Flavonoids from Glycyrrhiza pallidiflora hairy root cultures

Article abstract of DOI:10.1016/S0031-9422(02)00093-6

Three flavonoids named licoagrosides D, E and F together with four known flavonoids, medicarpin 3-O-glucoside, calycosin 7-O-glucoside, formononetin 7-O-(6″-malonylglucoside) and 2′-hydroxyformononetin 7-O-glucoside were isolated from Glycyrrhiza pallidiflora hairy root cultures. Their structures were determined on the basis of spectroscopic evidence. Licoagrosides E and F are the first examples of a 6a-hydroxypterocarpan glycoside and an α-O-glycosidic α-hydroxydihydrochalcone, respectively.

Full text of DOI:10.1016/S0031-9422(02)00093-6

Phytochemistry 60 (2002) 351–355
www.elsevier.com/locate/phytochem
Flavonoids from Glycyrrhiza pallidiflora hairy root cultures
Wei Lia, Kazuo Koikea, Yoshihisa Asadab, Masao Hirotanib, Hekai Ruib,
Takafumi Yoshikawab, Tamotsu Nikaidoa,*
aSchool of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-city, Chiba 274-8510, Japan
bSchool of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
Received 4 December 2001; received in revised form 22 February 2002
Abstract
Three flavonoids named licoagrosides D, E and F together with four known flavonoids, medicarpin 3-O-glucoside, calycosin 7-O-
glucoside, formononetin 7-O-(600-malonylglucoside) and 20-hydroxyformononetin 7-O-glucoside were isolated from Glycyrrhiza
pallidiflora hairy root cultures. Their structures were determined on the basis of spectroscopic evidence. Licoagrosides E and F are
the first examples of a 6a-hydroxypterocarpan glycoside and an a-O-glycosidic a-hydroxydihydrochalcone, respectively. # 2002
Elsevier Science Ltd. All rights reserved.
Keywords: Licorice; Glycyrrhiza pallidiflora; Leguminosae; Hairy root; Flavonoid; Licoagroside; a-Hydroxydihydrochalcone; 6a-Hydroxy-
pterocarpan; Glycoside
1. Introduction
2. Results and discussion
The roots of Glycyrrhiza species are one of the oldest
plant medicines. In studies on flavonoids produced by
hairy root cultures of Glycyrrhiza species, we have
reported 40 flavonoids, including ten newcompounds
from G. glabra hairy root cultures (Asada et al., 1998,
1999; Li et al., 1998, 2000). We also investigated the fla-
vonoid constituents of G. pallidiflora hairy root cultures
and reported nine flavonoids, including a newprenylated
isoflavone, licoagroisoflavone and a newcoumestan gly-
coside, licoagroside C (Li et al., 2001). Herein, we report
the isolation and structural determination of a new
pterocarpan glycoside, licoagroside D (1), a new
a-hydroxypterocarpan glycoside, licoagroside E (2) and
a new a-hydroxydihydrochalcone glycoside, licoagroside
F (3) together with 4 known flavonoids, medicarpin
3-O-glucoside (4), calycosin 7-O-glucoside (5), for-
mononetin 7-O-(600-malonylglucoside) (6), 20-hydroxy-
formononetin 7-O-glucoside (7) from the glycoside
fraction of G. pallidiflora hairy root cultures (see Fig. 1).
G. pallidiflora hairy root cultures were extracted with
methanol and then partitioned between ethyl acetate
and water according to the previous paper (Li et al.,
2001). The water layer was passed through a diaion HP-
20 column and washed with methanol. The methanol
eluate was evaporated, then applied to an ODS column
and purified by reverse-phase HPLC to give seven com-
pounds. The structures of known compounds 4–7 were
determined by analysis of the physical and spectroscopic
evidence, and confirmed by comparing with the litera-
ture data.
