Isoflavonoids from Belamcanda chinensis

Article abstract of DOI:10.1248/cpb.49.1229

Four new isoflavonoids were isolated along with six known related compounds from a rhizome of Belamcanda chinensis (Iridaceae), and their structures were characterized as 6″-O-p-hydroxybenzoyliridin, 6″-O-vanilloyliridin, 5, 6, 7, 3′-tetrahydroxy-4′-methoxyisoflavone and 2, 3-dihydroirigenin, respectively, on the basis of spectroscopic methods and chemical evidence.

Full text of DOI:10.1248/cpb.49.1229

September 2001
Notes
Chem. Pharm. Bull. 49(9) 1229—1231 (2001)
1229
Isoflavonoids from Belamcanda chinensis
Hideyuki ITO, Satomi ONOUE, and Takashi YOSHIDA*
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700—8530, Japan.
Received May 9, 2001; accepted June 19, 2001
Four new isoflavonoids were isolated along with six known related compounds from a rhizome of Belam-
canda chinensis (Iridaceae), and their structures were characterized as 6؆-O-p-hydroxybenzoyliridin, 6؆-O-vanil-
loyliridin, 5, 6, 7, 3؅-tetrahydroxy-4؅-methoxyisoflavone and 2, 3-dihydroirigenin, respectively, on the basis of
spectroscopic methods and chemical evidence.
Key words Belamcanda chinensis; Iridaceae; isoflavonoid glucoside; isoflavone; isoflavanone
The dried rhizomes of Belamcanda chinensis (L.) DC have with irigenin (5) and iridin (6). Based on these findings,
been used as folk medicine for the treatment of coughing and compound 1 was characterized as 6Љ-O-p-hydroxybenzoyl-
pharyngitis in China.1) As for the chemical constituents of iridin.
the plant, the occurrence of iridal-type triterpenoids2—4) and
Compound 2, a pale yellow amorphous powder, exhibited
isoflavonoids5,6) in the rhizomes, and phenols, benzoquinones a [MϩH]ϩ ion peak at m/z 673 in ESI-MS, and the molecular
and benzofurans3,7) in the seeds was reported. We also re- formula C32H32O16 was established by HR ESI-MS. The H-
1
ported the isolation and characterization of several iridals as and 13C-NMR (Table 1) spectra of 2 were almost superim-
ichthyotoxic components from the rhizomes of B. chinensis.8) posable on those of compound 1. The distinguishable feature
Further investigation of the rhizomes has resulted in the iso- of 2 from 1 in the 1H-NMR spectrum was the presence of an
lation of four new isoflavonoids, 6Љ-O-p-hydroxybenzoyliridin
(1), 6Љ-O-vanilloyliridin (2), 5,6,7,3Ј-tetrahydroxy-4Ј-methoxy
-
isoflavone (3) and 2,3-dihydroirigenin (4), together with six
known compounds. We report herein the structure elucida-
tion of these new compounds.
Fresh rhizomes of B. chinensis were soaked successively
in n-hexane and MeOH. The MeOH extract was partitioned
with ether and water. Column chromatography of the ether
soluble portion over silica and octadecylsilyl (ODS) gels
gave ten compounds including 1—4. Six among them were
identified as irigenin (5),9) iridin (6),9) tectorigenin (7),10) tec-
torigin (8),11) irisflorentin (9)12) and rhamnocitrin (10),13) by
comparison of their physicochemical data with those re-
ported in the literature.
Compound 1 was obtained as a pale yellow amorphous
powder. The presence of an isoflavone skeleton was sug-
gested from the UV spectrum (lmax 259, 320 nm). Its molec-
ular formula C31H30O15 was determined by high-resolution
electrospray ionization (HR-ESI) MS (m/z 643.1650
1
[MϩH]ϩ). The H-NMR spectrum of 1 showed two singlets
(d 8.16, 6.85), two meta-coupled doublets (Jϭ2 Hz) (d 6.80,
6.79) and a chelated hydroxyl proton signal (d 12.96) in the
aromatic region. The spectrum also indicated the presence of
three methoxyl groups (d 3.87, 3.81, 3.80) and an anomeric
proton of sugar [d 5.27 (d, Jϭ6.5 Hz)]. These signals were
very similar to those of iridin (6), except for extra AAЈBBЈ-
type signals at d 7.93 and 6.93 (each 2H, d, Jϭ8.5 Hz) in 1.
