Pyrano-isoflavans from Glycyrrhiza uralensis with antibacterial activity against Streptococcus mutans and Porphyromonas gingivalis

Article abstract of DOI:10.1021/np400788r

Continuing investigation of fractions from a supercritical fluid extract of Chinese licorice (Glycyrrhiza uralensis) roots has led to the isolation of 12 phenolic compounds, of which seven were described previously from this extract. In addition to these seven metabolites, four known components, 1-methoxyerythrabyssin II (4), 6,8-diprenylgenistein, gancaonin G (5), and isoglycyrol (6), and one new isoflavan, licorisoflavan C (7), were characterized from this material for the first time. Treatment of licoricidin (1) with palladium chloride afforded larger amounts of 7 and also yielded two new isoflavans, licorisoflavan D (8), which was subsequently detected in the licorice extract, and licorisoflavan E (9). Compounds 1-9 were evaluated for their antibacterial activities against the cariogenic Streptococcus mutans and the periodontopathogenic Porphyromonas gingivalis. Licoricidin (1), licorisoflavan A (2), and 7-9 showed antibacterial activity against P. gingivalis (MICs of 1.56-12.5 μg/mL). The most potent activity against S. mutans was obtained with 7 (MIC of 6.25 μg/mL), followed by 1 and 9 (MIC of 12.5 μg/mL). This study provides further evidence for the therapeutic potential of licorice extracts for the treatment and prevention of oral infections.

Full text of DOI:10.1021/np400788r

Article
pubs.acs.org/jnp
Pyrano-isoflavans from Glycyrrhiza uralensis with Antibacterial
Activity against Streptococcus mutans and Porphyromonas gingivalis
Jacquelyn R. Villinski,† Chantal Bergeron,† Joseph C. Cannistra,‡ James B. Gloer,‡ Christina M. Coleman,§
Daneel Ferreira,§ Jabrane Azelmat,⊥ Daniel Grenier,⊥ and Stefan Gafner*,†,∥
†Tom’s of Maine, 302 Lafayette Center, Kennebunk, Maine 04043, United States
‡Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States
§Department of Pharmacognosy and the Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi,
University, Mississippi 38677, United States
⊥
́
́ ́ ́
Groupe de Recherche en Ecologie Buccale, Faculte de Medecine Dentaire, Universite Laval, Quebec City, Quebec G1V 0A6, Canada
∥American Botanical Council, Austin, Texas 78714, United States
S
* Supporting Information
ABSTRACT: Continuing investigation of fractions from a supercritical fluid extract of Chinese licorice (Glycyrrhiza uralensis)
roots has led to the isolation of 12 phenolic compounds, of which seven were described previously from this extract. In addition
to these seven metabolites, four known components, 1-methoxyerythrabyssin II (4), 6,8-diprenylgenistein, gancaonin G (5), and
isoglycyrol (6), and one new isoflavan, licorisoflavan C (7), were characterized from this material for the first time. Treatment of
licoricidin (1) with palladium chloride afforded larger amounts of 7 and also yielded two new isoflavans, licorisoflavan D (8),
which was subsequently detected in the licorice extract, and licorisoflavan E (9). Compounds 1−9 were evaluated for their
antibacterial activities against the cariogenic Streptococcus mutans and the periodontopathogenic Porphyromonas gingivalis.
Licoricidin (1), licorisoflavan A (2), and 7−9 showed antibacterial activity against P. gingivalis (MICs of 1.56−12.5 μg/mL). The
most potent activity against S. mutans was obtained with 7 (MIC of 6.25 μg/mL), followed by 1 and 9 (MIC of 12.5 μg/mL).
This study provides further evidence for the therapeutic potential of licorice extracts for the treatment and prevention of oral
infections.
effectiveness of other ingredients, reduce toxicity, or improve
taste.2
hinese licorice (Glycyrrhiza uralensis Fisch., Fabaceae) is
Cthe main licorice species used in Asia and is called “gan-
Dental caries and periodontal diseases are oral infections that
affect a large proportion of the population throughout the
world. In an earlier study, the antibacterial activities of the main
isoflavonoids and coumarins of G. uralensis were evaluated
against major oral pathogens.3 As a continuation of this work to
characterize the metabolites present in the supercritical fluid
extract, the antibacterial activities of several minor compounds
cao” in Chinese, which means “sweet herb”. The name is due to
the presence of glycyrrhizin, a triterpene diglucuronate that
gives the root its typical sweet taste. Licorice has a long history
of use. The first recorded use was as a drug for the treatment of
wounds as described in the book Wu Shi Er Bing Fang, dating to
the second century B.C. According to Liu and Liu,1 the roots
and rhizomes of G. uralensis are used in traditional Chinese
medicine to strengthen the spleen and augment “Qi” (energy),
to moisten the lungs and stop cough, and to soothe spasms and
diminish pain. Licorice is used in as many as half of all
traditional Chinese medicine prescriptions to enhance the
Special Issue: Special Issue in Honor of Otto Sticher
Received: September 26, 2013
Published: January 30, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
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Journal of Natural Products
Article
were assessed against the cariogenic Streptococcus mutans and
the periodontopathogenic Porphyromonas gingivalis. S. mutans is
considered the principal etiological agent of dental caries
formation through its aciduric, acidogenic, and adhesion
properties.4 P. gingivalis has been identified as a key pathogen
contributing to chronic periodontitis,5 as the establishment of
P. gingivalis in subgingival sites induces an inflammatory
response that leads to gingival tissue destruction and
progressive loss of alveolar bone around the teeth.6
spectrum contained signals for three aromatic protons, of which
two were ortho-coupled, one methoxy group, one prenyl group,
and a 2,2-dimethyl-2H-pyran unit.
