Original Paper Received: November 19, 2013 Accepted: April 2, 2014 Published online: December 18, 2014

Caries Res 2015;49:78–89 DOI: 10.1159/000362676

In vitro Antimicrobial Activities of 1-Methoxyficifolinol, Licorisoflavan A, and 6,8-Diprenylgenistein against Streptococcus mutans Sug-Joon Ahn a Soon-Nang Park b Young Ju Lee c Eun-Jung Cho a Yun Kyong Lim b Xue Min Li d Mi-Hwa Choi b Young-Woo Seo c Joong-Ki Kook b   

 

 

a

 

 

 

 

 

 

Dental Research Institute, School of Dentistry, Seoul National University, Jongro-Gu, Seoul, b Korean Collection for Oral Microbiology and Department of Oral Biochemistry, School of Dentistry, Chosun University, and c Gwangju Center, Korea Basic Science Institute, Gwangju, Republic of Korea; d Department of Internal Medicine, Yantaishan Hospital, Yantai City, Shandong Province, China  

 

 

 

Abstract The objective of the study was to investigate the antimicrobial effects of purified single compounds from ethanol-extracted licorice root on Streptococcus mutans. The crude licorice root extract (CLE) was obtained from Glycyrrhiza uralensis, which was subjected to column chromatography to separate compounds. Purified compounds were identified by mass spectrometry and nuclear magnetic resonance. Antimicrobial activities of purified compounds from CLE were evaluated by determining the minimum inhibitory concentration and by performing time-kill kinetics. The inhibitory effects of the compounds on biofilm development were evaluated using crystal violet assay and confocal microsco-

S.-J. Ahn, S.-N. Park and Y.J. Lee contributed equally to this work.

© 2014 S. Karger AG, Basel 0008–6568/14/0491–0078$39.50/0 E-Mail [email protected] www.karger.com/cre

py. Cell toxicity of substances to normal human gingival fibroblast (NHGF) cells was tested using a methyl thiazolyl tetrazolium assay. Chlorhexidine digluconate (CHX) was used in the control group. Three antimicrobial flavonoids, 1-methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein, were isolated from the CLE. We found that the three flavonoids and CHX had bactericidal effects on S. mutans UA159 at the concentration of ≥4 and ≥1 μg/ml, respectively. The purified compounds completely inhibited biofilm development of S. mutans UA159 at concentrations over 4 μg/ml, which was equivalent to 2 μg/ml of CHX. Confocal analysis showed that biofilms were sparsely scattered in the presence of over 4 μg/ml of the purified compounds. However, the three compounds purified from CLE showed less cytotoxic effects on NHGF cells than CHX at these biofilm-inhibitory concentrations. Our results suggest that purified flavonoids from CLE can be useful in developing oral hygiene products, such as gargling solutions and dentifrices for preventing dental caries. © 2014 S. Karger AG, Basel

Sug-Joon Ahn, Professor Dental Research Institute, School of Dentistry Seoul National University, Daehakro 101 Jongro-Gu, Seoul 110–768 (Republic of Korea) E-Mail titoo @ snu.ac.kr Joong-Ki Kook, Professor Korean Collection for Oral Microbiology and Department of Oral Biochemistry School of Dentistry, Chosun University, 375 Seo-Suk Dong, Dong-Gu Gwangju 501–759 (Republic of Korea) E-Mail jkkook @ chosun.ac.kr

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Key Words Antimicrobial activity · 6,8-Diprenylgenistein · Licorisoflavan A · 1-Methoxyficifolinol · Streptococcus mutans

Dental plaque, oral biofilm formed on the tooth surface, plays an essential role in the etiology of dental caries. Although the oral flora is quite diverse and complex, Streptococcus mutans has been known to be a primary etiologic agent of human dental caries due to its specific ability to form biofilms on the tooth surfaces [Loesche, 1986; Takahashi and Nyvad, 2011]. Therefore, inhibition of the viability and biofilm formation of S. mutans is one of the strategies for the prevention of dental caries. Various antimicrobial agents are widely used for chemical plaque control and their inhibition of bacterial accumulation on tooth surfaces. Chlorhexidine (CHX) is a clinically proven antibacterial agent that is effective against a wide range of microorganisms in the oral cavity [Glassman, 2003; Paula et al., 2010]. In particular, CHX has been used to prevent and reduce carious lesions, because S. mutans species are particularly sensitive to CHX [Petti and Hausen, 2006]. However, CHX has some side effects, including bitter taste and the formation of extrinsic stains on the teeth and tongue [Van Strydonck et al., 2012]. Numerous attempts have been made to identify antimicrobial agents from natural products. Naturally occurring compounds possess structures that have high chemical diversity and biochemical specificity, as well as other molecular properties that make them attractive candidates for drug delivery and development [Koehn and Carter, 2005]. However, this approach can be challenging because of the complex chemistry and isolation procedures required to derive active compounds from natural products. Licorice root is one of the oldest botanicals in traditional Oriental medicine [Asl and Hosseinzadeh, 2008]. Over the past few decades, several research groups have investigated the chemical constituents and biological activities of licorice root. Previous chemical studies have led to the identification of about 100 phenolic compounds, many of which are isoprenoid-substituted phenols [Hatano et al., 2000a, b]. Some of these flavonoids have shown inhibitory activities against bacterial growth [Tsuchiya et al., 1996; Hatano et al., 2000b; Fukai et al., 2004]. In our previous study, we demonstrated that a crude licorice root extract (CLE) had strong antimicrobial effects against S. mutans [Ahn et al., 2012]. The purpose of the present study was to isolate and identify active components of CLE that showed potential anticariogenic activity without cytotoxic effects. In particular, we used time-kill kinetic assays in order to examine whether the antimicrobial activities of the isolated components are bactericidal

