International Journal of Engineering Research & Science (IJOER) ISSN [2395-6992] [Vol-2, Issue-1, January- 2016] Synergistic antimicrobial activity of apigenin against oral Pathogens Su-Mi Cha1, Gi-Ug Kim2, Jeong-Dan Cha3* 1Department of Oral Microbiology and Institute of Oral Bioscience, Chonbuk National University, Jeonju, South Korea 2Department of Dental Hygiene, Pohang College, Pohang, South Korea *3Institute of Jinan Red Ginseng, Jinan-gun, South Korea Abstract— Although a broad range of biological and pharmacological activities of apigenin have been reported, the mechanism(s) behind its antibacterial effects are not fully understood. In this study, we investigated the synergistic antibacterial activity of apigenin in combination with existing antimicrobial agents against oral bacteria. The combination effect of apigenin was evaluated against oral bacteria, either alone or with antibiotics, via broth dilution method and checkerboard and time kill assay. MIC/MBC values for apigenin, ampicillin, gentamicin, erythromycin, and vancomycin against all the tested bacteria ranged between 50-200/100-800 microg/mL, 0.0313-16/0.125-32 microg/mL, 2-256/4-512 microg/mL, 0.008-32/0.016-64 microg/mL, and 0.25-64/1-128 microg/mL, respectively. Checkerboard assay revealed synergistic activity in the combination of apigenin with antibiotics at fractional inhibitory concentration index (FICI) <0.5. 1-6 hours of treatment with 1/2 MIC of apigenin 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 apigenin is important in the antibacterial actions of oral pathogen agents. Keywords— Apigenin, antibacterial activity, oral pathogen bacteria, Synergistic effect, Minimum inhibitory concentrations (MICs), Minimum bactericidal concentrations (MBCs) I. 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, 4]. The association between oral diseases and the oral microbiota is well established [5]. Of the more than 750 species of bacteria that inhabit the oral cavity, a number are implicated in oral diseases [6-8]. The development of dental caries involves acidogenic and aciduric 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 [7, 9]. In contrast, periodontal diseases are subgingival conditions that have been linked to anaerobic gram-negative bacteria such as Porphyromonas gingivalis, Actinobacillus sp., Prevotella sp., and Fusobacterium sp. [10, 11]. In periodontal diseases, the areas at or below the gingival crevice become infected causing a cellular inflammatory response of the gingiva and surrounding connective tissue [12, 13]. These inflammatory responses can manifest as 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) [13, 14]. Many plant-derived medicines used in traditional medicinal 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 [15- 19]. Flavonoids have also been shown to exhibit broader bioactivities such as protection of vascular integrity, antihepatotoxicity, anti-inflammatory activity, antitumor effect, antiallergic properties, and antimicrobial effects [20-22]. Apigenin (4,5,7-trihydroxyflavone), a flavone subclass of flavonoid widely distributed in many herbs, fruits, and vegetables, is a substantial component of the human diet and has been shown to possess a variety of biological characteristics, including anticancer, antibacterial, antioxidant, anti-apoptosis, and anti-inflammatory [23-26]. The low antibacterial activity of apigenin alone in Gram-negative bacteria may be due to the presence of lipopolysaccharide and protein-rich outer membrane which covers and protects the internal peptidoglycan wall [27]. The in vitro anti-inflammatory effect