The presence of a para-substituted benzoyl unit in 1 was re-
vealed by the 13C-NMR spectrum (Table 1), which showed
the resonances of an ester carbonyl and six sp2 carbons be-
sides those due to the iridin moiety. Based on these spectral
data taking a [MϩH]ϩ ion peak in the ESI-MS, which is 104
mass units (C7H4O2) larger than that of 6 into consideration,
compound 1 was assumed to be p-hydroxybenzoyliridin. The
location of the acyl group in 1 was evidenced by downfield
shifts of H-6Љ signals of the glucose unit relative to those of
6. Acid hydrolysis of 1 afforded p-hydroxybenzoic acid along
Chart 1
To whom correspondence should be addressed. e-mail: yoshida@pheasant.pharm.okayama-u.ac.jp
© 2001 Pharmaceutical Society of Japan

1230
Vol. 49, No. 9
by HR-ESI-MS (m/z 363.1113 [MϩH]ϩ). The UV spectrum
of 4 displayed maxima at 253 and 288 nm indicative of an
isoflavanone skeleton. The H-NMR spectrum of 4 showed
Table 1. 13C-NMR Spectral Data of 1—4 and 6 (126 MHz, Acetone-d6)
Carbon
1
2
6
3
4
1
mutually coupled methine [d 3.92 (dd, Jϭ5.5, 7 Hz)] and
methylene proton signals [d 4.61 (dd, Jϭ7, 11.5 Hz), d 4.59
(dd, Jϭ5.5, 11.5 Hz)] as well as aromatic proton signals due
to H-8 (d 6.00), H-2Ј (d 6.51) and H-6Ј (d 6.56). The pres-
ence of three methoxyl signals (d 3.80, 3.76, 3.74) was also
indicated. Compound 4 was thus assumed to be 2, 3-dihy-
droirigenin and this assumption was substantiated by cat-
alytic hydrogenation of irigenin (5) with Pd/C to yield a di-
hydro derivative, which was shown to be identical with 4 by
HPLC and NMR. Based on these data, compound 4 was
characterized as racemic 2,3-dihydroirigenin (5,7,3Ј-trihy-
droxy-6,4Ј,5Ј-trimethoxyisoflavanone).16)
Aglycone
C-2
C-3
C-4
C-4a
C-5
C-6
C-7
C-8
155.2
122.3
181.8
108.0
154.8a)
134.1
157.7
94.9
155.2
122.3
181.7
107.8
154.8b)
133.7
157.7
94.8
155.5
123.4
181.6
107.7
153.9c)
133.4
157.3
95.0
154.5
123.5
182.0
106.5
146.3
123.6
157.9
94.4
154.2
121.5
116.0
145.6
146.3
111.5
117.3
72.0
51.6
198.1
103.3
160.0d)
130.0
156.7d)
95.3
C-8a
C-1Ј
C-2Ј
C-3Ј
C-4Ј
C-5Ј
C-6Ј
Glucose
C-1Љ
C-2Љ
C-3Љ
C-4Љ
C-5Љ
C-6Љ
Acyl group
C-1ٞ
C-2ٞ
C-3ٞ
C-4ٞ
C-5ٞ
C-6ٞ
C-7ٞ
OMe
153.9a)
127.4
105.9
151.1
137.4
153.6
110.7
153.9b)
127.2
105.8
151.0
137.4
153.6
110.7
153.8c)
127.1
105.6
150.9
137.3
153.6
110.6
159.5d)
132.3
110.0
151.3
136.8
154.2
105.5
Experimental
Optical rotations were measured with a JASCO DIP-1000 polarimeter.
UV spectra were measured on a HITACHI U-2001 spectrophotometer. ESI-
MS was performed with a Micromass Auto Spec OA-TOF spectrometer
using 50% MeOH containing 0.1% NH4OAc as a solvent. 1H- and 13C-NMR
101.4
74.4
77.9
71.5
75.3
64.6
101.3
74.1
77.5
71.3
75.2
64.7
101.2
73.8
77.6
70.4
76.9
61.8
1
spectra were recorded on a Varian VXR-500 instrument (500 MHz for H
and 126 MHz for 13C) and chemical shifts are given in d (ppm) values rela-
tive to that of the solvent [acetone-d6 (dH 2.04; dC 29.8)] on a tetramethylsi-
lane scale. The circular dichroism (CD) spectrum was recorded on a JASCO
J-720 W spectrometer. Normal-phase HPLC was conducted on a YMC-Pack
SIL A-003 (YMC Co., Ltd.) column (4.6 i.d.ϫ250 mm) developed with n-
hexane–MeOH–tetrahydrofuran–formic acid (55 : 33 : 11 : 1) containing ox-
alic acid (450 mg/l) (flow rate, 1.5 ml/min; detection 280 nm) at room tem-
perature. Reversed-phase HPLC was performed on a YMC-Pack ODS-A A-
302 (YMC Co., Ltd.) column (4.6 i.d.ϫ150 mm) developed with 0.01 M
H3PO4–0.01 M KH2PO4–CH3CN (42.5 : 42.5 : 15) (flow rate, 1.0 ml/min; de-
tection 280 nm) at 40 °C.
