Isoflavonoids and coumarins from Glycyrrhiza uralensis: Antibacterial activity against oral pathogens and conversion of isoflavans into isoflavan-quinones during purification

Article abstract of DOI:10.1021/np2004775

Phytochemical investigation of a supercritical fluid extract of Glycyrrhiza uralensis has led to the isolation of 20 known isoflavonoids and coumarins, and glycycarpan (7), a new pterocarpan. The presence of two isoflavan-quinones, licoriquinone A (8) and licoriquinone B (9), in a fraction subjected to gel filtration on Sephadex LH-20 is due to suspected metal-catalyzed oxidative degradation of licoricidin (1) and licorisoflavan A (2). The major compounds in the extract, as well as 8, were evaluated for their ability to inhibit the growth of several major oral pathogens. Compounds 1 and 2 showed the most potent antibacterial activities, causing a marked growth inhibition of the cariogenic species Streptococcus mutans and Streptococcus sobrinus at 10 μg/mL and the periodontopathogenic species Porphyromonas gingivalis (at 5 μg/mL) and Prevotella intermedia (at 5 μg/mL for 1 and 2.5 μg/mL for 2). Only 1 moderately inhibited growth of Fusobacterium nucleatum at the highest concentration tested (10 μg/mL).

Full text of DOI:10.1021/np2004775

Article
pubs.acs.org/jnp
Isoflavonoids and Coumarins from Glycyrrhiza uralensis: Antibacterial
Activity against Oral Pathogens and Conversion of Isoflavans into
Isoflavan-Quinones during Purification
Stefan Gafner,*,† Chantal Bergeron,† Jacquelyn R. Villinski,† Markus Godejohann,‡ Pavel Kessler,‡
John H. Cardellina II,§ Daneel Ferreira,⊥ Karine Feghali,∥ and Daniel Grenier∥
†Tom’s of Maine, 302 Lafayette Center, Kennebunk, Maine 04043, United States
‡Bruker-Biospin, Silberstreifen 4, 76287 Rheinstetten, Germany
§ReevesGroup, 9374 Highlander Boulevard, Walkersville, Maryland 21793, United States
⊥Department of Pharmacognosy and National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences,
School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States
∥
́
Groupe de Recherche en Ecologie Buccale, Faculte
́
de Med
́
ecine Dentaire, Universite
́
Laval, Quebec City, Quebec G1 V 0A6,
Canada
S
* Supporting Information
ABSTRACT: Phytochemical investigation of a supercritical
fluid extract of Glycyrrhiza uralensis has led to the isolation of
20 known isoflavonoids and coumarins, and glycycarpan (7), a
new pterocarpan. The presence of two isoflavan-quinones,
licoriquinone A (8) and licoriquinone B (9), in a fraction
subjected to gel filtration on Sephadex LH-20 is due to
suspected metal-catalyzed oxidative degradation of licoricidin
(1) and licorisoflavan A (2). The major compounds in the
extract, as well as 8, were evaluated for their ability to inhibit
the growth of several major oral pathogens. Compounds 1 and 2 showed the most potent antibacterial activities, causing a
marked growth inhibition of the cariogenic species Streptococcus mutans and Streptococcus sobrinus at 10 μg/mL and the
periodontopathogenic species Porphyromonas gingivalis (at 5 μg/mL) and Prevotella intermedia (at 5 μg/mL for 1 and 2.5 μg/mL
for 2). Only 1 moderately inhibited growth of Fusobacterium nucleatum at the highest concentration tested (10 μg/mL).
hinese licorice (Glycyrrhiza uralensis Fisch., Fabaceae) is
pterocarpans.1,2 Less common phenolic compounds include
coumarins, 2-arylbenzofurans, and coumestans. Polysaccharides
have been found in G. uralensis and G. glabra and reportedly
constitute up to 10% of the roots of G. glabra.2,3
C
found in steppe meadows, at lake and river sides, in
ravines, on slopes, and in depressions throughout much of
northern mainland China, especially the Asian steppes to the
west. While G. uralensis is the main species used in Asia,
European licorice (Glycyrrhiza glabra L.) also occurs in
northwest China, in wild desert regions, dry plains, grassy
plains with salty alkaline soil, and fallow wastelands that were
once used for producing rice, wheat, and millet. These two
species along with another Chinese native, Glycyrrhiza inflata
Bat., are official drug plants in the Chinese Pharmacopoeia. The
Chinese call licorice “gan-cao”, which means “sweet herb”.