Owing to the small amount of material isolated, only partial
13C NMR data could be obtained. In the HSQC data, the
aromatic proton resonances at δ 6.88, 6.40, and 6.17 correlated
with the 13C NMR resonances at δ 128.0, 108.4, and 99.9,
respectively, consistent with the pattern observed for previously
isolated licorice isoflavans, e.g., 1 and 2, with protons at C-5′,
C-6′, and C-8 and oxygen substituents at C-5, C-7, C-9, C-2′,
and C-4′. The positions of the prenyl moiety and dimethyl-2H-
pyran ring could not be established conclusively with the
available data. Attempts therefore were made to synthesize the
three possible regioisomers, 7,2′-dihydroxy-5-methoxy-6-pren-
yl-6‴,6‴-dimethylpyrano[2‴,3‴:4′,3′]isoflavan, 7,4′-dihydroxy-
5-methoxy-6-prenyl-6‴,6‴-dimethylpyrano[2‴,3‴:2′,3′]-
isoflavan, and 2′,4′-dihydroxy-5-methoxy-3′-prenyl-6″,6″-
dimethylpyrano[2″,3″:7,6]isoflavan, using licoricidin (1) as
the starting material. This material was abundantly available
to our research group from previous isolation efforts involving
G. uralensis (see Supporting Information).
A simple one-step process for oxidative cyclization of an
isoprenyl moiety to form a pyran ring using 2,3-dichloro-5,6-
dicyano-p-benzoquinone (DDQ) was reported by Jain and
Sharma in 1974.16 It was hypothesized that all three possible
oxidative cyclization products would be formed during
exposure of 1 to these conditions, but the resulting reaction
mixture contained only two products, both showing an [M +
H]+ ion at m/z 423 by ESIMS. After separation by preparative
HPLC, one of the products was identified as the pterocarpan 4,
based on comparison of spectroscopic and spectrometric
information with published data.12,13 The second compound
(8) was found to be an isomer of 4 based on HRESITOFMS
data, which showed an [M + H]+ ion at m/z 423.2191 (calcd
423.2172). The UV spectrum of 8 differed from that of 7, with
maxima at 277, 285, and 306 nm, as opposed to 224, 281, and
310 nm. The 1H NMR spectrum (Table 1) exhibited
resonances corresponding to the same subunits found in 7,
but the shifts did not match those observed for this isolate. The
most significant differences were observed in the resonances for
H-8 and the prenyl moiety. The structure was elucidated
ultimately by analysis of the HMBC data. In particular, the
correlations between OH-2′ (δ 7.20, br s) and C-1′ (δ 120.4),
C-2′ (δ 154.1), and C-3′ (δ 116.3) and between OH-4′ (8.17,
br s) and C-3′ were used to confirm the structure as the target
regioisomer, 2′,4′-dihydroxy-5-methoxy-3′-prenyl-6″,6″-
dimethylpyrano[2″,3″:7,6]isoflavan, licorisoflavan D (8). Com-
parison of the HPLC retention time and UV and MS data with
those of a minor component present in one of the licorice
fractions obtained during purification led to the conclusion that
8 is a naturally occurring constituent of the investigated licorice
root extract.
RESULTS AND DISCUSSION
■
Additional separation of fractions obtained earlier from a
supercritical fluid extract of G. uralensis roots led to the isolation
of the known licoricidin (1),7 licorisoflavan A (2),8 glyasperin
D,9 licoricone,10 1-methoxyficifolinol,11 glycyrin,9 and licoriqui-
none B (3),3 along with four additional known compounds, 1-
methoxyerythrabyssin II (4),12,13 6,8-diprenylgenistein,14 gan-
caonin G (5),10 and isoglycyrol (6),15 isolated for the first time
from this source. The structures of the known compounds and
the new isoflavan, licorisoflavan C (7), were determined based
on comparison of spectroscopic and spectrometric information
with published data.