Silica Gel Chromatography and High-Performance Liquid Chromatography CLE was fractionated by column chromatography over silica gel (Silica gel 60, Merck, Darmstadt, Germany) using nhexane:ethyl acetate (10:0, 1:9, 2:8, 3:7, 5:5, and 0:10) as a solvent. Twenty-one subfractions, (1) 10:0-1, (2) 10:0-2, (3) 10:0-3, (4) 9: 11, (5) 9:1-2, (6) 9:1-3, (7) 8:2-1, (8) 8:2-2, (9) 8:2-3, (10) 7:3-1, (11) 7:3-2, (12) 7:3-3, (13) 6:4-1, (14) 6:4-2, (15) 6:4-3, (16) 5:5-1, (17) 5: 5-2, (18) 5: 5-3, (19) 0: 10-1, (20) 0: 10-2, (21) 0: 10-3, were obtained. Each fraction solution was concentrated with a rotator evaporator (80 rpm, IKA, RV 10, Korea) at 40 ° C and lyophilized in a freeze dryer (Labconco, Kansas City, Mo., USA). Extracts were suspended in dimethyl sulfoxide (DMSO; Sigma, St. Louis, Mo., USA) to 10 mg/ml to test antimicrobial activity by determining minimum inhibitory concentration (MIC) using S. mutans UA159. Among these fractions, the ninth and tenth fractions, which showed the highest antimicrobial activity against S. mutans UA159, were further fractionated by high-performance liquid chromatography (HPLC, SPD-20A, STC-20A, LC-6AD, CBM-20A, Shimadzu, Tokyo, Japan). The ninth and tenth fractions were dried and lyophilized in a freeze dryer (Labconco, UK) and suspended in DMSO to a final concentration of 100 mg/ml. The extract was rediluted with 90% methanol to a concentration of 10 mg/ml and filtered through a 0.2-μm membrane. HPLC separation was per-

Antimicrobial Effect of Flavonoids on S. mutans UA159

Caries Res 2015;49:78–89 DOI: 10.1159/000362676

or bacteriostatic. These active compounds could be used in developing oral hygiene products for preventing dental caries in further studies. Materials and Methods Bacterial Strains and Growth Conditions S. mutans ATCC 25175T and Streptococcus sobrinus ATCC 33478T were purchased from the American Type Culture Collection (ATCC, Manassas, Va., USA). Clinical strains of S. mutans (KCOM 1054, KCOM 1111, KCOM 1113, KCOM 1116, KCOM 1126, KCOM 1128, KCOM 1136, KCOM 1197, KCOM 1202, KCOM 1207, and KCOM 1217) and S. sobrinus (KCOM 1157, KCOM 1196, and KCOM 1221) were obtained from the Korean Collection for Oral Microbiology (KCOM, Gwangju, Korea). S. mutans UA159 was a kind gift from Dr. Robert A. Burne, Department of Oral Biology, College of Dentistry, University of Florida. All strains were cultured in Todd-Hewitt (Difco, Detroit, Mich., USA) broth or on agar plates in a 37 ° C incubator in air containing 5% CO2.  

 

Preparation of Ethanol Extract of Licorice Root Licorice root from Glycyrrhiza uralensis was purchased from a herbal drug market (Youngju, Korea). Licorice root extract was prepared as described previously [Ahn et al., 2012] with modification (online suppl. fig. 1; for all online suppl. material, see www. karger.com/doi/10.1159/000362676). Briefly, dried roots were cut into thin slices, mixed with distilled water [distilled water:dried root ratio of 20:1 (v/w)] and heated for 2 h in a round-bottom flask. Distilled water was removed, and 95% ethanol was added to the flask [95% ethanol:residue at a 15:1 (v/w) ratio]. The mixture was heated at 78 ° C for 2 h, and the extract was evaporated and used for further purification.  

 

 

 

