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Structures of New Phenolics Isolated from Licorice, and the Effectiveness of Licorice Phenolics on Vancomycin-Resistant Enterococci

Department of Natural Product Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
Faculty of Pharmacy, Al-Azhar University, Assiut 71524, Egypt
Drug Discovery and Technology Center, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
Author to whom correspondence should be addressed.
Molecules 2014, 19(9), 13027-13041;
Submission received: 19 June 2014 / Revised: 19 August 2014 / Accepted: 20 August 2014 / Published: 25 August 2014
(This article belongs to the Section Natural Products Chemistry)


Licorice, which is the underground part of Glycyrrhiza species, has been used widely in Asian and Western countries as a traditional medicine and as a food additive. Our continuous investigation on the constituents of roots and stolons of Glycyrrhiza uralensis led to the isolation of two new phenolics, in addition to 14 known compounds. Structural studies including spectroscopic and simple chemical derivatizations revealed that both of the new compounds had 2-aryl-3-methylbenzofuran structures. An examination of the effectiveness of licorice phenolics obtained in this study on vancomycin-resistant strains Enterococcus faecium FN-1 and Enterococcus faecalis NCTC12201 revealed that licoricidin showed the most potent antibacterial effects against both of E. faecalis and E. faecium with a minimum inhibitory concentration (MIC) of 1.9 × 10−5 M. 8-(γ,γ-Dimethylallyl)-wighteone, isoangustone A, 3'-(γ,γ-dimethylallyl)-kievitone, glyasperin C, and one of the new 3-methyl-2-phenylbenzofuran named neoglycybenzofuran also showed potent anti-vancomycin-resistant Enterococci effects (MIC 1.9 × 10−5–4.5 × 10−5 M for E. faecium and E. faecalis). The HPLC condition for simultaneous detection of the phenolics in the extract was investigated to assess the quality control of the natural antibacterial resource, and quantitative estimation of several major phenolics in the extract with the established HPLC condition was also performed. The results showed individual contents of 0.08%–0.57% w/w of EtOAc extract for the major phenolics in the materials examined.

Graphical Abstract

1. Introduction

Infectious diseases caused by multidrug-resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) are serious problems worldwide [1]. Although Enterococcus bacteria are considered ordinary components in the healthy human intestinal flora, they are responsible for complicated urinary tract infections and serious endocarditis [2]. Enterococcus faecium and Enterococcus faecalis account for >95% of Enterococcus isolates from clinical cultures [3], and only a few drugs such as linezolid and a combination of quinupristin and dalfopristin are used clinically for VRE [4]. Since the adverse effects of these drugs have been revealed and drug resistance to them may appear soon, the development of a new group of low toxicity antibacterial agents is needed. Licorice has been used as a food sweetener and is one of the oldest and most frequently used crude drugs in traditional medicine, particularly in Asian countries. A variety of pharmaceutical functions, such as antiulcer, anti-inflammatory, antiviral, and anticarcinogenic activities have been reported for licorice constituents [5,6,7,8], and the antibacterial effects of licorice phenolics have been demosntrated for various bacterial species [9,10,11,12,13,14]. The effect of a compound isolated from licorice, gancaonin I (1), on VRE was also demonstrated in a previous study [15].
Our continuous studies have revealed the antibacterial effects of several licorice phenolics on MRSA, particularly those with both γ,γ-dimethylallyl (prenyl) and hydroxyl groups [16]. Licoricidin (2) has the same structural features and displays a suppressive effect on oxacillin resistance shown by MRSA [16]. We also reported the anti-VRE effects of several licorice phenolics in a previous study [17]. Our further investigations have led to the isolation of 16 phenolic compounds including two new compounds with rarely occurring 2-aryl-3-methylbenzofuran structures. This paper explains the structural determination of the new compounds and the effects of those phenolics on two VRE stains. In addition, an analytical condition for high-performance liquid chromatography (HPLC) to simultaneously analyze polyphenolic constituents in the EtOAc extract was established for quality control of the antibacterial resource, and several major phenolics in the extract were quantitated using the established HPLC condition.

2. Results and Discussion

A part of the EtOAc extract obtained from powdered licorice was subjected to column chromatography on ODS-gel and eluted with increasing concentrations of MeOH in H2O and then with increasing concentrations of CHCl3 in MeOH. The eluate with 50% CHCl3 in MeOH was subjected to column chromatography on MCI-gel CHP-20P with increasing concentrations of MeOH in H2O. Fractions from the column were purified by preparative HPLC, to give 16 licorice phenolics, including licoricidin (2) [18], 7-O-methylluteone (3) [19], glyasperin J trimethyl ether (4) [20], 3'-(γ,γ-dimethylallyl)-kievitone (5) [21], isoangustone A (6) [22], glyasperin J (7) [20], compound A (8), licoriphenone (9) [23], demethylhomopterocarpan (10) [24], glycyrrhisofavone (11) [25], licopyranocoumarin (12) [26], glyasperin C (13) [27], compound B (14), glycyrrhiza-isofavone B (15) [28], glycybenzofuran (16) [29], and 8-(γ,γ-dimethylallyl)-wighteone (17) [30] from the respective fractions. The structures of 27, 913, and 1517 (Figure 1) were identified by comparisons of spectral data with values reported in the literature [18,19,20,21,22,23,24,25,26,27,28,29,30], whereas the two arylbenzofurans, temporarily named compounds A (8) and B (14) (Figure 1), are new, and their structure elucidations are described below.
Figure 1. Structures of the compounds 125 found in the EtOAc extract of Glycyrrhiza uralensis roots and stolons (Tohoku licorice). Structures 217 are the compounds isolated in this study a.
Figure 1. Structures of the compounds 125 found in the EtOAc extract of Glycyrrhiza uralensis roots and stolons (Tohoku licorice). Structures 217 are the compounds isolated in this study a.
Molecules 19 13027 g001
a The configuration at C-3 in each of compounds 4, 5, and 7 was not determined in the previous papers [20,21].

