In Vitro Antibacterial Activity of Myrtus communis L. and Marrubium vulgare L. Leaves against Aggregatibacter actinomycetemcomitans and Eikenella corrodens

Materials and Methods Clinical strains of Aggregatibacter actinomycetemcomitans and Eikenella corrodens and two reference strains of A. actinomycetemcomitans were tested. The antibacterial activity of each studied plant extract was evaluated using agar diffusion and broth microdilution assays. Results Both aqueous and methanolic extracts of M. communis exhibited high antibacterial activity against periodontal pathogens as compared to M. vulgare extracts. At concentrations of 2.5-0.32 mg/disc, inhibition zones of the methanolic extract of M. communis ranged from 19.66 ± 0.57 to 12.33 ± 0.57 mm. The methanolic extract of M. vulgare showed at concentrations of 5-0.63 mg/disc inhibition zones ranging from 15.66 ± 0.57 to 12 ± 0.00 mm. Its aqueous extract at concentration of 0.63 mg/disc showed no antimicrobial activity against the clinical and reference strain of A. actinomycetemcomitans. Conclusion. This study showed that methanolic and aqueous extracts of M. communis and M. vulgare have in vitro an antibacterial activity against periodontal pathogens. They could be use as ingredients of an oral antimicrobial agent for prevention or treatment of periodontal diseases. Further research on isolating the compounds from these plant extracts and their toxicity effect could be conducted.


Introduction
Periodontitis is a chronic inflammatory disease that leads to periodontal attachment loss and alveolar bone destruction, which may gradually lead to tooth loss if left untreated. Periodontitis known as "chronic" and "aggressive" periodontitis is currently grouped in one category named "periodontitis" [1]. Many bacterial species associated with periodontitis have been identified in subgingival plaque samples such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, Campylobacter rectus, and Eikenella corrodens [2,3]. A. actinomycetemcomitans is strongly associated with periodontitis, and its pathogenicity is related to the production of numerous virulence factors [4]. E. corrodens has also been implicated in destructive periodontal diseases [5].
Management of this disease is based on mechanical periodontal debridement either none surgically or surgically. In some situations, adjunctive antibacterial treatments are necessary to control biofilm accumulation. However, because of the increasing bacterial resistance and the side effects of antimicrobial agents commonly used in periodontal treatment [6], natural antimicrobial agents have generated a great interest at the global level. Indeed, medicinal plants and their natural compounds may possess antibacterial, antioxidant, and anti-inflammatory activities, which thereby may have real benefits on oral health [7,8].
Among the wide variety of medicinal plants used worldwide, Myrtus communis L. (Myrtaceae), commonly known as myrtle, and Marrubium vulgare L. (Lamiaceae), known as "white horehound," are wide-spreading in the Mediterranean region [9]. It had been reported that these two plants possess several properties, inter alia, antibacterial, anti-inflammatory, analgesic, antioxidant, and antidiabetic [10][11][12][13]. In Morocco, M. communis and M. vulgare are known as "Rihane" and "Merriwut," respectively. Infusion and decoction from the leaves of M. communis are traditionally used for the treatment of respiratory diseases, diarrhoea [14], heart weakness, and digestive disturbances [15]. e leaves of myrtle have been reported to be used as a mouthwash for the treatment of halitosis [16], while its fruits are chewed for gingivitis and mouth ulcers [14]. A decoction from the leaves of M. vulgare is used as antidiabetic and can also be chewed for toothache [14]. Additionally, it had been reported that M. vulgare is prescribed by Moroccan traditional healers for halitosis [17].
Many studies reported the antibacterial activity of M. communis essential oil and M. vulgare essential oil against oral pathogens [18][19][20][21]. Nevertheless, insufficient data on the antimicrobial effect of the crude extracts of these plants, on periodontal pathogens, are available. Hence, this study aimed to test the antibacterial activity of M. communis and M. vulgare leaf extracts against selected periodontal pathogens, namely, A. actinomycetemcomitans and E. corrodens.

Materials and Methods
e research was conducted in accordance with the Ethical Principles for Medical Research involving human subjects. e approval was obtained from the Biomedical Research Ethics Committee (CERB) of the Faculty of Medicine and Pharmacy of the Mohammed V University in Rabat (n°54/2018). A total of 10 subjects aged between 20-50 years were recruited from the department of periodontology at the Center of Dental Consultations and Treatments in Rabat. e inclusion criteria were as follows: patients who had a periodontitis and who accepted to participate to the study and signed informant consent. e exclusion criteria were pregnant and lactating women, smokers, and patients who had received systemic antibiotic or periodontal treatment within the last 6 months.
In these patients, subgingival plaque samples were taken from the deepest periodontal pockets using sterile paper points. e pooled samples were placed in small vials with 1.5 ml of Phosphate Buffer Saline (PBS) and transferred for processing at the Laboratory of Oral Biology and Biotechnology, Faculty of Dental Medicine, Mohammed V University, Rabat. e plants were washed and shade dried for 2 weeks at room temperature. en, the dried leaves were milled in a mixer, and the powder was stored in an airtight container until use.

