Antibacterial Activity of Sophoraflavanone G Isolated from the Roots of Sophora Flavescens and Red Ginseng Extract

International Journal of Medicinal Plants. Photon 106 (2014) 418-429 https://sites.google.com/site/photonfoundationorganization/home/international-journal-of-medicinal-plants Original Research Article. 6672-4384

International Journal of Medicinal Plants Ph ton

Antibacterial activity of Sophoraflavanone G isolated from the roots of Sophora flavescens and Red Ginseng Extract

Jeong-Dan Cha a, Mi-Ran Jeong a, Seung-Mi Hwang a, Ji-Ye Lim a, Jea-Ran Kang a, Tae-Bum Lee b, Min-Jung Lee b, Hoo-Kil Jung c, Joo-Hee Lee c, Kyung-Min Choi a* a Institute of Jinan Red Ginseng, 520-9 Banwol-ri, Jinan-eup, Jinan-gun, Jeollabuk-do, 567-801 South Korea b Gochang Black Raspberry Research Institute, 558 Bokbunja-ro, Buan-myun, Gochang-gun, Jeollabuk-do, South Korea c Imsil Research Institute of Cheese Science, 50 Doin 2-gil, Seongsu-myeon, Imsil-gun, Jeollabuk-do 566-881, South Korea

The authors receive Thomas Edison Award-2014 in Lee M.J. Medicinal Plants for Inspiration and Knowledge Researcher Distribution among young research scholars. Email: [email protected]

Article history: Jung H.K. Received: 20 June, 2013 President Accepted: 26 June , 2013 Email: [email protected] Available online: 30 January, 2014 Lee J.H. Abbreviations: Researcher MICs: Minimum Inhibitory Concentrations, MBCs: Email: [email protected] Minimum Bactericidal Concentrations, CFU: Colony Forming Unit, FIC index: Fractional Inhibitory Abstract Concentration, FBC index: Fractional Bactericidal The objective of this study was to investigate the Concentration index, RGE: Red Ginseng Extract antibacterial activities of sophoraflavanone G from Sophora flavescens and Red Ginseng Extract Keywords: Sophora flavescens , Sophoraflavanone G, Red Ginseng (RGE) in combination with two antimicrobial agents extract, antibacterial activity, Checker board method, time against oral bacteria. The effects of kill method, synergic effect sophoraflavanone G + RGE combination was synergistic against all tested oral bacteria by FICI Corresponding Author: <0.5, except S. sanguinis and A. Choi K.M.* Ph.D actinomycetemcomitans and against S. sanguinis, Lab Head S. ratti, S. anginosus, F. nucleatum, P. intermedia, Email: [email protected] and P. gingivalis (FBCI <0.5). In particular, the Phone: +82634320913 Fax No: +82634320910 MICs/MBCs were reduced to one half ~ one sixteenth as a result of the combination for all Cha J.D. Ph.D bacteria. Furthermore, a time-kill study showed that Email: [email protected] the growth of the tested bacteria was completely attenuated after 2-6 h of treatment with the 1/2 MIC Jeong M.R. Ph.D of sophoraflavanone G and RGE, regardless of Email: [email protected] whether it was administered alone or combination or with ampicillin or gentamicin. The in vitro data Hwang S.M. Researcher suggest that sophoraflavanone G combined with Email: [email protected] RGE or other antibiotics may be microbiologically beneficial and not antagonistic. Lim J.Y. Researcher Citation: Email: [email protected] Cha J.D., Jeong M.R., Hwang S.M., Lim J.Y., Kang J.R., Lee T.B., Lee M.J., Jung H.K., Lee J.H., Choi K.M., 2014. Kang J.R. Antibacterial activity of Sophoraflavanone G isolated from Researcher the roots of Sophora flavescens and Red Ginseng Extract. Email: [email protected] International Journal of Medicinal Plants. Photon 106, 418-429. Lee T.B. Ph.D Email: [email protected]