Licorice is used in many Chinese herbal prescriptions as a guide
drug to enhance the activity of other ingredients, reduce
toxicity, and improve flavor.1 The major constituents of licorice
are the triterpene glycosides, which give the roots its typical
sweet taste. The aglycones are oleanane-type pentacyclic
triterpenes with glycyrrhetinic acid as the best known example.2
Another important class of compounds is the flavonoids, of
which more than 300 have been characterized so far in licorice
species. The major flavonoids belong to the flavanone and
chalcone type, but the lipophilic fraction also contains rather
unusual flavonoids such as isoflavans, isoflavenes, and
Despite the extensive use of licorice in traditional medicine,
there has been limited evidence for its use to treat ailments of
the oral cavity. Research by Martin and co-workers4 has focused
on the benefits of a water extract of licorice root for the
treatment of aphthous ulcers. There are also two reports on the
ability of glycyrrhizin, the sweet component of licorice root, to
reduce dental plaque formation;5,6 however, this effect could
not be confirmed in a small clinical trial.7 In addition, He and
co-workers8 reported that pterocarpenes isolated from G.
uralensis roots exerted antibacterial activity against Streptococcus
mutans, the major etiologic agent of dental caries. Our previous
studies have focused on a supercritical fluid extract of G.
uralensis roots, which has shown potent anti-inflammatory
properties in an in vitro macrophage model and an ex vivo
human whole blood model.9,10 More specifically, the extract
Received: June 8, 2011
Published: November 10, 2011
© 2011 American Chemical Society and
American Society of Pharmacognosy
2514
dx.doi.org/10.1021/np2004775|J. Nat. Prod. 2011, 74, 2514−2519

Journal of Natural Products
Article
Chart 1
inhibited lipopolysaccharide (LPS)-induced secretion of pro-
inflammatory cytokines, chemokines, and matrix metallopro-
teinases by macrophages. This effect was at least in part due to
the reduced phosphorylation of key intracellular kinases.
Licoricidin (1) and licorisoflavan A (2), the two predominant
compounds in the extract, were isolated previously and related
to at least part of the anti-inflammatory effect.10 Herein, the
identification of the minor compounds of the extract and of two
isoflavan-quinones formed from licoricidin (1) and licoriso-
flavan A (2) during the isolation process is described. In
addition, the antibacterial activities of the licorice isolates
toward important cariogenic and periodontopathogenic bacte-
rial species were compared.
spectrum, the signals at δ 4.14 (dd, J = 11.5, 5.0 Hz), 3.60 (t, J
= 11.1 Hz), 3.35 (m), and 5.61 (d, J = 6.7 Hz), corresponding
to H-6α, H-6β, H-6a, and H-11a, respectively, were similar to
the signals of the same protons present in the 1H NMR spectra
of known pterocarpans isolated from the extract. In addition,
1
the H NMR spectrum contained signals for three aromatic
protons (δ 6.93, 6.41, and 6.21, all s), one methoxy group (δ
3.95, s), and two oxygenated 3-methylbutyl units [δ 2.67 (m),
1.79 (m), 1.30 (s), 1.26 (s) and 2.79 (m), 1.79 (m), 1.36 (s),
1.32 (s), respectively].
The location of the substituents on the two aromatic rings of
7 was based mainly on the 13C NMR data and HMBC
correlations. HMBC correlations were observed between H-11a
and C-1 (δ 159.3) as well as the methoxy protons and C-1,
placing the methoxy group at C-1. On the basis of the 13C
NMR shift of the methoxy carbon, indicating a di-ortho
substitution, and the HMBC correlation between H-4′ (δ 2.79,
m) and C-1 and C-3 (δ 156.3), one of the isoprene groups had
to be attached at C-2 (δ 108.4), with C-3 found to bear an
oxygenated substituent. The incorporation of the 3-methylbutyl
side chain into a 6,6-dimethyl-4,5-dihydro-[6H]-pyran ring was
established on the basis of the 13C NMR shifts. One aromatic
proton was placed at C-4, while the two remaining aromatic
protons were located at C-7 and C-10 due to the lack of a
discernible coupling. The HMBC correlations between H-7 and
C-6a (δ 39.1), C-1″ (δ 24.2), C-9 (δ 154.9), and C-10a (δ
159.1) proved that the second 3-methylbutyl unit was attached
at C-8 (δ 120.6), with an oxygenated aromatic carbon adjacent
at C-9. The 13C NMR shifts of the side chain were consistent
with a 3-hydroxy-3-methylbutyl moiety. Thus, the structure of 7
was established as 9-hydroxy-1-methoxy-8-(3-hydroxy-3-meth-
ylbutyl)-6′,6′-dimethyl-4′,5′-dihydro-[6H]-pyrano[2′,3′:3,2]-
RESULTS AND DISCUSSION
■
Fractionation of the supercritical fluid extract of G. uralensis
roots led to the characterization of 20 known compounds and
one new pterocarpan. The known compounds were identified
as licoricidin (1),11 licorisoflavan A (2),12 vestitol,13 glyasperin
C,14 glyasperin D14 (3), glyasperin F,15 dihydrolicoisoflavone,15
allolicoisoflavone A (4),16 semilicoisoflavone B,17 licoricone,18
medicarpin,19 edudiol,20 1-methoxyficifolinol,17 kanzonol P,21
glycyrin (5),14 licocoumarone,14,18 gancaonin I,18 glycycoumar-
in (6),22 and licoarylcoumarin,23 based on comparison of
spectroscopic and spectrometric information with published
data. Liquiritigenin was identified on the basis of comparison of
HPLC retention time, UV, and MS data with a commercially
available standard compound.
