Novel phenolic glycosides, adenophorasides A-E, from Adenophora roots

Article abstract of DOI:10.1007/s11418-010-0398-5

Five novel phenolic glycosides, adenophorasides A (1), B (2), C (3), D (4), and E (5), were isolated from commercial Adenophora roots, together with vanilloloside (6), 3,4-dimethoxybenzyl alcohol 7-O-β-D-glucopyranoside (7), and lobetyolin (8). The structures of the new compounds (1-5) were characterized as 4-hydroxy-3-methoxyphenylacetonitrile 4-O-β-D- glucopyranoside (1), 4-hydroxy-3-methoxyphenylacetonitrile 4-O-β-D- glucopyranosyl-1→6)-b-D-glucopyranoside (2), 4-hydroxy-3- methoxyphenylacetonitrile 4-O-α-L-rhamnopyranosyl-(1→6)-β-D- glucopyranoside (3), 4-hydroxyphenylacetonitrile 4-O-β-D-glucopyranosyl- (1→6)-β-D-glucopyranoside (4),and 4-hydroxy-3-methoxybenzyl alcohol 4-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (5), respectively, by means of spectroscopic and chemical analyses. The Japanese Society of Pharmacognosy and Springer 2010.

Full text of DOI:10.1007/s11418-010-0398-5

J Nat Med (2010) 64:245–251
DOI 10.1007/s11418-010-0398-5
ORIGINAL PAPER
Novel phenolic glycosides, adenophorasides A–E,
from Adenophora roots
Yuka Koike
Yasuaki Hirai
Toshiyuki Atsumi
•
Motonori Fukumura
Yumiko Hori Shiho Usui
Kazuo Toriizuka
•
•
•
•
•
Received: 19 October 2009 / Accepted: 15 January 2010 / Published online: 16 March 2010
Ó The Japanese Society of Pharmacognosy and Springer 2010
Abstract Five novel phenolic glycosides, adenophora-
sides A (1), B (2), C (3), D (4), and E (5), were isolated
from commercial Adenophora roots, together with van-
illoloside (6), 3,4-dimethoxybenzyl alcohol 7-O-b-D-gluco-
pyranoside (7), and lobetyolin (8). The structures of the
new compounds (1–5) were characterized as 4-hydroxy-
activities of Platycodon roots in empirical medicine and
Kampo medicine [6]. Adenophora roots (the dried roots of
Adenophora species) have been used as antitussives and
expectorants in the same manner as Platycodon roots [7, 8].
However, in contrast to Platycodon roots, the chemical
constituents and bioactive components of Adenophora
roots have not yet been revealed. Therefore, we initiated
this study to elucidate the constituents of Adenophora
roots, specifically focusing on the fraction that included the
glycosides, in order to identify the bioactive components.
The origin of Adenophora roots is the dried roots of
Adenophora tetraphylla and the allied plants such as A.
triphylla and A. stricta [7–9]. As for the constituents of A.
tetraphylla, a-spinasterol [10], nonacosan-10-ol [10], 24-
methylene cycloartenol [10], lupenone [10], b-sitosterol
3
-methoxyphenylacetonitrile 4-O-b-D-glucopyranoside (1),
-hydroxy-3-methoxyphenylacetonitrile 4-O-b-D-glucopyr-
4
anosyl-(1?6)-b-D-glucopyranoside (2), 4-hydroxy-3-meth-
oxyphenylacetonitrile 4-O-a-L-rhamnopyranosyl-(1?6)-
b-D-glucopyranoside (3), 4-hydroxyphenylacetonitrile
4
-O-b-D-glucopyranosyl-(1?6)-b-D-glucopyranoside (4),
and 4-hydroxy-3-methoxybenzyl alcohol 4-O-b-D-gluco-
pyranosyl-(1?6)-b-D-glucopyranoside (5), respectively, by
means of spectroscopic and chemical analyses.
[
10], 3-O-palmitoyl-b-sitosterol [10], b-sitosterol 3-O-b-D-
Keywords Adenophora roots ꢀ Adenophorae Radix ꢀ
Campanulaceae ꢀ Phenolic glycoside ꢀ Phenylacetonitrile ꢀ
Adenophoraside
glucopyranoside [10, 11], eicosanoic acid [10], linoleic
acid [11], methyl stearate [11], syringinoside [11], and
shashenosides I–III [11] have been reported. And, essential
oils [12, 13], pyrrolidine and piperidine derivatives [14],
and triterpenoids [15] have been isolated from A. triphylla
and A. stricta. In this paper, we describe the isolation and
characterization of five novel compounds including phe-
nylacetonitriles, regarded as the precursors of cyanogenic
glycosides, from Adenophora roots.
