Structures of Two New Flavonoids and Effects of Licorice Phenolics on Vancomycin-Resistant Enterococcus Species

Since our previous study revealed that several licorice phenolics have antibacterial effects on methicillin-resistant Staphylococcus aureus (MRSA), and suppressive effects on the oxacillin resistance of MRSA, we further investigated effectiveness of licorice constituents on vancomycin-resistant Enterococcus (VRE) bacteria, and purified 32 phenolic compounds. Two flavonoids among them were characterized structurally, and identified their structures as demethylglycyrol (31) and 5,7-di-O-methylluteone (32), respectively. Examination of antibacterial effects of licorice phenolics showed that 3-arylcoumarins such as licoarylcoumarin (9) and glycycoumarin (26), and 2-arylcoumarones such as gancaonin I (17), have moderate to potent antibacterial effects on the VRE strains used in this study.


Introduction
Licorice is one of the most frequently used natural drugs in Asian traditional medicines. It produces various types of phenolic constituents, in addition to glycyrrhizin and related triterpene glycosides. Recently, the biological activities of licorice extracts and ingredients have attracted many researchers, and their effects in the treatment for different human diseases such as cancer, atherosclerosis, gastric ulcers, hepatitis and immunodeficiency have been summarized in some reviews [1][2][3]. Potential beneficial effects of licorice in common oro-dental diseases were also discussed in a review article [4]. Potent antibacterial activities of licorice phenolics against bacterial strains such as Helicobacter pylori, cariogenic bacterial species, Streptococcus mutans and Streptococcus sobrinus, and periodontopathogenic species, Porphyromonas gingivalis and Prevotella intermedia, were also reported [5][6][7][8]. Our previous investigation revealed that several naturally occurring compounds showed potent antibacterial effects on methicillin-resistant Staphylococcus aureus (MRSA) [9][10][11], and some of the licorice phenolics, such as licoricidin (1), showed suppressing effects on the oxacillin resistance of MRSA [11].
Among drug-resistant bacteria vancomycin-resistant Enterococci (VRE) is a serious menace for patients in hospitals. Just a few drugs such as linezolid show bacteriostatic activity against vancomycin-resistant strains of E. faecium and E. faecalis, and a combination of quinupristin and dalfopristin, which have bactericidal activity against most drug-resistant staphylococci, streptococci, and pneumococci, appears to be bacteriostatic against E. faecium, and is not active against Enterococcus faecalis [12]. Therefore, we have investigated on the effective constituents of licorice (licorice based on Glycyrrhiza uralensis) on VRE, and found that some phenolics among them had potent to moderate antibacterial effects on VRE. Since two compounds among the phenolics isolated from licorice have not yet been characterized, their structures were established in the present study. This paper describes structural evidence of the two compounds and effects of licorice phenolics on VRE. Worthy that antimicrobial activities of extracts of leaves and roots of Glycyrrhiza species were previously studied [13,14] against several bacterial strains including Enterococcus faecalis. Although a paper reported gancaonin I (17) as a compound with the anti-VRE effect [15], our study revealed several pure phenolic compounds from licorice should also be considered as lead compound candidates for new anti-VRE drugs, as shown below.
The signal pattern in the 1 H-NMR spectrum of 31 is similar to that of glycyrol (15) except that a methoxyl signal observed in the spectrum of 15 is absent in that of compound A. Therefore, the structure of demethylglycyrol (31) was assigned for this compound.  The 13 C-NMR spectrum showed five carbon signals ascribable to a prenyl group [δ C 17.9, 25.8 (dimethyl at C-3'), δ C 23.3 (C-1'), δ C 125.1 (C-2'), δ C 130.5 (C-3')], in addition to fifteen carbon signals assignable to the coumestan skeleton (see Table 1). Four carbon signals at δ C 104.1 (C-11b), δ C 113.9 (C-2), δ C 156.4 (C-4a), and δ C 158.5 (C-3) among the sp 2 carbon signals are correlated with the aromatic proton at δ H 6.25 (H-4) in the 1 H-detected multiple bond correlation (HMBC) spectrum ( Figure 3). On the other hand, correlations of the methylene proton signal at δ H 3.12 (H-1') with the carbon signals at δ C 113.9 (C-2), δ C 158.5 (C-3), and δ C 160.4 (C-1), along with the correlations with the allylic carbon signals at δ C 125.1 (C-2') and δ C 130.5 (C-3'), were also observed in the HMBC spectrum. These correlations are coincided with the location of the prenyl group at C-2.
The substitution pattern of the hydroxyl and prenyl groups on the coumestan skeleton was further confirmed by chemical evidence. Compound A (31) was methylated (see Experimental Section) to afford the methyl derivative 31a (Figure 2), which was identical with the compound obtained by methylation of the known compound glycyrol (15). The structure of demethylglycyrol (31) for compound A was thus established.