The molecular formula of 7, C26H30O5, was determined by
HRESITOFMS, which showed an [M + H]+ ion at m/z
423.2178, calcd for [C26H30O5 + H]+, m/z 423.2172. The
presence of an isoflavan skeleton was deduced from the NMR
data. In the 1H NMR spectrum, the signals corresponding to H-
2α, H-2β, H-3, H-4α, and H-4β (Table 1) were similar to the
signals of the C-ring protons present in the 1H NMR spectra of
known isoflavans isolated from the extract. In addition, the
A second synthetic approach was used to generate the two
remaining regioisomers, 7 and 9, as the use of DDQ did not
affect pyran-ring formation onto the B-ring. Initially, 1 was
acetylated using Ac2O−pyridine to prepare a mixture of 7,2′-
diacetyl-, 7,4′-diacetyl-, and 2′,4′-diacetyllicoricidin. These
products were separated by HPLC, and their structures
determined by NMR spectroscopy. 7,2′-Diacetyllicoricidin
was subjected to oxidative cyclization17 with PdCl2. Subsequent
deacetylation using 1.2 N NaOH and purification by
preparative HPLC afforded the new 7,2′-dihydroxy-5-me-
thoxy-6-prenyl-6‴,6‴-dimethylpyrano[2‴,3‴:4′,3′]isoflavan
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1
Table 1. H NMR Spectroscopic Data for Licorisoflavans C, D, and E (7−9)
a
licorisoflavan C (7)
licorisoflavan D (8)
licorisoflavan E (9)
b
c
c
c
c
position
δH, mult. (J in Hz)
δH, mult. (J in Hz)
HMBC
δH, mult. (J in Hz)
HMBC
d
2α (eq)
2β (ax)
3
4.16, ddd (2.0, 3.4, 10)
3.97, t (10)
4.22, ddd (2.2, 3.4, 10)
3.96, t (10)
3,4,9,10 ,1′
3,4,9,1′
4.17, dt (10, 2.2)
3.95, br t (10)
3.38, m
3,4,9,1′
3,4,9,1′
2,4,10,1′,2′,6′
3.35, m
3.39, dddd (3.4, 5.1, 10, 11)
2.91, ddd (2.2, 5.1, 16)
2.74, dd (11, 16)
6.04, s
2,4,10,1′,2′,6′
d
d
4α (eq)
4β (ax)
8
2.88, ddd (2.0, 5.3, 16)
2.80, dd (11, 16)
6.17, s
2,3,5,8 ,9, 10,OCH3-5 ,1′
2.89, ddd (1.4, 4.5, 16)
2.74, dd (11, 16)
6.17, s
2,3,5,9,10,1′
d d
d
d d
2,3,5,7 ,8 , 9,10,OCH3-5 ,1′
2,3,5,6 ,8 , 9,10,1′
d
d
d d
d
d
4 ,5,6,7,9,10,1″
4 ,5 ,6,7, 9,OCH3-5 ,1″
d
d
d
d
d
5′
6′
1″a
1″b
2″
4″
5″
1‴
2‴
4‴
5‴
OCH3-5
OH-7
OH-2′
OH-4′
6.40, d (8.4)
6.88, d (8.4)
3.30, dd (7.0, 14)
3.25, dd (7.0, 14)
5.25, m
6.46, d (8.4)
3 ,1′,2′ ,3′,4′,1‴
6.34, d (8.3)
6.95, d (8.3)
3.29, dd (7.2, 14)
3.23, dd (6.8, 14)
5.23, m
1′,2′ ,3′,4′,1‴
d
d
d
6.83, d (8.4)
2 ,3,2′,3′ ,4′
5,6,7,8,2″,3″,4″,5″, OCH3-5
3,2′,3′ ,4′
d
d
6.52, d (9.9)
5,6,7,2″,3″,5″
5,6,7,2″,3″,5″
6,1″,4″,5″
2″,3″,5″
d
d
d d
5.58, d (9.9)
5 ,6,7 ,8 , 3″,4″,5″
g
d
1.64, br s
1.36, s
1″ ,2″,3″, 5″
1″ ,2″,3″,4″
2′,3′,4′,2‴,3‴,4‴ ,5‴
3′,1‴,4‴,5‴
3′ ,2‴,3‴,5‴
3′ ,2‴,3‴,4‴
1.63, br s
g
d
1.75, br s
1.38, s
1.74, br s
2″,3″,4″
d
d
c
d
6.70, d (10)
3.45, br d (7.0)
5.26, m
6.79, d (10)
5.70, d (10)
1.37, s
2′,3′,4′,2‴,3‴,4‴ ,5‴
d d
5.66, d (10)
1′ ,3′,4′ ,3‴,4‴,5‴
e
d
1.42, s
1.66, br s
1.77, br s
3.72, s
2‴,3‴,5‴
2‴,3‴,4‴
5
e
d
1.43, s
1.37, s
3.69, s
5
3.67, s
f
e,f,g,h
8.39, br s
-
-
8.37, br s
e,f,g,h
-
7.20, br s
8.17, br s
1′,2′,3′
8.11, br s
f
8.08, br s
3′
-
a
b
c
No HMBC experiment was performed on this semisynthetic compound. Data collected using acetone-d6 at 500 MHz. Data collected using
acetone-d6 at 600 MHz. Indicates a correlation across more than three bonds.
d
e,f,g,h
These assignments are interchangeable.
