Phenolic glycosides from Rhodohypoxis baurii

Article abstract of DOI:10.1007/s11418-010-0465-y

Three new phenolic glycosides (1-3), together with a known glycoside (4), were isolated from the bulbs of Rhodohypoxis baurii (Hypoxidaceae). The structures of the new glycosides (1-3) were determined on the basis of extensive spectroscopic analyses, including two-dimensional (2D) NMR data, and of hydrolytic cleavage followed by chromatographic or spectroscopic analyses.

Full text of DOI:10.1007/s11418-010-0465-y

J Nat Med (2011) 65:224–228
DOI 10.1007/s11418-010-0465-y
NOTE
Phenolic glycosides from Rhodohypoxis baurii
•
Yukiko Matsuo Fumito Tachikawa
•
Yoshihiro Mimaki
Received: 18 June 2010 / Accepted: 2 August 2010 / Published online: 7 September 2010
Ó The Japanese Society of Pharmacognosy and Springer 2010
Abstract Three new phenolic glycosides (1–3), together
with a known glycoside (4), were isolated from the bulbs of
Rhodohypoxis baurii (Hypoxidaceae). The structures of the
new glycosides (1–3) were determined on the basis of
extensive spectroscopic analyses, including two-dimen-
sional (2D) NMR data, and of hydrolytic cleavage followed
by chromatographic or spectroscopic analyses.
Results and discussion
The bulbs of R. baurii (3.7 kg) were extracted with hot
MeOH. The MeOH extract was passed through a porous-
polymer polystyrene resin (Diaion HP-20) column, and the
MeOH-eluted fraction was subjected to column chroma-
tography (CC) using silica gel and octadecylsilanized
(ODS) silica gel, and to reversed-phase preparative HPLC,
giving compounds 1–4 (Fig. 1). Compound 4 was identi-
fied as (?)-pinoresinol 4-O-b-D-glucopyranoside (4), by
comparison of its physical and spectroscopic data with
literature values [5].
Keywords Rhodohypoxis baurii ꢀ Hypoxidaceae ꢀ
Phenolic glycoside ꢀ Structural elucidation
Introduction
Compound 1 was obtained as an amorphous solid, [a]D
-100.5°in MeOH, with a molecular formula of C24H28O9,
which was assigned on the basis of the high-resolution (HR)-
ESI-time of flight (TOF)-MS (m/z 483.1634 [M ? Na]?,
calcd. 483.1631) and 13C-NMR (24 carbon signals) data.
The IR spectrum of 1 suggested the presence of hydroxy
groups (3,370 cm-1) and aromatic rings (1,598 and
1,510 cm-1). The 1H-NMR spectrum of 1 showed signals of
an A2B2-type proton spin-coupling system at d 7.10 (d,
J = 8.4 Hz, H-200 and H-600) and 6.70 (d, J = 8.4 Hz, H-300
and H-500), assignable to a 1,4-disubstituted aromatic ring
(ring B), as well as signals for a 1,2,3,5-tetrasubstituted
aromatic ring (ring A) at d 6.33 (d, J = 2.8 Hz, H-40) and
6.01 (d, J = 2.8 Hz, H-60), a methoxy group, at d 3.81 (s),
and an anomeric proton at d 4.79 (d, J = 7.6 Hz, H-1000).
Enzymatic hydrolysis of 1 with b-D-glucosidase in HOAc/
NaOAc buffer (pH 5.0) gave D-glucose as the carbohydrate
moiety, whereas the labile genuine aglycone was decom-
posed during hydrolysis. On the other hand, treatment of 1
with Ac2O in pyridine produced the corresponding hexa-
acetate (1a). The 1H-NMR spectrum of 1a displayed signals
for two aromatic acetyl groups at d 2.25 and 2.19 (each 3H,
s), which were consistent with the presence of two hydroxy
Rhodohypoxis baurii (Bak.) Nel. belongs to the family
Hypoxidaceae and is indigenous to South Africa. It is low-
growing, up to 150 mm with sparsely hairy leaves and
colorful flowers, and is a popular container plant in Europe,
Japan, Australia, and New Zealand [1]. Although plants
belonging to the family Hypoxidaceae are well known to
contain a variety of phenolic compounds [2–4], no sys-
tematic chemical work has been carried out on R. baurii.