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Nuclear Magnetic Resonance Nuclear magnetic resonance (NMR) experiments were performed using a Varian 600 MHz NMR spectrometer (Varian, Calif., USA) equipped with a pulsed-field gradient triple-resonance cold probe. DMSO-d6 (Sigma) was used as a solvent, and chemical shifts for protons and carbon were reported in parts per million relative to DMSO at 2.50 and 39.51, respectively. All one-dimensional and two-dimensional NMR experiments were performed with standard pulse sequences in the VNMR (v.2.3) library and processed with the same software. 1H-NMR spectra were acquired with a 16-kHz sweep width using 32,000 time-domain points with an acquisition time of 3.4 s. 13C-NMR spectra were acquired with a 40-kHz sweep width using 64,000 time-domain points with an acquisition time of 1.37 s. gHSQC spectra were acquired using a 1H sweep width of 8 kHz and a 13C sweep width of 25 kHz, with 1,024 points in f2, 200 complex increments in f1, 4 scans per increment, and spectral editing (CH2 negative, CH/CH3 positive). The gHSQC experiments were optimized for a one-bond coupling constant of 140 Hz. gHMBC spectra were acquired using a 1H sweep width of 6 kHz and a 13C sweep width of 36 kHz, with 2,048 points in f2, 400 complex increments in f1, 8 scans per increment, and optimization for nJ (C,H) of 8 Hz. The phase-sensitive gHSQC was transformed with a Gaussian weighting function in both dimensions after applying zero-filling in f2 and either zero-filling or linear prediction in f1. Magnitude-mode gHMBC was transformed with a sine bell weighting function in both dimensions after applying zero-filling in f1. Electrospray ionization mass spectrometry (ESI-MS) was acquired using a Synapt HDMS TOF mass spectrometer (Waters, Milford, Mass., USA) in ESI-negative mode. Determination of MIC MIC values were determined using a microdilution assay according to the National Committee for Clinical Laboratory Standards [2003]. Briefly, S. mutans was grown in brain-heart infusion (BHI) broth (Difco) at 37 ° C in a 5% CO2 atmosphere to mid-exponential phase [OD600  = 0.5, approximately 6.5 × 107 colonyforming units (CFU)/ml] and added to a 96-well plate to a final concentration of 1 × 106 CFU/ml. To measure the MIC of CHX (20% chlorhexidine digluconate, Sigma) and purified compounds from CLE, these compounds were added to each well to final concentrations of 0.5, 1, 2, 4, 8, 16, and 32 μg/ml. The final DMSO concentration in each well was 1%. Culture medium containing 1% DMSO was used as negative control. After 24-hour incubation under the appropriate conditions, the lowest concentration of each component that inhibited visible growth was considered the MIC value.  

 

Time-Kill Kinetic Assay Time-kill kinetic assay was used to determine whether substances had either bactericidal or bacteriostatic effects against S. mutans UA159. Time-kill curves were assessed at the following concentrations of purified compounds from CLE and CHX: 0.5 × MIC, 1 × MIC, 2 × MIC, and 4 × MIC. The control curve was assessed in culture medium. Bacteria were inoculated in BHI broth

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Caries Res 2015;49:78–89 DOI: 10.1159/000362676

and incubated overnight in a 5% CO2 incubator. Liquid media containing the above-mentioned concentrations of each component were inoculated with 1 × 106 CFU/ml of an overnight culture and followed by incubation at 37 ° C in 5% CO2. At 0, 3, 6, 9, 12, and 24 h after bacterial inoculation, each bacterial culture solution was diluted 100- through 10,000-fold with phosphate-buffered saline (PBS; pH = 7.2) and plated onto BHI agar plates. Agar plates were incubated at 37 ° C in an atmosphere containing 5% CO2 for 48 h before bacterial colonies were counted.  

 

 

 

Determination of Cell Toxicity Normal human gingival fibroblast (NHGF) cells were a kind gift from Prof. Hyun-Sun Jang, Department of Oral Pathology, School of Dentistry, Chosun University (Gwangju, Korea). NHGF cells were grown in Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, N.Y., USA) supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories, Etobicoke, Ont., Canada), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL) at 37 ° C in a humidified 5% CO2 atmosphere. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, a tetrazole) assay was performed to evaluate the cytotoxicity of CHX and HPLC fractions of CLE to NHGF cells. The concentrations of the tested chemicals were determined dependent on their MIC values against S. mutans UA159. An 80% confluent monolayer of NHGF cells in a 24-well plate was incubated with 0.5 × MIC, 1 × MIC, 2 × MIC, and 4 × MIC of each HPLC fraction and CHX or 1% DMSO as a negative control in growth medium at 37 ° C in humidified air with 5% CO2 for 24 h. The medium in each well was then replaced with new culture medium containing 10% MTT solution (Sigma) and cultured for 3 h under the same culture conditions. Isopropanol (300 μl, Sigma) was added to each well to dissolve the formazan crystals. The culture plate was well shaken and samples (200 μl) were then transferred to a 96-well plate. Absorbance was measured at 595 nm.  

 

 

 

Biofilm Formation Assays To assess biofilm formation, S. mutans UA159 was grown in a semidefined biofilm medium (BM) with either 20 mM glucose (BM-glucose) or sucrose (BM-sucrose) as a carbohydrate source, as described previously [Ahn et al., 2008]. Biofilm assays were done using polystyrene 96-well (flat-bottom) cell culture clusters (Costar 3595; Corning Inc., Corning, N.Y., USA). Overnight cultures of S. mutans UA159 were transferred to prewarmed BHI and grown at 37 ° C in a 5% CO2 atmosphere to mid-exponential phase (OD600 = 0.5). Cultures were then diluted 100-fold in prewarmed BM containing the purified CLE compounds (0.5 × MIC, 1 × MIC, and 2 × MIC) or CHX (1 × MIC and 2 × MIC). A cell suspension containing only 1% DMSO was used as the negative control. Biofilm formation assays were performed with or without saliva coating. For saliva coating, unstimulated whole saliva (UWS) was collected by the spitting method from healthy volunteers as described previously [Ahn et al., 2012]. Subjects provided informed consent and the research protocol was reviewed and approved by the Institutional Review Board of the University Hospital. Saliva samples were centrifuged at 3,500 g for 10 min to remove any cellular debris, and the resulting supernatant was used after filter sterilization through a Stericup and Steritop (0.22-μm filter, Millipore, Billerica, Mass., USA). For experiments with saliva coating, each well was conditioned with 100 μl of UWS as previously described [Ahn et al., 2008, 2012]. Plates were incubated at 37 ° C  