123.7
132.6
116.0
162.7
116.0
132.6
166.3
56.3
123.7
113.6
154.3
152.2
115.6
124.7
166.5
56.2
56.2
60.6
61.1
60.6
56.2
60.6
60.7
60.7
60.8
56.3
60.6
60.9
Plant Material Rhizomes of B. chinensis cultivated at the herbarium of
the Faculty of Pharmaceutical Sciences, Okayama University, were collected
in January, 1995. A voucher specimen (OPH-I03) is kept at the same herbar-
ium.
a—d) Assignments interchangeable.
Extraction and Isolation The fresh rhizomes (1 kg) of B. chinensis
were chopped and soaked in hexane (2 l) three times (for each 24 h) at room
temperature to yield a hexane extract (2.5 g). The residue was further ex-
tracted with MeOH (2 lϫ3). The concentrated solution was diluted with H2O
and extracted successively with ether and n-BuOH to give Et2O (9.5 g), n-
BuOH (13 g), and H2O (36.8 g) extract. The Et2O extract was subjected to
column chromatography over silica gel using a solvent system CHCl3→
CHCl3–acetone→acetone–MeOH in stepwise gradient mode. The eluate
with CHCl3–acetone (4 : 1) was further purified by repeated column chro-
matography over silica gel using CHCl3–acetone to afford irigenin (5)
(22.9 mg), tectorigenin (7) (42.5 mg), and irisflorentin (9) (22.6 mg). The
acetone–MeOH (9 : 1) eluate was rechromatographed over silica gel with
CHCl3–MeOH to give 6Љ-O-p-hydroxyiridin (1) (7.6 mg), 5,6,7,3Ј-tetrahy-
droxy-4Ј-methoxyisoflavone (3) (2.6 mg), 5 (63 mg), 7 (13 mg), and 9
(12 mg). The eluate with acetone–MeOH (1 : 1) was purified by repeated col-
umn chromatography over YMC-gel ODS AQ 120 S50 (solvent: aqueous
MeOH) to furnish 1 (7.6 mg), 6Љ-vanilloyliridin (2) (3.3 mg), 3 (2.6 mg), and
iridin (6) (1.8 mg).
extra 3H singlet due to a methoxyl group, and the ABX-type
protons instead of the AAЈBBЈ-type protons of 1. Compound
2 was thus suggested to be a derivative of 1 possessing a 4-
hydroxy-3-methoxy or 3-hydroxy-4-methoxybenzoyl group
at position 6Љ of the glucose moiety in the molecule. The nu-
clear Overhauser effect spectroscopy (NOESY) spectrum of
2 showed a correlation between a signal at d 7.55 (d,
Jϭ2 Hz, H-2ٞ) and a methoxyl signal at d 3.84, establishing
the location of the methoxyl group at the C-3ٞ position. Con-
sequently, compound 2 was deduced to be 6Љ-O-vanil-
loyliridin, which was confirmed by acid hydrolysis yielding
vanillic acid besides 5 and 6.
Compound 3 showed a pseudo-molecular ion [MϩH]ϩ
peak at m/z 317 in the ESI-MS and its molecular formula
was determined to be C16H12O7 by HR-ESI-MS. The UV, 1H-,
and 13C-NMR spectra of 3 were similar to those of iristec-
torigenin A (11), which was isolated from Iris spuria.14) The
spectral comparison of 3 and 11 revealed the lack of a
methoxyl group in the former. The methoxyl group in 3 was
allocated to the C-4Ј position based on the NOESY spectrum
which showed a clear correlation between the methoxyl pro-
ton signal (d 3.87) and H-5Ј signal (d 6.87, d, Jϭ8 Hz). On
the basis of these data, compound 3 was characterized as
5,6,7,3Ј-tetrahydroxy-4Ј-methoxyisoflavone.15)
The residue after extraction with MeOH was further extracted with hot
MeOH. The extract was diluted with H2O and extracted with ether to give
ether and H2O extracts. The ether extract was purified in a way similar to
that described above to afford 1 (24.1 mg), 2 (1.4 mg), 3 (3.5 mg), 2,3-dihy-
droirigenin (4) (3.5 mg), tectoridin (8) (4 mg), 9 (17 mg), and rhamnocitrin
(10) (5.3 mg).