The present investigation of the bulbs of R. baurii led to the
isolation of three new phenolic glycosides (1–3), together
with a known glycoside (4). This paper is a report on the
structural determination of the new glycosides, on the basis
of extensive spectroscopic analyses, including two-
dimensional (2D) NMR data, and of hydrolytic cleavage
followed by chromatographic or spectroscopic analyses.
Y. Matsuo (&) ꢀ F. Tachikawa ꢀ Y. Mimaki
School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences,
1432-1 Horinouchi, Hachiouji, Tokyo 192-0392, Japan
e-mail: matsuoy@toyaku.ac.jp
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J Nat Med (2011) 65:224–228
225
and H-4 (3JH-3
,
= 10.8 Hz in 1, 3JH-3, H-4 = 15.4 Hz in
H-4
2) of the penta-1,3-diene group. These data indicated that 2
was the geometrical isomer of 1 with regard to C-3 of the
penta-1,3-diene moiety. The absolute configuration at C-5
also remains to be determined. The structure of 2 was
assigned as (3E)-5-[2-(b-D-glucopyranosyl)oxy-5-hydroxy-
3-methoxyphenyl]-5-(4-hydroxyphenyl)-penta-1,3-diene.
Compound 3 was deduced to be C27H34O15 from its
HR-ESI-MS data (m/z 621.1803 [M ? Na]?). The IR
spectrum of 3 was suggestive of a glycoside (3,394,
2,923 cm-1) and indicated the presence of a conjugated
ester carbonyl group (1,711 cm-1). The 1H-NMR spectrum
of 3 included signals for a 1,2,5-trisubstituted aromatic ring
at d 7.08 (d, J = 8.8 Hz, H-3), 6.93 (d, J = 3.1 Hz, H-6),
and 6.72 (dd, J = 8.8, 3.1 Hz, H-4), 1,2,6-trisubstituted
aromatic ring at d 7.34 (t, J = 8.4 Hz, H-40) and 6.67
(d, J = 8.4 Hz, H-50 and H-30), an oxymethylene group at d
5.43 (s, H-7), and two anomeric protons at d 4.98
(d, J = 2.3 Hz, H-1000 of Api) and 4.71 (d, J = 7.4 Hz, H-100
of Glc). In addition, an ester carbonyl carbon was observed
at d 168.6 in the 13C-NMR spectrum of 3. These spectral
properties of 3 were closely related to those of known
compound curculigoside [4, 6, 7]. However, the molecular
formula of 3 was higher than that of curculigoside by
C5H8O4, and enzymatic hydrolysis of 3 with naringinase
gave D-apiose and D-glucose. When the 13C-NMR spectrum
of 3 was compared with that of curculigoside [6], a set of five
additional signals corresponding a terminal b-D-apiofur-
anosyl unit (Api) [dH-1 4.98; dC 111.0 (CH), 78.1 (CH), 80.5
(C), 75.0 (CH2), and 65.7 (CH2)] appeared, and signal due to
C-6 of the inner b-D-glucopyranosyl moiety (Glc) shifted
down field by 6.3 ppm to d 67.7, suggesting that C-6 of Glc
was the glycosylated position to which the additional apiosyl
unit was attached. In the HMBC spectrum of 3, the anomeric
proton (H-1) of Api at d 4.98 showed a long-range correla-
tion with C-6 of Glc at d 68.7, of which H-1 at d 4.71
exhibited a correlation with C-2 of the anomeric moiety at d
149.6. The structure of 3 was shown to be 6-O-b-D-apio-
furanosyl curculigoside.