Introduction
The family of Campanulaceae comprises about 80 genera
such as Platycodon, Codonopsis, and Adenophora. Phyto-
chemical investigations of Platycodon roots (the dried roots
of Platycodon grandiflorum) have revealed many saponin
constituents [1–5]. It is widely thought that these saponins
play an important role in the antitussive and expectorant
Results and discussion
Adenophora roots (6.9 kg) were extracted with 70%
aqueous methanol at room temperature. After concentra-
tion, the extract was partitioned between water and diethyl
ether. The aqueous portion, after concentration, was sub-
jected to column chromatography over silica gel and oc-
tadecylsilanized silica gel (ODS) repeatedly to give eight
Y. Koike ꢀ M. Fukumura (&) ꢀ Y. Hirai ꢀ Y. Hori ꢀ S. Usui ꢀ
T. Atsumi ꢀ K. Toriizuka
School of Pharmacy, Showa University, 1-5-8 Hatanodai,
Shinagawa-ku, Tokyo 142-8555, Japan
e-mail: fukujy@pharm.showa-u.ac.jp
123

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compounds, as described in the ‘‘Experimental’’. Three of
these compounds were identified as vanilloloside (6), 3,4-
dimethoxybenzyl alcohol 7-O-b-D-glucopyranoside (7),
and lobetyolin (8) by comparison of their physical and
spectral data with those reported [16, 17]. The remaining
five compounds were found to be novel phenylacetonitrile
glycosides and a benzyl alcohol glycoside, as discussed
below (Fig. 1).
a phenolic hydroxyl group (d 9.07). In the HMBC spec-
H
trum of 1a, correlations were observed between a phenolic
hydroxyl proton (d 9.07) and C-3 (d 147.8), C-4 (d 146.0),
and C-5 (d 115.7); and H-7 (d 3.85) and C-1 (d 121.6), C-2
(d 112.3), C-6 (d 120.5), and C-8 (d 119.7); and a methoxyl
proton (d 3.75) and C-3 (d 147.8). These data suggested 1a
to be 4-hydroxy-3-methoxyphenylacetonitrile, and this was
proved by direct comparison (TLC, NMR, and MS) with an
authentic sample synthesized by Schwartz’s method [18].
On the other hand, the anomeric proton signal of 1
appeared as a doublet (J = 7.5 Hz) at d 4.89, and was
shown to correlate with the C-4 carbon signal (d 145.9)
in the HMBC spectrum. These results demonstrated
that adenophoraside A (1) was 4-hydroxy-3-methox-
yphenylacetonitrile 4-O-b-D-glucopyranoside.
Adenophoraside A (1), a white amorphous powder,
?
showed a [M?H] peak at m/z 326.1245 in HR FAB-MS,
suggesting its molecular formula to be C H NO . The IR
1
5
19
7
spectrum of 1 exhibited characteristic absorption bands
-
due to hydroxyl groups (3408 cm ), a nitrile group
1
-
2249 cm ), and an aromatic ring (1597 and 1518 cm ).
1
-1
(
1
3
The C-NMR spectroscopic features of 1 were close to those
of 6, except for the presence of the sp quaternary carbon
signal (d 119.5) and the methylene group signal (d 21.9)
instead of the hydroxymethyl carbon signal in 6 and some
differences in the chemical shifts of the C-1, C-2, and C-6
signals (-11.9, ?1.4, ?1.6 ppm, respectively) from that in 6
Adenophoraside B (2), a white amorphous powder,
?
showed a [M?H] peak at m/z 488.1755 in HR FAB-MS,
suggesting its molecular formula to be C H NO , 162
2
1
29
12
1
mass units (C H O ) larger than that of 1. The C-NMR
3
6
10 5
spectroscopic features of 2 were similar to those of 1,
except for the appearance of an additional six signals
(d 61.1, 70.1, 73.6, 76.6, 76.9, 103.3) and the observation
(
Table 1).