2.1. Structures of the New Compounds

Compound A (8): This compound was obtained as a light brown powder. Its molecular formula was C22H24O5, based on the [M + H]+ ion peak in the high-resolution fast-atom bombardment mass spectrometry (HR-FAB-MS). The ultraviolet (UV) spectrum of 8 showed absorption maxima at 214 (log ε 4.11), 238 (4.01), and 305 nm (4.52), indicating structural similarity to those of the known compounds glycybenzofuran (16) and licocoumarone (18) with 2-arylbenzofuran skeletons. The 1H nuclear magnetic resonance (NMR) spectrum of 8 (in acetone-d6) showed resonances of three aromatic protons at δH 7.30 (d, J = 8.4 Hz, H-4), 6.87 (d, J = 2.4 Hz, H-7), and 6.77 (dd, J = 2.4, 8.4 Hz, H-5), forming an ABX spin system, and a one-proton singlet at δH 6.41 (H-5'). The spectrum also showed four sets of proton resonances at δH 5.16 (1H, t, J = 6.6 HZ, H-2"), 3.32 (2H, d, J = 6.6 Hz, H-1"), 1.60 (3H, s), and 1.70 (3H, s) (2 × CH3 at C-3"), which are assignable to those of a γ,γ-dimethylallyl (prenyl) group. In addition, proton resonances characteristic of two methoxyl groups at δH 3.82 and 3.35 (3H each, s) and one methyl group at δH 1.89 (3H, s) were seen in the aliphatic region of the spectrum.
The 13C-NMR spectrum showed six carbon resonances due to oxygenated sp2 carbons (δC 160.0, 158.9, 156.2, 155.8, 153.1, and 145.7) and eight carbon resonances attributable to non-oxygenated sp2 carbons [δC 123.0, 119.6, 114.2 (2C) 111.5, 102.1 97.9, and 96.6]. The spectrum also showed five carbon resonances due to the prenyl group (δC 17.2, 22.5, 25.3, 124.3, and 129.9) and two methoxyl groups (δC 60.7 and 55.0). In addition to these resonances, the spectrum showed a methyl carbon resonance (δC 7.9), ascribable to the methyl group at C-3 of the 2-arylbenzofuran structure.
The assignments of these proton and carbon resonances were substantiated by the heteronuclear single quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) spectral data as summarized in Table 1. Key HMBC correlations among them and the nuclear Overhauser effect spectroscopy (NOESY) correlations indicating the locations of the respective substituents on the 2-arylbenzofuran skeleton are shown in Figure 2.
Figure 2. HMBC and NOESY correlations observed for compounds 8 and 14.
Figure 2. HMBC and NOESY correlations observed for compounds 8 and 14.
Molecules 19 13027 g002
Table 1. 1H- and 13C-NMR assignments, HSQC and HMBC correlations for compounds (8) and (14) (600 MHz for 1H and 151 MHz for 13C, acetone-d6, 27 °C) a,b.
Table 1. 1H- and 13C-NMR assignments, HSQC and HMBC correlations for compounds (8) and (14) (600 MHz for 1H and 151 MHz for 13C, acetone-d6, 27 °C) a,b.
Position4'-O-Methylglycybenzofuran (8)Neoglycybenzofuran (14)
δCHSQC aδH ( J in Hz)HMBC bδCHSQC aδH ( J in Hz)HMBC b
C-2145.7C H-10145.5C H-10
C-3114.2C H-10114.4C H-10
C-4119.6CH7.30, d (8.4) 119.3CH7.23, d (9.0)
C-5111.5CH6.77, dd (2.4, 8.4)H-4111.4CH6.71, dd (2.4, 9.0)
C-6155.8C H-5, 7155.7C H-4, 5, 7
C-797.9CH6.87, d (2.4) 97.8CH6.82, d (2.4)
C-8156.2C H-4, 7156.2CH H-4, 7
C-9123.0C H-5, 7, 10123.8CH H-5, 7, 10
C-107.9CH31.89 s 8.7CH31.97 s
C-1'102.1C H-5'103.5C H-5'
C-2'158.9C H-1"159.1C H-1"
C-3'114.2C H-5', H-2"112.9C H-5', 1", 2"
C-4'160.0C H-5', H-1"160.0C H-5', 1"
C-5'96.6CH6.41 s 98.7CH6.28 s
C-6'153.1C H-5'155.7C H-5'
C-1"22.5CH23.32, d (6.6) 22.9CH23.13, d (6.6)
C-2"124.3CH5.16, t (6.6) 124.6CH5.12, t (6.6)
C-3"129.9C H-4", H-5"129.7C H-4", 5"
C-4"17.2CH31.60 s 17.8CH31.69 s
C-5"25.3CH31.70 s 25.5CH31.63 s
-OCH360.7CH33.82 sH-1160.6CH33.28 sH-11
-OCH355.0CH33.35 sH-12
a HSQC, shows the relationship between a proton directly connected to a carbon. b HMBC correlations, optimized for 5 Hz, are from proton(s) stated to the optimized carbons.
The locations of the hydroxyl and methoxyl groups, and the prenyl group on the B-ring of this compound were shown by the following HMBC correlations. Correlation of the methylene proton resonance at δH 3.32 (H-1") of the prenyl moiety and an -OCH3 resonance (δH 3.82) with a common oxygenated aromatic carbon resonance at δC 160.0 (C-4') and also the correlations of the same methylene proton resonance (H-1") and a methoxyl proton resonance (δH 3.35) with a common oxygenated carbon resonance at δC 158.9 (C-2'). The carbon resonance at δC 160.0 (C-4') was also correlated with the singlet proton resonance at δH 6.41 (H-5', directly correlated with the C-5 carbon resonance at δC 96.6 in the HSQC spectrum), and this proton resonance was also correlated with an oxygenated aromatic carbon at δC 153.1 (C-6'), which was not correlated with any methoxyl proton resonance. The H-5' resonance was also correlated with the carbon resonances at δC 114.2 (C-3') and δC 102.1 (C-1') of the same aromatic ring. The last two carbon resonances were discriminated by a correlation of the methylene proton resonance (H-1") to the carbon resonance at δC 114.2 (C-3'). The sequence C-1' (δC 102.1)–C-2' (δC 158.9, with a methoxyl group)–C-3' (δC 114.2, with the prenyl group)–C-4' (δC 160.0, with a methoxyl group)–C-5' (δC 96.6)–C-6' (δC 153.1) was thus assigned for the B-ring. In addition, the NOESY spectrum of this compound showed correlations of the methine proton resonance at δH 5.16 (H-2" of the prenyl moiety) with the two methoxyl resonances at δH 3.82 (-OCH3 at C-4') and δH 3.35 (-OCH3 at C-2'), and the former methoxyl resonance also showed a correlation with the proton resonance at δH 6.41 (C-5'), in agreement with the sequence described above.