The molecular formula of a new pterocarpan (7), C26H32O6,
was determined by HREIMS, which showed a molecular ion
peak at m/z 440.2205 (calcd 440.2199). The pterocarpan
1
skeleton was deduced from the NMR data. In the H NMR
2515
dx.doi.org/10.1021/np2004775|J. Nat. Prod. 2011, 74, 2514−2519

Journal of Natural Products
Article
Table 1. NMR Spectroscopic Data of Licoriquinone A (8) and Licoriquinone B (9)
licoriquinone A (8)
licoriquinone B (9)
δH (J in Hz)
a
a
position
2
δC, mult.
δH (J in Hz)
3.92, dd (10.6, 7.9)
HMBC
3,4,9,1′
δC, mult.
HMBC
3,4,9,1′
68.2, CH2
68.1, CH2
3.88, dd (10.5, 7.7)
4.20, ddd (10.6, 3.1, 1.6)
3.33, ddddd (8.6, 7.9, 5.6, 3.1, 1.1)
2.65, dd (16.2, 8.6)
4.16, ddd (10.5, 3.1, 1.4)
3.31, ddddd (8.6, 7.7, 5.5, 3.1, 1.1)
2.63, dd (16.2, 8.6)
3
4
31.0, CH
24.5, CH2
2,4,6′
30.9, CH
24.3, CH2
2,4,10,1′
2,3,5,6,9,10,1′
2,3,5,9,10,1′
2.90, ddd (16.2, 5.6, 1.6)
2.88, ddd (16.2, 5.5, 1.4)
10
106.0, C
156.9, C
116.3, C
157.6, C
95.9, CH
153.2, C
150.5, C
186.4, C
120.9, C
151.5, C
183.4, C
128.0, CH
22.7, CH2
123.7, CH
130.9,
106.4, C
157.3, C
114.1, C
154.2, C
99.1, CH
153.0, C
150.3, C
186.4, C
120.5, C
151.4, C
183.5, C
128.4, CH
22.2, CH2
123.6, CH
130.7, C
25.2, CH3
16.8, CH3
21.7, CH2
120.3, CH
132.8, C
25.2, CH3
16.8, CH3
60.0, CH3
5
6
7
8
6.23, s
4,6,7,9,10,1″
6.09, s
4,5,6,7,9,10,1″
9
1′
2′
3′
4′
5′
6′
6.44, d (1.1)
2,3,1′,2′,4′
5,6,7,2″,3″
4″,5″
6.44, d (1.1)
2,3,1′,2′,4′
5,6,7,2″,3″
4″,5″
1″
3.21, d (7.3)
3.20, d (6.8)
2″
5.11, ddt (7.3, 2.9, 1.2)
5.13, dt (6.8, 1.2)
3″
4″
C25.8, CH3
17.8, CH3
22.3, CH2
119.6, CH
133.9, C
25.8, CH3
17.8, CH3
60.7, CH3
55.6, CH3
1.63, d (1.2)
1.73, s
2″,3″, 5″
2″,3″,4″
1.65, m
2″,3″, 5″
2″,3″,4″
5″
1.72, s
1‴
2‴
3‴
4‴
5‴
OCH3-5
3.10, d (7.1)
5.07, dt (7.1, 1.2)
4′,2′,2‴,3‴
4‴,5‴
3.09, d (7.1)
5.08, dt (7.1, 1.1)
4′,2′,2‴,3‴
4‴,5‴
1.65, d (1.2)
1.71, s
2‴,3‴,5‴
2‴,3‴,4‴
5
1.65, m
1.72, s
3.64, s
2‴,3‴,5‴
2‴,3‴,4‴
5
3.70, s
OCH3-7
3.77, s
7
a
HMBC correlations, optimized for 8 Hz, are from proton(s) to the indicated carbon.
pterocarpan. The compound has been named glycycarpan. The
absolute configuration of the compound was determined from
its optical rotation. Levorotary pterocarpans have the 6aR, 11aR
absolute configuration, and dextrorotary ones are 6aS, 11aS.24
The [α]D21 of −35.3 measured for 7 is consistent with a 6aR,
11aR configuration.
As part of our conducted stability studies, compounds 1 and
2 were subjected to stress tests under alkaline, acidic, and
oxidative conditions. Both compounds were unstable in 0.1 N
NaOH, yielding two major degradation products. One of the
degradation products derived from 2 had the same retention
time, UV, and MS data as 8. In order to confirm the structure
of 8, a larger amount of 2 was treated with 0.1 N NaOH for 2 h,
and the resulting mixture was analyzed by HPLC-NMR. The
13C NMR spectrum revealed the presence of two carbonyl
carbons at δ 186.4 and 183.4. The shifts of the two carbonyl
carbons are in good agreement with a para-quinone moiety.