6؆-Hydroxybenzoyliridin (1) A pale yellow amorphous powder, [a]D
ϩ3.4° (cϭ0.5, MeOH), ESI-MS m/z: 665 (MϩNa)ϩ, 643 (MϩH)ϩ. HR-
ESI-MS m/z: 643.1650 (MϩH)ϩ, Calcd for C31H30O15ϩH, 643.1663. UV
1
l max (MeOH) nm (log e): 259 (4.22), 320 (3.96). H-NMR (acetone-d6) d :
12.96 (1H, s, 5-OH), 8.16 (1H, s, H-2), 7.93 (2H, d, Jϭ8.5 Hz, H-2ٞ, 6ٞ),
6.93 (2H, d, Jϭ8.5 Hz, H-3ٞ, 5ٞ), 6.85 (1H, s, H-8), 6.80 (1H, d, Jϭ2 Hz, H-
2Ј or H-6Ј), 6.79 (1H, d, Jϭ2 Hz, H-2Ј or H-6Ј), 5.27 [1H, d, Jϭ6.5 Hz, glu-
cose (glc) H-1Љ], 4.74 (1H, dd, Jϭ2, 11.5 Hz, glc H-6Љ), 4.36 (1H, dd, Jϭ8,
11.5 Hz, glc H-6Љ), 4.04 (1H, dt, Jϭ2, 8 Hz, glc H-5Љ), 3.87, 3.81, 3.80 (each
Compound 4 was obtained as a pale yellow amorphous
powder and shown to have the molecular formula C18H18O8

September 2001
1231
of the 1H-NMR spectra.
3H, s, OCH3), 3.65 (2H, m, glc H-2Љ, 3Љ), 3.53 (1H, t, Jϭ9 Hz, glc H-4Љ).
13C-NMR: see Table 1.
Acknowledgements The authors are grateful to the SC-NMR Labora-
tory of Okayama University for the NMR experiments. This work was sup-
ported in part by a Grant-in-Aid for Scientific Research (No. 10557207)
from the Ministry of Education, Science, Sports and Culture of Japan.
Acid Hydrolysis of 1 A solution of 1 (0.7 mg) in 2.5% H2SO4 (1 ml)
was heated at 80 °C for 8 h. The reaction mixture was analyzed by normal-
and reversed-phase HPLC to detect peaks identical with those of irigenin
(5), iridin (6), and p-hydroxybenzoic acid.
6؆-Vanilloyliridin (2) A pale yellow amorphous powder, [a]D ϩ5.7°
(cϭ1.0, MeOH), ESI-MS m/z: 673 (MϩH)ϩ. HR-ESI-MS m/z: 673.1794
(MϩH)ϩ, Calcd for C32H32O16ϩH, 673.1769. UV nmax (MeOH) nm (log e):
References and Notes
1) Chang S., “Dictionary of Chinese Crude Drugs,” ed. by Shanghai Sci-
entific Technologic Publisher, Shanghai, 1977, p. 1883.
2) Takahashi K., Hoshino Y., Suzuki S., Hano Y., Nomura T., Phytochem-
istry, 53, 925—929 (2000).
3) Seki K., Haga K., Kaneko R., Phytochemistry, 38, 703—709 (1995).
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(1991).
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K., Agric. Biol. Chem., 46, 2595—2597 (1982).
7) Fukuyama Y., Okino J., Kodama M., Chem. Pharm. Bull., 39, 1877—
1879 (1991).
1
263 (4.03), 320 (3.40). H-NMR (acetone-d6) d: 12.96 (1H, s, 5-OH), 8.12
(1H, s, H-2), 7.61 (1H, dd, Jϭ1.5, 8.5 Hz, H-6ٞ), 7.55 (1H, d, Jϭ2 Hz. H-2ٞ),
6.95 (1H, d, Jϭ8.5 Hz, H-5ٞ), 6.81 (2H, br s, H-8, 2Ј), 6.79 (1H, d, Jϭ2 Hz,
H-6Ј), 5.25 (1H, d, Jϭ6.5 Hz, glc H-1Љ), 4.74 (1H, dd, Jϭ1.5, 12 Hz, glc H-
6Љ), 4.39 (1H, dd, Jϭ8, 12.5 Hz, glc H-6Љ), 4.04 (1H, dt, Jϭ2, 8 Hz, glc H-
5Љ), 3.88, 3.84, 3.81, 3.80 (each 3H, s, OCH3), 3.64 (2H, m, glc H-2Љ, 3Љ),
3.52 (1H, m, glc H-4Љ). 13C-NMR: see Table 1.