The presence of a methyl group at C-3 was clearly indicated by the HMBC correlations of the methyl proton resonance at δH 1.89 (H-10) with the carbon resonance at δC 145.7 (C-2), 114.2 (C-3), and 123.0 (C-9). The NOESY correlations of this proton resonance with the methoxyl proton resonance at δH 3.35 (-OCH3 at C-2'), and with the aromatic proton resonance at δH 7.30 (H-4), are also consistent with the location C-3 of the methyl group. The coupling patterns of H-4 [δH 7.30 (d, J = 8.4 Hz)], H-5 [δH 6.77 (dd, J = 2.4, 8.4 Hz)], and H-7 [δH 6.87 (d, J = 2.4 Hz)] resonances, forming an ABX system, indicated the location C-6 for the hydroxyl group. These data, and also the remaining HMBC correlations, satisfied the 2-aryl-3-methyl-6-hydroxybenzofuran structure.
Because structure 8 assigned to compound A was an analog of a compound reported previously, glycybenzofuran (16), the corresponding methylated products of compounds 8 and 16 were compared. As a result, product 8a from 8 was the same as that obtained by methylation of 16, as expected. The structure of compound A was thus substantiated to be 4'-O-methylglycybenzofuran (8).
Compound B (14): Compound B was obtained as a light brown powder. The molecular formula C21H22O5, which was the same as that of glycybenzofuran (16), was detected by HR-FAB-MS. The UV spectrum of 14 (in MeOH) showed absorption maxima at 210 (log ε 4.09), 238 (4.21), and 300 nm (4.30), where the spectral feature characteristic of the 2-arylbenzofuran skeleton was seen in the spectra of compounds 8 and 16. The aromatic region of the 1H-NMR spectrum of 14 (in acetone-d6) showed a one-proton singlet at δH 6.28 and the three proton resonances forming an ABX spin system at δH 7.23 (d, J = 8.4 Hz, H-4), 6.82 (d, J = 2.4 Hz, H-7), and 6.71 (dd, J = 2.4, 8.4 Hz, H-5). The spectrum also exhibited characteristic resonances of a prenyl moiety at δH 3.13 (2H, d, J = 6.6 Hz, H-1"), 5.12 (1H, t, J = 6.6 Hz, H-2"), 1.69 (3H, s), and 1.63 (3H, s) (gem-dimethyl at C-3"). In addition, the spectrum exhibited a methyl proton resonance at δH 1.97 (3H, s) and a methoxyl resonance at δH 3.28 (3H, s). These data indicate structural similarity of 14 to that of 8 except for the number of the methoxyl resonances. That is, 14 had a structure isomeric to 16, concerning the placement of the methoxyl group.
The 13C-NMR spectrum of 14 showed resonances of 14 sp2 carbons attributable to the 2-arylbenzofuran skeleton composed of six oxygenated carbons [δC 160.0, 159.1, 156.2, 155.7 (2C), and 145.5] and eight non-oxygenated carbons (δC 123.8, 119.3, 114.4, 112.9, 111.4, 103.5, 98.7, and 97.8). The spectrum also showed a methyl carbon resonance at δC 8.7, a methoxyl carbon resonance at δC 60.6, and five carbon resonances due to a prenyl unit (δC 17.8, 22.9, 25.5, 124.6, and 129.7).
The HMBC spectrum (Table 1 and Figure 2) showed correlations δC 159.1 (C-2')–δH 3.13 (H-1" of prenyl at C-3')–δC 160.0 (C-4')–δH 3.28 (OCH3 at C-4'), and also the correlations δC 160.0 (C-4')–δH 6.28 (H-5')–δC 155.7 (C-6'), and δH 6.28 (H-5')–δC 103.5 (C-1'). Furthermore, the NOESY spectrum showed correlations δH 5.12 (H-2" of prenyl at C-3')–δH 3.28 (-OCH3 at C-4')–δH 6.28 (H-5') (Figure 2). These correlations clearly indicate the sequence C-1'–C-6' (with -OH)–C-5'–C-4' (with -OCH3)–C-3' (with prenyl)–C-2' (with -OH) of the B-ring structure.
The presence of the methyl group at C-3 was indicated by the HMBC correlations from the methyl proton resonance (H-10) at δH 1.97 with C-2 (δC 145.5), C-3 (δC 114.4), and C-9 (δC 123.8) and the NOESY correlations between the methyl resonance at δH 1.97 and H-4 at δH 7.23. The resonances of H-4, H-5, and H-7, forming an ABX system as shown by the 1H-1H COSY spectrum, indicated the location of a hydroxyl group at C-6, and the HMBC correlations (Figure 2) concerning these aromatic proton resonances also satisfied the location C-6 of the hydroxyl group. Based on these findings, structure 14, which was isomeric to 16, was assigned to compound B which accordingly was named neoglycybenzofuran. Methylation of 14 afforded 8a and thus substantiated the structure 14 for neoglycybenzofuran.