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1. Introduction

Dental caries and periodontal disease are have a radioprotective effect against radiation- prevalent worldwide. Bacteria existing in the induced double-strand breaks in DNA and dental plaque or biofilm play an important role immunomodulatory activity, as evidenced by in the development of both dental caries and its stimulation of natural killer cells (Kenarova periodontal disease (Iwano et al., 2010; et al., 1990; Seo et al., 2013). Ginsenosides Marsh, 2005). Specific bacteria are also exhibit less potent but broad-spectrum closely related to specific dental diseases, for antibacterial activity against Gram-positive and example, Streptococcus mutans and Gram-negative bacterial strains, including the Porphyromonas gingivalis are associated with clinical isolates of MRSA (Kochan et al., 2013; dental caries and periodontal disease, Sung, Lee, 2008). respectively (Iwano et al., 2010; Burton et al., 2011; Sambunjak et al., 2011). Dental caries Sophorae radix , the dried root of Sophora and periodontitis are common oral diseases flavescens AITON (Leguminosae), is an caused by bacterial infection and the important herbal medicine, which has been development of dental plaque (Marsh, 1992; traditionally employed as an antipyretic, Loesche, 2007). Several antibacterial agents analgesic, antihelminthic, and stomachic, and including, fluorides, phenol derivatives, has been shown to exert antibacterial, anti- ampicillin, erythromycin, penicillin, tetracycline, inflammatory, antioxidant, and antimalarial and vancomycin have been used widely in effects (Kuroyanagi et al., 1999; Kim et al., dentistry to inhibit bacterial growth (Levi, 2004; Hsiang et al., 2001). S. flavescens is Eusterman, 2011; Nguyen, Marqui, 2011; De also known to harbor numerous flavonoids, Poi, 2001; Emilson, 1994). However, including formononetin, kushenol E, kushenol excessive use of these chemicals can result in B, sophoraflavanone G, kushenol L, kushenol derangements of the oral and intestinal flora M, kuraridin, kurarinone, kushenol N, and and cause side effects such as microorganism kushenol F (Jin et al., 2010; DeNaeyer et al., susceptibility, vomiting, diarrhoea and tooth 2004; Ryu et al., 1960; Shen et al., 2006). staining (Schaeken et al., 1991; Ekstrand et Recently, several researchers have reported al., 1998; Feres et al., 2010). These problems that these compounds have proven effective necessitate further search for natural against tumors, arrhythmia, and antibacterial agents that are safe for humans immunodeficiency, antitumor, antioxidant, and and specific for oral pathogens. Natural antibacterial activity and thus have generated products have recently been investigated more a great deal of attention and interest (Jin et al., thoroughly as promising agents to prevent oral 2010; DeNaeyer et al., 2004; Kim et al., 2002; diseases, especially plaque-related diseases Zhang et al., 2006). We reported previously such as dental caries (Feldman et al., 2011; that sophoraflavanone G isolated from the Greenberg et al., 2008). roots of Sophora flavescens exerts antibacterial effects against oral bacteria (Cha Ginseng root (Korean ginseng, Panax ginseng et al., 2007). C.A. Meyer) has been used for thousands of years in the traditional medical system in 2. Objective of Research oriental countries (Park, 1996; Sung, 1986). The main active components of P. ginseng are In this study, the antimicrobial activities of the triterpenoid saponins, called ginseng sophoraflavanone G isolated from Sophora saponins or ginsenosides, which have been flavescens roots and red ginseng extract shown to have a variety of biological against oral pathogens when used alone and properties including anti-inflammatory, in combination with antibiotics were assessed. antioxidant, and anticancer effects (Jung and Jin, 1996; Shibata et al., 1965; Kaneko and 3. Experimentals Nakanishi, 2004; Song, 2004). Red ginseng is made from steam heat treatment of fresh 3.1 Sample preparation ginseng roots. The red ginseng contains RGE derived from the root of P. ginseng was specific ginsenoside-Rh1, ginsenoside-Rh2, provided from KUNBO Corporation (Jinangun, 20(S)-ginsenoside Rg2, 20(S)-ginsenoside South Korea). RGE is made through a Rg3, and these are not detected or even as secondary process that concentrates the water trace amount in fresh and dried ginseng roots extract of six-year-old RG roots, and it was (Kim et al., 1991; Kim et al., 2013). These dissolved directly in 5% diluted ethanol, (1.0% specific ginsenosides of red ginseng can be as final concentration). The general used for important medicine. Ginsenosiedes composition of the product offered by KUNBO