The substitution pattern of the para-quinone ring was mainly
based on HMBC data (Table 1), in particular the correlations
between H-6′ and C-2 (δ 68.2), C-3 (δ 31.0), C-2′ (δ 186.4),
and C-4′ (δ 151.5) and between H-1‴ (δ 3.10) and C-2′ and C-
4′. Thus, compound 8 was identified as 4′-hydroxy-5,7-
dimethoxy-6,3′-diprenylisoflavanquinone (licoriquinone A).
Compound 1 was treated in a similar manner with 0.1 N
NaOH, and the resulting mixture was also subjected to HPLC-
NMR. The major degradation product from 1 was identified as
4′,7-dihydroxy-5-methoxy-6,3′-diprenylisoflavanquinone (9)
and named licoriquinone B. The configuration at C-3 of 8
and 9 was established as 3R, based on the specific rotation of 1
([α]D23 +12.8, c 0.3 in MeOH) and 2 ([α]D23 +11.9, c 0.4 in
MeOH). Fukai et al. established the configuration of licoricidin
([α]D21 +22.8) as 3R using CD data.11 As both 8 and 9 are
oxidative degradation products of 2 and 1, and the order of the
substituents at the stereogenic center does not change in the
In an attempt to isolate a larger amount of 2, a fraction rich
in this compound was subjected to gel filtration on Sephadex
LH-20. Besides a number of mixed fractions, there was one
fraction containing 2 and a small amount of compound 8, both
of which were purified by semipreparative HPLC. The UV
spectrum of 8, with maxima at 274 and 386 nm, suggested that
the compound is highly oxygenated. The molecular formula
was established as C27H32O6, on the basis of the HRTOFMS
data with an [M + H]+ peak at m/z 453.2241 (calcd 453.2272).
1
The H NMR signals at δ 4.20 (ddd, J = 10.6, 3.1, 1.6 Hz, H-
2α), 3.92 (dd, J = 10.6, 7.9 Hz, H-2β), 3.33 (ddddd, J = 8.6, 7.9,
5.6, 3.1, 1.1 Hz, H-3), 2.90 (ddd, J = 16.2, 5.6, 1.6 Hz, H-4α),
and 2.65 (dd, J = 16.2, 8.6 Hz, H-4β) are typical for the C ring
of an isoflavan. In addition, the spectrum revealed signals for
two aromatic protons, two methoxy groups, and two isoprenyl
moieties (Table 1). The HMBC data indicated that the
substitution pattern on the A ring was the same as in 2. The
correlation between H-6′ (δ 6.44) and C-3 (δ 31.0) and C-2′ (δ
186.4), the UV spectrum, and consideration of the molecular
formula suggested the presence of an isoflavan-quinone ring,
but the lack of proper 13C NMR data initially prevented the
unequivocal elucidation of the structure.
2516
dx.doi.org/10.1021/np2004775|J. Nat. Prod. 2011, 74, 2514−2519

Journal of Natural Products
Article
Figure 1. Effect of licorice compounds 1 (panel A), 2 (panel B), 3 (panel C), 4 (panel D), 5 (panel E), 6 (panel F), 8 (panel G), and penicillin G
(panel H) on growth of F. nucleatum, P. gingivalis, P. intermedia, S. mutans, and S. sobrinus. The data are means ± standard deviations of triplicate
§
#
assays. *, p < 0.001; , p < 0.005; and , p < 0.01 compared to control with no compounds.
oxidation process, they all have the same 3R configuration.
Despite considerable efforts, we were unable to get
unambiguous CD spectra for 1, 2, or 8.