2.2. Antibacterial Effects of Licorice Phenolics on VRE

The antibacterial effects of the licorice phenolics on the two species of VRE, E. faecium FN-1 and E. faecalis NCTC 12201, were estimated using the liquid dilution method as described previously [17]. The results summarized in Table 2 reveal that almost all of the licorice phenolics examined showed antibacterial effects [minimum inhibitory concentration (MIC), 1.9 × 10−5–3.5 × 10−4 M] on the VRE strains, and several ones among them showed noticeable anti-VRE effects (Table 2).
Table 2. Antibacterial effects of licorice phenolics on Enterococci (estimated minimum inhibitory concentrations, MIC) a.
Table 2. Antibacterial effects of licorice phenolics on Enterococci (estimated minimum inhibitory concentrations, MIC) a.
CompoundsNumber of -OH GroupsNumber of Prenyl GroupsMIC (10−5 M)
Enterococcus faecium FN-1Enterococcus faecalis NCTC12201
7-O-Methylluteone (3)318.78.7
Isoangustone A (6)423.83.8
Glycyrrhisofavone (11)419.09.0
Glycyrrhiza-isoflavone B (15)203535
8-(γ,γ-Dimethylallyl)-wighteone (17)321.93.8
Glicoricone (22) a31>35>35
6,8-Diprenylorobol (25) a423030
Licoricidin (2)321.91.9
Glyasperin C (13)314.54.5
Glyasperin J trimethyl ether (4)011414
3'-(γ,γ-Dimethylallyl)-kievitone (5)423.83.8
Glyasperin J (7)317.57.5
Licopyranocoumarin (12)20>3333
Glycyrin (20) a214.28.4
Glycycoumarin (21) a314.34.3
Glycyrol (18) a2135>35
Demethylhomopterocarpan (10)101212
Gancaonin I (1) a214.54.5
4'-O-Methylglycybenfuran (8)218.78.7
Noeglycybenzofuran (14)314.54.5
Glycybenzofuran (16)311818
Licoriphenone (9)31>3434
Standard antibacterial agents
Vancomycin a >6.9>6.9
Linezolid a 0.740.74
EtOAc extract from Tohoku licorice 16 µg/mL32 µg/mL
a Data taken from [17].
Among these compounds, licoricidin (2) (isoflavan) showed the most potent effects against both E. faecalis and E. faecium (MIC, 1.9 × 10−5 M). 8-(γ,γ-Dimethylallyl)-wighteone (17) (isoflavone), isoangustone A (6) (isoflavone), 3'-(γ,γ-dimethylallyl)-kievitone (5) (isoflavanone), glyasperin C (13) (isoflavan), and neoglycybenzofuran (14) (new, 2-aryl-3-methylbenzofuran) also showed anti-VRE effects with MICs of 1.9 × 10−5–4.5 × 10−5 M, respectively. All of these compounds have three or more phenolic hydroxyl groups and at least one prenyl group. In contrast, several other compounds such as licoriphenone (9) showed relatively weaker effects, and they had analogous structural features. Further experiments are required to clarify the structural factors responsible to the antibacterial effects.
The antibacterial effects of the EtOAc extract were comparable to those of potent anti-VRE constituents. Potential synergy and/or additive effects between the purified phenolics remain to be determined.

2.3. HPLC Analyses of Anti-VRE Phenolics for the Evaluation of EtOAc Extract from G. uralensis as a Source of Antibacterial Agent

Because of the important uses of licorice in traditional medicine, qualitative and quantitative analyses of licorice constituents and licorice products have been reported [31,32,33,34,35,36]. Remarkable anti-VRE effects of several licorice phenolics shown in our current and previous studies [17] suggested requirements of the identification and quantitation of those constituents. We therefore developed an HPLC-UV method for the simultaneous detection of major isolated phenolic constituents in EtOAc extract from G. uralensis, in order to evaluate the quality of the extract as a source of antibacterial agent.
The HPLC-UV profile of the EtOAc extract from Tohoku licorice used in the present study under the established condition is shown in Figure 3. Each constituent in the HPLC profile was identified by comparisons of its retention time, UV and MS spectra (data not shown) with those of the isolated one. The elution order of the identified constituents was as follows: demethylhomopterocarpan (10, tR 38.6 min), 7-O-methylluteone (3, tR 41.2 min), licopyranocoumarin (12, tR 46.0 min), glycybenzofuran (16, tR 46.6 min), glycyrol (18, tR 52.9 min), licoarylcoumarin (19, tR 68.0 min), licoriphenone (9, tR 73.2 min), glycyrin (20, tR 79.4 min), glycycoumarin (21, tR 85.9 min), glicoricone (22, tR 91.8 min), neoglycybenzofuran (14, tR 98.1 min), glycyrrhiza-isofavone B (15, tR 102.4 min), glyasperin D (23, tR 107.4 min), gancaonin G (24, tR 112.1 min), glyasperin C (13, tR 121.0 min), 8-(γ,γ-dimethylallyl)-wighteone (17, tR 141.1 min), licoricidin (2, tR 147.9 min), 4'-O-methylglycybenzofuran (8, tR 156.1 min), glyasperin J (7, tR 164.5 min), gancaonin I (1, tR 176.1 min), 6,8-diprenylorobol (25, tR 191.3 min), glycyrrhisofavone (11, tR 202.9 min), 3'-(γ,γ-dimethylallyl)-kievitone (5, tR 213.5 min), glyasperin J trimethyl ether (4, tR 222.8 min), and isoangustone A (6, tR 233.2 min).
Figure 3. HPLC-UV chromatogram of G. uralensis (Tohoku licorice) EtOAc extract at 280 nm a−c.
Figure 3. HPLC-UV chromatogram of G. uralensis (Tohoku licorice) EtOAc extract at 280 nm a−c.
Molecules 19 13027 g003
a Column, YMC-Pack Pro C18 (6.0 mm i.d. × 150); mobile phase, H2O/MeCN/MeCOOH (55:40:5, v/v/v); flow rate, 1.0 mL/min; oven temperature, 40 °C; detector, Hitachi L2455. b The numbers represent the isolated compounds, as displayed in Figure 1. c The configuration of each of the optically active constituents was not reflected in the present analytical conditions.
Quantitative analysis of several compounds was performed under the same HPLC condition, and the amounts of the major phenolic constituents are shown in Table 3. Among these major phenolics, gancaonin I (1) and isoangustone A (6) showed potent anti-VRE effects.
Table 3. Contents of major licorice phenolics in G. uralensis (Tohoku licorice) EtOAc extract.
Table 3. Contents of major licorice phenolics in G. uralensis (Tohoku licorice) EtOAc extract.
CompoundContent (% w/w) a
Glycyrol (18)0.54 ± 0.036
Gancaonin I (1)0.49 ± 0.025
Isoangustone A (6)0.34 ± 0.031
Glycyrin (20)0.26 ± 0.015
Glycycoumarin (21)0.24 ± 0.010
Glicoricone (22)0.18 ± 0.023
6,8-Diprenylorobol (25)0.094 ± 0.013
Licoriphenone (9)0.082 ± 0.017
a The value was given as the mean ± standard deviation (SD) based on the triplicate experiments.