Antibacterial Effects of Licorice Phenolics on VRE
E. faecium FN-1 and E. faecalis NCTC 12201 strains were used in this study. The antibacterial effects of the licorice phenolics on the VRE strains were estimated using the liquid dilution method. Although except for linezolid almost all of the tested antibacterial standard drugs showed high minimum inhibitory concentration (MIC) values against at least one of the two used strains as shown in Table 2, among the licorice phenolics examined in this study, a 2-arylcoumarin, gancaonin I (17) showed potent antibacterial effects against E. faecium (MIC of 8 μg/mL), and E. faecalis (MIC of 16 μg/mL). Additionally, two 3-arylcoumarins, licoarylcoumarin (9) and glycycoumarin (26) showed low MIC values (16 μg/mL) for E. faecium and E. faecalis. An isoflavone semilicoisoflavone B (14), and an isoflavan glyasperin D (29) showed potent (16 μg/mL) to moderate MIC (32 μg/mL) for E. faecium. Flavonols, flavanones, and chalcones showed no or weak (~128 μg/mL) antibacterial effects. Noticeable that the compounds with two or three phenolic hydroxyl groups accompanied by a prenyl group showed potent anti-VRE effects relative to those of the other structural features. Since coumestans such as isoglycyrol (18) and demethlglycyrol (31) showed weak antibacterial effects relative to those of 3-arylcoumarins, the structural rigidity may cause decrease of the effects.

General
UV spectra were recorded on a JASCO V-530 spectrometer. ESI-MS measurements were performed on an API-4000 instrument. HR-FAB-MS measurements were conducted on a JEOL JMS-700 MStation with a mixture of m-nitrobenzyl alcohol and dithiothreitol as the matrix. 1

Plant Material
The crude drug used in this study is Tohoku Licorice (root and stolon of Glycyrrhiza uralensis), purchased from Tochimoto-tenkai-do (Osaka, Japan) (Lot No. 002009037), and the specimen GU-07112011(NEL) was kept at the Medicinal Plant Garden, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences.
Separately, the ethyl acetate extract (40 g) from Tohoku Licorice was subjected to column chromatography on ODS-gel (2.2 i.d. × 75 cm) with increasing concentrations of CH 3 OH in H 2 O and then with increasing concentrations of CHCl 3 in CH 3 OH. The eluate with 10% CHCl 3 in CH 3 OH (4.1 g) was subjected to column chromatography on MCI-gel CHP-20P (2.2 i.d. × 45 cm) with increasing concentrations of CH 3 OH in H 2 O, and the eluate with 15% CH 3 OH in H 2 O (86 mg) was purified by preparative HPLC, to give liquiritin (12) (8.3 mg). The eluate with 30% CH 3 OH in H 2 O (53 mg) was purified by preparative TLC on silica gel with CHCl 3 -CH 3 OH, to give p-hydroxybenzoic acid (13) (6.9 mg) and semilicoisoflavone B (14) (2.0 mg). The eluate with 50% CHCl 3 in CH 3 OH (3.6 g) was subjected to column chromatography on MCI-gel CHP-20P (2.