Table 2. 13C NMR Spectroscopic Data for Licorisoflavans C,
D, and E (7−9)
(9) in low yield (1.5 mg, corresponding to 0.6%). The structure
1
was confirmed by analysis of H and 13C NMR data (Tables 1
and 2), and the product named licorisoflavan E (9).
The above approach involving acetylation, cyclization, and
licorisoflavan C (7) licorisoflavan D (8) licorisoflavan E (9)
a
b
c
position
δC, mult.
δC, mult.
δC, mult.
subsequent ester hydrolysis could not be used effectively for the
synthesis of 7,4′-dihydroxy-5-methoxy-6-prenyl-6‴,6‴-
dimethylpyrano[2‴,3‴:2′,3′]isoflavan (7), due to the initial
low yield of 7,4′-diacetyllicoricidin (6.7 mg; 2.2%) and the
representative overall low yield of 9. Subsequent attempts to
improve yields were unsuccessful. This prompted the use of a
third synthetic approach employing PdCl2 to effect oxidative
cyclization of only one prenyl moiety in licoricidin (1). Thus,
treatment of 1 with PdCl2 in EtOH for 4 h at room
temperature and subsequent preparative HPLC afforded the
mono(2,2-dimethyl-2H-pyrano)isoflavans 7−9 in 5.1%, 3.2%,
and 7.5% yields, respectively. The molecular formula of 7,
C26H30O5, was established by HRESITOFMS, which showed
an [M − H]− ion at m/z 421.2028 (calcd 421.2015), and the
1H NMR, retention time, UV, and mass data were identical to
those of the natural product isolated from the extract. The
structure of 7 could not be determined conclusively solely on
the basis of NMR data due to a lack of correlations between the
phenolic protons and the B-ring carbons and to an absence of
detectable NOE effects between the OH groups and aromatic
protons. The structure of 7, named licorisoflavan C, could,
however, be assigned as 7,4′-dihydroxy-5-methoxy-6-prenyl-
6‴,6‴-dimethylpyrano[2‴,3‴:2′,3′]isoflavan based on the
feasible outcomes of the synthesis and the established identities
of regioisomers 8 and 9. Isoflavan 7 was shown to be naturally
occurring based on comparison of its HPLC retention time and
UV and MS data with the identical compound in the licorice
extract.
2
70.3, CH2
32.4, CH
26.7, CH2
158.4, C
70.7, CH2
31.9, CH
26.7, CH2
155.5, C
69.8, CH2
31.6, CH
26.5, CH2
157.7, C
3
4
5
6
114.5, C
108.7, C
114.0, C
7
155.5, C
153.7, C
155.1, C
8
99.9, CH
154.5, C
100.9, CH
156.5, C
99.1, CH
153.8, C
9
10
108.3, C
109.5, C
107.7, C
1′
2′
3′
4′
5′
6′
121.1, C
120.4, C
121.5, C
152.8, C
154.1, C
150.6, C
110.4, C
116.3, C
110.6, C
152.2, C
155.4, C
152.8, C
108.4, CH
128.0, CH
23.4, CH2
125.3, CH
130.3, C
108.3, CH
125.2, CH
117.8, CH
128.5, CH
76.4, C
108.4, CH
127.2, CH
22.9, CH2
124.7, CH
129.9, C
1″
2″
3″
4″
5″
1‴
2‴
3‴
4‴
5‴
OCH3-5
25.9, CH3
17.9, CH3
118.1, CH
129.3, CH
76.5, C
27.9, CH3
27.8, CH3
23.3, CH2
123.9, CH
131.8, C
25.5, CH3
17.5, CH3
117.3, CH
129.8, CH
75.4, C
27.9, CH3
27.9, CH3
60.6, CH3
25.9, CH3
18.0, CH3
27.4, CH3
27.4, CH3
60.1, CH3
61.5, CH3
a
b
Data collected in acetone-d6 at 125 MHz. Data collected in acetone-
d6 at 100 MHz. Data (150 MHz) derived from HMBC experiments in
acetone-d6.
c
The absolute configuration at C-3 for naturally occurring 7
was established by electronic circular dichroism (ECD). The
positive Cotton effect (CE) near 300 nm and the negative CE
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Journal of Natural Products
Article
near 240 nm indicated the P-helicity of the C-ring and
corresponding (3R) absolute configuration by analogy to data
for similarly substituted isoflavan systems.18 The negative
specific rotation of 9 as compared to the positive specific
rotations of 7 and 8 reflects the unreliability of specific rotation
alone for assignment of absolute configuration for the
isoflavans.18 The absolute configurations for 8 and 9 could,
however, be assigned as above on the basis of their ECD
spectra and were established as (3R).18
MIC values of 7−9 were in the range 6.25−25.0 μg/mL.