3. Experimental Section

3.1. General Information

UV spectra were recorded on a V-530 spectrometer (JASCO, Tokyo, Japan). Measurements of electrospray ionization mass spectra were taken on an API-4000 instrument (AB Sciex, Framingham, MA, USA) and high-resolution fast atom bombardment-mass spectroscopy (HR-FAB-MS) was conducted on a JMS-700 MStation (JEOL, Tokyo, Japan) with a mixture of m-nitrobenzyl alcohol and dithiothreitol as the matrix. 1H and 13C-NMR spectra were recorded on an INOVA 600AS instrument (600 MHz for 1H and 151 MHz for 13C; Agilent, Santa Clara, CA, USA). Chemical shifts of the resonances in these spectra were adjusted using those of the solvent resonances [δH 2.04 and δC 29.8 for (CD3)2CO] and are given in δ (ppm) values. Analytical HPLC-DAD to monitor purification of the constituents was conducted on an ODS-A 302 (4.6 mm i.d. × 250 mm; YMC, Kyoto, Japan) column at 40 °C in an oven with 10 mM H3PO4/10 mM KH2PO4/MeCN (35:35:30, v/v/v, isocratic mode) as the eluent. A Hitachi L-2455 detector was used for monitoring UV absorption at 280 nm, and the flow rate was set at 1.0 mL/min. Preparative HPLC was performed on an ODS-A324 (10 mm i.d. × 300 mm: YMC) column at 40 °C in an oven with H2O/MeCN/MeCOOH (45:50:5, v/v/v) as the eluent. UV absorption at 280 nm was used for HPLC detection, and the flow rate was set at 2.0 mL/min. The procedure for the simultaneous HPLC analyses of the phenolic constituents in the EtOAc extract is described separately (see below). Silica gel (YMC), Toyopearl HW-40 (coarse grade; TOSOH, Tokyo, Japan), YMC-gel ODS-A (S, 75 μm; YMC), and MCI-gel CHP-20P (Mitsubishi Chemical, Tokyo, Japan) were used for column chromatography.

3.2. Plant Material

The crude drug used in this study was Tohoku licorice, which is the dried roots and stolons of Glycyrrhiza uralensis Fisch. ex DC, purchased from Tochimoto-tenkai-do (Osaka, Japan) (lot no. 002009037), and the GU-07112011(NEL) specimen was kept at the Medicinal Plant Garden, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences.

3.3. Extraction and Isolation

Licorice (1.0 kg) was pulverized and dipped in n-hexane (3 L × 2). Then, the defatted material was treated with EtOAc (3 L × 2) to give the extract (46.4 g). Part (40 g) of the EtOAc extract was subjected to column chromatography on ODS-gel (2.2 i.d. × 75 cm) with increasing concentrations of MeOH in H2O and then with increasing concentrations of CHCl3 in MeOH. The eluate with 50% CHCl3 in MeOH (3.6 g) was subjected to column chromatography on MCI-gel CHP-20P (2.2 i.d. × 45 cm) with increasing concentrations of MeOH in H2O. Fractions 94 (42 mg), 96 (40 mg), 161 (38 mg), 230 (31 mg), 231 (30 mg), 234 (28 mg), 327 (24 mg), and 337 (22 mg) were respectively purified by preparative HPLC on YMC-Pack A-324 (10 mm i.d. × 300 mm; 2.5 mL/min; H2O/MeCN/MeCOOH, 55:40:5, v/v/v; isocratic mode; monitored at 280 nm) to give the following phenolics: licoricidin (2, 3.1 mg), 7-O-methylluteone (3, 2 mg), and glyasperin J trimethyl ether (4, 1.5 mg) from fraction 94; 3'-(γ,γ-dimethylallyl)-kievitone (5, 4.0 mg), isoangustone A (6, 9.0 mg), glyasperin J (7, 2.3 mg), compound A (8, 3.2 mg), and licoriphenone (9, 1.9 mg) from fraction 96; demethylhomopterocarpan (10, 1.2 mg) from fraction 161; glycyrrhisofavone (11, 5.0 mg) from fraction 230; licopyranocoumarin (12, 4.9 mg), glyasperin C (13, 3.0 mg), and compound B (14, 2.0 mg) from fraction 231; glycyrrhiza-isofavone B (15, 1.6 mg) from fraction 234; glycybenzofuran (16, 1.8 mg) from fraction 327; and 8-(γ,γ-dimethylallyl)-wighteone (17, 1.5 mg) from fraction 337. The purity of each of the isolated compounds were >98%, as estimated by HPLC and 1H-NMR.