On enzymatic hydrolysis, 1 afforded a sugar portion and
an aglycone (1a), the latter as an orange solid. The sugar
portion was identified as D-glucose by HPLC. The aglycone
0
of the lower field shift (?7.8 ppm) of the C-6 carbon
signal from that in 1 because of the glycosylation shift
(Table 1). On enzymatic hydrolysis, 2 afforded a sugar
portion and an aglycone, which were identified as D-glu-
cose and 1a, respectively, by a similar method to that
?
a showed a [M] peak at m/z 163.0631 in HR FAB-MS
1
corresponding to the molecular formula of C H NO . The
9
9
2
IR spectrum of 1a exhibited absorption bands due to sim-
ilar functional groups as in 1. The NMR spectrum of 1a
indicated the presence of a methoxyl group (dH 3.75; dC
0
0
used for 1. A correlation was observed between H-1 (d
0
4.16, d, J = 8.1 Hz) and C-6 (d 68.4) in the HMBC
spectrum of 2 which indicated that the additional gluco-
5
5.6), a methylene group (dH 3.85; d 21.9), a 1,2,4-tri-
C
0
substituted benzene (d 6.72, 6.75, 6.87; d 112.3, 115.7,
pyranosyl moiety was attached to C-6 of 2. These facts
indicated that adenophoraside B (2) was 4-hydroxy-3-
H
C
1
20.5, 121.6, 146.0, 147.8), a nitrile group (d 119.7), and
C
Fig. 1 Structures of 1–8
isolated from Adenophora roots
1
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247
123

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J Nat Med (2010) 64:245–251
methoxyphenylacetonitrile 4-O-b-D-glucopyranosyl-(1?6)-
b-D-glucopyranoside.
AdenophorasideC (3), a white amorphouspowder,showed
suggesting its molecular formula to be C H O . This
20 30 13
result indicated that the molecular weight of 5 was 162
(C H O ) larger than that of vanilloloside (6). The IR
spectrum of 5 exhibited characteristic absorption bands due
6
10 5
?
a [M?H] peak at m/z 472.1868 in HR FAB-MS, suggesting
its molecular formula to be C H NO , 16 mass units
-
1
to hydroxyl groups (3408 cm ) and an aromatic ring (1597
2
1
29
11
1
3
-1
13
smaller than that of 2. The C-NMR spectrum of 3 exhibited
similar signals to that of 2, except for the presence of six
signals (d 17.9, 68.4, 70.4, 70.7, 72.0, 100.6) instead of the
terminal glucosyl moiety in 2 (Table 1). On enzymatic
hydrolysis, 3 afforded a sugar portion and an aglycone. The
former was identified as D-glucose and L-rhamnose, the latter
was identified as 1a by a similar method to that used for 1. The
and 1516 cm ). The C-NMR spectroscopic features of 5
were similar to those of 6, except for the appearance of
additional six signals (d 61.1, 70.1, 73.6, 76.6, 76.8, 103.2)
and the observation of the lower field shift (?7.5 ppm) of the
0
C-6 carbon signal from that in 6 because of the glycosylation
shift (Table 1). On enzymatic hydrolysis, 5 afforded an
aglycone, which was identified as vanillyl alcohol by com-
parison with commercial authentic samples, and D-glucose
identified by HPLC. On the other hand, the observation of the
00
configuration of C-1 was determined to be a from the
00
chemical shifts of C-5 (3: d 68.4; shatavaroside A [19]: d
1
8.7) in the C-NMR spectrum [19, 20]. The observation of
3
00
0
6
cross peak between H-1 (d 4.16, d, J = 8.1 Hz) and C-6 (d
68.2) in the HMBC spectrum of 5 indicated that the addi-
0
0
0
the cross peak between H-1 (d 4.51) and C-6 (d 66.5) in the
HMBC spectrum of 3 indicated that an a-L-rhamnopyranosyl
0
tional glucopyranosyl moiety was located to the C-6 . Thus,
0
moiety was located at C-6 of 3. Thus, the structure of
the structure of adenophoraside E (5) was characterized as 4-
hydroxy-3-methoxybenzyl alcohol 4-O-b-D-glucopyrano-
syl-(1?6)-b-D-glucopyranoside.
adenophoraside C (3) was determined to be 4-hydroxy-3-
methoxyphenylacetonitrile 4-O-a-L-rhamnopyranosyl-(1?6)-
b-D-glucopyranoside.