Compounds 7 and 9 gave MBC values of 6.25 and 12.5 μg/mL,
respectively, and the MBC value for 8 was >200 μg/mL. These
results suggest that isoflavans 7 and 9 may act via a bactericidal
mechanism of action against both bacteria, while 8 may be
bactericidal only against P. gingivalis.19 Weaker activity against
P. gingivalis was observed with isoflavan-quinone 3 and
pterocarpan 4. Compounds 3 and 4 were inactive against S.
mutans, and 5 and 6 were inactive in both assays. Although
prenylated pterocarpans reportedly have excellent antibacterial
properties against S. mutans,20 pterocarpan 4 showed less
potent antibacterial activity than the isoflavans 1, 2, and 7−9.
Previous reports suggest that some degree of lipophilicity and
phenolic hydroxy groups are required for the antibacterial
activity of isoflavonoids.21 Earlier results supported this premise
as isoflavans with two prenyl groups exhibited more potent
antibacterial activities than isoflavans bearing only one such
group.3 In the present study, cyclization of one prenyl group
onto the B-ring of compounds 7 and 9 as compared to 1 had
minimal effects on activity against S. mutans regardless of
whether ring formation involved C-2′ or C-4′. Cyclization of a
single prenyl group, however, did appear to reduce the activity
against P. gingivalis (compounds 7−9 as compared to 1 and 2),
irrespective of the prenyl group or aromatic ring involved.
A review on structure−activity relationships of isoflavanoids
reported that the presence of a C-7 and/or C-5 free hydroxy
group leads to isoflavonoids with more potent antibacterial
activity.22 As compounds 1, 2, and 7−9 all possess C-5 O-
methyl groups, the possible contribution of the C-7 hydroxy
group to antibacterial activity could be assessed. Compound 8,
with the C-7 hydroxy group involved in prenyl group
cyclization, was less active than 7 or 9 against S. mutans, but
was active against P. gingivalis. Compound 2, with a C-7 O-
methyl, also exhibited reduced activity against S. mutans, with
potent inhibition of P. gingivalis. These results support the
premise that a free C-7 hydroxy group is important for the
activity of isoflavans against S. mutans and also suggest that
additional factors besides lipophilicity and the presence of free
hydroxy groups at C-5 and/or C-7 may contribute to the
antibacterial activities of isoflavans against P. gingivalis.
Differences in the NMR spectra of compound 8 as compared
to 7 and 9 reflect the effects of prenyl group cyclization onto
the A-ring versus onto the B-ring, respectively. The cyclization
process shifts the prenyl double bond into conjugation with the
aromatic rings. Cyclization effects and such concomitant
double-bond changes were most apparent in the shifts of the
prenyl group protons (H-1″, H-2″, H-1‴, and H-2‴) and
carbons (C-1″, C-2″, C-3″, C-1‴, C-2‴, and C-3‴) and, to a
lesser extent, the aromatic protons, H-8, H-5′, and H-6′. The
conjugation associated with cyclization onto the B-ring in 7 and
9 resulted in distinct shielding of C-3′ (δ 110.4 and 110.6 in 7
and 9, respectively, versus 116.3 in 8) and deshielding of C-6′
(δ 128.0 and 127.2 in 7 and 9, respectively, versus 125.2 in 8),
while conjugation associated with cyclization onto the A-ring of
8 resulted in distinct shielding of C-6 (δ 108.7 in 8, versus
114.5 and 114.0 in 7 and 9, respectively). Cyclization involving
OH-2′ in 7 versus OH-4′ in 9 was reflected in the 13C NMR
spectra, with slight differences in chemical shifts for C-2′ (δ
152.8 and 150.6 for 7 and 9, respectively). Overall, 7, 9,
kanzonol I (7-O-methyllicorisoflavan C), and 7-O-methyllicor-
17
1
isoflavan E had similar H NMR characteristics, providing
further confirmation of the structural assignments made.