Fig. 1 Chemical structures of compounds 1–4 from Rhodohypoxis
baurii
groups on the aromatic rings, as well as signals for four
alcoholic acetyl groups at d 2.09, 2.00, 1.99, and 1.83 (each
3H, s). In the HMBC spectrum of 1, long-range correlations
were observed between OCH3 (d 3.81) and C-30 (d 153.8),
H-1000 (d 4.79) and C-20 (d 137.8), H-40 (d 6.33) and C-50
(d 155.6)/C-30 (d 153.8)/C-20 (d 137.8)/C-60 (d 108.1), H-60
(d 6.01) and C-50 (d 155.6)/C-30 (d 153.8)/C-10 (d 141.6)/C-20
(d 137.8)/C-40 (d 99.7)/C-5 (d 41.8), H-200, -600 (d 7.10) and
C-400 (d 156.5)/C-100 (d 136.6)/C-300, -500 (d 115.8)/C-5
(d 41.8), and between H-300, -500 (d 6.70) and C-400 (d 156.5)/
C-100 (d 136.6)/C-200, -600 (d 130.4), indicating that two
hydroxy groups, a methoxy group, and a b-D-glucopyranosyl
group were located at C-50, C-400, C-30, and C-20, respec-
tively. In addition, signals attributable to a penta-1,3-diene
group were observed at d 6.97 (ddd, J = 16.9, 10.8,
10.2 Hz, H-2), 6.06 (t, J = 10.8 Hz, H-3), 5.90 (d,
J = 10.3 Hz, H-5), 5.66 (dd, J = 10.8, 10.3 Hz, H-4,), 5.18
(dd, J = 16.9, 2.1 Hz, H-1a), and 5.11 (dd, J = 10.2,
1
2.1 Hz, H-1b), in the H-NMR spectrum of 1. Ring A and
ring B were revealed to be attached to C-5 of the penta-1,
3-diene group on the basis of HMBC correlations between
H-4/H-5 and C-100, H-200/H-600 and C-5, H-5 and C-10/C-20/
C-60, and between H-60 to C-5, respectively. The geometry at
C-3 was determined to be Z by a J value of 10.8 Hz between
H-3 and H-4. Accordingly, the structure of 1 was elucidated
as (3Z)-5-[2-(b-D-glucopyranosyl)oxy-5-hydroxy-3-methoxy-
phenyl]-5-(4-hydroxyphenyl)-penta-1,3-diene. The abso-
lute configuration at C-5 remains to be determined.
Three new phenolic glycosides (1–3), together with a
known glycoside (4), were isolated from the bulbs of
R. baurii. In particular, 1 and 2 are rare type of phenolic
glycosides, and the structure of 2 was the geometrical
isomer of 1 with regard to C-3 of the penta-1,3-diene
moiety. Although phenolic glycoside derivatives based on
3,3-diphenylprop-1-ene have been isolated from Dalbergia
species [8, 9], 5,5-diphenylpenta-1,3-diene derivatives
have not been reported previously.
Compound 2 had the same molecular formula of
C24H28O9 as 1 on the basis of the HR-ESI–MS data (m/z:
483.1632 [M ? Na]?). Comparison of the 1H- and
13C-NMR spectra of 2 with those of 1 showed their con-
1
siderable structural similarity. The H-NMR, HMQC, and
The isolated compounds 1–4 were evaluated for their
cytotoxic activities against HL-60 human promyelocytic
leukemia cells and A549 human lung adenocarcinoma
cells. Unfortunately, neither of them showed cytotoxic
activities at sample concentrations up to 20 lg/ml.
COSY spectra of 2 exhibited signals assignable to a 1,
4-disubstituted aromatic ring, a 1,2,3,5-tetrasubstituted
aromatic ring, and a penta-1,3-diene group as in 1. Com-
pound 2 was only different from 1 in the J value between H-3
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J Nat Med (2011) 65:224–228
Experimental
General
(7.0 mg). Fraction E9 was separated by an ODS silica gel
column eluted with MeCN–H2O (1:3) and MeOH–H2O
(1:2), and finally purified by HPLC using MeCN–H2O (1:3)
to give 3 (20.5 mg).
Optical rotations were obtained using a P-1030 (Jasco,
Tokyo, Japan) automatic digital polarimeter. IR and UV
spectra were recorded on a Jasco V-630 and a Jasco FT-IR
620 spectrophotometer, respectively. NMR spectra
(500 MHz for 1H-NMR) were recorded on a DRX-500
spectrometer (Bruker, Karlsruhe, Germany), using standard
Bruker pulse programs. Chemical shifts are given as d
values with reference to tetramethylsilane (TMS) as an
internal standard. HR-ESI-TOF–MS data were obtained on
an LCT mass spectrometer (Waters-Micromass, Manches-
ter, UK). Diaion HP-20 (Mitsubishi-Chemical, Tokyo,
Japan), BW-300 silica gel (Fuji-Silysia Chemical, Aichi,
Japan), and ODS silica gel (Nacalai Tesque, Kyoto, Japan)
were used for CC. TLC was carried out on precoated silica
gel 60 F254 (0.25 mm thick, Merck, Darmstadt, Germany)
and RP18 F254S plates (0.25 mm thick, Merck), and the
spots were visualized by spraying the plates with 10%
H2SO4 and then heating. HPLC was performed with a
system composed of a CCPM pump (Tosoh, Tokyo, Japan),
a CCP PX-8010 controller (Tosoh), RI-8010 (Tosoh), and
Shodex OR-2 (Showa-Denko, Tokyo, Japan) detectors, and
a Rheodyne injection port. A TSK gel ODS-100Z column
(10 mm i.d. 9 250 mm, 5 lm, Tosoh) was employed for
preparative HPLC.