of apigenin was studied in many cases. The apigenin decreased cytokines, TNF-α, IL-1β, and IL-6 in LPS-stimulated human peripheral blood mononuclear cells [28]. Apigenin has been reported to suppress cell proliferation in various cell types, and such an anti- proliferative effect has been shown to be associated with PI3K/Akt pathway [29, 30]. Although a broad range of biological and pharmacological activities of apigenin have been reported, the mechanism(s) behind its antibacterial effects are not fully Page | 27 International Journal of Engineering Research & Science (IJOER) ISSN [2395-6992] [Vol-2, Issue-1, January- 2016] understood. In this study, we investigated the synergistic antibacterial activity of apigenin in combination with existing antimicrobial agents against oral bacteria. II. MATERIALS AND METHODS 2.1 Bacterial strains The oral bacterial strains used in this study were: Streptococcus mutans ATCC 25175, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 27607, Streptococcus ratti KCTC (Korean collection for type cultures) 3294, Streptococcus criceti KCTC 3292, Streptococcus anginosus ATCC 31412, Streptococcus gordonii ATCC 10558, Actinobacillus actinomycetemcomitans ATCC 43717, Fusobacterium nucleatum ATCC 10953, Prevotella intermedia ATCC 25611, and Porphylomonas gingivalis ATCC 33277. Brain-Heart Infusion (Difco Laboratories, Detroit, MI) broth supplemented with 1% yeast extract (Difco) was used for all bacterial strains except P. intermedia and P. gingivalis. For P. intermedia and P. gingivalis, BHI broth containing hemin 1 μg/mL (Sigma, St. Louis, MO, USA) and menadione 1 μg/mL (Sigma) was used. 2.2 Minimum inhibitory concentrations/minimum bactericidal concentrations assay The minimum inhibitory concentrations (MICs) were determined for apigenin by the broth dilution method [18], and were carried out in triplicate. 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 s and MIC s, defined as MICs at which, 50 and 90%, respectively of oral bacteria were inhibited, were 50 90 determined. Following anaerobic incubation of MICs plates, the minimum bactericidal concentrations (MBCs) were determined on the basis of the lowest concentration of apigenin that kills 99.9% of the test bacteria by plating out onto each appropriate agar plate. Ampicillin, gentamicin, erythromycin, and vancomycin (Sigma) were used as standard antibiotics in order to compare the sensitivity of apigenin against oral bacteria. 2.3 Checker-board dilution test The antibacterial effects of a combination of apigenin, which exhibited the highest antimicrobial activity, and antibiotics were assessed by the checkerboard test as previously described [18]. The antimicrobial combinations assayed included apigenin with antibiotics, ampicillin, gentamicin, erythromycin, and vancomycin. 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 apigenin that kills 99.9% of the test bacteria by plating out onto each appropriate agar plate. The fractional inhibitory concentration (FIC)/ fractional bactericidal concentration (FBC) index was calculated according to the equation: FIC/FBC index=FIC/FBC +FIC/FBC =(MIC/MBC of drug A in combination/MIC/MBC of drug A B A alone)+(MIC/MBC of drug B in combination/MIC/MBC of drug B alone). The FIC and FBC index are the sum of the FICs and FBCs of each of the drugs, which in turn is defined as the MIC and MBC of each drug when it is used in combination divided by the MIC and MBC of the drug when it is used alone. The interaction was defined as synergistic if the FIC and FBC index was less than or equal to 0.5, additive if the FIC and FBC index was greater than 0.5 and less than or equal 1.0, indifferent if the FIC and FBC index was greater than 1.0 and less than or equal to 2.0, and antagonistic if the FIC and FBC index was greater than 2.0. 