3.4. Spectral Data

Compound A (4'-O-Methylglycybenzofuran, 8): This compound was obtained as a light brown powder; 1H- and 13C-NMR (see Table 1); HR-FAB-MS m/z 369.1702 ([M + H]+), (Calculated for C22H24O5, 369.1709).
Compound B (Neoglycybenzofuran, 14): This compound was obtained as a light brown powder; 1H- and 13C-NMR (see Table 1); HR-FAB-MS m/z 355.1546 ([M + H]+), (Calculated for C21H22O5, 355.1549).

3.5. Methylation of Compounds A and B, and Glycybenzofuran

Trimethylsilyldiazomethane solution (1 mL) was added to a solution of 8 (1 mg) in EtOH (0.1 mL), and the mixture was kept for 3 h at room temperature. After evaporating the solvent, the remaining product was purified by TLC on silica gel (Merck, silica gel F254) (CHCl3‒MeOH, 15:1, v/v) to give a methyl derivative of compound A (8a). Detection was effected by UV absorption at 254 nm. 1H-NMR (600 MHz, acetone-d6): δH 1.99, 1.74, 1.65 (each 3H, s, –CH3 × 3), 3.27 (2H, d, J = 6.6 Hz, H-1"), 3.38, 3.82 (each 3H, s, -OCH3 × 2), 5.17 (1H, t, H-2"), 6.40 (1H, s, H-5'), 6.80 (1H, dd, J = 2.4, 8.4 HZ, H-5), 6.88 (1H, d, J = 2.4 HZ, H-7),7.35 (1H, d, J = 8.4 Hz, H-4). The identical compound was obtained by treating compound B (14) and glycybenzofuran (16) with trimethylsilyldiazomethane in analogous ways.

3.6. Antibacterial Assay

Estimations of the antibacterial effects of licorice phenolics on the VRE E. faecium FN-1 and E. faecalis NCTC 12201 used in this study were conducted using VRE kindly provided by Y. Ike, Gunma University. The bacterial cells were precultured in Mueller-Hinton broth at 37 °C under aerobic conditions. They were incubated in the presence of compounds with the concentrations obtained by serial two-fold dilution at 37 °C without shaking in the same broth for 24 h on microplates as shown in a previous paper [17], and their MICs were estimated as the lowest concentrations where the bacterial cells were not observed visually as reported previously [16,17], and were given based on triplicate experiments. DMSO was used for dissolving compounds hardly soluble in water, and the final concentrations were set at <1%, where DMSO has no effect. The positive control, linezolid, was dissolved in water.

3.7. Simultaneous HPLC Analysis of Phenolic Constituents in the EtOAc Extract of Licorice

Simultaneous analysis of licorice phenolics was carried out on an HPLC-DAD D-2000 HSM system, composed of an L-2130 pump (Hitachi, Tokyo, Japan) and an L-2455 DAD (Hitachi). The DAD was set for obtaining UV spectral data from 200 to 400 nm, and chromatograms at 280 nm were used for the quantitative analyses. The column used was an YMC-Pack pro C18 (6.0 mm i.d. × 150 mm) and was set in an oven at 40 °C. The mobile phase consisted of H2O/MeCN/MeCOOH (55:40:5, v/v/v), and the flow rate was set at 1.0 mL/min. Quantitation of 1, 6, 9, 18, 20, 21, 22, and 25 was based on the HPLC profile monitored at 280 nm.
Licorice (10 g) was pulverized and extracted with EtOAc (100 mL × 3). Approximately 10 mg of the dried extract powder was dissolved in 10 mL of MeOH and filtered with a 0.45 µm PTFE membrane filter prior to injection (8 μL of the filtrate at 1 mg/mL) was applied to HPLC analysis. Stock solutions of eight licorice phenolics (1, 6, 9, 18, 20, 21, 22 and 25) were prepared at 0.1 mg/mL in MeOH, and diluted in series (from 0.1 to 0.001 mg/mL) to produce eight individual standard curves, for which the correlation coefficients were determined between 0.991 and 0.999 under the described HPLC conditions.

4. Conclusions

Our present investigation on the EtOAc extract of G. uralensis led to the purification of 16 compounds. Among the compounds obtained, two new compounds, 8 and 14, had 2-aryl-3-methylbenzofuran structures, which rarely occur in Nature. The isolated phenolics were categorized into isoflavones (3, 6, 11, 15, and 17), isoflavans (2 and 13), isoflavanones (4, 5, and 7), a 3-arylcoumarin (12), a pterocarpan (10), 2-aryl-3-methylbenzofurans (8, 14, and 16), and a benzylphenylketone (9). As shown in our previous studies, licorice phenolics possess remarkable antibacterial effects against MRSA [16] and VRE [17]. The effects of the licorice phenolics isolated in the present study on VRE were examined. Based on their MIC values (Table 2), the antibacterial activities of the isoflavans and the isoflavones, bearing prenyl and phenolic hydroxyl groups, were promising. Our previous study [17] also indicated that compounds with prenyl moieties, such as gancaonin I (1), licoarylcoumarin (19), and glycycoumarin (21), showed noticeable anti-VRE effects. Taken together, we conclude that licorice phenolics, particularly those with prenyl moieties, could be used for the development of anti-VRE agents. The mechanisms of action of these phenolics as well as their potential synergistic effects remain to be clarified and the possibility of presence of potential synergistic effects between these identified licorice constituents are remained to be clarified. With regard to their promising antibiotic activities, the phenolic constituents from licorice could be used as lead compounds for developing new antibacterial agents.


This study was supported in part by Drug Discovery Project for Intractable Infectious Diseases of Okayama University (IIDPO). Eerdunbayaer thanks Jinghao Qi, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, for his helpful advice. The NMR instrument used is the property of the Advanced Science Research Center, Okayama University.