9 was found in the supercritical fluid extract, and appeared only
after a gel filtration step, indicates that certain isoflavans are
prone to oxidative degradation into isoflavan-quinones. Usually,
only 1,2- and 1,4-dihydroxy-substituted aromatic rings are
subject to oxidation into ortho- and para-quinones, but Philipp
and Schink33 reported that resorcinol (1,3-dihydroxybenzene)
oxidized into 2-hydroxy-1,4-benzoquinone under alkaline
conditions with an electron acceptor having a high redox
potential, e.g., K3Fe(CN)6. Furthermore, Ling et al.34 reported
that tert-butylresorcinols tend to autoxidize in the presence of
copper(II) and that this reaction is favored by alkaline
conditions. Accordingly, the proposed mechanism of oxidation
of isoflavans 1 and 2 into the quinones 9 and 8 is outlined in
The isoflavan-quinones represent a rare group of plant
secondary metabolites, and only a few structures have been
reported thus far. Examples include claussequinone, astragalu-
quinone, pendulone, amorphaquinone, colutequinone, laurenti-
quinone, mucroquinone, and the abruquinones.25−32 It is not
clear as to whether isoflavan-quinones are genuine natural
products or oxidation products of related isoflavans. Grosvenor
and Gray29 showed that colutequinone was present in the
original extract, together with colutehydroquinone, which is
readily converted into the quinone. The fact that neither 8 nor
2517
dx.doi.org/10.1021/np2004775|J. Nat. Prod. 2011, 74, 2514−2519

Journal of Natural Products
Article
using the HPLC-SPE-NMR/TOFMS system described below. MPLC
Figure S1, Supporting Information. It is still not clear whether
the same mechanisms led to the formation of the isoflavan-
quinones during the isolation process. The fraction submitted
to gel filtration did not contain detectable amounts of isoflavan-
quinones; therefore, the conversion must have happened during
the separation or the subsequent drying process. Due to the fact
that the Sephadex LH-20 material was extensively used prior to
this separation, it is possible that metal ions such as copper(II)
were present on the column. Therefore, we hypothesize that
the reaction may occur under neutral conditions or that an
alkaline contaminant from previous use was present on the
stationary phase. In order to rule out an oxidation due to the
stationary phase itself or to the solvents, pure 1 or pure 2 was
again submitted to gel filtration using a new lot of Sephadex
LH-20. With the new material, none of the quinones were
found, neither when fractions were verified directly after eluting
off the column nor after one week in the CH2Cl2−MeOH
mixture. This supports the hypothesis that the degradation was
due to a contaminant from prior work with the old lot of
Sephadex LH-20 used in the initial purification step.
Interestingly, all the natural isoflavan-quinones possess either
a hydroxy or a methoxy group at C-3′ and/or C-4′ and,
therefore, may be produced from naturally occurring 2′,4′- or
3′,5′-dihydroxyisoflavans either in the plant or during isolation.
In continuation of our studies aimed to demonstrate the
beneficial properties of licorice for oral health, the effects of
compounds 1−6 and 8 (other isolates were not available in
sufficient quantities to perform the testing) were examined on
the growth of oral pathogens at concentrations between 1.25
and 10 μg/mL. Dental caries is the direct result of enamel
dissolution by acid-producing bacteria inhabiting dental biofilm,
especially when the biofilm reaches a critical mass due to poor
oral hygiene. Streptococcus mutans and S. sobrinus are known as
major cariogenic bacteria.35 While compounds 3−6 and 8 had
no effect on the growth of S. mutans and S. sobrinus,
compounds 1 and 2 at 10 μg/mL caused a marked growth
inhibition of both cariogenic bacterial species (Figure 1).
Periodontitis is a destructive inflammatory disorder that leads
to the loss of tooth support. It is initiated by a specific group of
Gram-negative anaerobic bacteria, including Porphyromonas
gingivalis, Prevotella intermedia, and Fusobacterium nucleatum,
which modulate periodontal tissue destruction through
complex interactions with mucosal and immune cells.36,37
Growth inhibition of F. nucleatum was observed only with
compound 1 (Figure 1A). On the other hand, the growth of P.
intermedia was inhibited by compounds 1−3, 5, and 6, and to a
lesser extent by 4 and 8 (Figure 1). Finally, complete growth
inhibition of P. gingivalis was obtained with 1 and 2 (Figures 1A
and B). Penicillin G was used as positive control and was found
to be active on all bacterial species tested (Figure 1G). In
conclusion, licorice compounds 1 and 2 were the most effective
in inhibiting growth of oral pathogens, thus suggesting that they
may have a therapeutic/preventive potential for oral infections.
was conducted on a Buchi C-615 system, equipped with two C-605
̈
pumps, a C-630 monitor, and a C-660 fraction collector (Buchi, Flawil,
̈
Switzerland). Semipreparative HPLC was carried out using an Agilent
1100 unit equipped with a DAD detector (Agilent, Burlington, MA,
USA). The HPLC-SPE-NMR/TOFMS system used for this study
consisted of an Agilent 1200 HPLC system (quaternary pump,
autosampler, diode array detector, Waldbronn, Germany), a Bruker/
Spark Prospekt 2 SPE cartridge exchanger (Emmen, The Nether-
lands), a MicrOTOF mass spectrometer (Bruker Daltonics, Bremen,
Germany), and a 600 MHz AVANCE II NMR spectrometer equipped
with a 5 mm TCI cryoprobe (Bruker Biospin, Rheinstetten, Germany).
Chromatography was performed on an XSelect CSH C18 column (250
× 4.6 mm, 5 μm). The solvent composition started with 50% A
(H2O−0.1% formic acid-d2) and 50% B (MeCN). On holding for 5
min, the composition changed to 100% B at 25 min using a linear
gradient. The reaction mixture (30 μL) reconstituted in 1 mL of EtOH
was injected onto the column. The mass spectrometer was operated in
the positive ionization mode using a scan range between m/z 50 and
1000. The calibration was done with a 20 mM lithium formate solution
that was introduced to the ion source with the help of a divert valve at
the beginning of each chromatographic run. SPE cartridges (10 × 2
mm) filled with Hysphere C18 HD material were used to trap the
chromatographic fractions. For the measurements of NMR data,
standard parameter sets created for the Bruker SELU program (SELU
Topspin 3.1) were employed uniformly (see Supporting Information).