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Corporation is as follows: moisture 34%, solid inhibited, were determined. Following volume 64%, ash 2.3%, total fat 0.02%, total anaerobic incubation of MICs plates, the crude saponin 70 mg/g, and ginsenosides minimum bactericidal concentrations (MBCs) (Rg1+Rb1) 20 mg/g. were determined on the basis of the lowest concentration of CT that kills 99.9% of the test The S. flavescens roots were collected in bacteria by plating out onto each appropriate October 2001, in Jinan, Jeonbuk Province, agar plate. Ampicillin (Sigma) and gentamicin Korea. The identity of the roots was confirmed (Sigma) were used as standard antibiotics in by Dr. Bong-Seop Kil at the College of Natural order to compare the sensitivity of Science, Wonkwang University. A voucher sophoraflavanone G and RGE against test specimen (JS01-3) has been deposited at the bacteria. Herbarium of the Department of Biomedicinal Chemistry and Cosmetics, at the College of 3.4 Checker-board dilution assay Technology and Natural Science of Mokwon The antibacterial effects of a combination of University. The structure of sophoraflavanone sophoraflavanone G and RGE, which G isolated from S. flavescens roots was exhibited the highest antimicrobial activity, and identified via comparisons of their spectral antibiotics were assessed by the checkerboard data (mp, MS, [ α], 1D NMR and 2D NMR) with test as previously described (Cha et al., 2007; those reported in the relevant literature (Cha et Chatterjee et al., 2009). The antimicrobial al., 2007; Ryu et al., 1960). combinations assayed included sophoraflavanone G and RGE with ampicillin 3.2 Bacterial strains or gentamicin. Serial dilutions of two different The cariogenic bacterial strains used in this antimicrobial agents were mixed in cation- study were: Streptococcus mutans ATCC supplemented Mueller-Hinton broth. After 24 h 25175, Streptococcus sanguinis ATCC 10556, of incubation at 37° C, the MIC was Streptococcus sobrinus ATCC 27607, determined to be the minimal concentration at Streptococcus ratti KCTC (Korean collection which there was no visible growth. The for type cultures) 3294, Streptococcus criceti fractional inhibitory concentration (FIC) index KCTC 3292, Streptococcus anginosus ATCC and fractional bactericidal concentration (FBC) 31412, and Streptococcus gordonii ATCC index are the sum of the FICs and FBCs of 10558 and the periodontopathogenic bacterial each of the drugs, which in turn is defined as strains used: Actinobacillus the MIC and MBC of each drug when it is used actinomycetemcomitans ATCC 43717, in combination divided by the MIC and MBC of Fusobacterium nucleatum ATCC 10953, the drug when it is used alone. The interaction Prevotella intermedia ATCC 25611, and was defined as synergistic if the FIC and FBC Porphylomonas gingivalis ATCC 33277. Brain- index was less than or equal to 0.5, additive if Heart Infusion (BHI) broth supplemented with the FIC and FBC index was greater than 0.5 1% yeast extract (Difco Laboratories, Detroit, and less than or equal 1.0, indifferent if the MI) was used for cariogenic bacterial strains. FIC and FBC index was greater than 1.0 and For periodontopathogenic bacterial strains, less than or equal to 2.0, and antagonistic if BHI broth containing hemin 1 µg/ml (Sigma, the FIC and FBC index was greater than 2.0 St. Louis, MO, USA) and menadione 1 µg/ml (Cha et al., 2007; Chatterjee et al., 2009). (Sigma) was used. 3.5 Time-kill assay 3.3 Minimum Inhibitory Concentrations/ A time-kill kinetic study against oral bacteria Minimum Bactericidal Concentrations assay was performed using the broth macrodilution The minimum inhibitory concentrations (MICs) method (Cha et al., 2007). The following were determined for sophoraflavanone G and samples were incubated in BHI medium at 37° RGE by the broth dilution method (Cha et al., C under anaerobic conditions: oral bacteria 5- 2007), and were carried out in triplicate. The 7×10 6 CFU/ml with sophoraflavanone G (MIC); antibacterial activities were examined after with sophoraflavanone G (1/2 MIC) and RGE incubation at 37° C for 18 h (facultative (1/2 MIC); with sophoraflavanone G (1/2 MIC) anaerobic bacteria), for 24 h (microaerophilic and Amp (1/2 MIC); and with bacteria), and for 1-2 days (obligate anaerobic sophoraflavanone G (1/2 MIC) and Gen (1/2 bacteria) under anaerobic conditions. MICs MIC) and bacteria 5-7×10 6 CFU/ml with RGE were determined as the lowest concentration (MIC); with RGE (1/2 MIC) and Amp (1/2 MIC); of test samples that resulted in a complete and with RGE (1/2 MIC) and Gen (1/2 MIC). At inhibition of visible growth in the broth. MIC 50s 0, 30 min and 1, 2, 3, 4, 5, 6, 12, and 24 h, and MIC 90s , defined as MICs at which, 50 and samples were taken and viable counts were 90%, respectively of oral bacteria were determined as follows. Colony counts were