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.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Horiuchi, K.; Shiota, S.; Hatano, T.; Yoshida, T.; Kuroda, T.; Tsuchiya, T. Antimicrobial activity of oleanolic acid from Salvia officinalis and related compounds on vancomycin-resistant Enterococci (VRE). Biol. Pharm. Bull. 2007, 30, 1147–1149. [Google Scholar] [CrossRef]
  2. Orsi, G.B.; Ciorba, V. Vancomycin resistant Enterococci healthcare associated infections. Ann. Ig. 2013, 25, 485–492. [Google Scholar]
  3. Rice, L.B. Emergence of vancomycin-resistant Enterococci. Emerg. Infect. Dis. 2001, 7, 183–187. [Google Scholar] [CrossRef]
  4. McNeil, S.A.; Clark, N.M.; Chandrasekar, P.H.; Kauffman, C.A. Successful treatment of vancomycin-resistant Enterococcus faecium bacteremia with linezolid after failure of treatment with synercid (quinupristin/dalfopristin). Clin. Infect. Dis. 2000, 30, 403–404. [Google Scholar] [CrossRef]
  5. Isbrucker, R.A.; Burdock, G.A. Risk and safety assessment on the consumption of licorice root (Glycyrrhiza sp.), its extract and powder as a food ingredient, with emphasis on the pharmacology and toxicology of glycyrrhizin. Regul. Toxicol. Pharmacol. 2006, 46, 167–192. [Google Scholar]
  6. Shen, X.-P.; Xiao, P.-G.; Liu, C.-X. Research and application of Radix Glycyrrhizae. Asian J. Pharmacodyn. Pharmacokinet. 2007, 7, 181–200. [Google Scholar]
  7. Asl, M.N.; Hosseinzadeh, H. Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds. Phytother. Res. 2008, 22, 709–724. [Google Scholar] [CrossRef]
  8. Messier, C.; Epifano, F.; Genovese, S.; Grenier, D. Licorice and its potential beneficial effects in common oro-dental diseases. Oral Dis. 2012, 18, 32–39. [Google Scholar] [CrossRef]
  9. Villinski, J.R.; Bergeron, C.; Cannistra, J.C.; Gloer, J.B.; Coleman, C.M.; Ferreira, D.; Gafner, S. Pyrano-isoflavans from Glycyrrhiza uralensis with antibacterial activity against Streptococcus mutans and Porphyromonas gingivalis. J. Nat. Prod. 2014, 77, 521–526. [Google Scholar] [CrossRef]
  10. Gafner, S.; Bergeron, C.; Villinski, J.R.; Godejohann, M.; Kessler, P.; Cardellina, J.H.; Grenier, D. Isoflavonoids and coumarins from Glycyrrhiza uralensis: Antibacterial activity against oral pathogens and conversion of isoflavans into isoflavan-quinones during purification. J. Nat. Prod. 2011, 74, 2514–2519. [Google Scholar] [CrossRef]
  11. He, J.; Chen, L.; Heber, D.; Shi, W.; Lu, Q.Y. Antibacterial compounds from Glycyrrhiza uralensis. J. Nat. Prod. 2006, 69, 121–124. [Google Scholar] [CrossRef]
  12. Fukai, T.; Marumo, A.; Kaitou, K.; Kanda, T.; Terada, S.; Nomura, T. Anti-Helicobacter pylori flavonoids from licorice extract. Life Sci. 2002, 71, 1449–1463. [Google Scholar] [CrossRef]
  13. Irani, M.; Sarmadi, M.; Bernard, F. Leaves antimicrobial activity of Glycyrrhiza glabra. Iran. J. Pharm. Res. 2010, 9, 425–428. [Google Scholar]
  14. Badr, A.E.; Omar, N.; Badria, F.A.A. Laboratory evaluation of the antibacterial and cytotoxic effect of liquorice when used as root canal medicament. Int. Endod. J. 2011, 44, 51–58. [Google Scholar] [CrossRef]
  15. Fukai, T.; Oku, Y.; Hano, Y.; Terada, S. Antimicrobial activities of hydrophobic 2-arylbenzofurans and an isoflavone against vancomycin-resistant Enterococci and methicillin-resistant Staphylococcus aureus. Planta Med. 2004, 70, 685–687. [Google Scholar] [CrossRef]
  16. Hatano, T.; Shintani, Y.; Aga, Y.; Shiota, S.; Tsuchiya, T.; Yoshida, T. Phenolic constituents of licorice. VIII. Structures of glicophenone and glicoisoflavanone, and effects of licorice phenolics on methicillin-resistant Staphylococcus aureus. Chem. Pharm. Bull. 2000, 48, 1286–1292. [Google Scholar] [CrossRef]
  17. Eerdunbayaer; Orabi, M.A.; Aoyama, H.; Kuroda, T.; Hatano, T. Structures of two new flavonoids and effects of licorice phenolics on vancomycin-resistant Enterococcus species. Molecules 2014, 19, 3883–3897. [Google Scholar] [CrossRef]
  18. Fukai, T.; Toyono, M.; Nomura, T. On the structure of licoricidin. Heterocycles 1988, 27, 2309–2313. [Google Scholar] [CrossRef]
  19. Tahara, S.; Ingham, J.L.; Mizutani, J. Metabolites of 7-O-methylluteone from Botrytis cinerea. Nippon Nogeikagaku Kagaku Kaishi 1989, 63, 999–1007. [Google Scholar] [CrossRef]
  20. Zeng, L.; Fukai, T.; Nomura, T.; Zhang, R.Y.; Lou, Z.C.; Fukai, T.; Nomura, T. Five new isoprenoid-substituted flavonoids, glyasperins F, G, H, I, and J from the roots of Glycyrrhiza aspera. Heterocycles 1992, 34, 1813–1828. [Google Scholar] [CrossRef]
  21. O’Neill, M.J.; Adesanya, S.A.; Roberts, M.F.; Inez, R.P. Inducible isoflavonoids from the lima bean, Phaseolus lunatus. Phytochemistry 1986, 25, 1315–1322. [Google Scholar] [CrossRef]