Gradient COSY and HMBC were acquired using 4 k complex data
points in F2 and 512 points in the F1 dimension. The multiplicity-
edited gradient HSQC was acquired with 2 k data points in F2 and
400 points in the F1 dimension. The number of scans per increment
differed according to sample and experimental requirements. For 9,
the COSY was acquired with four scans, the HSQC with eight scans,
and the HMBC with 32 scans per increment. For 8, the concentration
was lower, which resulted in longer acquisition times (COSY: 16
scans; HSQC: 32 scans; HMBC: 128 scans). For 9, a 13C NMR
spectrum with composite pulse decoupling on the proton channel was
acquired on an AVANCE III 600 MHz NMR spectrometer equipped
with a 5 mm QNP cryoprobe head (Bruker Biospin, Rheinstetten,
Germany). A total of 24 576 scans were collected in 131 072 complex
data points at a sweep width of 36 057 Hz with a relaxation delay of 5
s. The experiment time was 26.5 h.
Reagents. LiChrosolv LCMS-grade MeCN and H2O were
obtained from Merck (Darmstadt, Germany). All other solvents for
HPLC, MPLC, or forced degradation studies were from Fisher
Scientific (Pittsburgh, PA, USA). TFA was purchased from Sigma (St.
Louis, MO, USA). Formic acid was from Fluka (Buchs, Switzerland).
Formic acid-d2 (95%) and methanol-d4 (99.8%) were obtained from
Deutero GmbH (Kastellaun, Germany).
Plant Material. Commercially available dried and cut Glycyrrhiza
uralensis roots (lot #770407, Flavex, Rehlingen, Germany) were used
for this project. The identity of the material was based on comparison
of the HPLC-UV trace with authentic G. uralensis material obtained
from the American Herbal Pharmacopeia. A voucher specimen (TOM
10001) was deposited at the Tom’s of Maine Herbarium in
Kennebunk, ME, USA.
Extraction and Isolation. The plant material was ground and
extracted using supercritical CO2 with 5% EtOH as a modifier. For the
isolation process, see Supporting Information.
Glycycarpan (7): white powder; [α]D21 −35.3 (c 1.1, CHCl3); UV
1
(EtOH) λmax (log ε) 209 (4.49), 275 (3.61) nm; H NMR (CDCl3,
EXPERIMENTAL SECTION
600 MHz) δ 6.93 (1H, s, H-7), 6.41 (1H, s, H-10), 6.21 (1H, s, H-4),
5.61 (d, J = 6.7 Hz, H-11a), 4.14 (dd, J = 11.5, 5.0 Hz, H-6α), 3.95
(3H, s, OCH3), 3.60 (t, J = 11.1 Hz, H-6β), 3.35 (1H, m, H-6a), 2.79
(2H, m, H-4′), 2.67 (2H, m, H-1″), 1.79 (2H, m, H-5′), 1.79 (2H, m,
H-2″), 1.36a (3H, s, H-7′), 1.32a (3H, s, H-8′), 1.30b (3H, s, H-4″),
1.26b (3H, s, H-5″); 13C NMR (CDCl3, 125 MHz) δ 159.3 (C, C-1),
159.1 (C, C-10a), 156.3 (C, C-3), 155.1 (C, C-5), 154.9 (C, C-9),
125.0 (CH, C-7), 120.6 (C, C-8), 118.7 (C, C-6b), 108.4 (C, C-2),
106.6 (C, C-11b), 100.8 (CH, C-4), 98.8 (CH, C-10), 75.5 (CH, C-
11a), 74.6 (C, C-6′), 71.2 (C, C-3″), 66.3 (CH2, C-6), 61.4 (CH3,
■
General Experimental Procedures. Optical rotations were
measured on a JASCO P-1010 polarimeter (compound 7) or on a
589−546 Rudolph Research Analytical Autopol IV automatic
polarimeter (compounds 1 and 2). UV data were measured on a
Cary 50 UV-spectrophotometer (Varian, Palo Alto, CA, USA). NMR
spectra were obtained on an AVANCE II 500 MHz or an AVANCE
600 MHz instrument (Bruker Biospin, Billerica, MA, USA). The
HREIMS data for 7 were obtained with an MSRoute instrument
(JEOL, Peabody, MA, USA). HRMS data for 8 and 9 were obtained
2518
dx.doi.org/10.1021/np2004775|J. Nat. Prod. 2011, 74, 2514−2519

Journal of Natural Products
Article
OCH3), 43.3 (CH2, C-2″), 39.1 (CH, C-6a), 32.3 (CH2, C-5′), 29.5
(CH3, C-4″), 29.5 (CH3, C-5″), 26.9 (CH3, C-7′ or C-8′), 26.2 (CH3,
C-7′ or C-8′), 24.2 (CH2, C-1″), 16.8 (CH2, C-4′). a,bSignals with the
same superscript are interchangeable
(4) Martin, M. D.; Sherman, J.; Van der Ven, P.; Burgess, J. Gen.