Methylation of Compound A and Glycyrol
Methylation of compound A (31) was carried out as shown in the literature [42]. Briefly, a solution of compound A (1.5 mg) in EtOH was treated by TMS-diazomethane at room temperature 3 h. The reaction mixture was concentrated under reduced pressure to a residue which was purified by TLC on silica gel (CHCl 3 -MeOH, 15:1, v/v), to give three compounds: glycyrol (0.5 mg), the monomethyl derivative of compound A (0.3 mg) (identified by 1 H-NMR), and the corresponding trimethyl derivative (0.3 mg) (31', Figure 2 1H, s, H-2). This compound is identical with that obtained by analogous treatment of glycyrol (15).

Antibacterial Assay
Estimation of antibacterial effects of licorice phenolics on vancomycin-resistant Enterococcus strains was carried out as has been described in the literature [9,43,44]. Enterococcus faecium FN-1 and E. faecalis NCTC 12201 used in this study were vancomycin resistant ones which were kindly provided by Dr. Y. Ike, Gunma University. The bacterial cells, pre-cultured in Mueller-Hinton broth at 37 °C under aerobic condition, were incubated in the presence of compounds with the concentrations obtained by serial two-fold dilution at 37 °C without shaking in 96-well plates in the same broth for 24 h. The inocula were adjusted to yield a final cell density of about 10 5 CFU. The standard antibacterial drugs erythromycin, norfloxacin, vancomycin, linezolid, imipenem, tetracycline, oxacillin and gentamycin were used as reference compounds for the tested strains Enterococcus faecium FN-1 and E. faecalis NCTC 12201 in the present study. The minimum inhibitory concentrations (MICs) were estimated as the lowest concentrations where the bacterial cells were not observed visually.
The MIC values were determined based on triplicate experiments.

Conclusions
Previous reports have shown that phenolics from licorice are potent antibacterial against MRSA [11,45], and some of them showed suppressing effects on the oxacillin resistance of MRSA [11]. To discover bioactive natural compounds from natural source, Glycyrrhiza uralensis was investigated, affording a new coumestan 31 and an isoflavone 32, together with three known flavanols 5-7, three flavanones 11, 12 and 19, a chalcone 23, eight isoflavones 2, 10, 14, 20, 22, 25, 28 and 32, one isoflavan 29, four 3-arylcoumarins 8, 9, 16 and 26, three coumestans 15, 18 and 31, two 2-arylcoumarins 17 and 27 and p-hydroxybenzoic acid (13). Vancomycin-resistant Enterococci (VRE) is a serious drug-resistant bacteria, and just a few compounds such as linezolid, or a combination of quinupristin and dalfopristin have been used for treatments of diseases caused by them [12]. Therefore we have also investigated the effectiveness of the thirty two licorice phenolics isolated in this study on VRE, and we found that several compounds possesses moderate to potent antibacterial activity against VRE, and the 2-arylcoumarone gancaonin I (17) have the highest potency against the tested strains E. faecium (MIC of 8 μg/mL), and E. faecalis (MIC of 16 μg/mL), which is in agreement with the previously reported potent activity for a 2-arylcoumarin, gancaonin I (17) [15]. In addition to that, two 3-arylcoumarins, licoarylcoumarin (9) and glycycoumarin (26), also showed comparable antibacterial effects on E. faecalis (16 μg /mL). These findings could be useful in developing antibacterial agents from licorice and its various active phenolics. Besides the well-known traditional uses of licorice and the various reported biological effects [1][2][3][4][5][6][7][8], a recent study has added that several licorice phenolics exhibit higher tumor-specific cytotoxic effects [46]. However, further specific investigations on the safety of the pure licorice phenolics for human are awaited.

Author Contributions
The contributions of the respective authors are as follows: Eerdunbayaer performed isolation, identification, and structure elucidation of the constituents, and prepared the manuscript. M. A. A. Orabi contributed to checking and confirming all of the procedures of the isolation and structural identification, especially interpretation of the NMR spectra, and also to preparing the manuscript. H. Aoyama contributed to the MS measurements and interpretation of those spectra. T. Kuroda contributed to the antibacterial experiments. This study was performed based on the planning of T. Hatano, the corresponding author.