  22. Sil Lee, Y.; Ha Kim, S.; Kyu Kim, J.; Shin, H.K.; Kang, Y.H.; Park, Y.; Lim, S.S. Rapid identification and preparative isolation of antioxidant components in licorice. J. Sept. Sci. 2010, 33, 664–671. [Google Scholar] [CrossRef]
  23. Kiuchi, F.; Chen, X.; Tsuda, Y. Four new phenolic constituents from licorice (root of Glycyrrhiza sp.). Heterocycles 1990, 31, 629–636. [Google Scholar] [CrossRef]
  24. Sasaki, H.; Kashiwada, Y.; Shibatav, H.; Takaishi, Y. Prenylated flavonoids from the roots of Desmodium caudatum and evaluation of their antifungal activity. Planta Med. 2012, 78, 1851–1856. [Google Scholar] [CrossRef]
  25. Hatano, T.; Kagawa, H.; Yasuhara, T.; Okuda, T. Two new flavonoids and other constituents in licorice root: Their relative astringency and radical scavenging effects. Chem. Pharm. Bull. 1988, 36, 2090–2097. [Google Scholar] [CrossRef]
  26. Hatano, T.; Yasuhara, T.; Fukuda, T.; Noro, T.; Okuda, T. Phenolic constituents of licorice. II. Structures of licopyranocoumarin, licoarylcoumarin and glisoflavone, and glisoflavone, and inhibitory effects of licorice phenolics on xanthine oxidase. Chem. Pharm. Bull. 1989, 37, 3005–3009. [Google Scholar] [CrossRef]
  27. Kwon, H.J.; Kim, H.H.; Ryu, Y.B.; Kim, J.H.; Jeong, H.J.; Lee, S.W.; Lee, W.S. In vitro anti-rotavirus activity of polyphenol compounds isolated from the roots of Glycyrrhiza uralensis. Bioorg. Med. Chem. 2010, 18, 7668–7674. [Google Scholar]
  28. Hatano, T.; Takagi, M.; Ito, H.; Yoshida, T. Phenolic constituents of liquorice. VII. A new chalcone with a potent radical scavenging activity and accompanying phenolics from liquorice. Chem. Pharm. Bull. 1997, 45, 1485–1492. [Google Scholar] [CrossRef]
  29. Li, S.; Li, W.; Wang, Y.; Asada, Y.; Koike, K. Prenyl flavonoids from Glycyrrhiza uralensis and their protein tyrosine phosphatase-1B inhibitory activities. Bioorg. Med. Chem. Lett. 2010, 20, 5398–5401. [Google Scholar] [CrossRef]
  30. Singhal, A.K.; Sharma, R.P.; Thyagarajan, G.; Herz, W.; Govindan, S.V. New prenylated isoflavones and a prenylated dihydroflavonol from Millettia pachycarpa. Phytochemistry 1980, 9, 929–934. [Google Scholar]
  31. Zhang, Q.; Ye, M. Chemical analysis of the Chinese herbal medicine Gan-Cao (licorice). J. Chromatogr. A 2009, 1216, 1954–1969. [Google Scholar] [CrossRef]
  32. Chen, X.J.; Zhao, J.; Meng, Q.; Li, S.P.; Wang, Y.T. Simultaneous determination of five flavonoids in licorice using pressurized liquid extraction and capillary electrochromatography coupled with peak suppression diode array detection. J. Chromatogr. A 2009, 1216, 7329–7335. [Google Scholar] [CrossRef]
  33. Liang, X.; Zhang, L.; Zhang, X.; Dai, W.; Li, H.; Hu, L.; Zhang, W. Qualitative and quantitative analysis of traditional Chinese medicine Niu Huang Jie Du Pill using ultra performance liquid chromatography coupled with tunable UV detector and rapid resolution liquid chromatography coupled with time-of-flight tandem mass spectrometry. J. Pharm. Biomed. Anal. 2010, 51, 565–571. [Google Scholar] [CrossRef]
  34. Seo, C.S.; Lee, J.A.; Jung, D.; Lee, H.Y.; Lee, J.K.; Ha, H.; Shin, H.K. Simultaneous determination of liquiritin, hesperidin, and glycyrrhizin by HPLC-photodiode array detection and the anti-inflammatory effect of Pyungwi-san. Arch. Pharm. Res. 2011, 34, 203–210. [Google Scholar] [CrossRef]
  35. Wen, J.; Qiao, Y.; Yang, J.; Liu, X.; Song, Y.; Liu, Z.; Li, F. UPLC-MS/MS determination of paeoniflorin, naringin, naringenin and glycyrrhetinic acid in rat plasma and its application to a pharmacokinetic study after oral administration of SiNiSan decoction. J. Pharm. Biomed. Anal. 2012, 66, 271–277. [Google Scholar]
  36. Zhou, S.; Cao, J.; Qiu, F.; Kong, W.; Yang, S.; Yang, M. Simultaneous determination of five bioactive components in radix glycyrrhizae by pressurised liquid extraction combined with UPLC-PDA and UPLC/ESI-QTOF-MS confirmation. Phytochem. Anal. 2013, 24, 527–533. [Google Scholar] [CrossRef]
  • Sample Availability: Samples of all of the compounds are unavailable.

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MDPI and ACS Style

Eerdunbayaer; Orabi, M.A.A.; Aoyama, H.; Kuroda, T.; Hatano, T. Structures of New Phenolics Isolated from Licorice, and the Effectiveness of Licorice Phenolics on Vancomycin-Resistant Enterococci. Molecules 2014, 19, 13027-13041.

AMA Style

Eerdunbayaer, Orabi MAA, Aoyama H, Kuroda T, Hatano T. Structures of New Phenolics Isolated from Licorice, and the Effectiveness of Licorice Phenolics on Vancomycin-Resistant Enterococci. Molecules. 2014; 19(9):13027-13041.

Chicago/Turabian Style

Eerdunbayaer, Mohamed A. A. Orabi, Hiroe Aoyama, Teruo Kuroda, and Tsutomu Hatano. 2014. "Structures of New Phenolics Isolated from Licorice, and the Effectiveness of Licorice Phenolics on Vancomycin-Resistant Enterococci" Molecules 19, no. 9: 13027-13041.

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