 

 

Ahn/Park/Lee/Cho/Lim/Li/Choi/Seo/ Kook

 

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formed using YMC-Pack ODS-A column (250 × 10 mm, YMC Co., Ltd., Kyoto, Japan) with UV detection at 254 nm and a temperature of 40 ° C. The sample was eluted at a flow rate of 2 ml/min using the following methanol gradient: 90–100% over 10 min and then held at 100% for 30 min.

 

 

Confocal Microscopy S. mutans UA159 biofilms for confocal microscopy were generated on 8-well Lab-Tek Chamber Permanox slides (Nalge Nunc International, Rochester, N.Y., USA). Overnight cultures were transferred to fresh BHI broth and allowed to grow to mid-exponential phase (OD600 = 0.5). Cultures were then diluted 1: 100 in BM-glucose or BM-sucrose containing the purified compounds (F9-0, 0.5 × MIC, 1 × MIC, and 2 × MIC; F9-1 and 10-3, 0.5 × MIC and 1 × MIC) or CHX (1 × MIC and 2 × MIC) in the absence or presence of saliva coating, as described above. Culture medium containing only 1% DMSO was used as the negative control. Following 24 h of growth, each well was washed twice with 400 μl of Tris-buffered saline (TBS; pH 7.2), stained for 20 min in the dark with 300 μl TBS containing 10 μM SYTO 13 (Invitrogen, Carlsbad, Calif., USA), and washed once with 400 μl TBS. Chamber walls were then gently removed, 120 μl TBS was deposited on each biofilm, and chamber slides were covered with a coverslip that was secured. Biofilms were examined under a Carl Zeiss LSM 700 Laser Scanning Microscope (Carl Zeiss, Jena, Germany). Images were obtained at a 200× magnification. Three independent biofilm experiments were performed, and at least three image stacks per experiment were collected. Simulated xyz three-dimensional images were generated using ZEN software (Carl Zeiss).

Table 1. MIC of silica gel fractions of ethanol extracts of licorice

root derived from Glycyrrhiza uralensis against the S. mutans UA159 Silica gel fractions

MIC, μg/ml

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21)

>32 >32 >32 >32 >32 >32 >32 16 8 8 8 16 16 16 >32 >32 >32 >32 >32 >32 >32

10:0-1 (SG-F1) 10:0-2 (SG-F2) 10:0-3 (SG-F3) 9:1-1 (SG-F4) 9:1-2 (SG-F5) 9:1-3 (SG-F6) 8:2-1 (SG-F7) 8:2-2 (SG-F8) 8:2-3 (SG-F9) 7:3-1 (SG-F10) 7:3-2 (SG-F11) 7:3-3 (SG-F12) 6:4-1 (SG-F13) 6:4-2 (SG-F14) 6:4-3 (SG-F15) 5:5-1 (SG-F16) 5:5-2 (SG-F17) 5:5-3 (SG-F18) 0:10-1 (SG-F19) 0:10-2 (SG-F20) 0:10-3 (SG-F21)

Purification of Single Compounds with Antimicrobial Activity against S. mutans UA159 and Determination of Their Chemical Structures Column chromatographic separation over silica gel was carried out to find fractions with antimicrobial activity through bioassays using S. mutans UA159. Twenty-

one fractions from the CLE were obtained using nhexane:ethyl acetate eluent. Among these, the ninth, tenth, and eleventh fractions had the highest antimicrobial activity against S. mutans UA159 (table  1). In this study, the ninth and tenth fractions were subjected to further purification to identify active compounds. The eleventh fraction was not used because the tenth and eleventh fractions were eluted using the same chemical composition of eluent, n-hexane:ethyl acetate (7:3) and their MIC were the same as 8 μg/ml (table 1). Two (HPLC-F9-0 and HPLC-F9-1) and seven (HPLC-F10-0 to HPLC-F10-6) fractions were obtained from the ninth and tenth fractions by HPLC, respectively (fig.  1). MIC values of the eight HPLC fractions ranged from 2 to 16 μg/ml (table 2). In this study, HPLC-F9-0, HPLC-F9-1, and HPLC-F10-3 were chosen for further evaluation of antimicrobial activities and identification of their chemical structures because the antimicrobial activities of these three fractions were higher than those of other fractions and the area of these three HPLC fractions was larger than those of others. The MIC value of CLE against S. mutans UA159 was reported to be 8 μg/ml in a previous study [Ahn et al., 2012]. In the current study, the three isolated compounds had 2–4 times higher antimicrobial activity (HPLC-F9-0,

Antimicrobial Effect of Flavonoids on S. mutans UA159

Caries Res 2015;49:78–89 DOI: 10.1159/000362676

Statistical Analysis All assays were performed in triplicate and independently repeated 3 times. Factorial analysis of variance was used to analyze biofilm formation with respect to saliva coating, type of antimicrobial agent, and its concentration. Multiple comparisons were performed using the Bonferroni correction. Values were considered statistically significant when p < 0.05.

