Chemical structures of constituents from the leaves of Polyscias balfouriana

Article abstract of DOI:10.1007/s11418-017-1081-x

A new pyrrolidine derivative, (5S)-hydroxyethyl 2-oxopyrrolidine-5-carboxylate (1), a new flavonol glycoside, tamaraxetin 3,7-di-O-α-l-rhamnopyranoside (2), and a new triterpene saponin, polyscioside A methyl ester (3), along with six known compounds (4–9) were isolated from the leaves of Polyscias balfouriana. Their chemical structures were elucidated on the basis of extensive spectroscopic analysis.

Full text of DOI:10.1007/s11418-017-1081-x

J Nat Med
DOI 10.1007/s11418-017-1081-x
NOTE
Chemical structures of constituents from the leaves of Polyscias
balfouriana
Sachiko Sugimoto1 Yoshi Yamano1 Hany Ezzat Khalil2 Hideaki Otsuka3
•
•
•
•
Mohamed Salah Kamel2 Katsuyoshi Matsunami1
•
Received: 16 November 2016 / Accepted: 15 February 2017
Ó The Japanese Society of Pharmacognosy and Springer Japan 2017
Abstract A new pyrrolidine derivative, (5S)-hydroxyethyl
2-oxopyrrolidine-5-carboxylate (1), a new flavonol gly-
coside, tamaraxetin 3,7-di-O-a-L-rhamnopyranoside (2),
and a new triterpene saponin, polyscioside A methyl ester
(3), along with six known compounds (4–9) were isolated
from the leaves of Polyscias balfouriana. Their chemical
structures were elucidated on the basis of extensive spec-
troscopic analysis.
the leaves of P. fruticosa are used as a diuretic, febrifuge,
and anti-dysentery agent [3]. Plants of the genus Polyscias
are known as a rich source of saponins and several species
have already been studied chemically [3, 6–8]. Neverthe-
less, no study on the metabolites of the plant under
investigation has been reported so far. The present study
focused on the constituents of P. balfouriana, collected in
Zohria Botanical Garden in Giza, Egypt, which afforded
one pyrrolidine derivative, (5S)-hydroxyethyl 2-oxopyrro-
lidine-5-carboxylate (1), a new flavonol glycoside, tama-
raxetin 3,7-di-O-a-L-rhamnopyranoside (2), and a new
triterpene saponin, polyscioside A methyl ester (3). In this
paper, we describe the isolation and structural elucidation
of the new compounds.
Keywords Polyscias balfouriana Á Araliaceae Á
Pyrrolidine Á Tamaraxetin
Introduction
Polyscias balfouriana is an ornamental plant which
belongs to the family Araliaceae. It is also known as dinner
plate Aralia or Balfour Polyscias [1]. This plant is culti-
vated in Egypt for ornamental purposes as it gives good
shade in gardens [2, 3]. It grows to a height of 6 m and the
leaves are leathery and coarsely toothed glossy green with
white margins. Traditionally, a related species, P. fulva,
has been used to treat fever, mental illness, malaria,
venereal infections, and obesity [4, 5]. In Asian countries,
Results and discussion
Air-dried leaves of P. balfouriana (3.5 kg) were extracted
with methanol (MeOH) three times at room temperature.