Table 3 collates the minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC) values
Table 3. Minimum Inhibitory Concentration (MIC) and
Minimum Bactericidal Concentration (MBC) Values for 1−
9 against P. gingivalis and S. mutans
P. gingivalis ATCC 33277
S. mutans ATCC 35668
a
a
a
a
compound
MIC
MBC
MIC
MBC
This study demonstrates the potential benefits of licorice
root extract for the treatment or prevention of major oral
diseases due to the presence of isoflavans 1, 2, 7, and 8. One of
the challenges with products for the oral cavity is the short time
of exposure of an agent to tooth and gum surfaces. While this
time period may be extended by the use of hard lozenges,
candies, or teas, it remains short for products such as
mouthwash or toothpaste, as exemplified by a study of 173
U.S. adults, who indicated the average time to brush the teeth
was 46 s.23 More studies are therefore needed to confirm the
abilities of licorice-containing products to alter the composition
of oral microflora in humans to improve oral health.
1
3.125
1.56
3.125
1.56
12.5
50
25
100
2
3
25
25
>200
>200
>200
>200
>200
4
50
200
200
50
200
200
200
>200
>200
5
6
7
6.25
6.25
12.5
1.56
6.25
6.25
12.5
12.5
6.25
6.25
>200
12.5
8
25
9
12.5
tetracycline
penicillin G
0.05
1.56
a
MIC and MBC in μg/mL.
EXPERIMENTAL SECTION
of the nine compounds tested against P. gingivalis and S.
mutans. Compounds 1 and 2 were reported previously as
inhibitors of the oral pathogens P. gingivalis and Prevotella
intermedia at a concentration of 5 μg/mL and S. mutans and S.
sobrinus at 10 μg/mL.3 Both compounds therefore were
included in the present study as additional positive controls
and to assess the MIC/MBC values for these compounds. The
isoflavans 7−9 showed marked antibacterial activities against
both oral pathogens tested. MIC and MBC values of 6.25−12.5
μg/mL against P. gingivalis were obtained. For S. mutans, the
■
General Experimental Procedures. Optical rotations were
recorded on a 589-546 Rudolph Research Analytical Autopol III
automatic polarimeter. UV data were measured on a Cary 60 UV
spectrophotometer (Agilent, Burlington, MA, USA). ECD data were
collected using an Olis Cary-17 spectrophotometer (1 cm path length
cell). NMR spectra were obtained using an AVANCE II 500 MHz or
an AVANCE 600 MHz instrument (Bruker Biospin, Billerica, MA,
USA). Analytical and semipreparative HPLC was carried out using an
Agilent 1100 unit consisting of an Agilent quaternary pump, UV/vis
detector (DAD), degasser, automatic sample injector, and Agilent
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dx.doi.org/10.1021/np400788r | J. Nat. Prod. 2014, 77, 521−526

Journal of Natural Products
Article
1100 series LC/MSD trap (Agilent, Burlington, MA, USA). HPLC
analysis of fractions and isolates was performed on a Zorbax Eclipse
XDB C18 column (250 × 4.6 mm i.d., 5 μm; Agilent, Santa Clara, CA,
USA), with a Zorbax C18 guard column (12.5 × 4.6 mm i.d., 5 μm)
using a flow rate of 1.0 mL/min, a column temperature of 30 °C,
detection at 280 nm, and a mobile phase of MeCN (0.05% TFA)−
H2O (0.05% TFA) with 0−3 min (45:55); 3−25 min (45:55 to
100:0). All solvents for HPLC were from Fisher Scientific (Pittsburgh,
PA, USA). Trifluoroacetic acid, Ac2O, benzene, DDQ, PdCl2, and
pyridine were obtained from Sigma (St. Louis, MO, USA).
J = 11, 16 Hz, H-4β), 2.32 (3H, s, CH3COO-2′), 2.23 (3H, s,
CH3COO-7), 1.76 (3H, s, H3-5‴), 1.74 (3H, s, H3-5″), 1.65 (6H, s,
H3-4″, H3-4‴); 13C NMR (150 MHz, acetone-d6) 169.9 (C,
CH3COO-2′), 169.5 (C, CH3COO-7), 158.0 (C, C-5), 155.6 (C, C-
4′), 154.1 (C, C-9), 149.4 (C, C-7), 149.1 (C, C-2′), 131.6 (CH, C-
2‴), 131.3 (CH, C-2″), 125.8 (CH, C-6′), 125.7 (C, C-1′), 124.0 (C,
C-3″), 123.1 (C, C-3‴), 121.9 (C, C-3′), 119.7 (C, C-6), 115.0 (C, C-
10), 114.0 (CH, C-5′), 107.6 (CH, C-8), 70.4 (CH2, C-2), 60.7 (CH3,
OCH3-5), 31.9 (CH, C-3), 27.8 (CH2, C-4), 25.8 (CH3, C-4‴), 25.7
(CH3, C-4″), 24.2 (CH2, C-1‴), 24.0 (CH2, C-1″), 20.6 (CH3,
CH3COO-7, CH3COO-2′) 17.9 (CH3, C-5″, C-5‴).
Plant Material. Commercially available dried and cut G. 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.