Adenophoraside D (4), a white amorphous powder,
?
showed a [M?H] peak at m/z 458.1611 in HR FAB-MS,
As mentioned above, we isolated eight compounds from
Adenophora roots, including five novel compounds (1–5).
Among them, 1–4 are phenylacetonitrile glycosides. It is
well known that cyanogenic glycosides are found in many
natural sources such as plants of the family Rosaceae and
Gramineae and are biosynthesized from their correspond-
ing amino acids via phenylacetonitrile. To our knowledge,
only a few phenylacetonitriles have been isolated from
natural sources: phenylacetonitrile [21], 4-[b-D-glucopyr-
anosyl-(1?3)-a-L-rhamnopyranosyl]-phenylacetonitrile [22],
niazirin [23], niazirinin [23], and niaziridin [24]. This is the
first report of phenylacetonitrile isolation from plants of the
family Campanulaceae. In this study, saponin was not
isolated from Adenophora roots. This is mere conjecture,
but the antitussive and expectorant effects of Adenophora
roots might therefore not be due to saponins.
suggesting its molecular formula to be C H NO , 30
2
0
27
11
1
mass units (CH O) smaller than that of 2. The C-NMR
3
2
spectrum of 4 showed 20 signals, twelve of which were
similar to those due to the disaccharide moiety of 2
(Table 1). On enzymatic hydrolysis, 4 afforded a sugar
portion and an aglycone (4a), the latter as an orange solid.
The sugar portion was identified as D-glucose by HPLC.
?
The aglycone 4a showed a [M] peak at m/z 133.0534
(
C H NO) in HR FAB-MS. The NMR spectrum of 4a
8 7
indicated the presence of a methylene group (d 3.85; d
H
C
2
1.5), a 1,4-disubstituted benzene (dH 6.75, 6.75, 7.12,
.12; d 115.6, 115.6, 121.1, 129.2, 129.2, 156.8), a nitrile
7
C
group (d 119.7), and a phenolic hydroxyl group (d 9.49).
C
H
The HMBC spectrum of 4a revealed correlations between
H-7 (d 3.85) and C-1 (d 121.1), C-2 (d 129.2), C-6 (d
Additionally, we evaluated the cytotoxic activity of
novel phenolic glycosides (1–5) against human breast
cancer (MCF-7) and leukemia (U937) cell lines by using
the MTT assay. The results demonstrated that these com-
pounds had little inhibitory effect on cell growth even at
100 lM (data not shown).
1
29.2), and C-8 (d 119.7); and a phenolic hydroxyl proton
d 9.49) and C-3 (d 115.6), C-4 (d 156.8), and C-5 (d
15.6). These results indicated that 4a was 4-hydro-
(
1
xyphenylacetonitrile, i.e., corresponding to the demethoxyl
derivative of 1a, and this was proved by direct comparison
(
TLC, NMR, and MS) with an authentic sample synthe-
sized by Schwartz’s method [18]. On the other hand, the
0
Experimental
observation of the cross peak between H-1 (d 4.82) and
C-4 (d 156.9) in the HMBC spectrum of 4 indicated that
the sugar moiety was located at C-4 of 4. Consequently,
adenophoraside D (4) was characterized as 4-hydro-
xyphenylacetonitrile 4-O-b-D-glucopyranosyl-(1?6)-b-D-
glucopyranoside.
General
Optical rotations were obtained on a P-1020 polarimeter
(JASCO, Tokyo, Japan). IR spectra were measured on an
FT/IR-410 spectrometer (JASCO). NMR spectra were
recorded on a JNM ECA-500 spectrometer (500 MHz for
Adenophoraside E (5), a white amorphous powder,
?
showed a [M?H] peak at m/z 479.1750 in HR FAB-MS,
1
13
H, 125 MHz for C; JEOL, Tokyo, Japan), and the
1
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J Nat Med (2010) 64:245–251
249
chemical shifts were given in ppm scale from TMS used as
an internal standard. The signals were assigned by DEPT
was purified by ODS CC, eluting with water containing
increasing amounts of methanol, to give five fractions: Frs.
1
1
and 2D NMR techniques ( H– H COSY, HMQC, HMBC).