Compound 1 was obtained as a powder. The FAB
mass spectrum of 1 showed a pseudo molecular ion peak
at m/z 449 [M+H]+ which is larger by 18 mass units
than that of medicarpin 3-O-glucoside (4). Its molecular
formula, C22H24O10, was established by the HR-FAB
mass spectrum. The 1H NMR spectrum of 1 was similar
1
to that of 4, except for B ring protons. Namely, the H
NMR spectrum of 1 showed a set of proton signals [3.73
(1H, dd, J=11.0, 11.0 Hz, Hax-6), 4.30 (1H, dd, J=11.0,
5.0 Hz, Heq-6), 3.59 (1H, ddd, J=11.0, 7.0, 5.0 Hz, H-
6a), 5.59 (1H, d, J=7.0 Hz, H-11a)] characteristic of
pterocapan. The spectrum also exhibited the presence of
an ABX-type aromatic proton system appearing at ꢀ
7.52 (1H, d, J=9.2 Hz), 7.06 (1H, dd, J=9.2, 2.8 Hz)
* Corresponding author. Tel.: +81-474-721391; fax: +81-474-
721404.
E-mail address: nikaido@phar.toho-u.ac.jp (T. Nikaido).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00093-6

352
W. Li et al. / Phytochemistry 60 (2002) 351–355
Fig. 1. Flavonoids from Glycyrrhiza pallidiflora hairy root cultures.
and 7.08 (1H, d, J=2.8 Hz) due to A ring protons, an
AB-type aromatic proton signals resonating at ꢀ 6.82
(1H, d, J=7.8 Hz) and 6.22 (1H, d, J=7.8 Hz) due to B
ring protons as well as a methoxyl proton signal at ꢀ
3.78 and the protons of a b-glucosyl group with the
anomeric proton signal resonating at ꢀ 5.66 (1H, d,
J=7.3 Hz). The positions of the methoxyl and b-gluco-
pyranosyloxy groups in 1 were determined from the
NOESY spectrum as shown in Fig. 2. Namely, NOE’s
observed between the methoxy and C-8 protons,
anomeric proton of b-glucopyranosyloxy groups and C-
2, C-4 protons suggested that the methoxy and b-glu-
copyranosyloxy groups are located at C-9 and C-3,
respectively. The proton and carbon signals were fur-
ther assigned as shown in Table 1 by the analysis of
HMQC and HMBC spectra (Fig. 3). The vicinal cou-
pling constants of 7.0 Hz between protons of C-6a and
C-11a indicated they are cis- configuration (Pachler et
al., 1967) and the absolute configuration was deter-
mined as 6a-R and 11a-R by CD spectrum analysis: a
negative Cotton effect at 228 nm and a positive Cotton
effect at 284 nm were observed (Kitakawa et al., 1994).
Enzymatic hydrolysis of 1 with naringinase gave an
aglycone 1a and glucose. 1a was confirmed to be 3, 10-
Compound 2 was obtained as a powder. The FAB
mass spectrum of 2 showed a molecular ion peak at m/z
448 [M]+. HR-FAB mass spectrum suggested it has the
same molecular formula, C22H24O10 as 1. A comparison
1
of the H NMR spectrum of 2 with that of 1, showed
that H-6a proton signal of 1 disappeared, and H-11a
proton signal changed to a singlet, H-6 protons changed
to an AB quartet. Also, in the 13C NMR spectrum, the
1
dihydroxy-9-methoxypterocarpan by its H NMR spec-
trum (Kurosawa et al., 1978). Glucose was determined
as having a d- form by HPLC analysis of its 1-[(S)-N-
acetyl - a - methylbenzylamino] - 1 - deoxyalditol acetate
derivative (Oshima and Kumanotani, 1981; Oshima et
al., 1982). Thus, the structure of compound 1 was deter-
mined as (6a-R, 11a-R)-3-O-b-d-glucopyranosyl-10-
hydroxy-9-methoxypterocarpan, named as licoagroside D.
Fig. 2. NOE’s detected by the NOESY spectra of flavonoids 1 and 2.