Compound 1
Amorphous solid, [a]2D5 -100.5° (c 0.40, MeOH). IR mmax
(film) cm-1: 3,370 (OH), 2,923 (CH), 1,598 and 1,510
(aromatic rings). UV kmax (MeOH) nm (log e): 228 (4.23),
1
281 (3.36). H- and 13C-NMR, see Table 1. HR-ESI-TOF-
MS m/z: 483.1634 [M ? Na]? (calcd. for C24H28O9Na,
483.1631).
Enzymatic hydrolysis of 1
Compound 1 (3.0 mg) was treated with b-D-glucosidase
(Sigma EC 232-589-7; 50 mg) in HOAc/NaOAc buffer (pH
5.0, 1.5 ml) at room temperature for 20 h. The reaction
mixture was subjected to CC on silica gel using CHCl3–
MeOH–H2O (7:4:1) followed by passage through a Sep-Pak
C18 cartridge (Waters, Milford, MA) to give a sugar fraction,
which was then analyzed by HPLC under the following
conditions: column, Capcell Pak NH2 UG80 (4.6 mm
i.d. 9 250 mm, 5 lm, Shiseido, Tokyo, Japan); solvent,
MeCN-H2O (85:15); detection, RI and OR; flow rate, 0.8 ml/
min. Identification of D-glucose was carried out by com-
parison of its retention time and optical rotation with those of
an authentic sample. tR (min): 17.6 (D-glucose, positive
optical rotation).
Plant material
The bulbs of R. baurii were purchased from a garden center
in Shunnkouenn, Tokyo, Japan. The bulbs were cultivated,
and the flowered plants were identified by one of the
authors (Y.M.). A voucher specimen has been deposited in
our laboratory (voucher no. 10-004-Rb, Laboratory of
Medicinal Pharmacognosy).
Acetylation of 1
A mixture of 1 (0.4 mg) and Ac2O (0.5 ml) in pyridine
(0.5 ml) was stirred at room temperature for 24 h. The
reaction mixture was evaporated to give the corresponding
hexaacetate (1a, 0.3 mg).
Extraction and isolation
Compound 1a
The bulbs of R. baurii (fresh weight, 3.7 kg) were extracted
with MeOH (18 l). After removing the solvent, the MeOH
extract (110 g) was passed through a Diaion HP-20 column
and successively eluted with 30% MeOH, 50% MeOH,
MeOH, EtOH, and EtOAc (each 5 l). CC of the MeOH-
eluted fraction (3.0 g) on silica gel eluted with a stepwise
gradient mixture of CHCl3–MeOH–H2O (30:10:1; 15:10:1),
and finally with MeOH alone, provided six fractions (A–F).
Fraction E was chromatographed on ODS silica gel eluted
with MeOH–H2O (5:1) into 11 subfractions (frs. E1–E11). Fr.
E1 was subjected to CC on silica gel eluted with CHCl3–
MeOH–H2O (50:10:1; 30:10:1) and on ODS silica gel eluted
with MeOH–H2O (2:3) to yield 1 (6.1 mg), 2 (4.7 mg), and 4
Amorphous solid, [a]2D5 -52.3°(c 0.02, MeOH). IR mmax
(film) cm-1: 3,434 (OH), 2,922 (CH), 1,747 (C = O),
1,557 (aromatic ring). 1H-NMR (CD3OD, 500 MHz) d:
7.24 (d, J = 8.4 Hz, H-200 and H-600), 6.96 (d, J = 8.4 Hz,
H-300 and H-500), 6.71 (d, J = 2.6 Hz, H-40), 6.26 (d,
J = 2.6 Hz, H-60), 3.86, 2.25, 2.19, 2.09, 2.00, 1.99, and
1.83 (each 3H, s). HR-ESI-TOF–MS m/z: 693.2165
[M ? Na-COCH3]? (calcd. for C34H38O14Na, 693.2159).