Extraction and Isolation. Plant material (0.41 g) was ground and
extracted using supercritical CO2 with 5% EtOH as a modifier. The
compounds described here were isolated from two fractions previously
obtained by gel filtration of MPLC fraction 11 on Sephadex LH-20.3
Sephadex fraction 3 (106 mg) was separated by semipreparative
HPLC on a Zorbax SB C18 column (250 × 9.4 mm, 5 μm) using
MeOH (0.001% TFA)−H2O (0.001% TFA) (74:26) at 45 °C and a
flow rate of 3 mL/min, yielding 1 (1.3 mg), 3 (0.9 mg), 4 (0.2 mg), 5
(0.6 mg), licoricone (0.4 mg), glyasperin D (0.6 mg), 1-
methoxyficifolinol (0.2 mg), and 6,8-diprenylgenistein (1.0 mg).
Sephadex fraction 4 (31 mg) was separated by semipreparative HPLC
with the same column and mobile phase (ratio 73:27) at 50 °C,
yielding 1 (1.9 mg), 2 (1.5 mg), 6 (0.8 mg), 7 (0.9 mg), glycyrin (1.0
mg), and glyasperin D (1.0 mg).
7,4′-Diacetyllicoricidin and 2′,4′-Diacetyllicoricidin. See Support-
ing Information.
Synthesis of Licorisoflavan E (9). 7,2′-Diacetyllicoricidin (16.2 mg)
was dissolved in 90% aqueous EtOH (1.0 mL), and PdCl2 (5.9 mg)
was added. The mixture was stirred at room temperature for 48 h, the
solvent was evaporated, and the residue was dissolved in 1.2 N NaOH
(1 mL) in 90% aqueous EtOH for 30 min to hydrolyze the acetate
groups. Licorisoflavan E (9; 1.5 mg) was obtained after purification by
semipreparative HPLC on an Agilent PrepHT XDB C18 column (250
× 21.2 mm, 7 μm) using MeOH−H2O (91:9), at a flow rate of 6 mL/
min and a temperature of 50 °C.
Licorisoflavan E (9): white powder; [α]2D1 −9 (c 0.1, MeOH); UV
(EtOH) λmax (log ε) 207 (4.73), 227 (4.64), 279 (4.09), 309 sh (3.43)
nm; ECD (c 0.004, MeOH) λ (Δε) 218 (+10.0), 239 (−3.9), 262
1
(+1.7), 273 (+1.7), 286 (+1.8) nm; H (600 MHz, acetone-d6) and
13C (150 MHz, acetone-d6) NMR data, see Tables 1 and 2; HPLC-
ESIMS m/z 423 [M + H]+, MS2 m/z 367 [M + H − 56]+, 221 [M + H
− 202]+, 189 [M + H − 234]+.
Synthesis of Licorisoflavan C (7). Licoricidin (1, 102 mg) was
dissolved in EtOH (2.0 mL), to which PdCl2 (18.4 mg) was added.
The mixture was stirred at room temperature for 4 h and directly
subjected to semipreparative fractionation by HPLC on an Agilent
PrepHT XDB C18 column (250 × 21.2 mm, 7 μm) using MeCN−
H2O (88:12), at a flow rate of 5.6 mL/min and a temperature of 40
°C, to yield 7 (5.2 mg), 8 (3.2 mg), and 9 (7.6 mg).
Licorisoflavan C (7): white powder; [α]2D1 +18 (c 0.1, MeOH); UV
(EtOH) λmax (log ε) 207 (4.71), 224 sh (4.59), 281 (4.04), 310 sh
(3.40) nm; ECD (c 0.004, MeOH) λ (Δε) 210 (+8.8), 238 (−5.2),
1
265 sh (+1.8), 289 (+4.5) nm; H (600 MHz, acetone-d6) and 13C
(150 MHz, acetone-d6) NMR data, see Tables 1 and 2;
HRESITOFMS m/z 423.2178 [M + H]+ (calcd for [C26H30O5
+
H]+, 423.2172); HPLC-ESIMS m/z 423 [M + H]+, MS2 m/z 367 [M
Bioassays. Stock solutions of compounds were prepared in EtOH
and kept at 4 °C protected from light. Minimal inhibitory
concentrations and minimal bactericidal concentrations were deter-
mined using a microbroth dilution assay. Briefly, S. mutans (ATCC
35668) and P. gingivalis (ATCC 33277) were grown at 37 °C in Todd-
Hewitt broth (THB) (BBL Microbiology Systems, Cokeysville, MD,
USA) supplemented with hemin (10 μg/mL) and vitamin K (10 μg/
mL). P. gingivalis and S. mutans were incubated under anaerobic (N2/
H2/CO2: 80/10/10) and aerobic conditions, respectively. Overnight
cultures were diluted in fresh THB medium to obtain an optical
density at 660 nm (OD660) of 0.2. Equal volumes (100 μL) of bacteria
and a series of 2-fold dilutions of compounds (200 μg/mL to 0.78 μg/
mL) in culture medium were mixed into the wells of 96-well plates
(Sarstedt, Newton, NC, USA). Control wells with no bacteria or no
compounds were also prepared. Penicillin G and tetracycline were
used as reference compounds for S. mutans and P. gingivalis,
respectively. After an incubation of 24 h at 37 °C, bacterial growth
was recorded visually. MIC values (μg/mL) of compounds for each
bacterial species were determined as the lowest concentration at which
no growth occurred. To determine MBC values (μg/mL), aliquots (5
μL) of each well showing no visible growth were spread on THB agar
plates, which were incubated for 3 days at 37 °C. MBCs of compounds
for each bacterial species were determined as the lowest concentration
at which no colony formation occurred. The MIC and MBC values
were determined in three independent experiments to assess
reproducibility.