Then, the concentrated MeOH extract was partitioned with n-
hexane. The MeOH extracts (150 g) were subjected to Diaion
HP-20 column chromatography to give H2O-, MeOH-, and
acetone-eluted fractions. The MeOH-eluted fraction was
subjected to normal and reversed-phase silica gel column
chromatography (CC) and repetitive HPLC separation to give
three new compounds, (5S)-hydroxyethyl 2-oxopyrrolidine-
5-carboxylate (1), a new flavonol glycoside, tamaraxetin 3,7-
di-O-a-L-rhamnopyranoside(2), and anew triterpenesaponin,
polyscioside A methyl ester (3) (Fig. 1), together with six
known compounds, (5S)-2-oxopyrrolidine-5-carboxylic acid
(4) [9], (5S)-methyl 2-oxopyrrolidine-5-carboxylate (5) [10],
oleanolic acid 3-O-b-D-glucopyranosyl-(100 ? 20)-b-D-glu-
curonopyranoside60-methylester(6) [11], oleanolicacid3-O-
b-D-glucopyranosyl-(100 ? 30)-b-D-glucuronopyranoside (60-
& Katsuyoshi Matsunami
matunami@hiroshima-u.ac.jp
1
Department of Pharmacognosy, Graduate School of
Biomedical and Health Sciences, Hiroshima University, 1-2-
3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
2
Pharmacognosy Department, Faculty of Pharmacy, Minia
University, Minia 61519, Egypt
3
Faculty of Pharmacy, Yasuda Women’s University, 6-13-1
Yasuhigashi, Asaminami-ku, Hiroshima 731-0153, Japan
123

J Nat Med
Fig. 1 Structures of
constituents isolated from
Polyscias balfouriana
methyl ester) (7) [12], chikusetsusaponin V methyl ester (8)
[13], and oleanolic acid 3-O-b-D-glucopyranosyl (100 ? 30)-
b-D-glucuronopyranoside 60-methyl ester, 28-b-D-glucopyra-
nosyl ester (9) [12]. As shown in Fig. 1, their structures were
elucidated by extensive inspection of spectroscopic data,
including high-resolution (HR) electrospray ionization (ESI)-
MS, 1D- and 2D-NMR spectroscopy, and chemical and bio-
chemical methods.
correlations between the following protons and carbons: H-1
and C-3, 4, 5; H-3 and C-2, 5; H-4 and C-2, 5, 6; H-5 and C-
2, 6; H-7 and C-6. This result suggested that the structure of
compound 1 is not a tetrahydrofuran type, but a pyrrolidine
type. The absolute configuration of 1 was guessed to be S by
comparison of the optical rotation sign with that reported for
L(S)-pyroglutamic acid ([a]2D0 -8.0*12.0, in H2O; CAS no.
98-79-3) or D(R)-pyroglutamic acid ([a]2D0 ?8.0*12.0, in
H2O; CAS no. 4042-36-8) in the literature. Consequently,
the chemical structure of 1 was determined as shown in
Fig. 1.
Compound 1 was isolated as an amorphous powder with
negative optical rotation ([a]2D7 -15.0, in H2O). The IR
spectrum showed absorption bands at 3362, 1649, and
1048 cm-1 due to hydroxy, amide, and ether functional
groups, respectively. HR-ESI-MS revealed a quasi-molecu-
lar ion [M?Na]? at 196.0578, from which the molecular
formula was determined to be C7H11NO4. The 13C-NMR
spectrum of 1 displayed two carbonyl carbons (dc 173.9 and
178.1) and two oxymethylene carbons (dc 60.4 and 67.9),
one methine carbon (dc 56.3), and two methylene carbons
(dc 25.8 and 30.1). The 13C-NMR spectra of 1 were very
similar to those of 4, except for the presence of oxymethy-
lene signals. As shown in Fig. 2, the double quantum filter
correlation spectroscopy (DQF-COSY) experiment of 1
indicated the presence of certain structures (highlighted by
bold lines). The heteronuclear multiple bond connectivity
(HMBC) spectroscopy experiment revealed long-range
Compound 2 was isolated as a pale yellow powder with
negative optical rotation ([a]2D6 -147.5 in MeOH). Its
elemental composition was determined to be C28H32O15 by
positive-ion HR-ESI-MS measurement ([M?Na]? m/
z 631.1638). Acid hydrolysis of 2 with 1 M HCl afforded L-
rhamnose [14]. The carbon and proton signals due to the
aglycone moiety in the 1H- and 13C-NMR spectra of 2 were
superimposable on those of tamaraxetin 3-O-glycosides
1
[15]. The H-NMR spectra of 2 showed five downfield
doublets at dH 7.45 (1H, dd, J = 8.8, 2.1 Hz), 7.42 (1H, d,
J = 2.1 Hz), 7.13 (1H, d, J = 8.8 Hz), 6.52 (1H,
J = 2.1 Hz), and 6.84 (1H, J = 2.1 Hz), corresponding to
H-60, 20, 50, 6, and 8, and the presence of a methoxy group
at dH 3.91 (3H, s) of the tamaraxetin aglycone, respec-
tively. In the HMBC spectrum (Fig. 3), the methoxy pro-
tons showed a correlation peak to dC 150.2 (C-40), and two
anomeric protons at dH 5.30 (1H, d, J = 1.1 Hz, H-100) and
5.61 (1H, d, J = 1.6 Hz, H-1000) showed correlation peaks
to dC 134.9 (C-3) and 161.7 (C-7), respectively. Enzymatic
hydrolysis of 2 with crude naringinase afforded tamarax-
etin [16]. On the basis of those findings, the structure of
compound 2 was determined to be tamaraxetin 3,7-di-O-a-
L-rhamnopyranoside (Fig. 1).