Compound 2
Amorphous solid, [a]2D5 -91.4°(c 0.04, MeOH). IR tmax
(film) cm-1: 3,465 (OH), 1,650 (aromatic ring). UV kmax
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J Nat Med (2011) 65:224–228
227
Table 1 1H- and 13C-NMR data for 1 and 2 in CD3OD at 300 K
Table 2 1H- and 13C-NMR data for 3 in CD3OD at 300 K
Position
1
2
Position
1H
J (Hz)
13C
1H
J (Hz)
13C
1H
J (Hz)
13C
1
128.9
149.6
119.2
116.3
154.0
116.4
63.2
2
1a
1b
2
5.18 dd 16.9, 2.1
5.11 d 10.2
118.0 5.02 dd 17.0, 2.1
4.94 dd 10.2, 2.1
115.8
3
7.08 d
8.8
4
6.72 dd
8.8, 3.1
6.97 ddd 16.9, 10.8, 134.5 6.38 ddd 17.0, 10.7, 138.5
10.2
5
10.2
6
6.93 d
5.43 s
3.1
3
6.06 t
10.8
130.2 5.85 dd 15.4, 10.7 132.9
7
10
20, 60
30, 50
40
4
5.66 dd 10.8, 10.3 135.5 5.99 dd 15.4, 6.9
138.5
46.2
114.2
158.8
105.2
132.6
168.6
56.5
5
10
20
30
40
50
60
100
5.90 d
10.3
41.8 5.50 d
141.6
6.9
141.6
137.8
153.8
99.8
6.67 d
7.34 t
8.4
8.4
137.8
153.8
70
6.33 d
6.01 d
2.8
2.8
99.7 6.35 d
155.6
2.8
2.8
OMe
Glc 100
Glc 200
Glc 300
Glc 400
Glc 500
3.81 s
155.3
108.6
136.6
130.2
115.8
156.7
56.4
4.71 d
7.4
104.5
75.0
108.1 5.98 d
136.6
3.47 dd
3.43 dd
3.37 dd
3.49 m
4.00 dd
3.63 dd
4.98 d
8.7, 7.4
9.0, 8.7
9.0, 8.7
200, 600
300, 500
400
7.10 d
6.70 d
8.4
8.4
130.4 7.09 d
115.8 6.70 d
156.5
7.8
8.6
78.0
71.6
76.9
Glc 600
Glc 600
a
11.1, 1.8
11.1, 6.3
2.3
68.7
OMe
3.81 s
Glc 1000 4.79 d
56.4 3.82 s
105.7 4.81 d
b
7.6
7.6
105.5
75.9
Api 1000
Api 2000
Api 3000
Api 4000
Api 4000
Api 5000
111.0
78.1
80.5
75.0
Glc 2000 3.50 dd 8.8, 7.6
Glc 3000 3.43 dd 8.9, 8.8
Glc 4000 3.39 dd 8.9, 8.8
Glc 5000 3.14 m
Glc 6000a 3.66 dd 11.7, 2.6
Glc 6000b 3.59 dd 11.7, 4.9
76.0 3.48 dd 8.9, 7.6
78.0 3.43 dd 8.9, 8.9
71.6 3.39 dd 8.9, 8.7
78.1 3.14 m
3.92 d
2.3
78.0
71.5
a
3.94 d
3.76 d
3.59 s
9.7
9.7
78.2
b
62.7 3.73 dd 11.8, 2.4
3.62 dd 11.8, 5.0
62.6
65.7
the presence of D-apiose and D-glucose. Authentic D-apiose
was prepared from acid hydrolysis of 1,2,3,5-di-O-isopro-
pylidiene-a-D-apiose (Sigma, St. Louis, MO). tR(min): 4.1
(D-apiose, positive optical rotation), 17.3 (D-glucose, positive
optical rotation).
1
(MeOH) nm (log e): 228 (3.81), 280 (2.87). H- and 13C-
NMR, see Table 1. HR-ESI-TOF-MS m/z: 483.1632
[M ? Na]? (calcd. for C24H28O9Na, 483.1631).
Compound 3
Acknowledgments We are grateful to Dr. Y. Shida and Mr.
H. Fukaya, Tokyo University of Pharmacy and Life Sciences, for the
mass spectra and elemental analyses.
Amorphous solid, [a]2D5 -46.5°(c 0.50, MeOH). IR mmax
(film) cm-1: 3,394 (OH), 2,923 (CH), 1,711 (C=O), 1,596
and 1,499 (aromatic rings). UV kmax (MeOH) nm (log e):
1
257 (3.31), 282 (3.32). H- and 13C-NMR, see Table 2.
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C27H34O15Na, 621.1795).
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