The absolute stereostructures of cyanogenic glycosides, hydracyanosides A, B, and C, from the leaves and stems of Hydrangea macrophylla

Article abstract of DOI:10.1016/j.tetlet.2009.05.111

Three new cyanogenic glycosides named hydracyanosides A (1), B (2), and C (3) were isolated from the leaves and/or stems of Hydrangea macrophylla in China. The absolute stereostructures of hydracyanosides were characterized on the basis of chemical and ph

Full text of DOI:10.1016/j.tetlet.2009.05.111

Tetrahedron Letters 50 (2009) 4639–4642
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Tetrahedron Letters
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The absolute stereostructures of cyanogenic glycosides, hydracyanosides A,
B, and C, from the leaves and stems of Hydrangea macrophylla
Seikou Nakamura a, Zhibin Wang a,b, Fengming Xu a, Hisashi Matsuda a, Lijun Wu b, Masayuki Yoshikawa a,
*
a Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
b School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new cyanogenic glycosides named hydracyanosides A (1), B (2), and C (3) were isolated from the
leaves and/or stems of Hydrangea macrophylla in China. The absolute stereostructures of hydracyanosides
were characterized on the basis of chemical and physiochemical evidence including single crystal X-ray
crystallographic analysis. To the best of our knowledge, this is the first scientific report of cyanogenic
glycosides from Hydrangea plants.
Received 20 April 2009
Revised 25 May 2009
Accepted 28 May 2009
Available online 3 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Hydrangea macrophylla
Saxifragaceae
Cyanogenic glycoside
Hydracyanoside
1. Introduction
2. Results and discussion
A Saxifragaceae plant, Hydrangea macrophylla (Thunb.) Ser., is
native to Japan and widely cultivated in many countries includ-
ing Japan and China. The blossoms are mainly used for ornamen-
tal purposes. In June 2008, the cases of food poisoning that
present symptoms of vomiting, etc. after eating the leaves of H.
macrophylla were generated in Osaka and Ibaraki prefectures of
Japan. At first, the Ministry of Health, Labor, and Welfare of Japan
advised the possibility that the food poisoning was caused by
cyanogenic glycosides in the leaves of H. macrophylla. However,
since the scientific information for the cyanogenic glycoside con-
stituents in the leaves of H. macrophylla are insufficient, the no-
tice was corrected. Actually, to the best of our knowledge,
there is no scientific evidence concerning cyanogenic glycoside
constituents of Hydrangea plants. Thus, the findings of cyanogenic
glycosides from H. macrophylla were ambiguous, so that the
cause of the food poisoning remained to be not clarified. In the
course of our chemical and pharmacological studies on Hydrangea
plants,1 we tried to examine the chemical constituents from the
leaves and stems of H. macrophylla and isolated three new cyano-
genic glycosides named hydracyanosides A (1), B (2), and C (3)
together with a known cyanogenic glycoside, taxiphyllin (5).2,3
This Letter deals with the absolute stereostructure elucidation
of these three new constituents (1–3).
The fresh leaves (2.2 kg) and stems (3.0 kg) of H. macrophylla
cultivated in Sichuan province of China4 were finely cut and ex-
tracted with MeOH under reflux to provide a MeOH extract
[573 g (26.0%) from leaves, 361 g (12.0%) from stems]. The MeOH
extract was partitioned into an EtOAc–H2O (1:1, v/v) mixture to
furnish an EtOAc-soluble fraction (8.4% from leaves, 2.6% from
stems) and an aqueous phase. The aqueous phase was further
extracted with n-BuOH to give an n-BuOH-soluble fraction (6.5%
from leaves, 3.3% from stems) and a H2O-soluble fraction (11.1%
from leaves, 6.1% from stems). The n-BuOH-soluble fractions from
the leaves and stems were subjected to normal- and reversed-
phase silica gel column chromatographies, and finally HPLC to give
hydracyanosides A [1, 1.92 g (0.089%) from leaves, 3.89 g (0.13%)
from stems], B [2, 11 mg (0.00037%) from stems], and C [3,
12 mg (0.00041%) from stems] together with taxiphyllin [5, 6 mg
(0.00027%) from leaves]. In a similar method, hydracyanoside A
(1, 201 mg, 0.0079%) was obtained from the EtOAc-soluble fraction
of the stems (Fig. 1).
Hydracyanoside A (1),5
½
a 2D5
ꢀ
ꢁ67.4 (MeOH), was isolated as a
white powder. The IR spectrum of 1 showed an absorption band
at 2365 cmꢁ1 ascribable to cyano group and broad bands at 3380
and 1080 cmꢁ1 suggestive of an oligoglycoside structure. In the
positive-ion FABMS of 1, a quasimolecular ion peak was observed
at m/z 364 (M+Na)+. The HRFABMS analysis revealed the molecular
formula of 1 to be C15H19NO8. Acid hydrolysis of 1 liberated D-glu-
cose, which was identified by HPLC analysis using an optical rota-
* Corresponding author. Tel.: +81 75 595 4633; fax: +81 75 595 4768.
E-mail address: myoshika@mb.kyoto-phu.ac.jp (M. Yoshikawa).
tion detector.6 The 1H (CD3OD) and 13C NMR (Table 1) spectra7 of 1
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.05.111