MS spectra were obtained on a JMS-700 spectrometer
C-1 (H O, 2.1 g), C-2 (30% MeOH, 6.6 g), C-3 (30%
2
MeOH, 5.8 g), C-4 (70% MeOH, 630 mg), and C-5 (MeOH,
86 mg). Fr. C-2 was purified by silica gel CC [CHCl3–
MeOH–H O (14:6:1)] and then ODS CC (15% CH CN) to
(
JEOL), with the matrix used for FAB-MS shown in
parentheses. HPLC was carried out on a Mightysil RP-
8GP (20 mm i.d. 9 250 mm, 5 lm; Kanto Chemical,
2
3
1
give 3 (26 mg) and 7 (6 mg). Fr. D was purified by silica gel
CC [CHCl –MeOH–H O (38:12:1)] to give four fractions:
Tokyo, Japan) with a UV-8011 (Tosoh, Tokyo, Japan) for
preparative HPLC or a COSMOSIL Sugar-D (4.6 mm
i.d. 9 250 mm; Nacalai Tesque, Kyoto, Japan) with an RI-
3
2
Frs. D-1 (183 mg), D-2 (241 mg), D-3 (291 mg), and D-4
(1.34 g). Fr. D-2 was purified by ODS CC (40% MeOH) to
give 8 (56 mg).
2
031 Plus and an OR-2090 Plus detector (JASCO) for the
D/L determination of monosaccharides. TLC was performed
on pre-coated silica gel 60 F254 or RP-18 WF254 plates
Adenophoraside A (1)
(
Merck Ltd., Darmstadt, Germany) with detection achieved
2
D
5
by spraying with 10% H SO followed by heating. Column
4
A white amorphous powder. [a] -55.9° (c 1.00, MeOH).
2
-
IR (KBr) cm : 3408, 2916, 2249, 1597, 1518. Positive HR
1
chromatography (CC) was performed on silica gel 60 (63–
00 lm; Merck Ltd.) or ODS (chromatorex DM-1020T;
?
FAB-MS (NBA) m/z: 326.1245 ([M?H] , C H NO :
2
1
5
20
7
1
326.1240). C- and H-NMR: Table 1. Selected HMBC
3
1
Fuji-Silysia Chemical Ltd., Aichi, Japan). Other reagents,
unless otherwise stated, were obtained from Wako Pure
Chemical Industries (Osaka, Japan).
correlations: H-7/C-1, C-2, C-6, C-8; OCH /C-3; H-6/C-4;
3
0
H-1 /C-4.
Extraction and isolation
Adenophoraside B (2)
2
5
Commercial Adenophora roots (6.90 kg, Lot. No.
A white amorphous powder. [a]D -57.6° (c 1.00, H O).
2
?
Positive HR FAB-MS (NBA) m/z: 488.1755 ([M?H] ,
0
28107003: imported from Hubei Province in China; To-
-
1
chimoto-Tenkaido Co., Osaka, Japan; voucher specimens
are deposited at the Herbarium of Showa University) were
powdered and percolated with 70% MeOH (67 L) at room
temperature. After being concentrated in vacuo at 40°C, the
C H NO : 488.1768). IR (KBr) cm : 3396, 2941,
21 30 12
1
2251, 1599, 1520. C- and H-NMR: Table 1. Selected
3
1
HMBC correlations: H-7/C-1, C-2, C-6, C-8; OCH /C-3;
3
0
00
0
H-6/C-4; H-1 /C-4; H-1 /C-6 .
7
0% MeOH extract (1.75 kg) was suspended in H O
2
(
(
4.8 L) and successfully extracted with diethyl ether
Adenophoraside C (3)
3 9 1.10 L) to yield a diethyl ether extract (20.9 g). The
residual H O layer (1.73 kg) was purified by ODS CC,
2
25
A white amorphous powder. [a]D -35.4° (c 0.13, H O).
2
eluting with water containing increasing amounts of
?
Positive HR FAB-MS (NBA) m/z: 472.1868 ([M?H] ,
methanol, to give seven fractions (Fr): Frs. A (H O,
2
-1
C H NO : 472.1819). IR (KBr) cm : 3433, 2924,
2
1
30
11
1
.68 kg), B (H O, 10.5 g), C (30% MeOH, 17.2 g), D
2
13
252, 1595, 1514. C- and H-NMR: Table 1. Selected
1
2
(
30% MeOH, 2.5 g), E (70% MeOH, 8.2 g), F (70%
HMBC correlations: H-7/C-1, C-2, C-6, C-8; OCH /C-3;
3
MeOH, 1.4 g), and G (MeOH, 0.7 g).