Fig. 2 2D-NMR correlations of compound 1
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J Nat Med
1
Scientific LTQ Orbitrap XL spectrometer; H- and 13C-
NMR spectra: Bruker Avance 600 spectrometer at
600 MHz and 150 MHz, respectively, with tetramethylsi-
lane as an internal standard. Highly porous synthetic resin,
Diaion HP-20, was purchased from Mitsubishi Chemical
Co., Ltd. (Tokyo, Japan). Silica gel CC was performed on
silica gel 60 (E. Merck, Darmstadt, Germany; 70–230
mesh). Reversed-phase ODS open CC (RPCC) was per-
formed on a Cosmosil 75C18-OPN column (Nacalai Tes-
que, Kyoto, Japan; U = 50 mm, L = 25 cm, linear
gradient: MeOH–H2O). HPLC was performed on an ODS-
3
column (Inertsil; GL Science, Tokyo, Japan;
U = 10 mm, L = 25 cm, flow rate 2.00 mL/min) and the
eluate was monitored with a refractive index monitor, RID-
6A (Shimadzu, Kyoto, Japan). Precoated silica gel 60 F254
plates (E. Merck; 0.25 mm in thickness) were used for
TLC monitoring visualized by spraying with a 10% solu-
tion of H2SO4 in ethanol and heated to around 150 °C on a
hotplate. Crude naringinase was provided courtesy of
Amano Enzyme Inc. (Aichi, Japan).
Fig. 3 2D-NMR correlations of compound 2
Compound 3, [a]2D6 ?10.9, was isolated as an amorphous
powder and its elemental composition was determined to
be C49H78O19 by HR-ESI-MS. Other spectroscopic data
were similar to polyscioside A [3], except for the presence
1
of methyl signals in the H-(dH 3.86) and 13C-(dc 52.0)
NMR spectra. The position of the methyl group was
expected on the carbonic acid group at C-60 of the glu-
curonic acid moiety from the obvious upfield shift of the
carbonyl carbon by 2.1 ppm compared with polyscioside A
[3] in the 13C-NMR spectrum. In the HMBC spectrum,
significant correlations from dH 3.86 to dC 170.1 (C-60)
were observed. Compound 3 may be an artifact produced
during extraction. Acid hydrolysis of 3 with 1 M HCl
afforded D-glucose and D-glucuronolactone [17]. Therefore,
the structure of compound 3 was elucidated as shown in
Fig. 1.
Plant material
The leaves of P. balfouriana were collected in Al Zohria
Botanical Garden, Giza, Egypt in 2010. The plant was
kindly identified by Dr. Mamdouh Shokry, Botanist and
director of Al-Zohria Botanical Garden. A voucher speci-
men (Minia-10-July-PB) was kept in the Herbarium of the
Pharmacognosy Department, Faculty of Pharmacy, Minia
University, Minia, Egypt.