Preparation of Aqueous
Extracts. 50 g of dry powdered leaves of M. communis and M. vulgare were mixed with 500 ml of sterile distilled water and boiled for 15 min at 100°C. Each extract was filtered by using Wattman filter paper No. 1, and the filtrate was dried under reduced pressure by using a rotary evaporator at 60°C [22]. en, the aqueous extracts were lyophilized and stored at −20°C until use.

Preparation of Methanolic Extracts.
For M. communis, the powder from dry leaves was extracted using Soxhlet extraction with 95% methanol for 6 hours with the ratio of 10 : 1(v/w) of solvent [23]. Dry leaves of M. vulgare were extracted by maceration in 95% methanol for 48 hours. Extracts of both plants were then filtered by using Wattman filter paper No. 1 and concentrated under reduced pressure using a rotary evaporator at 40°C [24]. Dried methanolic extracts were stored at −20°C for further use.

Microbial Strains.
e antibacterial activity of M. communis and M. vulgare leaf extracts was tested on the following bacteria: clinical strains of Aggregatibacter actinomycetemcomitans, Eikenella corrodens and 2 reference strains of A. actinomycetemcomitans «ATCC 43718» and Y4 «ATCC43718».
A selective medium "Dentaid-1" was prepared to isolate A. actinomycetemcomitans by using Brain Heart Infusion agar supplemented with 5 g of yeast extract, 1.5 g of sodium fumarate, and 1 g of sodium formate per litre. e medium was autoclaved for 15 min at 121°C. Vancomycin was added to a final concentration of 9 µg/mL [25]. e samples were vortexed for 2 min and seeded into the culture media Dentaid-1 for isolation of A. actinomycetemcomitans and blood agar culture media for isolation of E. corrodens. e inoculated plates were incubated into an anaerobic environment at 37°C under 5% CO 2 for 3 to 5 days. Identification of the isolates was performed based on guidelines in the manual of Clinical Microbiology [26], i.e., colony morphology, gram staining, and biochemical tests.
Strains were stored at −80°C in Brain Heart Infusion broth with 20% glycerol at the Laboratory of Oral Biology 2 Evidence-Based Complementary and Alternative Medicine and Biotechnology, Faculty of Dental Medicine, Mohammed V University, Rabat.

Antibacterial Assays.
e antibacterial activity of aqueous and methanolic extracts of M. communis and M. vulgare was evaluated qualitatively and quantitatively by standard agar disk diffusion and broth microdilution according to the Clinical and Laboratory Standards Institute (CLSI) recommendations [27,28]. e aqueous and methanolic extracts were dissolved in sterile distilled water and 10% dimethyl sulfoxide (DMSO), to determine the concentration of 100 mg/ml and 200 mg/ml as a stock solution for M. communis and M. vulgare, respectively.

Agar Disk Diffusion.
e antimicrobial activity of plant extracts was evaluated by agar disk diffusion as previously described [23]. e stock solutions were diluted to 12.5-100 mg/ml at a final concentration for M. communis and 25-200 mg/ml for M. vulgare and used for antimicrobial testing.
Colonies of bacterial strains were picked from overnight pure culture on a nonselective medium and were suspended in 0.85% NaCl. e suspension of each bacterial strain was standardized with a 0.5 Mc Farland density (1 × 10 8 colonyforming unit (CFU)/ml) using Sensititre®Nephelometer. e inoculum of each bacterial suspension was used within 15 min of preparation and was swabbed in three directions over the entire surface of blood agar plates.
Blank filter paper disks (6 mm in diameter, Oxoid) were impregnated with 25 µl of each plant extract at various concentrations and left to dry for 2 hours before applying them over the test plates. Disks impregnated with 25 µl of sterilized distilled water and 10% DMSO were used as negative controls for aqueous and methanolic extracts, respectively, whereas Doxycycline (30 µg/disc, Oxoid) was used as a positive control. Doxycycline, as a reference antibiotic, showed an efficacy either on A. actinomycetemcomitans or E. corrodens [29,30] in in vitro studies. e plates were incubated at 37°C, 5% CO 2 for 48 hr, and after incubation, we measured the diameters of the zone of inhibition in millimetres. All experiments were repeated three times at each concentration of extracts.