2.4 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 were inoculated with a 50 suspension of the test strain, giving a final bacterial count between 5~6.6×106 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. Page | 28 International Journal of Engineering Research & Science (IJOER) ISSN [2395-6992] [Vol-2, Issue-1, January- 2016] III. RESULTS AND DISCUSSION The main etiological factor of dental caries and periodontal disease is dental plaque [31-33]. Therefore, it is reasonable to search for natural products that have antiplaque properties and antimicrobial activity against oral pathogens [16, 19, 34-35]. Apigenin was evaluated for their antimicrobial activities against eleven common bacterial species present in the oral cavity. The results of the antimicrobial activity showed that apigenin exhibited antimicrobial activities against cariogenic bacteria (MICs, 25 to 200 µg/mL; MBCs, 100 to 800 µg/mL), against periodontopathogenic bacteria (MICs, 100 to 200 µg/mL; MBCs, 200 to 400 µg/mL) and for ampicillin, either 0.0313/0.125 or 16/32 μg/mL; for gentamicin, either 2/4 or 256/512 μg/mL; for erythromycin, either 0.008/0.016 or 32/64; for vancomycin, either 0.25/1 or 64/128 on tested all bacteria (Table 1). The MIC and MIC ranges of apigenin were from 6.25 to 12.5 µg/mL and 25 to 200 µg/mL, respectively. The apigenin 50 90 showed stronger antimicrobial activity against S. ratti, S. gordonii, and P. intermedia than another bacteria (MIC/MBC, 25/50-100 µg/mL) and the range of MIC and MIC were 6.25 µg/mL and 25 µg/mL. 50 90 TABLE 1 ANTIBACTERIAL ACTIVITY OF APIGENIN AND ANTIBIOTICS IN ORAL BACTERIA Apigenin Ampicillin Gentamicin Erythromycin Vancomycin Samples MIC MIC MIC/MBC 50< 90< MIC/MBC S. mutans 25 100 100/200 0.125/0.25 8/16 0.063/0.125 1/2 ATCC 251751 S. sanguinis 12.5 50 50/200 0.25/0.5 16/32 0.016/0.031 0.25/1 ATCC 10556 S. sobrinus 25 100 100/200 0.0313/0.125 16/32 0.031/0.063 1/2 ATCC 27607 S. ratti 12.5 50 50/100 0.125/0.5 8/16 0.008/0.016 0.5/1 KCTC 32942 S. criceti 6.25 25 25/100 0.0313/0.125 8/16 0.125/0.25 1/4 KCTC 3292 S. anginosus 50 200 200/800 0.0625/0.25 8/16 0.125/0.5 1/4 ATCC 31412 S. gordonii 12.5 50 50/100 0.0625/0.25 16/32 0.031/0.063 0.5/1 ATCC 10558 A. actinomycetemcomitans 50 200 200/400 16/32 8/16 0.125/0.25 2/4 ATCC 43717 F. nucleatum 25 100 100/200 8/16 2/4 32/64 64/128 ATCC 51190 P. intermedia 50 200 200/400 1/2 32/32 16/32 16/36 ATCC 49049 P. gingivalis 25 200 200/400 0.5/0.5 256/512 2/8 8/16 ATCC 33277 1American Type Culture Collection (ATCC) 2Korean collection for type cultures (KCTC) Page | 29 International Journal of Engineering Research & Science (IJOER) ISSN [2395-6992] [Vol-2, Issue-1, January- 2016] Natural products, polyphenols are a major source of chemical diversity and have provided important therapeutic agents for many oral bacterial diseases [36-38]. Combinations of some herbal materials and different antibiotics might affect the inhibitory effect of these antibiotics [18, 39, 40]. The synergistic effects of apigenin alone or with antibiotics were evaluted in oral bacteria (Table 2 and 3). In combination with apigenin, the MIC for ampicillin was reduced ≥4-fold in tested bacteria, except S. anginosus, producing a synergistic effect as defined by FICI ≤ 0.5. The MBC for ampicillin was shown synergistic effects in S. mutans, S. sanguinis, S. ratti, S. gordonii, F. nucleatum, P. intermedia, and P. gingivalis by FBCI ≤ 0.5 (Table 2). In combination with apigenin, the MIC for gentamicin was reduced ≥4-8-fold in all tested bacteria expect S. ratti by FICI ≤ 0.5 and MBC in S. mutans, S. sobrinus, S. criceti, S. anginosus, S. gordonii, F. nucleatum, P. intermedia, and P. gingivalis by FBCI ≤ 0.5 (Table 3). Moreover, the MIC for erythromycin with apigenin was reduced ≥4-fold in tested bacteria, except S. sanguinis, S. rattii, and S. anginosus, producing a synergistic effect as defined by FICI ≤ 0.5 and the MBC for erythromycin was shown synergistic effects in S. mutans, S. criceti, S. anginosus, S. gordonii, A. actinomycetemcomitans, and P. gingivalis by FBCI ≤ 0.5 (Table 2). TABLE 2 SYNERGISTIC EFFECTS OF APIGENIN WITH AMPICILLIN AGAINST ORAL BACTERIA. MIC/MBC (μg/ml) FIC/FBC FICI/FBCI2 Outcome Strains Agent Combination1 Alone S. mutans Apigenin 100/200 12.5/50 0.125/0.125 Synergistic/ 0.375/0.375 Synergistic ATCC 251753 Ampicillin 0.125/0.25 0.0313/0.0625 0.25/0.25 S. sanguinis Apigenin 50/200 12.5/50 0.25/0.25 Synergistic/ 0.5/0.5 Synergistic Ampicillin 0.25/0.5 0.0625/0.125 0.25/0.25 ATCC 10556 S. sobrinus Apigenin 100/200 25/50 0.25/0.25 Synergistic/ 0.5/0.75 Additive Ampicillin 0.0625/0.125 0.0156/0.0625 0.25/0.5 ATCC 27607 S. ratti Apigenin 50/100 12.5/25 0.25/0.25 Synergistic/ 0.5/0.5 Synergistic KCTC 32944 Ampicillin 0.25/0.5 0.0625/0.125 0.25/0.25 S. criceti Apigenin 25/100 6.25/12.5 0.25/0.125 Synergistic/ 0.5/0.625 Additive Ampicillin 0.0625/0.125 0.0156/0.0625 0.25/0.5 KCTC 3292 S. anginosus Apigenin 200/800 50/200 0.25/0.25 Additive/ 0.75/0.75 Additive Ampicillin 0.125/0.25 0.0625/0.125 0.5/0.5 ATCC 31412 S. gordonii Apigenin 50/100 12.5/12.5 0.25/0.125 Synergistic/ 0.5/0.375 Synergistic Ampicillin 0.0625/0.25 0.0156/0.0625 0.25/0.25 ATCC 10558 A. actinomycetemcomitans Apigenin 200/400 25/100 0.125/0.25 Synergistic/ 0.25/0.75 Additive Ampicillin 16/32 2/16 0.125/0.5 ATCC 43717 F. nucleatum Apigenin 100/200 25/50 0.25/0.25 Synergistic/ 0.5/0.5 Synergistic Ampicillin 16/32 4/8 0.25/0.25 ATCC 51190 P. intermedia Apigenin 200/400 50/100 0.25/0.25 Synergistic/ 0.375/0.5 Synergistic Ampicillin 2/4 0.25/1 0.125/0.25 ATCC 49049 P. gingivalis Apigenin 200/400 50/100 0.25/0.25 Synergistic/ 0.5/0.5 Synergistic ATCC 33277 Ampicillin 0.5/1 0.125/0.25 0.25/0.25 1The MIC and MBC of apigenin with ampicillin 2 The fractional inhibitory concentration index (FIC index) 3American Type Culture Collection (ATCC) 4Korean collection for type cultures (KCTC) Page | 30 International Journal of Engineering Research & Science (IJOER) ISSN [2395-6992] [Vol-2, Issue-1, January- 2016] TABLE 3 SYNERGISTIC EFFECTS OF APIGENIN WITH GENTAMICIN AGAINST ORAL BACTERIA. MIC/MBC (μg/ml) FIC FICI2 Outcome Strains Agent Combination1 Alone S. mutans Apigenin 100/200 12.5/50 0.125/0.25 Synergistic/ 0.375/0.5 Synergistic ATCC 251753 Gentamicin 8/16 2/4 0.25/0.25 S. sanguinis Apigenin 50/200 12.5/50 0.25/0.25 Synergistic/ 0.5/0.75 Additive ATCC 10556 Gentamicin 16/32 4/16 0.25/0.5 S. sobrinus Apigenin 100/200 12.5/25 0.125/0.125 Synergistic/ 0.375/0.375 Synergistic ATCC 27607 Gentamicin 16/32 4/16 0.25/0.5 S. ratti Apigenin 50/100 12.5/25 0.5/0.25 Additive/ 0.75/0.75 Additive KCTC 32944 Gentamicin 16/16 4/8 0.25/0.5 S. criceti Apigenin 25/100 6.25/12.5 0.25/0.125 Synergistic/ 0.5/0.375 Synergistic KCTC 3292 Gentamicin 8/16 2/4 0.25/0.25 S. anginosus Apigenin 200/800 50/200 0.25/0.25 Synergistic/ 0.375/0.5 Synergistic ATCC 31412 Gentamicin 16/32 2/8 0.125/0.25 S. gordonii Apigenin 50/100 6.25/25 0.125/0.25 Synergistic/ 0.375/0.5 Synergistic ATCC 10558 Gentamicin 16/32 4/8 0.25/0.25 A. actinomycetemcomitans