0
00
0
H-6/C-4; H-1 /C-4; H-1 /C-6 .
Fr. B was purified by ODS CC, eluting with water con-
taining increasing amounts of methanol, to give five frac-
Adenophoraside D (4)
tions: Frs. B-1 (H O, 3.8 g), B-2 (30% MeOH, 3.1 g), B-3
2
(
(
30% MeOH, 248 mg), B-4 (70% MeOH, 91 mg), and B-5
MeOH, 131 mg). Fr. B-2 was purified by ODS CC (25%
2
5
A white amorphous powder. [a]D -66.1° (c 1.00, H O).
2
?
Positive HR FAB-MS (NBA) m/z: 458.1611 ([M?H] ,
MeOH) to give six fractions: Frs. B-2-1 (377 mg), B-2-2
150 mg), B-2-3 (59 mg), B-2-4 (557 mg), B-2-5 (299 mg),
and B-2-6 (1.0 g). Fr. B-2-1 was purified by silica gel CC
CHCl –MeOH (4:1)] to give 1 (67 mg). B-2-2 was purified
-
C H NO : 458.1662). IR (KBr) cm : 3435, 2920,
1
2
0
28
11
(
1
3
1
251, 1591, 1514. C- and H-NMR: Table 1. Selected
2
0
HMBC correlations: H-7/C-1, C-2, C-6, C-8; H-1 /C-4;
00
[
3
0
H-1 /C-6 .
by silica gel CC [CHCl –MeOH–H O (38:12:1)] to give 6
3
2
(
56 mg). Fr. B-2-4 was purified by silica gel CC [CHCl3–
Adenophoraside E (5)
MeOH–H O (34:16:3)] and then ODS CC (10% MeOH) to
2
give 2 (307 mg). Frs. B-2-5 and B-2-6 were purified by silica
gel CC [CHCl –MeOH–H O (14:6:1 and 16:9:2, respec-
2
5
A white amorphous powder. [a]D -60.5° (c 1.00, H O).
2
3
2
?
Positive HR FAB-MS (NBA) m/z: 479.1750 ([M?H] ,
tively)] to give 4 (79 mg) and 5 (109 mg), respectively. Fr. C
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J Nat Med (2010) 64:245–251
-
C H O : 479.1765). IR (KBr) cm : 3408, 2925, 1597,
1
4-Hydroxy-3-methoxyphenylacetonitrile (1a)
An orange solid. Positive HR FAB-MS (NBA) m/z:
2
0 31 13
1
3
1
516. C- and H-NMR: Table 1. Selected HMBC cor-
1
0
relations: H-7/C-1, C-2, C-6; OCH /C-3; H-6/C-4; H-1 /C-
3
0
0
0
?
-1
4
; H-1 /C-6 .
163.0631 ([M] , C
9
H
9
NO
390, 2925, 2252, 1603, 1518. C-NMR (DMSO-d6,
25 MHz) d: 21.9 (C-7), 55.6 (OCH ), 112.3 (C-2), 115.7
2
:163.0633). IR (Nujor) cm
:
1
3
3
1
Known compounds isolated
3
(
C-5), 119.7 (C-8), 120.5 (C-6), 121.6 (C-1), 146.0 (C-4),
1
2
5
147.8 (C-3). H-NMR (DMSO-d , 500 MHz) d: 3.75 (3H,
Vanilloloside (6) [16]: A white amorphous powder. [a]D
47.7° (c 0.50, MeOH). Positive HR FAB-MS (NBA) m/z:
6
s, OCH ), 3.85 (2H, s, H -7), 6.72 (1H, dd, J = 1.7,
3
-
2
?
16.1148 ([M] , C H O : 316.1158). IR (KBr) cm
-1
8.0 Hz, H-6), 6.75 (1H, d, J = 8.0 Hz, H-5), 6.87 (1H, d,
J = 1.7 Hz, H-2), 9.07 (1H, s, 4-OH). Selected HMBC
correlations: H-7/C-1, C-2, C-6, C-8; OCH /C-3; H-6/C-4;
3
:
1
4 20 8
1
433, 2922, 1597, 1514. C-NMR (DMSO-d , 125 MHz)
3
3
6
0
0
d: 55.6 (OCH ), 60.7 (C-6 ), 62.7 (C-7), 69.7 (C-4 ), 73.3
3
3
0
0
0
0
OH/C-3, C-4, C-5.