W. Li et al. / Phytochemistry 60 (2002) 351–355
353
Table 1
1H and 13C NMR spectral data for licoagroside D (1) and E (2) in pyridine-d5
Position
1
2
13C NMR
1H NMR
13C NMR
1H NMR
1
132.5
110.9
159.6
105.0
157.0
66.8
7.52 (1H, d, J=9.2 Hz)
7.06 (1H, d, J=9.2, 2.8 Hz)
132.8
111.4
160.0
105.1
156.8
70.7
7.59 (1H, d, J=8.5 Hz)
7.08 (1H, dd, J=8.5, 2.2 Hz)
2
3
4
7.08 (1H, d, J=2.8 Hz)
7.07 (1H, d, J=2.2 Hz)
4a
6
3.73 (1H, dd, J=11.0, 11.0 Hz, Hax)
4.30 (1H, dd, J=11.0,5.0 Hz, Heq)
3.59 (1H, ddd, J=11.0, 7.0, 5.0 Hz)
4.42 (1H, d, J=11.0 Hz)
4.55 (1H, d, J=11.0 Hz)
6a
6b
40.6
121.7
114.2
105.7
150.2
133.3
148.3
78.7
76.3
123.1
124.9
107.6
162.6
97.4
7
6.82 (1H, d, J=7.8 Hz)
6.22 (1H, d, J=7.8 Hz)
7.54 (1H, d, J=8.5 Hz)
6.65 (1H, dd, J=8.5, 2.2 Hz)
8
9
10
6.66 (1H, d, J=2.2 Hz)
5.80 (1H, s)
10a
11a
11b
OCH3
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
161.7
86.2
5.59 (1H, d, J=7.0 Hz)
114.7
56.4
115.5
55.5
3.78 (3H, s)
5.66 (1H, d, J=7.3 Hz)
3.64 (3H, s)
5.62 (1H, d, J=7.4 Hz)
101.8
74.8
78.3
102.1
74.9
78.5
4.29ꢀ4.43 (4H, m, Glc-2,3,4-H and Glc-6-Ha)
4.29ꢀ4.43 (4H, m, Glc-2,3,4-H and Glc-6-Ha)
71.0
78.8
71.1
78.9
4.10 (1H, ddd, J=10.1, 5.5, 2.0 Hz)
4.50 (1H, dd, J=12.0, 2.0 Hz, Glc-6-Hb)
4.08 (1H, m)
4.49 (1H, dd, J=12.0, 2.3 Hz, Glc-6-Hb)
62.1
62.2
chemical shift of C-6a was deshielded by 35.7 ppm
relative to that of 1. All these data indicated that 2 is a
6a-hydroxypterocarpan derivative. In the 1H NMR
spectrum, 2 also showed two ABX-type aromatic pro-
ton signals appearing at d 7.59 (1H, d, J=8.5 Hz), 7.08
(1H, dd, J=8.5, 2.2 Hz) and 7.07 (1H, d, J=2.2 Hz) due
to A ring protons, and ꢀ 7.54 (1H, d, J=8.5 Hz), 6.65
(1H, dd, J=8.5, 2.2 Hz) and 6.66 (1H, d, J=2.2 Hz) due
to B ring protons, a methoxy proton signal at ꢀ 3.64 and
an anomeric proton signal at ꢀ 5.62 (1H, d, J=7.4 Hz),
suggesting a b-d-glucopyranoside. The positions of the
methoxy and b-glucopyranosyloxy groups in 2 were
determined from the NOESYspectrum as shown in
Fig. 2. The absolute configuration was determined as
6a-S and 11a-S by CD spectrum analysis: a negative
Cotton effect at 228 nm and a positive Cotton effect at
286 nm were observed (van Aardt et al., 2001).
Furthermore, enzymatic hydrolysis gave an aglycone
which was confirmed to be 3, 6a-dihydroxy-9-methox-
ypterocarpan (2a) by its 1H NMR spectrum (Bilton
et al., 1976). The b-glucose was determined as having a
d- form by HPLC analysis performed as described for 1.
Thus, the structure of compound 2 was determined as
(6a-S, 11a-S)-3-O-b-d-glucopyranosyl-6a-hydroxy-9-
methoxypterocarpan, named as licoagroside E.