Dent. 2008, 56, 206−210, quiz 211−212, 224.
(5) Segal, R.; Pisanty, S.; Wormser, R.; Azaz, E.; Sela, M. N. J. Pharm.
Sci. 1985, 74, 79−81.
Licoriquinone A (8): orange powder; UV (EtOH) λmax (log ε) 209
(6) Steinberg, D.; Sgan-Cohen, H. D.; Stabholz, A.; Pizanty, S.; Segal,
R.; Sela, M. N. Isr. J. Dent. Sci. 1989, 2, 153−157.
(7) Goultschin, J.; Palmon, S.; Shapira, L.; Brayer, L.; Gedalia, I. J.
Clin. Periodontol. 1991, 18, 210−212.
1
(4.52), 274 (3.72), 386 (2.83) nm; H and 13C NMR data, see Table
1.
Licoriquinone B (9): 1H and 13C NMR data, see Table 1.
Forced Degradation Study of 2 in 0.1 N NaOH. Compound 2
(15.9 mg) was dissolved in 9.0 mL of EtOH, to which 1.0 mL of 1 N
NaOH was added. After 2 h, the reaction was quenched by adding 1.0
mL of 1 N HCl, and the solution was evaporated to dryness. The dry
mixture containing 8 as the predominant degradation product was
washed three times with 10 mL of deionized H2O to remove NaCl,
then freeze-dried, and finally analyzed by HPLC-NMR/MS.
Compound 1 (45.1 mg) was subjected to the same treatment in 0.1
N NaOH to give a mixture of degradation products with 9 as the
predominant compound. This mixture was also subjected to HPLC-
NMR/MS analysis as outlined above.
Effects on Bacterial Growth. The effects of licorice compounds
1−6 and 8 on the growth of Gram-negative (P. gingivalis ATCC
33277, P. intermedia ATCC 25611, F. nucleatum ATCC 25586) and
Gram-positive (S. mutans ATCC 25175, S. sobrinus ATCC 33478) oral
pathogens were determined in a microplate dilution assay. Twenty-
four-hour cultures of bacteria in Todd Hewitt Broth (BBL
Microbiology Systems, Cockeysville, MD, USA), supplemented with
hemin (10 μg/mL) and vitamin K (1 μg/mL), were diluted in fresh
broth medium to obtain an optical density at 660 nm (OD660) of 0.2.
Samples (100 μL) were added to the wells of a 96-well tissue culture
plate (Sarstedt, Newton, NC, USA) containing 100 μL of serial
dilutions of sterile compounds (10 to 1.25 μg/mL) or penicillin G (2.5
to 0.00625 μg/mL) in broth medium. Control wells with no
compounds were also inoculated. After incubation for 24 h at 37 °C
under anaerobic (N2−H2−CO2 75:10:15) (P. gingivalis, P. intermedia,
F. nucleatum) or aerobic (S. mutans, S. sobrinus) conditions, the OD660
was recorded. The means ± standard deviations of triplicate assays
were calculated. When the growth inhibition was ≥20%, differences
between the control and test values were analyzed for statistical
significance using the Student’s t test and were considered significant
at p < 0.01, 0.005, or 0.001.
(8) He, J.; Chen, L.; Heber, D.; Shi, W.; Lu, Q. Y. J. Nat. Prod. 2006,
69, 121−124.
(9) Bodet, C.; La, V. D.; Gafner, S.; Bergeron, C.; Grenier, D. J.
Periodontol. 2008, 79, 1752−1761.
(10) La, V. D.; Tanabe, S. I.; Bergeron, C.; Gafner, S.; Grenier, D. J.
Periodontol. 2011, 82, 122−128.
(11) Fukai, T.; Toyono, M.; Nomura, T. Heterocycles 1988, 27,
2309−2313.
(12) Shih, T. L.; Wyvratt, M. J.; Mrozik, H. J. Org. Chem. 1987, 52,
2029−2033.
(13) Takashima, Y.; Kaneko, Y.; Kobayashi, Y. Tetrahedron 2010, 66,
197−207.
(14) Kwon, H. J.; Kim, H. H.; Ryu, Y. B.; Kim, J. H.; Joeng, H. J.;
Lee, S. W.; Chang, J. S.; Cho, K. O.; Rho, M. C.; Park, S. J.; Lee, W. S.
Bioorg. Med. Chem. 2010, 18, 7668−7674.
(15) McKee, T. C.; Bokesch, H. R.; McCormick, J. L.; Rashid, M. A.;
Spielvogel, D.; Gustafson, K. R.; Alavanja, M. M.; Cardellina, J. H. II;
Boyd, M. R. J. Nat. Prod. 1997, 60, 431−438.