Results and Discussion

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for 2 h with gentle shaking and then washed 3 times with PBS (pH = 7.2). Immediately after air drying for 30 min, wells were inoculated with 200 μl of cell suspensions containing various concentrations of purified compounds from CLE or CHX. For experiments without saliva coating, the same procedure was performed with sterile PBS instead of UWS. After inoculation, all plates were incubated at 37 ° C in a 5% CO2 atmosphere for 24 h. Culture medium was then decanted and plates were washed twice with 200 μl of sterile distilled water to remove planktonic and loosely bound cells. Adherent bacteria were stained with 50 μl of 0.1% crystal violet for 15 min. After rinsing twice with 200 μl of water, bound dye was extracted from stained cells using 200 μl of 99% ethanol. Biofilm formation was then quantified by measuring the absorbance of the solution at 600 nm using a spectrophotometer (Helios Beta, Thermo Scientific, Madison, Wis., USA).

Detector A 254 nm

B. Conc. (Method)

HPLC-F9-1

30.0

HPLC-F9-0

50

20.0 25

10.0

0

A

Color version available online

Mpa

mV 75

0 20

10

0

min

30

Mpa

mV Detector A 254 nm

400

B. Conc. (Method) HPLC-F10-3

30.0

300 20.0

200

semipreparative HPLC separation of CLE. Peak HPLC-F9-0 (A), HPLC-F9-1 (A), and HPLC-F10-3 (B) were collected for microprobe NMR analysis. Mpa = Megapascal.

10.0

100 0

B

0

2 μg/ml; HPLC-F9-1, 4 μg/ml, and HPLC-F10-3, 4 μg/ml) than CLE against S. mutans UA159 (table 2). The MIC value of CHX was 1 μg/ml. Chemical structures of HPLC-F9-0, HPLC-F9-1, and HPLC-F10-3 were assigned based on NMR and mass spectral data (online suppl. tables 1–3). These compounds were identified as 3,9-dihydroxy-1-methoxy-2,8-diprenyl pterocarpan (1-methoxyficifolinol) [Kiuchi et al., 1990], 7-O-methyllicoricidin (licorisoflavan A) [Shih et al., 1987], and 6,8-diisoprenyl-5,7,4’-trihydroxyisoflavone (6,8-diprenylgenistein) [He et al., 2006], respectively (online suppl. fig. 2). 1-Methoxyficifolinol (HPLC-F9-0) exhibited an ESIMS [M-H]– peak at m/z 421 and its molecular formula was determined to be C26H30O5. The pterocarpan skeleton was deduced from 1H-NMR data from signals at δ 4.11 (dd, J = 10.8, 4.8 Hz), 3.42 (t, J = 10.2 Hz), 3.35 (m), and 5.48 (d, J = 7.2 Hz), corresponding to H-6α, H-6β, H-6a, and H-11a, respectively. 1H-NMR spectrum contained signals for three aromatic protons (δ 6.94, 6.24, and 6.16, s), one methoxy group (δ 3.80, s), and two prenyl groups [δ 5.14 (t, J = 6.8 Hz), 3.18 (m), 1.70 (s), 1.62 (s) and 5.23 (t, J = 7.3 Hz), 3.11 (m), 1.66 (s), 1.67 (s)] (online suppl. table 1). The locations of the substituents were determined based mainly on the 13 C-NMR data and HMBC correlations. HMBC correla82

Caries Res 2015;49:78–89 DOI: 10.1159/000362676

10

20

30

0 min

Table 2. MIC of HPLC fractions of ethanol extracts of licorice root derived from Glycyrrhiza uralensis and chlorhexidine against S. mutans UA159

HPLC fractions

MIC, μg/ml

HPLC-F9-0 HPLC-F9-1 HPLC-F10-0 HPLC-F10-1 HPLC-F10-2 HPLC-F10-3 HPLC-F10-4 HPLC-F10-5 HPLC-F10-6 CHX

2 4 16 16 8 4 8 16 8 1

tions between H-11a (δ 5.48) and C-1 (δ 159.3) as well as the methoxy protons and C-1 indicated that the methoxy group was present at C-1. In addition, the position of the two prenyl substituents was determined to be at C-2 and C-8, which was supported by HMBC correlations (H-1′/C-1, 2, 3, H-1′′/C-7, 8, 9). HPLC-F9-1 (licorisoflavan A) is a prenylated isoflavone with a molecular formula of C27H34O5, as determined by the [M-H]– peak at m/z 437 of ESI-MS. 1HAhn/Park/Lee/Cho/Lim/Li/Choi/Seo/ Kook

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Fig. 1. Chromatogram (254 nm) of the

Table 3. MIC of HPLC fractions of ethanol extracts of licorice root derived from Glycyrrhiza uralensis against

type strains and clinical isolates of S. mutans and S. sobrinus

S. mutans ATCC 25175T S. mutans KCOM 1054 S. mutans KCOM 1111 S. mutans KCOM 1113 S. mutans KCOM 1116 S. mutans KCOM 1126 S. mutans KCOM 1128 S. mutans KCOM 1136 S. mutans KCOM 1197 S. mutans KCOM 1202 S. mutans KCOM 1207 S. mutans KCOM 1217 S. sobrinus ATCC 33478T S. sobrinus KCOM 1157 S. sobrinus KCOM 1196 S. sobrinus KCOM 1221