Extraction and isolation
Conclusion
Air-dried leaves of P. balfouriana (3.5 kg) were extracted
three times with MeOH (5 L 9 3) at room temperature and
then concentrated. Then, the concentrated MeOH extract
was partitioned with n-hexane. The MeOH extracts (150 g)
were subjected to Diaion HP-20 CC [2.0 kg, H2O (20
L) ? MeOH (16 L) ? acetone (6 L)] to give H2O- (98 g),
MeOH- (35 g), and acetone-eluted fractions (3 g). The
MeOH-soluble fraction (35 g) was subjected to silica gel
CC [1.0 kg, CHCl3 (6 L) ? CHCl3–MeOH {[9:1 (6
L) ? 7:1 (6 L) ? 5:1 (6 L) ? 3:1 (6 L)] ? CHCl3–
MeOH–H2O (15:6:1) (6 L) ? MeOH (6 L)}] to give 15
fractions [Fr. 1 (3.7 g), Fr. 2 (1.0 g), Fr. 3 (1.2 g), Fr. 4
(0.83 g), Fr. 5 (0.90 g), Fr. 6 (0.83 g), Fr. 7 (1.6 g), Fr. 8
(2.4 g), Fr. 9 (1.7 g), Fr. 10 (1.0 g), Fr. 11 (0.68 g), Fr. 12
(1.4 g), Fr. 13 (2.4 g), Fr. 14 (9.3 g), and Fr. 15 (3.9 g)].
Fraction 5 (0.90 g) was separated by reversed-phase silica
gel CC [20 g, MeOH–H2O (3:7 ? 2:3 ? 1:1 ? 3:2
? 7:3 ? 4:1 ? 9:1) ? MeOH] to yield nine fractions
[Fr. 5-1 (172.7 mg), Fr. 5-2 (57.4 mg), Fr. 5-3 (80.5 mg),
Fr. 5-4 (80.4 mg), Fr. 5-5 (31.5 mg), Fr. 5-6 (60.0 mg), Fr.
In conclusion, one pyrrolidine derivative, hydroxyethyl
(5S)-2-oxopyrrolidine-5-carboxylate (1), a new flavonol
glycoside, tamaraxetin 3,7-di-O-a-L-rhamnopyranoside (2),
and a new triterpene saponin, polyscioside A methyl ester
(3), were isolated from the leaves of P. balfouriana and
their structures were determined on the basis of chemical
and physicochemical evidence.
Experimental
General methods
The following instruments were used to obtain physical
and spectroscopic data: specific rotations: JASCO P-1030
polarimeter; IR spectra: Shimadzu FT-710 spectropho-
tometer; UV spectrum: JASCO V-520 UV/Vis spec-
trophotometer; HR-ESI mass spectra: Thermo Fisher
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1
Table 1 NMR spectral data for compound 1 (150 MHz for 13C, 600 MHz for H, pyridine-d5)
Position
13C
1H
Position
7
13C
1H
1
2
3
–
9.22 (s)
67.9
4.43 (1H, ddd, J = 4.6, 5.4, 12.6 Hz)
4.46 (1H, ddd, J = 4.6, 5.4, 12.6 Hz)
3.96 (1H, ddd, J = 4.6, 5.4, 12.2 Hz)
3.99 (1H, ddd, J = 4.6, 5.4, 12.2 Hz)
178.1
30.1
–
2.24 (1H, dd-like, J = 9.4, 15.6 Hz)
2.38 (1H, dd-like, J = 5.9, 15.6 Hz)
2.18 (1H, ddd-like, J = 4.8, 9.4, 12.2 Hz)
2.29 (1H, ddd-like, J = 5.9, 8.8, 12.2 Hz)