+ H − 56]+, 221 [M + H − 202]+, 189 [M + H − 234]+.
Synthesis of 8. Licoricidin (1, 40 mg) and DDQ (25 mg) were
dissolved in benzene (6 mL) and heated under reflux for 5 min. After
cooling, the sample was evaporated to dryness and separated by
semipreparative HPLC on a Zorbax SB C18 column (250 × 9.4 mm, 5
μm) using MeOH−H2O (82:18), at a flow rate of 3 mL/min at 40 °C,
yielding the starting material (1, 2.9 mg), pterocarpan 4 (1.0 mg), and
8 (9.9 mg).
Licorisoflavan D (8): white powder; [α]2D1 +39 (c 0.3, MeOH); UV
(EtOH) λmax (log ε) 205 (4.58), 277 (3.96), 285 (2.95), 306 (3.65)
nm; ECD (c 0.005, MeOH) λ (Δε) 221 (+12.1), 242 (−4.3), 277
(+6.4), 286 (+7.0) nm; 1H (600 MHz, acetone-d6) and 13C (150
MHz, acetone-d6) NMR data, see Tables 1 and 2; HRESITOFMS m/z
423.2191 [M + H]+ (calcd for [C26H30O5 + H]+, 423.2172); HPLC-
ESIMS m/z 423 [M + H]+, MS2 m/z 367 [M + H − 56]+, 231 [M + H
− 192]+, 219 [M + H − 204]+, 191 [M + H − 232]+.
Acetylation of 1. Licoricidin (1, 254 mg) was dissolved in pyridine
(25 mL), to which Ac2O (130 mg) was added. The mixture was stirred
in an airtight vial for 16 h, as preliminary studies showed that this
amount of time was optimum for obtaining high yields of diacetylated
licoricidin isomers. After evaporation to dryness, the residue was
separated by semipreparative HPLC on an Agilent PrepHT XDB C18
column (250 × 21.2 mm, 7 μm) using MeOH−H2O (88:12), at a flow
rate of 6 mL/min and a temperature of 50 °C, yielding 7,2′-
diacetyllicoricidin (24.2 mg), 7,4′-diacetyllicoricidin (6.7 mg), and
2′,4′-diacetyllicoricidin (54.6 mg).
7,2′-Diacetyllicoricidin: white powder; 1H NMR (600 MHz,
acetone-d6) δ 8.57 (1H, br s, OH-4′), 7.05 (1H, d, J = 8.4 Hz, H-
6′), 6.83 (1H, d, J = 8.4 Hz, H-5′), 6.37 (1H, s, H-8), 5.17 (1H, m, H-
2‴), 5.08 (1H, m, H-2″), 4.13 (1H, ddd, J = 2.0, 3.0, 10 Hz, H-2α),
3.94 (1H, m, H-2β), 3.70 (3H, s, OCH3-5), 3.23 (2H, dd, J = 6.9, 15
Hz, H-1″b, H-1‴b), 3.17 (2H, dd, J = 6.5, 15 Hz, H-1″a, H-1‴a), 3.07
(1H, m, H-3), 2.96 (1H, ddd, J = 1.9, 5.0, 16 Hz, H-4α), 2.80 (1H, dd,
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR spectra of 7,2′-diacetyllicoricidin, and 7−9, as well as
NMR data and spectra of 7,4′-diacetyllicoricidin and 2′,4′-
diacetyllicoricidinare available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +1 207-985-2944. Fax: +1 207-985-2196. E-mail:
sgafner99@gmail.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful to Dr. D. Wiemer (University of Iowa)
for discussions on the synthesis of 7 and to Dr. C. Schneider
(Memorial University of Newfoundland, St. John’s, Canada) for
collecting NMR data for some of the licorice isolates.
DEDICATION
■
Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich,
Switzerland, for his pioneering work in pharmacognosy and
phytochemistry.
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