(
C-2 ), 76.9 (C-5 ), 77.0 (C-3 ), 100.2 (C-1 ), 111.1 (C-2),
1
15.2 (C-5), 118.6 (C-6), 136.4 (C-1), 145.3 (C-4), 148.8
1
(
C-3). H-NMR (DMSO-d , 500 MHz) d: 3.15 (1H, m,
4-Hydroxyphenylacetonitrile (4a)
6
0
0
H-4 ), 3.23 (1H, overlapped, H-2 ), 3.23 (1H, overlapped,
0
0
H-3 ), 3.26 (1H, overlapped, H-5 ), 3.43 (1H, ddd,
0
An orange solid. Positive HR FAB-MS (NBA) m/z:
?
-1
133.0534 ([M] , C H NO:133.0528). IR (Nujor) cm :
8 7
J = 5.8, 5.8, 12.1 Hz, H-6 a), 3.64 (1H, br dd, J = 4.6,
0
13
1
2.1 Hz, H-6 b), 3.73 (3H, s, OCH ), 4.40 (2H, d,
3
3326, 2925, 2252, 1610, 1516. C-NMR (DMSO-d6,
125 MHz) d: 21.5 (C-7), 115.6 (C-3), 115.6 (C-5), 119.7
(C-8), 121.1 (C-1), 129.2 (C-2), 129.2 (C-6), 156.8 (C-4).
0
J = 5.2 Hz, H-7), 4.85 (1H, d, J = 7.5 Hz, H-1 ), 5.09
(
1H, t, J = 5.2 Hz, 7-OH), 6.78 (1H, dd, J = 1.8,
.0 Hz, H-6), 6.92 (1H, d, J = 8.0 Hz, H-5), 7.01 (1H, d,
J = 1.8 Hz, H-2). Selected HMBC correlations: H-7/C-1,
1
8
H-NMR (DMSO-d , 500 MHz) d: 3.85 (2H, s, H -7), 6.75
6
2
(2H, d, J = 8.6 Hz, H-3 and H-5), 7.12 (2H, d, J = 8.6 Hz,
H-2 and H-6), 9.49 (1H, s, 4-OH). Selected HMBC
correlations: H-7/C-1, C-2, C-6, C-8; OH/C-3, C-4, C-5.
C-2, C-6; OCH /C-3; H-6/C-4; H-2/C-4; 7-OH/C-1, C-7;
3
0
H-1 /C-4.
3
,4-Dimethoxybenzyl alcohol 7-O-b-D-glucopyranoside
2
5
(
7) [16]: A white amorphous powder. [a] -39.9° (c 0.33,
Synthesis of 4-hydroxy-3-methoxyphenylacetonitrile (1a)
D
MeOH).
Lobetyolin (8) [17]: A brown syrup. [a]D -26.6° (c 1.89,
MeOH).
2
5
4-Hydroxy-3-methoxyphenylacetonitrile (1a) was synthe-
sized as described elsewhere but with slight modifications
[
18]. Briefly, a mixture of 4-hydroxy-3-methoxybenzyl
Enzymatic hydrolysis of 1–5
alcohol (1.23 g, 8.0 mmol) and NaCN (472 mg, 9.6 mmol)
in DMF (20 ml) was stirred under nitrogen at 120°C for
24 h. The reaction mixture was cooled and 0.4 ml of water
was added, the mixture was then alkalized with solid
NaOH to more than pH 10, followed by evaporation under
vacuum. Water (1 ml) was added and neutralized with
acetic acid. After the mixture was extracted with CHCl3,
Each glycoside (10.0 mg) was incubated with emulsin
(
b-glucosidase from almonds: G-0395: Lot No. 119H4029;
.8 units; Sigma, MO, USA) in McIlvaine buffer (pH 5.0)
6
at 37°C for 4 h. The reaction mixture was diluted with
H O and passed through a Sep-pak ODS cartridge
2
(
Waters, MA, USA), eluting with H O and MeOH, to
2
the extract was washed with H O three times and dried
2
afford an H O eluate and a MeOH eluate, respectively. In
2
over anhydrous Na SO , followed by evaporation under
2
4
the case of 1–4, the MeOH eluate was purified by reverse-
phase preparative HPLC (40% MeOH). Finally, 1a was
obtained as aglycone in the case of 1–3, and 4a and 5a
were obtained in the case of 4 and 5, respectively. The
vacuum. The extract (1.26 g) was purified by silica gel CC
[hexane–acetone (2:1)] and then ODS CC (30% MeOH) to
give 1a (315 mg, 1.9 mmol) as an orange solid.