Compound 3 was obtained as a powder. The FAB
mass spectrum of 3 showed a pseudo molecular ion
peak at m/z 437 [M+H]+ which is less by 12 mass units
than that of 1. Its molecular formula, C21H24O10, w as
Fig. 3. 1H–13C long-range correlations by the HMBC spectra of
flavonoids 1–3.

354
W. Li et al. / Phytochemistry 60 (2002) 351–355
established by the HR-FAB mass spectrum. The UV
spectrum of 3 showed maximum absorption at 214
(4.27), 283 (4.11), 327 (3.93) nmwhich suggested 3 is a
dihydrochalcone derivative (Mabry et al., 1970). The 1H
NMR spectrum of 1 showed methylene proton signals
appearing at ꢀ 3.49 (1H, dd, J=16.0, 7.1 Hz), 3.51 (1H,
dd, J=16.0, 7.1 Hz) and an oxymethine proton reso-
nating at ꢀ 5.98 (1H, dd, J=7.1, 7.1 Hz). In addition,
ABX-type aromatic proton signals appearing at d 6.68
(1H, dd, J=8.9, 2.3 Hz), 6.70 (1H, d, J=2.3 Hz) and
8.37 (1H, d, J=2.3 Hz), AA0BB0-type aromatic proton
signals appearing at ꢀ 7.01 (1H, d, J=8.3 Hz) and 7.26
(1H, d, J=8.3 Hz), and the protons of a b-glucosyl
group with the anomeric proton signal appearing at ꢀ
5.10 (1H, d, J=7.6 Hz) indicated that 3 is an
a-hydroxydihydrochalcone glucoside. Comparing the
13C NMR spectra of 3 and kanzonol Y (Li et al., 2000;
Fukai et al., 1996), an a-hydroxydihydrochalcone
isolated from G. glabra hairy root cultures, the signal
of C-3 showed a 5.2 ppm down-field shift, suggesting
that the a-hydroxy was glycosylated. This was further
confirmed by analysis of the HMBC spectrum. Stereo-
chemistry of C-a was determined as R by enzymatic
hydrolysis, and comparing the value of the [a]D of its
aglycone 3a with the literature (Ferrari et al., 1983;
Bezuidenhoudt et al., 1987). The glucose subunit was
confirmed as having a d- form by the same method as
1 and 2. Thus, the structure of 3 was determined as a-R,
a-O-b-d-glucopyranosyl-4, 40, 70-trihydroxydihydro-
chalcone, and named as licoagroside F.
on a JEOL JMS-AX505HA. The 1H and 13C NMR
spectra were measured with a JEOL ECP-500 spectro-
meter in pyridine-d5 solution and chemical shifts are
expressed in ꢀ (ppm) referring to TMS. For HPLC,
JASCO HPLC system was used. Column chromato-
graphy was carried out using Chromatorex DM1020T
ODS. TLC was conducted on Kieselgel 60 F254 plates
(Merck).
3.2. Extraction and separation of flavonoids
The MeOH extract obtained previously was parti-
tioned between EtOAc and H2O (Li et al., 2001). The
water phase was evaporated under reduced pressure
below40 ꢁC to remove EtOAc, and then subjected to a
Diaion HP-20 column and further washed with MeOH
to give the crude glycoside fraction (GPH, 2.63 g). The
GPH was next subjected to ODS chromatography and
eluted by aqueous MeOH (0–100%) to give five fractions.
Further purification of fractions 3 (283 mg) and 4 (200
mg) was achieved by repeated RP–HPLC (SenshuPak
Pegasil ODS, 5 mm C18À120 A, 20 mm Â150 mm) to give
1 (11 mg), 2 (7 mg), 3 (7 mg), 4 (2 mg), 5 (11 mg), 6 (5 mg)
and 7 (1 mg).