Acid Hydrolysis of 2 A solution of 2 (1.0 mg) in 2.5% H2SO4 (1 ml)
was heated at 80 °C for 4 h . Normal- and reversed-phase HPLC of the reac-
tion mixture showed peaks identical with those of irigenin (5), iridin (6), and
vanillic acid.
8) Ito H., Onoue S., Miyake Y., Yoshida T., J. Nat. Prod., 62, 89—93
(1999).
9) Ali A. A., El-Emary N. A., El-Moghazi M. A., Darwish F. M., Frahm
A. W., Phytochemistry, 22, 2061—2063 (1983).
5,6,7,3؅-Tetrahydroxy-4؅-methoxyisoflavone (3) A pale yellow amor-
phous powder, ESI-MS m/z: 317 (MϩH)ϩ. HR-ESI-MS m/z: 317.0683
(MϩH)ϩ, Calcd for C16H12O7ϩH, 317.0661. UV nmax (MeOH) nm (log e):
1
267 (4.29), 294 (4.02). H-NMR (acetone-d6) d: 13.25 (1H, s, 5-OH), 8.14
10) Mabry T. J., Kagan J., Resler H., Phytochemistry, 4, 177—183 (1965).
11) Shawl A., Kumar T., Phytochemistry, 31, 1399—1401 (1992).
12) Morita N., Arisawa M., Kondo Y., Takemoto T., Chem. Pharm. Bull.,
21, 600—603 (1973).
13) Barbera O., Sanz J. F., Sanchez-Parareda J., Alberto Marco J., Phyto-
chemistry, 25, 2361—2365 (1986).
14) Shawl A. S., Vishwapaul, Zaman A., Kalla A. K., Phytochemistry, 23,
2405—2406 (1984).
15) This compound was reported as a synthetic product. Horie T.,
Sasagawa M., Torii F., Kawamura Y., Yamashita K., Chem. Pharm.
Bull., 44, 486—491 (1996).
(1H, s, H-2), 7.14 (1H, d, Jϭ2 Hz, H-2Ј), 6.94 (1H, dd, Jϭ2, 8 Hz, H-6Ј),
6.87 (1H, d, Jϭ8 Hz, H-5Ј), 6.48 (1H, s, H-8), 3.87 (3H, s, OCH3). 13C-
NMR: see Table 1.
2,3-Dihydroirigenin (4) A pale yellow amorphous powder, [a]D Ϯ0°
(cϭ0.5, MeOH), ESI-MS m/z: 363 (MϩH)ϩ. HR-ESI-MS m/z: 363.1113
(MϩH)ϩ, Calcd for C18H18O8ϩH, 363.1080. UV l max (MeOH) nm (log e):
1
253 (3.77), 288 (4.09). H-NMR (acetone-d6) d: 12.35 (1H, s, 5-OH), 6.56
(1H, d, Jϭ2 Hz, H-6Ј), 6.51 (1H, d, Jϭ2 Hz, H-2Ј), 6.00 (1H, s, H-8), 4.61
(1H, dd, Jϭ7, 11.5 Hz, H-2a), 4.59 (1H, dd, Jϭ5.5, 11.5 Hz. H-2b), 3.92
(1H, dd, Jϭ5.5, 7 Hz, H-3), 3.80, 3.76, 3.74 (each 3H, s, OCH3). 13C-NMR:
see Table 1.
16) Although this compound was registered in Chemical Abstracts
(121928—03—8) as noted by reviewer of the manuscript, it is based
on an erroneous structural drawing as isoflavanone for isoflavone, iri-
genin, in the literature cited in the Chemical Abtracts. Therefore, this
is regarded as a new natural product.
Preparation of 4 from Irigenin (5) A solution of 5 (15 mg) in MeOH
(2 ml) containing 10% Pd/C was stirred overnight at room temperature under
hydrogen atmosphere. The catalyst was filtered off, and the reaction mixture
was evaporated in vacuo. The product was purified by preparative TLC
(CHCl3–MeOH, 5 : 1) to yield the hydrogenated derivative (0.7 mg), which
was identified with 4 by co-chromatography (HPLC) and direct comparison