Apigenin 200/400 50/100 0.25/0.25 Synergistic/ 0.5/0.75 Additive ATCC 43717 Gentamicin 8/16 2/8 0.25/0.5 F. nucleatum Apigenin 100/200 12.5/25 0.125/0.125 Synergistic/ 0.375/0.375 Synergistic ATCC 51190 Gentamicin 4/8 1/2 0.25/0.25 P. intermedia Apigenin 200/400 50/100 0.25/0.25 Synergistic/ 0.375/0.75 Additive ATCC 25611 Gentamicin 32/32 4/16 0.125/0.5 P. gingivalis Apigenin 200/400 50/100 0.25/0.25 Synergistic/ 0.375/0.5 Synergistic ATCC 33277 Gentamicin 256/512 32/128 0.125/0.25 1The MIC and MBC of apigenin with gentamicin 2 The fractional inhibitory concentration index (FIC index) 3American Type Culture Collection (ATCC) 4Korean collection for type cultures (KCTC) Page | 31 International Journal of Engineering Research & Science (IJOER) ISSN [2395-6992] [Vol-2, Issue-1, January- 2016] TABLE 4 SYNERGISTIC EFFECTS OF APIGENIN WITH ERYTHROMYCIN AGAINST ORAL BACTERIA MIC/MBC (μg/ml) FIC FICI2 Outcome Strains Agent Combination1 Alone S. mutans Apigenin 100/200 25/50 0.25/0.25 Synergistic/ 0.375/0.5 Synergistic ATCC 251753 Erythromycin 0.063/0.125 0.008/0.031 0.125/0.25 S. sanguinis Apigenin 50/200 12.5/50 0.25/0.25 Additive/ 0.75/0.75 Additive ATCC 10556 Erythromycin 0.016/0.031 0.008/0.016 0.5/0.5 S. sobrinus Apigenin 100/200 25/100 0.25/0.5 Synergistic/ 0.5/0.75 Additive ATCC 27607 Erythromycin 0.031/0.063 0.008/0.016 0.25/0.25 S. ratti Apigenin 50/100 12.5/25 0.25/0.25 Additive/ 0.75/0.75 Additive KCTC 32944 Erythromycin 0.008/0.016 0.004/0.008 0.5/0.5 S. criceti Apigenin 25/100 6.25/25 0.25/0.25 Synergistic/ 0.5/0.5 Synergistic KCTC 3292 Erythromycin 0.125/0.25 0.031/0.063 0.25/0.25 S. anginosus Apigenin 200/800 50/100 0.25/0.125 Additive/ 0.75/0.375 Synergistic ATCC 31412 Erythromycin 0.125/0.5 0.063/0.0125 0.5/0.25 S. gordonii Apigenin 50/100 12.5/25 0.25/0.25 Synergistic/ 0.5/0.5 Synergistic ATCC 10558 Erythromycin 0.031/0.063 0.008/0.016 0.25/0.25 A. actinomycetemcomitans Apigenin 200/400 50/100 0.25/0.25 Synergistic/ 0.5/0.5 Synergistic ATCC 43717 Erythromycin 0.125/0.25 0.031/0.063 0.25/0.25 F. nucleatum Apigenin 100/200 25/50 0.25/0.25 Synergistic/ 0.5/0.75 Additive ATCC 51190 Erythromycin 32/64 8/32 0.25/0.5 P. intermedia Apigenin 200/400 50/200 0.25/0.5 Synergistic/ 0.5/0.75 Additive ATCC 25611 Erythromycin 16/32 4/8 0.25/0.25 P. gingivalis Apigenin 200/400 50/100 0.25/0.25 Synergistic/ 0.375/0.5 Synergistic ATCC 33277 Erythromycin 2/8 0.25/2 0.125/0.25 1The MIC and MBC of apigenin with erythromycin 2 The fractional inhibitory concentration index (FIC index) 3American Type Culture Collection (ATCC) 4Korean collection for type cultures (KCTC) Page | 32 International Journal of Engineering Research & Science (IJOER) ISSN [2395-6992] [Vol-2, Issue-1, January- 2016] TABLE 5 SYNERGISTIC EFFECTS OF APIGENIN WITH VANCOMYCIN AGAINST ORAL BACTERIA. MIC/MBC (μg/ml) FIC FICI2 Outcome Strains Agent Combination1 Alone S. mutans Apigenin 100/200 25/100 0.25/0.5 Synergistic/ 0.5/0.75 Additive ATCC 251753 Vancomycin 1/2 0.25/0.5 0.25/0.25 S. sanguinis Apigenin 50/200 12.5/50 0.25/0.25 Synergistic/ 0.5/0.375 Synergistic ATCC 10556 Vancomycin 0.25/1 0.063/0.125 0.25/0.125 S. sobrinus Apigenin 100/200 25/50 0.25/0.25 Synergistic/ 0.5/0.5 Synergistic ATCC 27607 Vancomycin 1/2 0.25/0.5 0.25/0.25 S. ratti Apigenin 50/100 12.5/50 0.25/0.5 Synergistic/ 0.5/0.75 Additive KCTC 32944 Vancomycin 0.5/1 0.125/0.25 0.25/0.25 S. criceti Apigenin 25/100 6.25/25 0.25/0.25 Synergistic/ 0.5/0.5 Synergistic KCTC 3292 Vancomycin 1/4 0.25/1 0.25/0.25 S. anginosus Apigenin 200/800 50/100 0.25/0.125 Synergistic/ 0.5/0.25 Synergistic ATCC 31412 Vancomycin 1/4 0.25/0.5 0.25/0.125 