Ph ton 420 performed in duplicate, and means were (MICs, 0.2 to 6.4 µg/ml; MBCs, 0.8 to 12.8 taken. The solid media used for colony counts µg/ml) and RGE against cariogenic bacteria were Brain-Heart Infusion (BHI) agar for (MICs, 10 to 40 mg/ml; MBCs, 20 to 80 mg/ml) streptococci and Brain-Heart Infusion agar and periodontopathogenic bacteria (MICs, 20 containing hemin and menadione for P. to 40 mg/ml; MBCs, 40 to 80 mg/ml) and for intermedia and P. gingivalis . ampicillin, either 0.0313/0.125 or 16/32 µg/ml; for gentamicin, either 2/4 or 256/512 µg/ml on tested all bacteria (Table 1, Fig. 1-4). The 4. Results and Discussion MIC 50 and MIC 90 determinations for cariogenic and periodontopathogenic bacteria were With the increase in the incidence of confirmed similar antibacterial activity of resistance to antibiotics, alternative natural sophoraflavaone G and RGE. The range of products of plants could be of interest. Some MIC 50 and MIC 90 of sophoraflavanone G were plant extracts and phytochemicals are known from 0.05 to 1.6 µg/ml and 0.2 to 6.4 µg/ml to have antimicrobial properties, which could and RGE from 1.25 to 10 mg/ml and 10 to 40 be of great importance in the therapeutic mg/ml, respectively. The sophoraflavanone G treatments (Song et al., 2004; Sung WS, Lee, showed the strongest antimicrobial activity 2008; Hsiang et al., 2001; Kim et al., 2002; against cariogenic bacteria, S. gordonii Chen et al., 2000; Mahady, 2005). Many (MIC/MBC, 0.8/0.8 µg/ml) and plants have been evaluated not only for direct periodontopathogenic bacteria (MIC/MBC, antimicrobial activity but also as resistance- 0.2/0.8 µg/ml) and the range of MIC 50 and modifying agents (Mahady, 2005; Garvey et MIC 90 were 0.1 µg/ml and 0.8 µg/ml and 0.05 al., 2011; Radhika et al., 2011). In this study, µg/ ml and 0.8 µg/ml, respectively (Table 1). sophoraflavanone G and RGE were evaluated Phytochemical constituents such as alkaloids, for their antimicrobial activities against eleven flavonoids, tannins, phenols, saponins, and common bacterial species present in the oral several other aromatic compounds are cavity. The results of the antimicrobial activity secondary metabolites of plants that serve a showed that sophoraflavanone G exhibited defence mechanism against prediction by antimicrobial activities against cariogenic many microorganisms, insects and other bacteria (MICs, 0.8 to 3.2 µg/ml; MBCs, 0.8 to herbivores (Konaté et al., 2012; Chen et al., 6.4 µg/ml) and periodontopathogenic bacteria 2000; Wang et al., 2012; Hsu et al., 2003).

Table 1: Antibacterial activity of sophoraflavanone G, RGE, and antibiotics in oral bacteria Samples Sophoraflavanone G (µg/ml) RGE (mg/ml) Ampicillin Gentamicin MIC/MB MIC MIC MIC/MBC MIC MIC MIC/MBC ( µg/ml) 50< 90< 50< 90< C S. mutans 0.4 3.2 3.2/3.2 1.25 10 10/40 0.0625/0.25 8/16 ATCC 25175 1 S. sanguinis 0.8 3.2 3.2/6.4 2.5 10 10/40 0.25/0.5 16/32 ATCC 10556 S. sobrinus 0.4 3.2 3.2/3.2 1.25 10 10/20 0.0313/0.125 16/32 ATCC 27607 S. ratti 2 0.2 1.6 1.6/3.2 5 20 20/40 0.125/0.5 8/16 KCTC 3294 S. criceti 0.4 1.6 1.6/3.2 1.25 10 10/20 0.0313/0.125 8/16 KCTC 3292 S. anginosus 0.4 3.2 3.2/6.4 5 40 40/80 0.0625/0.25 8/16 ATCC 31412 S. gordonii 0.1 0.8 0.8/0.8 2.5 10 10/20 0.0625/0.25 16/32 ATCC 10558 A. actinomycetemco 0.8 3.2 3.2/3.2 5 20 20/40 16/32 8/16 mitans ATCC 43717 F. nucleatum 1.6 6.4 6.4/12.8 10 40 40/80 8/16 2/4 ATCC 51190 P. intermedia 0.8 3.2 3.2/6.4 2.5 20 20/40 1/2 32/32 ATCC 49049 P. gingivalis 0.05 0.2 0.2/0.8 5 20 20/40 0.5/0.5 256/512 ATCC 33277 1 American Type Culture Collection (ATCC); 2 Korean collection for type cultures (KCTC)