MIC, μg/ml 1-methoxyficifolinol (HPLC-F9-0)

licorisoflavan A (HPLC-F9-1)

6,8-diprenylgenistein (HPLC-F10-3)

2 4 2 2 2 1 2 2 1 2 4 2 1 1 2 1

2 2 4 2 4 1 2 2 4 4 2 2 4 2 4 1

2 4 4 2 4 2 4 4 1 4 2 4 4 4 4 2

NMR signals at δ 4.09 (dt, J = 10.0, 3.0 Hz, H-2α), 3.86 (t, J  = 10.0 Hz, H-2β), 3.28 (m, H-3), 2.75 (dd, J  = 16.2, 4.8 Hz, H-4α), and 2.62 (dd, J = 16.2, 10.8 Hz, H-4β) are typical for the C ring of an isoflavan (online suppl. table 2). In addition, the spectrum revealed signals for three aromatic protons, two methoxy groups, and two isoprenyl moieties. The position of the prenyl group was determined to be at C-6, C-3′, which was supported by the HMBC correlations (H-1′′/C-6, H-1′′/C-3′). HPLC-F10-3 (6,8-diprenylgenistein) was also a prenylated isoflavone. Its molecular formula was established to be C25H26O5 based on ESI-HR-MS data with an [M-H]– peak at m/z 405.1700 (calculated 405.1702). The typical isoflavone skeleton was deduced from the presence of one methylene (δ 2.81, δ 3.10, H-3) and one oxygen-substituted methine (δ 5.29, H-2) on the 1H-NMR spectrum and 14 aromatic carbons and a carbonyl at δ 199.5, one oxygensubstituted methine at δ 75.6 and one methylene in the aliphatic region on the 13C-NMR spectrum. Four aromatic protons of δ 7.37 (d, J = 6.6 Hz, H-2′/H-6′) and δ 6.8 (d, J = 6.6 Hz, H-3′/H-5′) and HMBC correlations indicated the hydroxyl substitution positions (H-5, H-7, H-4′) on the A and B rings of the isoflavone. Moreover, the chemical shift of C-4 at δ 180.4 indicated that there was a hydroxyl substitution at C-5 (online suppl. table 3). The position of the prenyl group was directly assigned to be C-6, C-8 by HMBC correlation (H-1′′/C-6, H-1′′/C-7, H-1′′′/C-8, H-1′′′/C-9).

1-Methoxyficifolinol, Licorisoflavan A, and 6,8-Diprenylgenistein Have Antimicrobial Activity against Clinical Isolates of S. mutans and S. sobrinus with Little Cytotoxic Effect on NHGF Cells 1-Methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein had antibacterial activity against type strains and 14 clinical strains of S. mutans and S. sobrinus isolated from a Korean population that were introduced as a bacterial model system to determine the optimal concentrations for developing oral hygiene products [Kim et al., 2011]. The MIC values of 1-methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein against the type strains and clinical strains of mutans streptococci ranged from 1 to 4 μg/ml (table 3). The MIC90 values of the three compounds were 4 μg/ml. Licorisoflavan A has been reported to inhibit growth of S. mutans ATCC 25175T and S. sobrinus ATCC 33478T by about 30 and 70%, respectively, at 10 μg/ml [Gafner et al., 2011]. In that study, the growth media for S. mutans and S. sobrinus was ToddHewitt broth supplemented with hemin (10 μg/ml) and vitamin K (1 μg/ml), which may explain the discrepancy in antimicrobial efficacy of licorisoflavan A on S. mutans ATCC 25175T and S. sobrinus ATCC 33478T between our study and the previous study. The S. mutans ATCC 25175T and S. sobrinus ATCC 33478T strains also become clinical isolates and some important phenotypic characteristics might be lost with repeated manipulation of the strains,

Antimicrobial Effect of Flavonoids on S. mutans UA159

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which might also cause the discrepancy. 6,8-Diprenylgenistein has been reported to have antimicrobial activity against S. mutans ATCC 2517T with MIC of 2 μg/ml [He et al., 2006], which is identical to our finding. 6,8-Diprenylgenistein has also been shown to have antimicrobial activity against Staphylococcus aureus (ATCC 12600T), Escherichia coli (ATCC 11775T), Klebsiella pneumoniae (ATCC 13883T), and Bacillus subtilis (ATCC 6051T) with MIC ranging from 7.8 to 15.6 μg/ml [Chukwujekwu et al., 2011]. These results indicate that 6,8-diprenylgenistein has antimicrobial activity against both Gram-positive and Gram-negative bacteria. 1-Methoxyficifolinol has been previously purified and identified from licorice [Kiuchi et al., 1990]. However, there are no reports about the antimicrobial activity of 1-methoxyficifolinol. To the best of our knowledge, this is the first study to report the antimicrobial activity of 1-methoxyficifolinol. The antimicrobial activities of 1-methoxyficifolinol, licorisoflavan A, 6,8-diprenylgenistein, and CHX were 84

