Synergistic Effect of the Ethanol Extract of Alismatis rhizoma against Oral Pathogens

Background: Alismatis rhizoma or Alisma orientale (Zexie in Chinese), the dried rhizome of Alisma orientale Juzepzuk (Alismataceae), is known to have diuretic and damp-heat clearing actions and has been used for the treatment of dysuria, edema, urinary tract infections, the retention of fluid and phlegm, and vertigo in Asia and Europe. Methods: In this study, the combination effect of the ethanol extract of Alismatis rhizoma (ARE) was evaluated against oral bacteria, either alone or with antibiotics, via broth dilution method and checkerboard and time kill assay. Results: In these results, MIC/MBC values for ARE against all the tested bacteria ranged between 313-5000/625-5000 microg/mL, for ampicillin 0.25-32/0.5-64 microg/mL and for gentamicin 4-32/864 microg/mL respectively. Furthermore, the MIC and MBC were reduced to one half-twelve as a result of the combination of ARE with antibiotics. 1-2 hours of treatment with 1/2 MIC of ARE 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. Conclusions: These results suggest that the ARE is important in the antibacterial actions of oral pathogen agents. in preventing dental caries [9]. Of the selected putative periodontal species, strains of Prevotella intermedia, Fusobacterium nucleatum and to the first time, Tannerella forsythia, were β-lactamase positive, with P. intermedia being the most frequently detected enzyme positive species [10]. Sub gingival isolates of P. gingivalis, P. intermedia and F. nucleatum in a group of subjects increase in the MIC values of tetracycline [11]. This clinical observation led to studies that established metronidazole as an important antibiotic for anaerobic infection. Since then, this compound has also played an important role in treating anaerobe related infection in the oral cavity, abdomen, and female genital tract, among others [12]. Oral bacteria have been reported to show increased resistance towards common antibiotics such as penicillin, cephalosporin, erythromycin, tetracycline, and metronidazole which have been used therapeutically for the treatment of oral infection [13,14]. The increase in resistance and adverse effects has lead researchers to explore novel anti-infective herbal compounds which could be used for effective treatment of oral diseases [15,16]. Natural medicine is a precious resource of therapeutically active compounds and has increasingly attracted the attention of researchers [17]. Alismatis rhizoma or Alisma orientale (Zexie in Chinese), the dried rhizome of Alisma orientale Juzepzuk (Alismataceae), is known to have diuretic and damp-heat clearing actions and has been used for the treatment of dysuria, edema, urinary tract infections, the retention of fluid and phlegm, and vertigo in Asia and Europe [18]. Phytochemistry studies have shown that protostane triterpenes, guaiane sesquiterpenes, alisol B, polysaccharides, and kaurane diterpenes are the major chemical components [19,20], its components have been imported antbioactivities, including anti-hepatitis, antiinflammatory, hypo-glycemic, and immunological effects [20-23]. A new triterpenoid from Alisma orientale exhibits their antibacterial effect against antibiotic resistant strains, Enterococcus faecium, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa [23]. Many plant-derived medicines used in traditional medicinal Su-Mi Cha1, Su-Beom Han1, Young-Soo Lee2 and Jeong Dan Cha3* 1Department of Oral Microbiology and Institute of Oral Bioscience, Chonbuk National University, Jeonju, South Korea 2Department of Dental Hygiene, Sun Moon University, Republic of Korea 3Department of Efficacy Research, Institute of Jinan Red Ginseng, South Korea *Address for Correspondence Jeong-Dan Cha, Professor, Institute of Jinan Red Ginseng, 520-9 Banwolri, Jinan-eup, Jinan-gun, Jeollabuk-do, 567-801 South Korea, Tel: +82-63432-0913; Fax: +82-63-432-0910; E-mail: joungdan@ijrg.re.kr Submission: 11 February 2015 Accepted: 06 March 2015 Published: 10 March 2015 Research Article Open Access Journal of Oral Biology


Introduction
Oral diseases are major health problems with dental caries and periodontal diseases among the most important preventable global infectious diseases [1,2]. Oral health influences the general quality of life and poor oral health is linked to chronic conditions and systemic diseases [3]. The association between oral diseases and the oral microbiota is well established [4]. The development of dental caries involves acidogenic and acid uric gram-positive bacteria, primarily the mutans streptococci (Streptococcus mutans and S. sobrinus), lactobacilli, and actinomycetes, which metabolize sucrose to organic acids (mainly lactic acid) that dissolve the calcium phosphate in teeth, causing decalcification and eventual decay [5,6]. In contrast, periodontal diseases are sub gingival conditions that have been linked to anaerobic gram-negative bacteria such as Porphyromonas gingivalis, Actinobacillus sp, Prevotella sp, and Fusobacterium sp. [7,8]. Antibiotics such as ampicillin, chlorhexidine, erythromycin, penicillin, tetracycline, and vanco-mycin have been very effective systems have been recorded in pharmacopeias as agents used to treat infections and a number of these have been recently investigated for their efficacy against oral microbial pathogens [24][25][26].
In this study, we investigated the synergistic antibacterial activity of the ethanol extact of Alismatis rhizoma (ARE) in combination with existing antimicrobial agents against oral bacteria.