4.40 (1H, dd, J = 4.8, 8.8 Hz)
–
8
60.4
4
25.8
5
6
56.3
173.9
Table 2 NMR spectral data for
compound 2 (150 MHz for 13C,
Position 13C
1H
Position 13C
1H
1
600 MHz for H, DMSO-d6)
2
156.1
–
–
–
–
100
102.0
5.30 (1H, d, J = 1.1 Hz)
3
134.9
178.0
160.9
200
70.02 4.03 (1H, brs)
4
300
70.32 3.56 (1H, brd, J = 9.4 Hz)
5
400
71.2
3.20 (1H, dd, J = 9.0, 9.4 Hz)
6
99.5 6.52 (1H, d, J = 2.1 Hz)
161.7
500
600
70.07 3.49 (1H, dd, J = 6.1, 9.0 Hz)
7
–
17.5
98.4
69.8
0.88 (1H, d, J = 6.1 Hz)
5.61 (1H, d, J = 1.6 Hz)
3.89 (1H, brs)
8
94.5 6.84 (1H, d, J = 2.1 Hz)
100’
200’
300’
400’
500’
600’
9
157.5
105.7
10
10
20
30
40
70.20 3.70 (1H, brd, J = 9.0 Hz)
71.6 3.36 (1H, m)
70.61 3.25 (1H, dd, J = 6.2, 9.1 Hz)
17.9 1.18 (1H, d, J = 6.2 Hz)
122.1
–
115.4 7.42 (1H, d, J = 2.1 Hz)
146.4
150.2
–
–
50
60
111.7 7.13 (1H, d, J = 8.8 Hz)
120.9 7.45 (1H, dd, J = 2.1, 8.8 Hz)
55.7 3.91 (3H, s)
40-OMe
m multiplet
5-7 (45.3 mg), Fr. 5-8 (109.1 mg), and Fr. 5-9 (124.2 mg)].
Fr. 5-1 (172.7 mg) was purified by HPLC [MeOH–H2O
(5:95, v/v)] to give (5S)-2-oxopyrrolidine-5-carboxylic
acid (4, 7.9 mg, 0.00022%), (5S)-hydroxyethyl 2-oxopy-
rrolidine-5-carboxylate (1, 2.6 mg, 0.000074%), and (5S)-
methyl 2-oxopyrrolidine-5-carboxylate (5, 5.3 mg,
0.00015%) from the peaks at 12, 35, and 45 min, respec-
tively. Fr. 8 (2.4 g) was separated by reversed-phase silica
gel CC [120 g, MeOH–H2O (2:3 ? 1:1 ? 3:2 ? 7:3
? 4:1 ? 9:1) ? MeOH] to yield eight fractions [Fr. 8-1
(927.9 mg), Fr. 8-2 (40.5 mg), Fr. 8-3 (65.0 mg), Fr. 8-4
(88.4 mg), Fr. 8-5 (442 mg), Fr. 8-6 (346.4 mg), Fr. 8-7
(155 mg), and Fr. 8-8 (359 mg)]. Fr. 8-4 (88.4 mg) was
purified by HPLC [MeOH–H2O (80:20, v/v)] to give
polyscioside A methyl ester (3, 12.7 mg, 0.00036%) from
the peak at 16 min. Fr. 8-5 (400 mg) was purified by HPLC
[MeOH–H2O (17:3, v/v)] to give oleanolic acid 3-O-b-D-
methyl ester (6, 17.5 mg, 0.00050%) and oleanolic acid
3-O-b-D-glucopyranosyl-(100 ? 30)-b-D-glucuronopyranoside
60-methyl ester (7, 11.8 mg, 0.00034%) from the peaks at 26
and 28 min, respectively. Fr. 10 (1.0 g) was separated by
reversed-phase silica gel CC [120 g, MeOH–H2O (1:9 ?
1:4 ? 3:7 ? 2:3 ? 1:1 ? 3:2 ? 7:3 ? 4:1) ? MeOH]
to yield seven fractions [Fr. 10-1 (309 mg), Fr. 10-2
(61.6 mg), Fr. 10-3 (27.3 mg), Fr. 10-2 (74.4 mg), Fr. 10-5
(83.5 mg), Fr. 10-6 (415 mg), and Fr. 10-7 (35.6 mg)]. Fr.