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further evaluated using a time-kill kinetic assay of S. mutans UA159. Three flavonoids and CHX had bactericidal effects on S. mutans UA159 at the concentration of ≥4 and ≥1 μg/ml, respectively (fig.  2). The MIC value of 1-methoxyficifolinol was 2 μg/ml, at which concentration 1-methoxyficifolinol had bacteriostatic rather than bactericidal effect. We performed an MTT assay to examine whether CHX and the three HPLC fractions of CLE were cytotoxic to NHGF cells at the concentrations that had antimicrobial effects on S. mutans UA159. The 1-methoxyficifolinol, licorisoflavan A, 6,8-diprenylgenistein, and CHX up to 1 × MIC (2, 4, 4, and 1 μg/ml, respectively) had no cytotoxic effects on NHGF cells (about 95% cell viability; fig. 3). Cell viability was still maintained at 74.8, 61.9, and 77.9% at 2 × MIC (4, 8, and 8 μg/ml) of 1-methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein, respectively (fig. 3A–C). In particular, 6,8-diprenylgenistein was the least cytotoxic compound among the three isolated Ahn/Park/Lee/Cho/Lim/Li/Choi/Seo/ Kook

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UA159.

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compounds to NHGF cells at 2 × MIC. In contrast, the cell viability obtained at 2 × MIC (2 μg/ml) of CHX was 52.1% (fig. 3D). A classification of cytotoxicity has been previously proposed; extracts were rated as severely, moderately, or slightly cytotoxic when the activity relative to controls was less than 30%, between 30 and 60%, or greater than 60%, respectively [Sletten and Dahl, 1999]. Considering the classification, the concentration at ≤2 × MIC of three purified compounds and ≤1 × MIC of CHX are slightly cytotoxic. The implications of using the slightly cytotoxic compounds are dependent on the cytotoxic mechanisms. If three purified compounds act as mutagens, they must not be used as components of oral hygiene product despite noncytotoxic concentration. However, the cytotoxic mechanisms of the three purified compounds are not known yet. Therefore, cytotoxic mechanisms of the three purified compounds on human oral tissue cells should be identified in further studies. Human oral tissue cells might be more resistant to chemicals in vivo than in vitro because oral tissue cells in vivo are continuously supplied with nutrients through the blood [Ahn

et al., 2012]. Considering these results, 1-methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein may be suitable for use in vivo at concentrations up to 8 μg/ml after evaluating cytotoxicity of three purified compounds by in vivo test.

Antimicrobial Effect of Flavonoids on S. mutans UA159

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Effects of 1-Methoxyficifolinol, Licorisoflavan A, 6,8-Diprenylgenistein, and CHX on Biofilm Formation of S. mutans UA159 We next investigated the effects of the three CLE flavonoids and CHX on biofilm formation of S. mutans UA159 with respect to carbohydrate source and saliva coating (fig. 4). We indirectly measured the amounts of biofilm using crystal violet staining. Although this method does not directly measure the amounts of adhered cells, this system is easy, simple, and cost-effective to quantify the amounts of biofilm and it also allows for quantification of glucan-rich matrices. In this study, biofilm was grown with 200 μl of biofilm media after coating with 100 μl of saliva. In our previous studies [Ahn et al., 2008, 2012], the results with using 85

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Fig. 3. Effects of 1-methoxyficifolinol (A), licorisoflavan A (B), 6,8-diprenylgenistein (C), and CHX (D) on the cell viability of NHGF cells. The 1 × MICs of 1-methoxyficifolinol, licorisoflavan A, 6,8-diprenylgenistein, and CHX against NHGF cells were 2, 4, 4, and 1 μg/ ml, respectively.

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Glucose Biofilm formation (OD600)

Biofilm formation (OD600)

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bohydrate source or the presence of saliva coating (fig. 4A, B). Confocal analysis of biofilms in the absence and presence of the three flavonoids and CHX was performed to examine the effects of these compounds on biofilm architecture (fig. 5). Although there was some variation in cell volumes, the microscopic image findings were similar to those of the quantitative biofilm assays. When glucose was used as a sole carbohydrate source, biofilms were less spread out and were disturbed in the presence of certain levels of 1-methoxyficifolinol, licorisoflavan A, 6,8-diprenylgenistein, and CHX compared to the longer chains and adherent clusters present in the vehicle control (1% DMSO, v/v; fig. 5A). Biofilms in sucrose medium showed thick cell aggregates with an average thickness of over 40 μm in the vehicle control (fig. 5B). In contrast, biofilms were thin and sparsely scattered along the substratum in the presence of 1-methoxyficifolinol, licorisoflavan A, 6,8-diprenylgenistein, and CHX at 4, 4, 4, and 2 μg/ml, respectively. Inhibition of biofilm formation by the purified flavonoids and CHX was due mainly to growth alterations, because 1-methoxyficifolinol, licorisoflavan A, 6,8-diprenylgenistein, and CHX showed significant growthinhibitory effects against S. mutans at 2, 4, 4, and 1 μg/ ml, respectively (table 2; fig. 2). These results are consistent with previous studies showing that flavonoids purified from natural products are effective inhibitors of the growth of microorganisms [Wen et al., 2004]. This study has some limitations. Bacteria in the oral cavity would never be exposed to a constant concentration of antimicrobial compounds. In addition, S. mutans cells do not occur in monoculture in vivo, but rather in a diverse microbial consortium comprised of several hundred species of bacteria. Further study will be needed using an in vivo model to develop a promising approach for the prevention of dental caries. In summary, three flavonoids, 1-methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein, which we isolated from ethanol-extracted licorice root, reduced bacterial viability in both suspension and biofilm states with minimal cytotoxicity at 4, 4, and 4 μg/ml, respec-