Broth Microdilution.
e Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) values of extracts were determined by broth microdilution as previously described [31]. From stock solutions of each extract, two-fold serial dilutions were prepared in Brain Heart Infusion broth (BHI) to obtain concentrations ranging from 50 to 0.19 mg/ml for M. communis and 100 to 0,39 mg/ml for M. vulgare. 50 µl of each dilution was dispensed into the wells 1 to 9 of a 96-well microtitre plate (Figure 1). e inoculum of each microorganism was prepared by direct suspension of overnight colonies into BHI broth. e suspension was adjusted to equal the turbidity of a 0.5 Mc Farland standard using a nephelometer. en, 50 µl of each microorganism suspension was added to wells 1 to 10 for a final volume of 0.1 ml.
Control wells were included. An inoculated well with broth but not extracts was used as a positive control to check growth control (well 10), whereas uninoculated wells filled with the extracts and BHI broth served as negative controls. Solvent and medium controls were also used. After incubation at 37°C, 5% CO 2 for 48 hr, we added 20 µl of Triphenyl Tetrazolium Chloride (TTC, Oxoid) 0.1% solution, and we incubated the plate again for 2 hours [32]. en, the growth in wells was observed to determine the minimal inhibitory concentration which is the lowest concentration of the extract that inhibits the visible growth of the tested bacteria. e minimal bactericidal concentration was determined by plating an aliquot of each well that had no color change of indicator, onto blood agar plates, and incubated for 48 hr at 37°C under 5% CO 2 . After incubation, the MBC was recorded at the lowest concentration of the extract that could kill 99.9% of each microorganism on the agar plate. e MIC and MBC assays were repeated three times for each extract.

Statistical Analysis.
e data were analyzed using the Statistical Package for the Social Sciences (SPSS). e zone of inhibition values of all extracts at different concentrations were expressed as mean ± standard deviation (SD). Descriptive statistics were performed, and data were analyzed using one-way analysis of variance (ANOVA) and Bonferroni post hoc. Differences between means of inhibition zone diameters of the extracts of both plants were considered statistically significant when the p value was less than 5% (p < 0, 05).  Clinical strains and reference strains of A. actinomycetemcomitans had the same susceptibility to extracts of both plants, and there was no statistically significant difference (p < 0, 05). All bacterial strains showed susceptibility to doxycycline. e inhibition zone data of two plant extracts are summarized in Tables 1 and 2 . e results of the Minimum Inhibitory Concentrations (MICs) determined by microbroth dilution and the Minimum Bactericidal Concentrations (MBCs) obtained from the plates are showed in Table 3. e MICs of the methanolic and aqueous extract of M. communis ranged between 0.19-3.12 mg/ml against all periodontal pathogens. e MICs of the methanolic extract of M. vulgare ranged between 1.56-3.12 mg/ml, whereas the MICs of its aqueous extract ranged between 25-50 mg/ml.