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Table 2: Synergistic effects of sophoraflavanone G with ampicillin against oral bacteria Strains Agent MIC/MBC 1 FIC/FBC 3 FICI/FBCI 4 Outcome Alone Combination 2 S. mutans Sophoraflavanone G 3.2/3.2 0.4 /0.4 0.125/0.125 0.375/0.375 Synergistic/ ATCC 25175 5 Ampicillin 0.25/0.5 0.0625/0.125 0.25/0.25 Synergistic S. sanguinis Sophoraflavanone G 3.2/3.2 0.8/1.6 0.25/0.5 0.5/0.75 Synergistic ATCC 10556 Ampicillin 1/2 0.25/0.5 0.25/0.25 / Additive S. sobrinus Sophoraflavanone G 3.2/3.2 0.8/1.6 0.25/0.5 0.313/0.75 Synergistic/ ATCC 27607 Ampicillin 0.25/0.5 0.0313/0.125 0.063/0.25 Additive S. ratti Sophoraflavanone G 1.6/3.2 0.8/0.8 0.5/0.25 1/0.5 Additive / KCTC 3294 6 Ampicillin 1/2 0.5/0.5 0.5/0.25 Synergistic S. criceti Sophoraflavanone G 1.6/3.2 0.4/0.8 0.25/0.25 0.75/0.5 Additive/ KCTC 3292 Ampicillin 0.5/1 0.25/0.25 0.5/0.25 Synergistic S. anginosus Sophoraflavanone G 3.2/6.4 0.8/0.8 0.25/0.125 0.75/0.625 Additive/ ATCC 31412 Ampicillin 1/1 0.5/0.5 0.5/0.5 Additive S. gordonii Sophoraflavanone G 0.8/0.8 0.1/0.2 0.125/0.25 0.375/0.5 Synergistic/ ATCC 10558 Ampicillin 1/2 0.025/0.5 0.25/0.25 Synergistic A. Sophoraflavanone G 3.2/3.2 0.4/0.8 0.125/0.25 0.31/0.5 Synergistic/ actinomycete Ampicillin 32/64 8/16 0.25/0.25 Synergistic mcomitans ATCC 43717 F. nucleatum Sophoraflavanone G 6.4/12.8 0.8/1.6 0.125/0.125 0.375/0.375 Synergistic ATCC 51190 Ampicillin 4/4 1/1 0.25/0.25 / Synergistic P. intermedia Sophoraflavanone G 3.2/6.4 0.8/1.6 0.25/0.25 0.5/0.5 Synergistic/ ATCC 49049 Ampicillin 4/8 1/2 0.25/0.25 Synergistic P. gingivalis Sophoraflavanone G 0.2 /0.8 0.05/0.1 0.25/0.125 0.375/0.25 Synergistic/ ATCC 33277 Ampicillin 0.25/0.5 0.0313/0.062 0.125/0.125 Synergistic 5 1 µg/ml; 2 The MIC and MBC of the sophoraflavanone G with ampicillin; 3 The fractional inhibitory concentration (FIC)/ the fractional bactericidal concentration (FBC); 4 The fractional inhibitory concentration index (FIC index)/ the fractional bactericidal concentration index (FBC index); 5 American Type Culture Collection (ATCC); 6 Korean collection for type cultures (KCTC)

Table 3: Synergistic effects of sophoraflavanone G with gentamicin against oral bacteria Strains Agent MIC/MBC 1 FIC/FBC 3 FICI/ Outcome 4 Alone Combination 2 FBCI S. mutans Sophoraflavanone G 3.2/3.2 0.8/0.8 0.25/0.25 0.75/0.5 Additive/ 5 ATCC 25175 Gentamicin 8/16 4/4 0.5/0.25 Synergistic S. sanguinis Sophoraflavanone G 3.2/6.4 0.4/0.8 0.125/0.125 0.375/ Synergistic/ ATCC 10556 Gentamicin 64/64 16/16 0.25/0.25 0.375 Synergistic S. sobrinus Sophoraflavanone G 3.2/3.2 1.6/1.6 0.5/0.5 0.75/0.7 Additive/ ATCC 27607 Gentamicin 4/8 1/2 0.25/0.25 5 Additive S. ratti Sophoraflavanone G 1.6/3.2 0.8/0.8 0.5/0.25 1/0.5 Additive / 6 KCTC 3294 Gentamicin 16/32 8/8 0.5/0.25 Synergistic S. criceti Sophoraflavanone G 1.6/3.2 0.4/0.4 0.25/0.125 0.375/ Synergistic/ KCTC 3292 Gentamicin 8/16 1/2 0.125/0.125 0.25 Synergistic S. anginosus Sophoraflavanone G 3.2/6.4 0.8/0.8 0.25/0.125 0.281/ Synergistic/ ATCC 31412 Gentamicin 32/32 1/4 0.031/0.125 0.25 Synergistic S. gordonii Sophoraflavanone G 0.8/0.8 0.1/0.1 0.125/0.125 0.625/ Additive/ ATCC 10558 Gentamicin 32/32 16/16 0.5/0.5 0.625 Additive A. Sophoraflavanone G 3.2/3.2 0.8 /1.6 0.25/0.5 0.5/ Synergistic/ actinomycetemc Gentamicin 4/8 1/2 0.25/0.25 0.75 Additive omitans ATCC 43717 F. nucleatum Sophoraflavanone G 6.4/12.8 1.6/1.6 0.25/0.125 0.5/0.25 Synergistic/ ATCC 51190 Gentamicin 2/4 0.5/0.5 0.25/0.125 Synergistic P. intermedia Sophoraflavanone G 3.2/6.4 0.4/0.8 0.125/0.125 0.375/ Synergistic/ ATCC 25611 Gentamicin 16/32 4/4 0.25/0.125 0.25 Synergistic P. gingivalis Sophoraflavanone G 0.2 /0.8 0.05/0.1 0.25/0.125 0.31/ Synergistic/ ATCC 33277 Gentamicin 256/512 16/64 0.063/0.125 0.25 Synergistic 1 µg/ml; 2 The MIC and MBC of the sophoraflavanone G with gentamicin; 3 The fractional inhibitory concentration (FIC)/ the fractional bactericidal concentration (FBC); 4 The fractional inhibitory concentration index (FIC index)/

