Antibacterial activity and synergistic effects between Machilus thunbergii ethanol extract and antibiotics against oral pathogens

The cortex of Machilus thunbergii, which has been consumed as traditional herbal medicine for treatment of leg edema and abdominal distension and pain for a long period of time. In this study, the combination effect of M. thunbergii ethanol extract (MTEE) was evaluated against oral bacteria, either alone or with antibiotics, via broth dilution method and checkerboard and time kill assay. MIC/MBC values for MTEE against all the tested bacteria ranged between 12.5-50/50-200 microg/ mL, for ampicillin 0.0625-8/0.125-32 microg/mL and for gentamicin 4-128/8-512 microg/mL respectively. Furthermore, the MIC and MBC were reduced to one half-eighth as a result of the combination of MTEE with antibiotics. 1-6 hours of treatment with 1/2 MIC of MTEE with 1/2 MIC of antibiotics resulted from an increase of the rate of killing in units of CFU/mL to a greater degree than was observed with alone. These results suggest that the MTEE is important in the antibacterial actions of oral pathogens agents. *Correspondence to: Jeong-Dan Cha, Research Manager, Material Development Team, R&D Center, General Bio Co., Ltd., 254 Yongtusna-ro, Songdong-myeon, Namwon-si, Jeollabuk-do, 55793 Republic of Korea, Tel. +82-63-263-0001, Fax: +82-70-5101-1563, E-mail: cjd@generalbio.co.kr


Introduction
Oral disease is one of the most important preventable infectious diseases, a major health problem in dental caries and periodontal disease [1,2]. Oral health affects the general quality of life and poor oral health is associated with chronic conditions and systemic diseases [3][4][5]. There are more than 750 bacteria in the oral cavity, many of which are related to oral diseases [6]. The development of dental caries includes acidogenic and aciduric gram-positive bacteria, mainly mutans streptococci (Streptococcus mutans and S. sobrinus), lactobacilli and actinomycetes, which metabolize sucrose into organic acids that dissolve the calcium phosphate in teeth, causing decalcification and eventual decay [6][7][8]. In contrast, periodontal disease is subgingival and gum diseases associated with anaerobic gram-negative bacteria such as Porphyromonas gingivalis, Actinobacillus sp., Prevotella sp., and Fusobacterium sp. [9][10][11]. In periodontal disease, gingival crevices or areas beneath the gingiva are infected, causing cellular inflammatory response of the gingiva and surrounding connective tissue [10,11]. These inflammatory reactions can be caused by gingivitis (extremely common and seen as bleeding of the gingival or gum tissues) or periodontitis (the inflammatory response results in loss of collagen attachment of the tooth to the bone and in loss of bone) [12][13][14].
Many plant-derived medicines used in traditional medicinal systems have been documented in pharmacopeias for the treatment of infections and a number of these have been recently proved effective against oral microbial pathogens [15][16][17][18]. Machilus thunbergii (Lauraceae) is widely distributed in Korea. The cortex of M. thunbergii, which has been consumed as traditional herbal medicine for treatment of leg edema and abdominal distension and pain for a long period of time [19][20][21]. Isoquinoline alkaloids have been obtained from the root , lignin, catechin and polysaccharides from the heartwood, polysaccharides, and essential oils from the leaves, volatile components from the fruits, and lignans and neolignans from the cortex [22][23][24]. Some of these compounds are antioxidants with hepatoprotective, and anti-bacterial activities, while a few other show inhibitory effects on nitric oxide synthesis inactivated macrophages and neuroprotective activity against glutamate-induced neurotoxicity [22,25,26]. Machilin A (MA), one of the lignans shows biological activities, including stimulation of osteoblast differentiation via activation of p38 mitogenactivated protein(MAP) kinases in an in vitro osteoblasts [20].
In this study, we investigated the synergistic antibacterial activity of M. thunbergii ethanol extract (MTEE) in combination with existing antimicrobial agents against oral bacteria.

Bacterial strains
The oral bacterial strains used in this study were :

Minimum inhibitory concentrations/minimum bactericidal concentrations assay
The minimum inhibitory concentrations (MICs) were determined for M. thunbergiii ethanol extract (MTEE) by the broth dilution method, and were carried out in triplicate (27). The antibacterial activities were examined after incubation at 37℃ for 18 h (facultative anaerobic bacteria), for 24 h (microaerophilic bacteria), and for 1-2 days (obligate anaerobic bacteria) under anaerobic conditions. MICs were determined as the lowest concentration of test samples that resulted in a complete inhibition of visible growth in the broth. MIC 50 s and MIC 90 s, defined as MICs at which, 50 and 90%, respectively of oral bacteria were inhibited, were determined. Following anaerobic incubation of MICs plates, the minimum bactericidal concentrations (MBCs) were determined on the basis of the lowest concentration of MTEE that kills 99.9% of the test bacteria by plating out onto each appropriate agar plate. Ampicillin (Sigma) and gentamicin (Sigma) were used as standard antibiotics in order to compare the sensitivity of MTEE against oral bacteria.