3.3. Licoagroside D (1)
ꢁ
Powder, [ꢁ]D À117.4ꢁ (c=1.09, MeOH, 22 C). CD
ꢁ
(c=2.23Â10À4, MeOH, 23 C): [y]284 13 409.8, [y]228
À
36358.4. UV l nm (log "): 227 (4.98), 277 (4.54). IR u
cmÀ1: 3390, 1621, 1502. FAB–MS (positive) m/z: 449
[(M+H)+]. HR–FAB–MS (positive) m/z: Found:
449.1483; Calc. for C22H25O10 [(M+H)+]: 449.1447. 1H
NMR (500 MHz, pyridine-d5): see Table 1. 13C NMR
(125 MHz, pyridine-d5): see Table 1.
In conclusion, we have isolated three new flavonoids
and four known flavonoids from the hairy roots of G.
pallidiflora. To our knowledge, licoagroside E (2) is the
first example of the glycoside of 6a-hydroxypterocarpan
and licoagroside F (3) is the first example of a-hydro-
xydihydrochalcone which is glycosylated at the a-posi-
tion. The known flavonoids, calycosin 7-O-glucoside
(5), formononetin 7-O-(600-malonylglucoside) (6) have
been isolated from the hairy root cultures of G. glabra,
but none of the known flavonoids has been reported
from roots of G. pallidiflora.
3.4. Licoagroside E (2)
ꢁ
Powder, [ꢁ]D À176.4ꢁ (c=0.66, MeOH, 22 C). CD
ꢁ
(c=2.23Â10À4, MeOH, 23 C): [y]286 28632.8, [y]228
À
89228.0. UV l nm (log "): 224 (4.95), 283 (4.78). IR u
cmÀ1: 3394, 1622, 1500. FAB–MS (positive) m/z: 471
[(M+Na)+], 448 [M+]. HR–FAB–MS (positive) m/z:
Found: 448.1366; Calc. for C22H24O10 [M+]: 448.1370.
1H NMR (500 MHz, pyridine-d5): see Table 1. 13C
NMR (125 MHz, pyridine-d5): see Table 1.
3. Experimental
3.1. General experimental procedures
3.5. Licoagroside F (3)
The UV spectra were obtained with a Shimadzu UV-
160 spectrophotometer, whereas the IR spectra were
measured with a JASCO FT/IR-300E (by a KBr disk
method) spectrometer. The optical rotations were mea-
sured with a JASCO DIP-370 digital polarimeter in a
0.5 dm length cell, while the CD spectra were recorded
on a JASCO J-720W spectropolarimeter. The EI–MS,
FAB–MS, HR–EI–MS and HR–FAB–MS were taken
Powder, [ꢁ]D À2.6ꢁ (c=0.91, MeOH, 22 ꢁC). CD (c=
ꢁ
2.29Â10À5, MeOH, 23 C): [y]294 4166.6, [y]275À4324.7,
[y]237 5447.6. UV l nm (log "): 214 (4.27), 283 (4.11),
327 (3.93). IR u cmÀ1: 3414, 1619, 1525. FAB–MS
(positive) m/z: 437 [(M+H)+], 459 [(M+Na)+]. HR–
FAB–MS (positive) m/z: Found: 459.1276; Calc. for
C21H24O10Na [(M+Na)+]: 459.1267. 1H NMR (500

W. Li et al. / Phytochemistry 60 (2002) 351–355
355
Table 2
1H and 13C NMR spectral data for licoagroside F (3) in pyridine-d5
Pak Pegasil ODS, 4.6Â150 mm; Solvent: 33% aqueous
CH3CN; flowrate, 0.8 ml/min; detection, UV 230 nm.
The derivatives of d-glucose were detected with the tR
of 38.3 min.
Position
13C NMR
1H NMR
C¼O
202.6
79.3
39.4
a
b
5.98 (1H, dd, J=7.1, 7.1 Hz)
3.49 (1H, dd, J=16.0, 7.1 Hz)
3.51 (1H, dd, J=16.0, 7.1 Hz)
References
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127.4
131.1
116.0
157.5
113.3
167.1
103.6
166.6
109.4
134.0
103.2
75.4
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6.68 (1H, dd, J=8.9, 2.3 Hz)
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Glc-1
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78.3
71.5
78.6
62.7
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