Ph ton 422 the fractional bactericidal concentration index (FBC index); 5 American Type Culture Collection (ATCC); 6 Korean collection for type cultures (KCTC)

Table 4: Synergistic effects of sophoraflavanone G with RGE against oral bacteria Strains Agent 1 MIC/MBC 1 FIC/FBC 3 FICI/FBCI 4 Outcome Alone Combination2 S. mutans Sophoraflavanone G 3.2/3.2 0.8/1.6 0.25/0.5 0.5/0.75 Synergistic/ 5 ATCC 25175 RGE 10/40 2.5/10 0.25/0.25 Additive S. sanguinis Sophoraflavanone G 3.2/6.4 0.8/1.6 0.2/0.25 0.75/0.5 Additive/ ATCC 10556 RGE 10/40 2.5/10 0.25/0.25 Synergistic S. sobrinus Sophoraflavanone G 3.2/3.2 0.8/1.6 0.25/0.5 0.5/0.75 Synergistic/ ATCC 27607 RGE 10/20 2.5/5 0.25/0.25 Additive S. ratti Sophoraflavanone G 1.6/3.2 0.4/0.8 0.25/0.25 0.5/0.5 Synergistic/ KCTC 32946 RGE 20/40 5/10 0.25/0.25 Synergistic S. criceti Sophoraflavanone G 1.6/3.2 0.8/1.6 0.25/0.5 0.5/0.75 Synergistic/ KCTC 3292 RGE 10/20 2.5/5 0.25/0.25 Additive S. anginosus Sophoraflavanone G 3.2/6.4 0.4/1.6 0.125/0.25 0.375/0.5 Synergistic/ ATCC 31412 RGE 40/80 10/20 0.25/0.25 Synergistic S. gordonii Sophoraflavanone G 0.8/0.8 0.2/0.4 0.25/0.5 0.5/0.75 Synergistic/ ATCC 10558 RGE 10/20 2.5/5 0.25/0.25 Additive A. Sophoraflavanone G 3.2/3.2 1.6/1.6 0.5/0.5 0.75/0.75 Additive/ actinomycete RGE 20/40 5/10 0.25/0.25 Additive mcomitans ATCC 43717 F. nucleatum Sophoraflavanone G 6.4/12.8 1.6/3.2 0.25/0.25 0.5/0.5 Synergistic/ ATCC 51190 RGE 40/80 10/20 0.25/0.25 Synergistic P. intermedia Sophoraflavanone G 3.2/6.4 0.8/1.6 0.25/0.25 0.5/0.5 Synergistic/ ATCC 25611 RGE 20/40 5/10 0.25/0.25 Synergistic P. gingivalis Sophoraflavanone G 0.2 /0.8 0.05/0.1 0.25/0.125 0.5/0.375 Synergistic/ ATCC 33277 RGE 20/40 5/10 0.25/0.25 Synergistic 1 Sophoraflavanone G: µg/ml, RGE: mg/ml; 2The MIC and MBC of sophoraflavanone G with RGE; 3 The fractional inhibitory concentration (FIC)/ the fractional bactericidal concentration (FBC); 4 The fractional inhibitory concentration index (FIC index)/ the fractional bactericidal concentration index (FBC index); 5 American Type Culture Collection (ATCC); 6 Korean collection for type cultures (KCTC)