S. gordonii Apigenin 50/100 12.5/25 0.25/0.25 Synergistic/ 0.5/0.5 Synergistic ATCC 10558 Vancomycin 0.5/1 0.125/0.25 0.25/0.25 A. actinomycetemcomitans Apigenin 200/400 50/100 0.25/0.25 Synergistic/ 0.5/0.75 Additive ATCC 43717 Vancomycin 2/4 0.5/2 0.25/0.5 F. nucleatum Apigenin 100/200 25/100 0.25/0.5 Synergistic/ 0.5/0.75 Additive ATCC 51190 Vancomycin 64/128 16/32 0.25/0.25 P. intermedia Apigenin 200/400 50/100 0.25/0.25 Synergistic/ 0.5/0.75 Additive ATCC 25611 Vancomycin 16/36 4/16 0.25/0.5 P. gingivalis Apigenin 200/400 50/100 0.25/0.25 Synergistic/ 0.5/0.5 Synergistic ATCC 33277 Vancomycin 8/16 2/4 0.25/0.25 1The MIC and MBC of apigenin with vancomycin 2 The fractional inhibitory concentration index (FIC index) 3American Type Culture Collection (ATCC) 4Korean collection for type cultures (KCTC) Page | 33 International Journal of Engineering Research & Science (IJOER) ISSN [2395-6992] [Vol-2, Issue-1, January- 2016] Flavonoid complexes attach with extra cellular soluble protein and with bacterial cell wall. Thus they exhibit antibacterial activity [41, 42]. Naturally derived apigenin was reported to have potential antimicrobial activity [26, 27, 43]. Trihydroxyflavone or apigenin is a naturally occurring bioflavonoid abundantly present in fruits and vegetables whose antibacterial activity against certain strains of Gram-negative and Gram-positive bacteria like Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Pseudomonas aeruginosa has been reported recently [26,27,43,44]. In this study, apigenin, a flavone compound found in several plants also shows susceptibility on gram-positive bacteria as well as gram-negative bacteria. The bacterial effect of apigenin with ampicillin or gentamicin against oral bacteria was confirmed by time-kill curve experiments. The apigenin (MIC or MIC ) alone resulted rate of killing increasing or not 50 changing in CFU/ml at time dependent manner, with a more rapid rate of killing by apigenin (MIC ) with ampicillin or/and 50 gentamicin (MIC ) (Fig 1-2). A strong bactericidal effect was exerted in drug combinations. 50 FIG. 1. TIME-KILL CURVES OF MIC OF APIGENIN ALONE AND ITS COMBINATION WITH MIC50 OF AMP OR GEN AGAINST. S. MUTANS, S. SANGUINIS, S. SOBRINUS, S. ANGINOSUS, S. CRICETI, AND S. RATTI. BACTERIA WERE INCUBATED WITH APIGENIN (●), APIGENIN + AMP (○), AND APIGENIN + GEN (▼) OVER TIME. DATA POINTS ARE THE MEAN VALUES±S.E.M. OF SIX EXPERIMENTS. CFU, COLONY-FORMING UNITS. Page | 34 International Journal of Engineering Research & Science (IJOER) ISSN [2395-6992] [Vol-2, Issue-1, January- 2016] FIG. 2. TIME-KILL CURVES OF MIC OF APIGENIN ALONE AND ITS COMBINATION WITH MIC50 OF AMP OR GEN AGAINST S. GORDONII, A. ACTINOMYCETEMCOMITANS, F. NUCLEATUM, P. INTERMEDIA, AND P. GINGIVALIS. BACTERIA WERE INCUBATED WITH APIGENIN (●), APIGENIN + AMP (○), AND APIGENIN + GEN (▼) OVER TIME. DATA POINTS ARE THE MEAN VALUES±S.E.M. OF SIX EXPERIMENTS. CFU, COLONY- FORMING UNITS. Page | 35 International Journal of Engineering Research & Science (IJOER) ISSN [2395-6992] [Vol-2, Issue-1, January- 2016] IV. CONCLUSION In conclusion, these findings suggest that apigenin, a flavone compound found in several plants 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. ACKNOWLEDGEMENTS This work was supported by a Korea Research Foundation Grant funded by the Korean Government (KRF-20110023479). CONFLICT OF INTEREST STATEMENT The authors have declared no conflict of interest. AUTHORS’ CONTRIBUTION Jeong-Dan Cha has substantial contributions to conception and design and drafting and revising it. 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