(16) Tsukayama, M.; Li, H.; Tsurumoto, K.; Nishiuchi, M.;
Kawamura, Y. Bull. Chem. Soc. Jpn. 1998, 71, 2673−2680.
(17) Kiuchi, F.; Xing, C.; Tsuda, Y. Heterocycles 1990, 31, 629−636.
(18) Fukai, T.; Wang, Q. H.; Kitagawa, T.; Kusano, K.; Nomura, T.;
Iitaka, Y. Heterocycles 1989, 29, 1761−1772.
(19) Herath, H. M. T. B.; Dassanayake, R. S.; Priyadarshani, A. M.
A.; De Silva, S.; Wannigama, G. P.; Jamie, J. Phytochemistry 1998, 47,
117−119.
(20) Guchu, S. M.; Yenesew, A.; Tsanuo, M. K.; Gikonyo, N. K.;
Pickett, J. A.; Hooper, A. M.; Hassanali, A. Phytochemistry 2007, 68,
646−651.
(21) Fukai, T.; Nishizawa, J.; Yokoyama, M.; Tantai, L; Nomura, T.
Heterocycles 1994, 38, 1089−1098.
(22) Sato, Y.; Akao, T.; He, J. X.; Nojima, H.; Kuraishi, Y.; Morota,
T.; Asano, T.; Tani, T. J. Ethnopharmacol. 2006, 105, 409−414.
(23) Hatano, T.; Yasuhara, T.; Fukuda, T.; Noro, T.; Okuda, T.
Chem. Pharm. Bull. 1987, 37, 3005−3009.
(24) Slade, D.; Ferreira, D.; Marais, J. P. J. Phytochemistry 2005, 66,
2177−2215.
(25) Kurosawa, K.; Ollis, W. D.; Redman, B. T.; Sutherland, I. O.; de
Oliveira, A. B.; Gottlieb, O. R.; Alves, H. M. Chem. Commun. 1968, 20,
1263.
ASSOCIATED CONTENT
■
S
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
(26) Hayashi, Y.; Shirato, T.; Sakurai, K.; Takahashi, T. Mokuzai
Gakkaishi 1978, 24, 898−901.
Corresponding Author
*Tel: +1-207-467-2227. Fax: +1-207-985-2196. E-mail:
stefang@tomsofmaine.com.
(27) Shibata, H.; Shimizu, S. Heterocycles 1978, 10, 85−86.
(28) El-Sebakhy, N. A.; Asaad, A. M.; Abdallah, R. M.; Toaima, S.
M.; Abdel-Kader, M. S.; Stermitz, F. R. Phytochemistry 1994, 36,
1387−1389.
ACKNOWLEDGMENTS
■
(29) Grosvenor, P. W.; Gray, D. O. Phytochemistry 1996, 43, 377−
380.
The authors are grateful to Dr. K. L. McPhail (Oregon State
University, Corvallis, OR, USA) for measuring the optical
rotation of 7 and to Drs. C. Schneider (Memorial University of
Newfoundland, St. John’s, Canada) and K. L. Colson (Bruker-
Biospin, Billerica, MA, USA) for the NMR data of the licorice
isolates.
(30) Song, C. Q.; Hu, Z. B. Acta Bot. Sin. 1998, 40, 734−739.
(31) Kamnaing, P.; Fanso Free, S. N. Y.; Nkengfack, A. E.; Folefoc,
G.; Tanee Fomum, Z. Phytochemistry 1999, 51, 829−832.
(32) Takahashi, M.; Fuchino, H.; Sekita, S.; Satake, M.; Kiuchi, F.
Chem. Pharm. Bull. 2006, 54, 915−917.
(33) Philipp, B.; Schink, B. J. Bacteriol. 1998, 180, 3644−3649.
(34) Ling, K. L.; Lee, Y.; Macikenas, D.; Protasiewicz, J. D.; Sayre, L.
M. J. Org. Chem. 2003, 68, 1358−1366.
(35) Selwitz, R. H.; Ismail, A.; Pitts, N. B. Lancet 2007, 369, 51−59.
(36) Haffajee, A. D.; Socransky, S. S. Periodontol. 2000 1994, 5, 78−
111.
REFERENCES
■
(1) Zhang, Q.; Ye, M. J. Chromatogr. A 2009, 1216, 1954−1969.
(2) Willuhn, G. In Teedrogen und Phytopharmaka 4th ed.; Wichtl, M.,
Ed.; Wissenschaftliche Verlagsgesellschaft m.b.H.: Stuttgart, 2002; pp
347−351.
(3) Zhao, J. F.; Kiyohara, H.; Yamada, H.; Takemoto, N.; Kawamura,
H. Carbohydr. Res. 1991, 219, 149−172.
(37) Madianos, P. N.; Bobetsis, Y. A.; Kinane, D. F. J. Clin.
Periodontol. 2005, 32, 57−71.
2519
dx.doi.org/10.1021/np2004775|J. Nat. Prod. 2011, 74, 2514−2519