Discussion
e present study showed that the aqueous and methanolic extracts of M. communis and M. vulgare had an antibacterial activity against the tested strains, i.e., A. actinomycetemcomitans and E. corrodens.
Although aqueous and methanolic extracts of M. communis were active against all microorganisms tested in all concentrations, methanolic extract was significantly more effective than aqueous extract. Both extracts exhibited high antibacterial activity as compared to the methanolic and aqueous extract of M. vulgare. e methanolic extract of M. vulgare was found to be more effective when compared with its aqueous extract. ese results may be attributed to a better solubility of the active compounds in organic solvents such as methanol and ethanol. Furthermore, it was shown that the highest extraction of total phenolic compounds from M. vulgare leaves was obtained by methanol [33].
An antibacterial agent is considered to have a bactericidal effect if the ratio of MBC/MIC is ≤4 and having a bacteriostatic effect if the ratio is >4 [34,35]. For the tested plant extracts, the MBC/MIC ratio was less than four except for the aqueous extract of M. vulgare. us, the aqueous and methanolic extracts of M. communis and the methanolic extract of M. vulgare showed a bactericidal activity, while the aqueous extract of M. vulgare showed a bacteriostatic effect. e antimicrobial activity of M. communis and M. vulgare against periodontal pathogens may be attributed to their active components. Indeed, leaves of M. communis are known to contain polyphenolic compounds, tannins, and flavonoid glycosides [36]. A chromatographic HPLC profile of the myrtle leaves identified 15 new phenolic compounds [37]. It had shown that these different compounds possess an antibacterial activity [38][39][40][41]. Regarding M. vulgare, the principal component is diterpenes labdane marrubium, and it contains also a variety of compounds, e.g., alkaloids, steroids, terpenes, and tannins [12]. e antibacterial activity of these various compounds has been reported in some studies [42][43][44].
ese plant extracts may probably act as an antimicrobial agent that involves specific mechanisms toward bacterial cells. Indeed, they may interfere with cell wall synthesis and protein synthesis and can also affect cytoplasmic membrane and cell metabolism [45]. e antibacterial activity of M. communis leaf extracts has been reported against Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, and Listeria monocytogenes) and Gram-negative bacteria (Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, and Campylobacter jejuni) [46]. It had been also reported that the hydroalcoholic extract of myrtle leaves had the great effect on Staphylococcus aureus and Vibrio chloreae [47]. Additionally, Rahimvand et al. [48] showed that the aqueous and methanolic extracts of myrtle leaves had an antimicrobial effect against the reference strains of A. actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia. Other studies reported the antibacterial activity in vitro of polyherbal toothpaste containing leaf extracts of M. communis against oral pathogens (Streptococcus mutans, Lactobacillus casei, S. sanguis, S. salivarius, and Candida albicans) and the clinical efficacy of a paste containing M. communis leaves in the treatment of recurrent aphthous stomatitis [49,50].
Previous studies have shown an antimicrobial activity of M. vulgare extracts against pathogenic microorganisms, e.g., Escherichia coli, Bacillus cereus, Proteus mirabilis, and other bacterial and fungal strains, e.g., Staphylococcus aureus, Klebsiella pneumoniae, Candida albicans, Aspergillus flavus, and M. tuberculosis [51][52][53]. We assume that this study is the first one to assess the antimicrobial efficacy of M. vulgare against periodontal pathogens.
rough this study, we acquire attention to the traditional use of medicinal plants for oral health in Morocco. Indeed, during the last ten years, many studies were particularly interested in the biological activities of medicinal plants on general health, but published national ethnobotanical studies on oral health are limited, and some of them reported that only 2% of medicinal plants are traditionally used for oral hygiene [54,55]. Compared with the rate of the therapeutic use of plants for other infectious diseases, dental use remains low in most regions of Morocco. For example, in Taounate Province (north of Morocco), only 2.3% of medicinal plants were used traditionally for the mouth and dental hygiene, while 24.9% was used for digestive disorders and 9.8% for the bronchopulmonary system [15]. Diverse plants used in traditional medicine may be interesting for the identification of new antimicrobial agents against relevant periodontal pathogens. Nevertheless, good oral hygiene is a major factor in maintaining periodontal health, and the use of adjuvant agents based on medicinal plants may be a good help for plaque control. Indeed, many herbal drugs and natural products are used as antimicrobial agents in kinds of toothpaste and mouth rinses to reduce plaque accumulation and gingival inflammation [56].
is study showed that M. communis and M. vulgare possess in vitro antibacterial activity against periodontal pathogens. However, we have only studied the crude extracts of the leaves of these plants, and the active compounds of   Evidence-Based Complementary and Alternative Medicine each plant must be isolated and tested. e bacteria tested were in planktonic culture; thereby, the antimicrobial activity of these plants should be verified against subgingival bacterial biofilm communities. e relationship between different subgingival microorganisms may interfere with their susceptibility to antimicrobial agents. Previous studies have shown that most subgingival bacteria species were resistant to antimicrobials when they are protected by a biofilm structure in comparison with the planktonic bacteria [57,58].
As periodontitis is a polymicrobial infection, we suggest further analysis of the antibacterial effect of the tested plant extracts against other periodontal pathogens such as bacteria of the red complex or bacteria newly associated with periodontitis and also against periodontal biofilm. e synergistic action of these plant extracts should be considered too. Finally, cells and animal toxicity studies of these plants and their active compounds are to achieve before clinical trials.

Conclusions
With the limit of the present study, the extracts of M. communis and M. vulgare showed an antibacterial effect on A. actinomycetemcomitans and E. corrodens. is antimicrobial action of natural compounds could serve as a suitable adjunctive agent for the prevention or treatment of periodontal diseases associated with these microorganisms. As periodontitis is associated with a complex multimicrobial biofilm, further analysis is needed for conclusive statement about the potential benefit of the extracts of M. communis and M. vulgare.

Data Availability
Data supporting the results of our study are included in the manuscript.
Disclosure e funder had no role in the study's design, data collection and analysis, the decision to publish, or the preparation of the manuscript.

Conflicts of Interest
e authors declare no conflicts of interest in connection with this article.