Table 5: Synergistic effects of RGE with ampicillin against oral bacteria Strains Agent MIC/MBC 1 FIC/FBC 3 FICI/FBCI 4 Outcome Alone Combination 2 S. mutans RGE 10/40 2.5/10 0.5/0.5 Additive / 5 0.75/0.75 ATCC 25175 Ampicillin 0.25/0.5 0.063/0.125 0.25/0.25 Additive S. sanguinis RGE 10/40 2.5/10 0.25/0.25 Synergistic / 0.5/0.5 ATCC 10556 Ampicillin 1/2 0.25/0.5 0.25/0.25 Synergistic S. sobrinus RGE 10/20 2.5/10 0.25/0.5 Synergistic/ 0.5/0.75 ATCC 27607 Ampicillin 0.25/0.5 0.063/0.125 0.25/0.25 Additive S. ratti RGE 20/40 10/20 0.5/0.5 Additive / 6 0.75/0.75 KCTC 3294 Ampicillin 1/2 0.25/0.5 0.25/0.25 Additive S. criceti RGE 10/20 2.5/5 0.25/0.25 Synergistic / 0.5/0.5 KCTC 3292 Ampicillin 0.5/1 0.125/0.25 0.25/0.25 Synergistic S. anginosus RGE 40/80 10/20 0.25/0.25 Synergistic / 0.5/0.75 ATCC 31412 Ampicillin 1/1 0.25/0.5 0.25/0.5 Additive S. gordonii RGE 10/20 2.5/10 0.25/0.5 Synergistic/ 0.5/0.75 ATCC 10558 Ampicillin 1/2 0.25/0.5 0.25/0.25 Additive A. RGE 20/40 10/10 0.5/0.25 actinomycetemc Additive / 1.0/0.5 omitans Ampicillin 32/64 16/16 0.5/0.25 Synergistic ATCC 43717 F. nucleatum RGE 40/80 20/40 0.5/0.5 Additive / 0.75/1.0 ATCC 51190 Ampicillin 4/4 1/2 0.25/0.5 Additive P. intermedia RGE 20/40 2.5/5 0.25/0.125 Synergistic/ 0.5/0.375 ATCC 49049 Ampicillin 4/8 1/2 0.25/0.25 Synergistic P. gingivalis RGE 20/40 2.5/10 0.25/0.25 Synergistic/ 0.5/0.75 ATCC 33277 Ampicillin 0.25/0.5 0.063/0.25 0.25/0.5 Additive 1 RGE: mg/ml, ampicillin: µg/ml; 2 The MIC and MBC of the RGE with ampicillin; 3 The fractional inhibitory concentration (FIC)/ the fractional bactericidal concentration (FBC); 4 The fractional inhibitory concentration index

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(FIC index)/ the fractional bactericidal concentration index (FBC index); 5 American Type Culture Collection (ATCC); 6 Korean collection for type cultures (KCTC)

Table 6: Synergistic effects of RGE with gentamicin against oral bacteria MIC/MBC 1 Strains Agent FIC/FBC 3 FICI/FBCI 4 Outcome Alone Combination 2 S. mutans RGE 10/40 5/20 0.5/0.5 Additive/ 5 0.75/0.5 ATCC 25175 Gentamicin 8/16 2/4 0.25/0.25 Synergistic S. sanguinis RGE 10/40 2.5/10 0.25/0.25 Synergistic/ 0.375/0.5 ATCC 10556 Gentamicin 64/64 8/16 0.125/0.25 Synergistic S. sobrinus RGE 10/20 2.5/10 0.25/0.5 Synergistic/ 0.5/0.75 ATCC 27607 Gentamicin 4/8 1/2 0.25/0.25 Additive S. ratti RGE 20/40 2.5/10 0.125/0.25 Additive / 6 0.625/0.75 KCTC 3294 Gentamicin 16/32 8/16 0.5/0.5 Additive S. criceti RGE 10/20 2.5/5 0.25/0.25 Synergistic/ 0.5/0.5 KCTC 3292 Gentamicin 8/16 2/4 0.25/0.25 Synergistic S. anginosus RGE 40/80 10/20 0.25/0.25 Synergistic/ 0.5/0.75 ATCC 31412 Gentamicin 32/32 8/16 0.25/0.5 Additive S. gordonii RGE 10/20 2.5/5 0.25/0.25 Synergistic/ 0.5/0.75 ATCC 10558 Gentamicin 32/32 8/16 0.25/0.5 Additive A. RGE 20/40 5/10 0.25/0.25 Synergistic/ actinomycetemcomitans 0.5/0.5 Gentamicin 4/8 1/2 0.25/0.25 Synergistic ATCC 43717 F. nucleatum RGE 40/80 10/20 0.25/0.25 Synergistic/ 0.5/0.5 ATCC 51190 Gentamicin 2/4 0.5/1 0.25/0.25 Synergistic P. intermedia RGE 20/40 5/10 0.25/0.25 Additive / 0.75/0.75 ATCC 25611 Gentamicin 16/32 8/16 0.5/0.5 Additive P. gingivalis RGE 20/40 10/10 0.5/0.25 Additive / 0.75/0.5 ATCC 33277 Gentamicin 256/512 64/128 0.25/0.25 Synergistic 1 RGE: mg/ml, gentamicin: µg/ml; 2The MIC and MBC of the RGE with gentamicin; 3 The fractional inhibitory concentration (FIC)/ the fractional bactericidal concentration (FBC); 4 The fractional inhibitory concentration index (FIC index)/ the fractional bactericidal concentration index (FBC index); 5 American Type Culture Collection (ATCC); 6 Korean collection for type cultures (KCTC)