Minimum inhibitory concentrations/minimum bactericidal concentrations assay
The minimum inhibitory concentrations (MICs) were determined for ARE by the broth dilution method [27], and were carried out in triplicate. The antibacterial activities were examined after incubation at 37 °C 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 acacetin 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 ARE against oral bacteria.

Checker-board dilution test
The antibacterial effects of a combination of ARE, which exhibited the highest antimicrobial activity, and antibiotics were assessed by the checkerboard test as previously described [27,28]. The antimicrobial combinations assayed included ARE with ampicillin or gentamicin. Serial dilutions of two different antimicrobial agents were mixed in cation-supplemented Mueller-Hinton broth, which is the medium usually used for dilution antimicrobial susceptibility tests. This medium is supplemented with calcium and magnesium salts to produce correct MICs with aminoglycosides and Pseudomonas aeruginosa [29]. 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 ARE 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 1/2 MIC were inoculated with a suspension of the test strain, giving a final bacterial count between 6~7×10 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, 6, 9, 12, 18 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. gingivalis.

Results and Discussion
The ARE was evaluated for their antimicrobial activities against twelve common bacterial species present in the oral cavity. The results of the antimicrobial activity showed that the ARE exhibited antimicrobial activities against cariogenic bacteria (MICs, 313 to 2500 µg/mL; MBCs, 625 to 5000 µg/mL), against periodonto pathogenic ISSN: 2377-987X bacteria (MICs, 2500 µg/mL; MBCs, 5000 µg/mL) and for ampicillin, either 0.25/0.5 or 32/64 μg/mL; for gentamicin, either 4/8 or 32/64 μg/mL on tested all bacteria ( Table 1). The range of MIC 50 and MIC 90 of the ARE were from 313 to 2500 µg/mL and 313 to 2500 µg/mL, respectively. The ARE showed stronger antimicrobial activity agains S. mutans, S. creceti, S. parasanguinis, and S. gordonii than another bacteria and the range of MIC 50 and MIC 90 were 313-625 µg/mL and 313-625 µg/mL. Natural products are a major source of chemical diversity and have provided important therapeutic agents for many bacterial diseases [27,28,30,31]. Combinations of some herbal materials and different antibiotics might affect the inhibitory effect of these antibiotics [30,31]. The synergistic effects of the ARE alone or with antibiotics were evaluted in oral bacteria ( Table 2 and Table 3). In combination with the ARE, the MIC for ampicillin was reduced ≥4-fold in cariogenic bacteria, S. mutans, S. sobrinus, S. criceti, S. parasanguinis, S. anginosus, and S. gordonii, producing a synergistic effect as defined by FICI ≤ 0.5. The MBC for ampicillin was shown synergistic effects in all cariogenic bacteria by FBCI ≤ 0.5, but not the periodontopathogenic bacteria by FBCI ≥ 0.75 (additive) (able 2). In combination with the ARE, the MIC for gentamicin was reduced     (Table 3).
Recently, interest in A. rhizoma is growing, because many of its components, such as triterpene, alisol B, and especially polysaccharides, have been reported to have important bioactivities, including anti-hepatitis, anti-inflammatory, hypoglycemic and immunological effects [20][21][22]. Four triterpenes (alisol A, alisol A monoacetate, alisol B, and alisol B monoacetate) isolated from the A. rhizoma have show anti-complementary and antibacterial activity against antibiotic resistant strains [20,32]. The polysaccharides of A. rhizoma have been documented for their effects of against lipid peroxidation, which are potentially important for human health [33]. Conventional methods, such as those by hot water, immersion or Soxhlet, are often time-consuming and expensive, with low yield of the polysaccharides of A. rhizoma or even loss of some of their pharmacological activities [33,34].
The bacterial effect of the ARE with ampicillin or gentamicin against oral bacteria was confirmed by time-kill curve experiments.
The ARE (MIC or 1/2 MIC) alone resulted rate of killing increasing or not changing in CFU/ml at time-dependent manner, with a more rapid rate of killing by ARE (1/2 MIC) with ampicillin (1/2 MIC) or gentamicin (1/2 MIC) (Figures 1 and 2). The ARE with ampicillin (1/2 MIC) combination was bactericidal effect up to and beyond 12 h exposure with all killing compared to ARE alone in S. sobrinus ISSN: 2377-987X

Conclusion
In conclusion, these findings suggest that a strong bactericidal effect of ARE was exerted in drug combinations and fulfills the ISSN: 2377-987X 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.