Fig. 4. Effects of 1-methoxyficifolinol (a), licorisoflavan A (b), 6,8-diprenylgenistein (c), and CHX (d) on biofilm development by S. mutans UA159 in glucose (A) or sucrose (B) medium in the

of flavonoids and CHX were 0, 1, 2, 4, or 8 μg/ml. Biofilm formation was assayed on polystyrene microtiter plates after staining with crystal violet. Data were obtained from three independent experiments performed in triplicate. Error bars represent standard deviations.

absence (white bar, control) or presence (black bar) of saliva coating. For biofilm formation, S. mutans was grown in a semi-defined BM with 20 mM glucose or sucrose for 24 h. Concentrations

Antimicrobial Effect of Flavonoids on S. mutans UA159

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more than 100 μl of saliva for coating were not significantly different from those with using 100 μl of saliva for coating. This may be due to the fact that S. mutans biofilm forms mainly on the bottom of the polystyrene cell culture plates. As a result, we decided to use the minimal volume of saliva. In the presence of glucose, the inhibitory effect was dependent on the concentration of the compound. Although saliva coating significantly inhibits biofilm formation of S. mutans in a glucose medium (p < 0.001), purified compounds inhibited biofilm formation of S. mutans UA159 at sub-MIC concentrations (p < 0.001; fig. 4A). The inhibition of biofilm formation of S. mutans in the presence of glucose may be explained in several ways. First, salivary constituents binding to bacterial surface structures that are needed for interbacterial interactions that support biofilm formation could also block the maturation of the biofilms [Pecharki et al., 2005]. Second, saliva coating can inhibit bacterial adhesion by decreasing the surface free energy of the underlying materials as previously reported [Quirynen and Bollen, 1995]. Such surface modification by saliva coating may reduce the strength of bacterial adhesion to the substratum, resulting in a decrease in the amount of adherent bacteria. In contrast to glucose-containing medium, sucrosedependent biofilm development was not inhibited at sub-MIC levels, but was inhibited at the MIC level (licorisoflavan A, 6,8-diprenylgenistein) or 2 × MIC level (1-methoxyficifolinol and CHX, p < 0.001). The higher concentration required for inhibition in the presence of sucrose may be due to the fact that S. mutans strains synthesize sticky exopolysaccharides when using sucrose as a substrate [Ahn et al., 2008]. These exopolysaccharides mediate adherence of the cells and promote vigorous biofilm formation, preventing direct contact between antibacterial agents and bacteria and interfering with the diffusion of antibacterial substance into the biofilm matrix. However, 1-methoxyficifolinol, licorisoflavan A, 6,8-diprenylgenistein, and CHX completely inhibited biofilm formation of S. mutans UA159 at concentrations of 4, 4, 4, and 2 μg/ml, respectively, regardless of the car-

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in the presence of 1-methoxyficifolinol (F9-0), licorisoflavan A (F9-1), 6,8-diprenylgenistein (F10-3), and CHX in semidefined BM with 20 mM glucose (A) or 20 mM sucrose (B) for 24 h. Con-

tively. In addition, the purified compounds completely inhibited growth of clinical isolates of Korean mutans streptococci at these concentrations. Furthermore, the three purified compounds were less cytotoxic to NHGF cells than CHX. Considering that CHX is widely used for chemical plaque control because of its antibacterial effects on both Gram-positive and Gram-negative organisms and its inhibition of bacterial accumulation on tooth surfaces, these results suggest that these purified flavonoids may be useful alternatives to CHX for the prevention of dental caries when used in oral hygiene products, such as gargling solutions and dentifrices.

centrations of three flavonoids were 0, 1, 2, or 4 μg/ml and the concentration of CHX was 0, 1, or 2 μg/ml in the medium. Cells were stained with SYTO 13 for 20 min.

Doctor and Food Co., Ltd., for generously providing crude licorice root extract for this work. This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2000750) and in part by research funds from Chosun University, 2013.

Author Contributions Conception and design of the study: Sug-Joon Ahn, Joong-Ki Kook; acquisition of data: Soon-Nang Park, Young Ju Lee, EunJung Cho, Yun Kyong Lim, Xue Min Li, Mi-Hwa Choi, YoungWoo Seo; drafting and finishing the manuscript: Sug-Joon Ahn, Soon-Nang Park, Young Ju Lee, Joong-Ki Kook; revising the manuscript: Sug-Joon Ahn, Joong-Ki Kook.

Acknowledgments

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There is no conflict of interest for any of the authors that might introduce bias or affect their judgment.

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Disclosure Statement We would like to thank Dr. Robert A. Burne, Department of Oral Biology, College of Dentistry, University of Florida, for generously providing S. mutans UA159 for this work. We also thank

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Antimicrobial Effect of Flavonoids on S. mutans UA159

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