Activity of antibiotics plus the plant extract was combination of sophoraflavanone G with determined using the checkerboard technique gentamicin was observed resulted in the (Cha et al., 2007; Hemaiswarya et al., 2008). decrease ≥4-fold in MIC for all tested The synergistic effect of sophoraflavanone G periodontopathogenic bacteria by FICI ≤ 0.31- and RGE with ampicillin or gentamicin in oral 0.5 and in MBC except additive effect in A. bacteria was presented in Tables 2-6, actinomycetemcomitans by FBCI ≤ 0.75 respectively. In combination of (Table 4). In combination with RGE, the MIC sophoraflavanone G with RGE, the MIC ranges for ampicillin was reduced ≥4-fold in ranges were observed in cariogenic bacteria at most of tested cariogenic bacteria, producing a 0.2 µg/ml to 0.8 µg/ml and reduced ≥4-fold, synergistic effect as defined by FICI ≤ 0.5, producing a synergistic effect as defined by except additive effect in S. mutans and S. ratti FICI ≤ 0.375-0.5, except additive effect in S. by FICI ≤ 0.75 and producing a synergistic sanguinis by FICI ≤ 0.75. The MBC ranges effect by FICI ≤ 0.31-0.5 in (0.4 µg/ml to 1.6 µg/ml) of sophoraflavanone G periodontopathogenic bacteria, P. intermedica with RGE were reduced ≥4-fold in S. and P. gingivalis . Then the MBC ranges for sanguinis, S. ratti , and S. anginosus by FBCI ≤ ampicillin was reduced ≥4-fold in cariogenic 0.5 (Table 2). In periodontopathogenic bacteria, S. sanguinis and S. criceti , producing bacteria, the MIC and MBC values (0.05 µg/ml a synergistic effect as defined by FBCI ≤ 0.5 to 1.6 µg/ml and 0.1 µg/ml to 3.2 µg/ml) of and producing a synergistic effect by FBCI ≤ sophoraflavanone G with RGE were also 0.375-0.5 in periodontopathogenic bacteria, A. observed by ≥4-fold, producing a synergistic actinomycetemcomitans and P. Intermedia effect as defined by FICI/FBCI ≤ 0.5/0.5, (Table 5). The combination of gentamicin and except additive effect in A. RGE resulted in the decrease in MIC/MBC for actinomycetemcomitans by FICI/FBCI ≤ all bacteria, with the MIC/MBC of 2.5-10/5-20 0.75/0.75 (Table 2), with ampicillin, the mg/ml by FICI/FBCI ≤ 0.375-0.5 in S. MIC/MBC ranges were observed in sanguinis, S. sobrinus, S. Criceti preventing periodontopathogenic bacteria reduced ≥4- dental caries (Bedeni ć et al., 2012; Radhika et fold, producing a synergistic effect as defined al., 2011; Greenberg et al., 2008; Sofrata et by FICI/FBCI ≤ 0.375-5/0.25/0.5 (Table 3). The al., 2008; Kouidhi et al., 2011). The main

Ph ton 424 active components, Ginsenosides of P. quercetin, sophoraflavanone G and ginseng exhibit similar killing potency to kaempferol are previously demonstrated to propionic acid, which is known to be a cidal have antimicrobial and antimalaria activity agent toward bacteria; the antibacterial activity (Cha et al., 2007; Kim et al., 2004). We of ginsenosides toward MRSA is due to the previously reported that the sophoraflavanone killing action, as described by time-killing plots G is related to gram-positive bacteria, and with MRSA (Sung, Lee, 2008). Some flavonoid fulfills the criteria associated with a novel derivatives from S. flavescens such as MRSA (Cha et al., 2009).

Figure 1: Time–kill curves of MIC or 1/2 MIC of sophoraflavanone G and RGE alone and its combination against S. mutans, S. sanguinis, S. sobrinus, S. anginosus, S. criceti, and S. ratti . Bacteria were incubated with sophoraflavanone G (●), RGE (○), and sophoraflavanone G + RGE (▼) over time. Data points are the mean values±S.E.M. of six experiments. CFU, colony-forming units

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Figure 2: Time–kill curves of MIC or 1/2 MIC of sophoraflavanone G and RGE alone and its combination against S. gordonii, A. actinomycetemcomitans, F. nucleatum, P. intermedia, and P. gingivalis . Bacteria were incubated with sophoraflavanone G (●), RGE (○), and sophoraflavanone G + RGE (▼) over time. Data points are the mean values±S.E.M. of six experiments. CFU, colony-forming units

The synergistic effect of sophoraflavanone G complete killing against S. sobrinus and S. and RGE with ampicillin or gentamicin against anginosus for 3h and 4h, respectively and the oral bacteria was confirmed by time-kill curve other bacteria for up to 24 h (Fig. 1, 2). experiments. The cultures of all bacteria, with a cell density of 10 5 CFU/ml, were exposed to References MIC or 1/2 MIC of sophoraflavanone G and RGE alone and combination. We observed Bedeni ć B., Budimir A., Gveri ć A., Plecko V., that the combination of sophoraflavanone G Vranes J., Bubonja-Sonje M., Kaleni ć S., 2012. and RGE resulted rate of killing increasing in Comparative urinary bactericidal activity of oral antibiotics against gram-positive pathogens. CFU/ml at time-dependent manner. However, Lijecnicki vjesnik, 134(5-6), 148-155. sophoraflavanone G with RGE resulted in

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