10-2 (61.6 mg) was purified by HPLC [MeOH–H2O (50:50,
v/v)] to give tamaraxetin 3,7-di-O-a-L-rhamnopyranoside (2,
8.4 mg, 0.00024%) from the peak at 16 min. Fr. 10-5
(83.5 mg) was purified by HPLC [MeOH–H2O (3:1, v/v)] to
give chikusetsusaponin
V methyl ester (8, 6.9 mg,
0.00020%) and oleanolic acid 3-O-b-D-glucopyranosyl
(1 ? 3)-b-D-glucuronopyranoside 60-methyl ester, 28-O-b-
D-glucopyranosyl ester (9, 5.3 mg, 0.00015%) from the
peaks at 27 and 35 min, respectively.
glucopyranosyl-(100 ? 20)-b-D-glucuronopyranoside
60-
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1
Table 3 NMR spectral data for compound 3 (150 MHz for 13C, 600 MHz for H, pyridine-d5)
Position
1
13C
1H
Position
13C
1H
39.0
0.73 (1H, m)
10
20
30
40
50
60
105.6
81.2
76.2
82.5
75.2
170.1
53.0
105.7
77.4
78.4
72.7
78.74
63.3
4.90 (1H, d, J = 7.3 Hz)
4.32 (1H, dd, J = 7.3, 8.6 Hz)
4.30 (1H, dd, J = 8.6, 9.0 Hz)
4.38 (1H, dd, J = 9.0, 9.2 Hz)
4.54 (1H, d, J = 9.2 Hz)
–
1.30 (1H, m)
2
26.9
1.73 (1H, m)
1.99 (1H, dd, J = 3.8, 13.6 Hz)
3
4
5
6
89.9
39.9
56.2
18.9
3.15 (1H, dd, J = 4.3, 11.7 Hz)
–
0.65 (1H, d, J = 11.8 Hz)
–COOCH3
3.85 (3H, s)
1.23 (1H, m)
100
200
300
400
500
600
5.40 (1H, d, J = 7.6 Hz)
4.05 (1H, dd, J = 7.6, 8.5 Hz)
4.21 (1H, brd, J = 8.9 Hz)
4.28 (1H, brd, J = 8.9 Hz)
3.89 (1H, ddd, J = 2.6, 4.4, 9.3 Hz)
4.42 (1H, dd, J = 4.4, 11.3 Hz)
4.49 (1H, dd, J = 2.6, 11.3 Hz)
4.96 (1H, d, J = 7.9 Hz)
3.93 (1H, m)
1.41 (1H, m)
7
33.62
1.24 (1H, m)
1.40 (1H, m)
8
40.2
48.4
–
9
1.55 (1H, m)
10
11
12
13
14
15
37.4
–
24.1
1.85–1.95 (2H, m)
100’
200’
300’
400’
500’
600’
105.5
74.9
78.67
72.0
78.9
62.8
123.0
145.3
42.4
5.44 (1H, brd, J = 3.8 Hz)
–
4.15 (1H, m)
–
4.17 (1H, m)
28.8
1.14 (1H, m)
3.96 (1H, ddd, J = 2.4, 4.6, 9.0 Hz)
4.25 (1H, m)
2.15 (1H, dd, J = 3.9, 14.8 Hz)
2.10 (2H, m)
16
17
18
19
24.1
47.1
42.4
46.9
4.50 (1H, dd, J = 2.6, 11.2 Hz)
–
–
3.27 (1H, dd, J = 4.0, 13.6 Hz)
1.28 (1H, m)
1.77 (1H, m)
20
21
31.4
33.66
1.80 (1H, dd, J = 6.4, 13.4 Hz)
2.03 (1H, dd, J = 4.8, 13.4 Hz)
1.19 (1H, m)
22
34.7
1.85 (1H, dd, J = 2.9, 8.5 Hz)
1.19 (3H, s)
23
24
25
26
27
28
29
30
28.5
17.1
15.9
17.8
26.6
180.6
24.2
33.8
1.00 (3H, s)
0.76 (3H, s)
0.95 (3H, s)
1.27 (3H, s)
–
0.99 (3H, s)
0.94 (3H, s)
m multiplet
The known compounds were identified by comparison
1
ESI-MS (positive-ion mode) m/z: 196.0578 [M?Na]?
(calcd. for C7H11NO4Na: 196.0580).
of their physical data ([a]D, IR, MS, H-, and 13C-NMR)
with reported values.