H O eluate was analyzed by HPLC under the following
2
Synthesis of 4-hydroxyphenylacetonitrile (4a)
conditions: column, COSMOSIL Sugar-D; solvent, 70%
acetonitrile; flow rate, 1.0 ml/min; detectors, RI-2031 and
4-Hydroxyphenylacetonitrile (4a) was synthesized simi-
larly to 1a but with slight modifications. Briefly, a mixture
of 4-hydroxybenzyl alcohol (1.01 g, 8.15 mmol) and
NaCN (483 mg, 9.9 mmol) in DMF (25 ml) was stirred
under nitrogen at 120°C for 26 h. The reaction mixture was
cooled and 0.5 ml of water was added, then the mixture
OR-2090 plus. Consequently, the constituent of the H O
2
eluate was detected to be D-glucose (t 5.9 min, positive)
R
in the case of 1, 2, 4, and 5, and D-glucose and L-rhamnose
(tR 4.2 min, negative) in the case of 3 by comparison with
an authentic sample.
1
23

J Nat Med (2010) 64:245–251
251
was alkalized with solid NaOH to more than pH 10, fol-
lowed by evaporation under vacuum. After the addition of
water (17.5 ml) and neutralization with acetic acid, the
10. Yao S, Liu R, Huang X, Kong L (2007) Preparative isolation and
purification of chemical constituents from the root of Adenophora
tetraphylla by high-speed counter-current chromatography with
evaporative light scattering detection. J Chromatogr A 1139:254–
262
mixture was extracted with CHCl . The extract was washed
3
with H O three times and dried over anhydrous Na SO ,
2
11. Kuang HX, Shao CJ, Kasai R, Ohtani K, Tian ZK, Xu JD, Tanaka
O (1991) Phenolic glycosides from roots of Adenophora tetra-
phylla collected in Heilongjiang, China. Chem Pharm Bull
2
4
followed by evaporation under vacuum. The extract
(
(
964 mg) was purified by silica gel CC [hexane–acetone
3
9:2440–2442
2:1)] to give 4a (546 mg, 4.1 mmol) as an orange solid.
12. Miyazawa M, Horiuchi E, Kawata J (2007) Components of the
essential oil from Adenophora triphylla var japonica. J Essent Oil
Res 20:125–127
13. Ueyama Y, Furukawa K (1987) Volatile components of shajin.
Nippon Nogeikagaku Kaishi 61:1577–1582
Acknowledgments We express our appreciation to Ms. S. Matsu-
bayashi and Y. Odanaka of Showa University for the MS and NMR
spectra measurements. This study was financially supported by a
‘
‘High-Tech Research Center’’ Project for Private Universities:
14. Asano N, Nishida M, Miyauchi M, Ikeda K, Yamamoto M, Kizu
H, Kameda Y, Watson AA, Nash RJ, Fleet GWJ (2000) Poly-
hydroxylated pyrrolidine and piperidine alkaloids from Adeno-
phora triphylla var japonica (Campanulaceae). Phytochemistry
matching fund subsidy from MEXT (Ministry of Education, Culture,
Sports, Science and Technology) of Japan, 2007–2009.
5
3:379–382
1
1
5. Konno C, Saito T, Oshima Y, Hikino H, Kabuto C (1981)
Structure of methyl adenophorate and triphyllol, triterpenoids of
Adenophora triphylla var japonica roots. Planta Med 42:268–274
6. Kanho H, Yaoya S, Kawahara N, Nakane T, Takase Y, Masuda
K, Kuroyanagi M (2005) Biotransformation of benzaldehyde-
type and acetophenone-type derivatives by Pharbitis nil hairy
roots. Chem Pharm Bull 53:361–365
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