonoids, demonstrated both a higher antimicrobial activity and inhibition of glucosyltransferase activity [12].
The results of Gebara et al. [14] showed that propolis extract demonstrated antimicrobial activity in vitro not only against some periodontophatic bacteria (F. nucleatum, P. gingivalis, P. intermedia, P. melaninogenica, A. actinomycetemcomitans and C. gingivalis), but also against some organisms able to cause superinfection (S. aureus, P. aeruginosa, E. coli and Candida albicans). Interestingly, the MIC of propolis was 0.25 µg/mL for Fusobacterium nucleatum, which meant that the tested microorganism was susceptible to propolis at a lower MIC than the strain in the present study (12.5 µg/mL). Regarding susceptibility of the tested microorganisms to propolis, it was worth mentioning that they seemed to be more susceptible to propolis than to some antibiotics [14,15].
Similar to the results of present study, Koo et al. [6], Topcuoglu et al. [16] and Kim et al. [17] reported a greater anti-Streptococcus mutans effect, with minimum inhibitory concentrations of 14-35 µg/mL of propolis.
Considerable variability of the chemical composition of propolis (due to its geographical distribution) may be a limitation in terms of its quality control, comparability and effect reproducibility [18]. That could affect the determination of MIC values which depends on technical details that may vary between laboratories and on bacterial inherent virulence and susceptibility [19].
Four different propolis solutions exhibited equal effectiveness against investigated strains in the study, but one of them -the one dissolved in acetone -had the outstanding MIC of 6.3 µg/mL only for L. acidophilus. Comparing the antimicrobial effect of Egyptian propolis with propolis from New Zealand on S. mutans and Lactobacillus spp., the propolis hexane fraction from New Zealand was reported to have the strongest antimicrobial action [20]. Although it was capable of inhibiting the development of cariogenic bacteria Lactobacillus fermentum, the activity of Chilean propolis was variable and depended on the chemical composition of the propolis used [21].
The exhibited antimicrobial activity of the essential oils is supposed to be due to the synergism of the compounds [22]. The antimicrobial activities of essential oils of different Satureja species have been extensively studied because of their very low minimal inhibitory concentrations [23]. The minimal inhibitory concentrations of S. kitaibelii essential oil ranged from 0.097 µg/mL (C. albicans) to 25 µg/mL (Enterococcus faecalis) [8]. The same MIC of 12.5 μg/mL for P. gingivalis and P. aeruginosa was established in the present and in one earlier work [8], respectively. Moreover, for other Satureja species, many authors reported antibacterial effectiveness of S. hortensis [24,25] and Satureja intermedia [26] against cariogenic bacteria F. nucleatum, S. mutans, S. salivarius and S. sanguis. Contemporary investigations have confirmed antibacterial activity of S. officinalis essential oils against S. mutans [27]. In order to develop novel and effective agents against oral bacteria responsible for dental caries, Moreira et al. [28] emphasized that manool and manool-rich S. officinalis extract (SODH2) were important and selective plant-derived products that could be potentially used in the control of caries disease. A very promising anti-Streptococcus mutans effect with MIC values of 6.24 µg/mL of manool and especially of 15.68 µg/mL of SODH2 has been obtained, which is similar to the results of this work (12.5 µg/mL). Regarding minimal inhibitory concentrations of essential oil, the results of the present study for P. gingivalis (25 µg/mL) corresponded to those for S. salivarius and S. sobrinus (24.96 µg/mL) [28].

Conclusions
This study showed a positive inhibitory influence of different propolis solutions and essential oils on the growth of investigated oral microorganisms. The observed reduction in oral flora counts may provide an alternative preventive and therapeutic approach for individuals at high risk for dental caries and other oral diseases.