Compound 2: Pale yellow powder, [a]2D6 -147.5
(c = 0.68, MeOH); IR mmax (film) cm-1: 3349, 2917, 1658,
1600, 1446, 1257, 1126, 1059, 1024; UV kmax (MeOH) nm
Compound 1: Amorphous powder, [a]2D7 -15.0
(c = 0.26, H2O); IR mmax (film) cm-1: 3356, 2935, 1664,
1407, 1284, 1236, 1083; 1H-NMR (pyridine-d5, 600 MHz):
Table 1; 13C-NMR (pyridine-d5, 150 MHz): Table 1; HR-
1
(log e): 230 (3.99), 257 (4.26), 376 (3.83), 415 (3.64); H-
NMR (DMSO-d6, 600 MHz): Table 2; 13C-NMR (DMSO-
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Foundation for Pharmaceutical Sciences and the Cosmetology
Research Foundation for financial support.
d6, 150 MHz): Table 2; HR-ESI-MS (positive-ion mode)
m/z: 631.1638 [M?Na]? (calcd. for C28H32O15Na:
631.1633).
Compound 3: Amorphous powder, [a]2D6 ?10.9
(c = 0.70, MeOH); IR mmax (film) cm-1: 3377, 2942, 1727,
1695, 1363, 1162, 1071, 1036; 1H-NMR (pyridine-d5,
600 MHz): Table 3; 13C-NMR (pyridine-d5, 150 MHz):
Table 3; HR-ESI-MS (positive-ion mode) m/z: 993.5033
[M?Na]? (calcd. for C49H78O19Na: 993.5030).
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Acid hydrolysis of compounds 2 and 3
Solutions of 2 and 3 (1 mg each) were hydrolyzed with
1 M HCl (1.0 mL) at 80 °C for 3 h. After cooling, the
reaction mixtures were neutralized with Amberlite IRA-
400 (OH- form) and the resin was removed by filtration.
Then, the filtrates were extracted with ethyl acetate. The
aqueous layers were subjected to HPLC analysis [column:
Shodex Asahipak NH2P-50 4E, 250 9 4.6 mm i.d.; mobile
phase: MeCN–H2O (3:1, v/v); detection: optical rotation
(JASCO 2090Plus Chiral); flow rate: 1.0 mL/min] to
identify L-rhamnose (from 2), D-glucose (from 3), and D-
glucuronolactone (from 3), which were confirmed by
comparison of their retention times with those of the
authentic samples: tR: 6.5 min (L-rhamnose, negative
optical rotation); tR: 8.2 min (D-glucose, positive optical
rotation); tR: 5.3 min (D-glucuronolactone, positive optical
rotation).
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A solution of 2 (3.5 mg) in 0.2 M acetate buffer (1.0 mL,
pH 3.8) was treated with naringinase (10.5 mg) and the
solution was stirred at 37 °C for 6 h. The reaction mixture
was added to 1.0 mL of ethanol and then centrifuged at
4000 rpm for 10 min, and the supernatant solution was
concentrated under reduced pressure to give a residue. The
residue was purified by reversed-phase silica gel CC [3 g,
MeOH–H2O (0:10 ? 1:1) ? MeOH] to yield tamaraxetin
(2a, 0.9 mg). Compound 2a was identified by comparison
of its physical data (MS, 1H-, and 13C-NMR) with reported
values [15].
Acknowledgements The authors are grateful for access to the
superconducting NMR instrument (Bruker Avance 600) at the Ana-
lytical Center of Molecular Medicine of the Hiroshima University
Faculty of Medicine. The measurement of ESI-MS was made using a
Thermo Fisher Scientific LTQ Orbitrap XL spectrometer at the Nat-
ural Science Center for Basic Research and Development (N-BARD),
Hiroshima University. This work was supported in part by Grants-in-
Aid from the Ministry of Education, Culture, Sports, Science and
Technology of Japan (Nos. 16K18896, 26460122, and 15H04651),
the Japan Society for the Promotion of Science, and the Ministry of
Health, Labour and Welfare. Thanks are also due to the Research
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