Checker-board dilution test
The antibacterial effects of a combination of MTEE, which exhibited the highest antimicrobial activity, and antibiotics were assessed by the checkerboard test as previously described (27). The antimicrobial combinations assayed included MTEE with ampicillin or gentamicin. Serial dilutions of two different antimicrobial agents were mixed in cation-supplemented Mueller-Hinton broth. After 24-48 h of incubation at 37°C, the MICs were determined to be the minimal concentration at which there was no visible growth and MBCs were determined on the basis of the lowest concentration of MTEE that kills 99.9% of the test bacteria by plating out onto each appropriate agar plate.

Time-kill curves
Bactericidal activities of the drugs under study were also evaluated using time-kill curves on oral bacteria. Tubes containing Mueller-Hinton supplemented to which antibiotics had been added at concentrations of the MIC 50 were inoculated with a suspension of the test strain, giving a final bacterial count between 5~6.6×10 6 CFU/ml. The tubes were thereafter incubated at 37°C in an anaerobic chamber and viable counts were performed at 0, 0.5, 1, 2, 3, 4, 5, 6, 12 and 24 h after addition of antimicrobial agents, on agar plates incubated for up to 48 h in anaerobic chamber at 37°C. Antibiotic carryover was minimized by washings by centrifugation and serial 10-fold dilution in sterile phosphate-buffered saline, pH 7.3. Colony counts were performed in duplicate, and means were taken. The solid media used for colony counts were BHI agar for streptococci and BHI agar containing hemin and menadione for P. intermedia and P. gingivalis.

Minimum inhibitory concentrations/minimum bactericidal concentrations of MTEE and antibiotics
MTEE evaluated the antimicrobial activities against eleven bacterial species present in the oral cavity.

Synergistic effect of MTEE with antibiotics
Natural products are a major source of chemical diversity and have provided important treatment agents for many bacterial diseases [16,[27][28][29]. The combination of some natural products and antibiotics can increase the antimicrobial activity of antibiotics [30,31]. The synergistic effects of MTEE alone or with antibiotics were evaluated in oral bacteria (Tables 2 and 3). In combination with MTEE, the MIC for ampicillin was reduced ≥4-fold in all tested bacteria, producing a synergistic effect as defined by FICI ≤ 0.5, except S. gordonii by FICI≤0.75 and additive. The MBC for ampicillin was shown synergistic effects in all tested bacteria by FBCI ≤ 0.5, except S. ratti, S. criceti, and P. gingivalis by FICI≤0.75 and additive ( Table 2). In combination with MTEE, the MIC for gentamicin was reduced ≥4-8-fold in all tested bacteria, except S. criceti and P. gingivalis by FICI ≥ 0.75 and MBC in all tested bacteria by FBCI ≤ 0.5, except S. sanguinis, S. ratti, S. anginosus, and F. nucleatum by FBCI ≤ 0.75 (Table 3).
Phytochemical constituents such as alkaloids, flavonoids, tannins, phenols, saponins, and several other aromatic compounds are secondary metabolites of plants that serve a defence mechanism against prediction by many microorganisms, insects and other herbivores [32][33][34][35]. Flavonoid complexes attach with extra cellular soluble protein and with bacterial cell wall [36,37]. Lignans, alkaloids, flavonoids, butanolides, and essential oils have been derived from M. thunbergii; some of these compounds are antioxidants with hepatoprotective and antibacterial activities [23,25,26]. Both the ethyl acetate fraction and water fraction  [38]. In this study, M. thunbergii ethanol extract shows susceptibility on gram-positive bacteria as well as gram-negative bacteria [38].

Time kill of MTEE with antibiotics
The bacterial effect of MTEE with ampicillin or gentamicin against oral bacteria was confirmed by time-kill curve experiments. The MTEE (MIC or MIC 50 ) alone resulted rate of killing increasing or not   (Figures 1-3). A strong bactericidal effect was exerted in drug combinations.
In conclusion, these findings suggest that MTEE fulfills the conditions required of a novel cariogenic bacteria and periodontal pathogens, particularly bacteroides species drug and may be useful in the future in the treatment of oral bacteria.