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S. Nakamura et al. / Tetrahedron Letters 50 (2009) 4639–4642
Figure 1. Structures of hydracyanosides A (1), B (2), and C (3).
Table 1
C-2. In addition, the NOE correlation between methoxy proton
13C NMR data for 1–3
and H-50 was observed in the difference NOESY experiment on 1.
Thus, 1 was characterized to be a cyanogenic glycoside with a
3-hydroxy-4-methoxyphenyl moiety. Next, the absolute configura-
tion at the C-2 position of 1 was characterized by comparison of
the 2- and 100-proton signals of 1 with those of known cyanogenic
glycosides on the 1H NMR (CD3OD) spectrum (Fig. 3, Table 2).
Namely, Seigler et al. reported that the 2- and 100-proton signals
in the 1H NMR (CD3OD) spectrum of prunasin [4, d 5.89 (H-2),
4.25 (H-100)]8 and taxiphyllin [5, d 5.78 (H-2), 4.17 (H-100)]3 having
Position
1
2
3
1
119.6
68.1
126.8
115.7
147.9
150.4
112.6
121.0
56.5
101.2
74.5
77.6
71.3
78.0
62.6
—
119.7
68.7
119.6
68.1
116.8
130.9
125.1
160.4
125.1
130.9
—
100.7
74.1
87.4
70.0
2
10
127.4
115.8
148.2
150.4
112.6
120.8
56.4
20
30
40
50
60
40-O-Me
100
102.2
74.7
200
300
77.6
71.5
77.9
69.9
400
500
78.0
600
62.7
105.2
75.7
77.8
71.6
78.1
62.6
1000
2000
3000
4000
5000
6000
104.9
75.3
—
—
—
—
77.8
71.6
77.9
62.7
—
Measured in CD3OD at 150 MHz.
indicated the presence of a methoxy group [d 3.85 (3H, s)], a
methine bearing an oxygen function [d 5.77 (1H, s, H-2)], an
ABX-type aromatic ring [d 6.94 (1H, d, J = 8.2 Hz, H-50), 7.02 (1H,
Figure 3. Known cyanogenic glycosides (4–7).
dd, J = 2.0, 8.2 Hz, H-60), 7.05 (1H, d, J = 2.0 Hz, H-20)], a b-
D-gluco-
Table 2
pyranosyl moiety [d 4.27 (1H, d, J = 7.6 Hz, H-100)], and a cyano
group (dc 119.6, C-1). As shown in Figure 2, the DQF COSY experi-
ment on 1 indicated the presence of partial structures written in
bold lines, and in the HMBC experiment, long-range correlations
were observed between the following protons and carbons: H-2
and C-1, 10, 20, 60; H-20 and C-40, 60; H-5 and C-10, 30; H-100 and
Comparison of the chemical shifts of 1ꢁ3 and known cyanogenic glycosides on the 1H
NMR (CD3OD) spectrum
H
1
2
3
4
5
6
7
2
100
5.77
4.27
5.73
4.29
5.79
4.23
5.89
4.25
5.78
4.17
6.03
4.67
5.90
4.67
Figure 2. DQF COSY, HMBC, and NOE correlations of hydracyanosides A (1), B (2), and C (3).

S. Nakamura et al. / Tetrahedron Letters 50 (2009) 4639–4642
4641
Figure 4. Absolute stereostructure of hydracyanoside A (1).
R orientation were shifted upfield relative to those of sambunigrin
[6, d 6.03 (H-2), 4.67 (H-100)]8 and dhurrin [7, d 5.90 (H-2), 4.67
(H-100)]3 having S orientation. The 2- and 100-proton signals of 1
were observed at d 5.77 and 4.27, respectively, so that the C-2 ori-
entation of 1 was characterized to be R. Finally, the absolute ste-
In conclusion, a cyanogenic glycoside, hydracyanoside A (1),
was isolated as a major constituent from the leaves and stems of
H. macrophylla cultivated in Sichuan province of China. In addition,
two cyanogenic glycosides, hydracyanosides B (2) and C (3), were
also isolated from the stems. To the best of our knowledge, this
is the first scientific report of the isolation of cyanogenic glycosides
from Hydrangea plants. In the cases of food poisoning after eating
the leaves of H. macrophylla, the possibility of the involvement of
cyanogenic glycosides was suggested. Continuously, toxicity assay
of these cyanogenic glycosides should be subjected further.
reostructure at the 2-position in
1 was confirmed to be R
orientation by the single crystal X-ray crystallographic analysis of
the penta-O-acetyl derivative (1a) from 1 upon acetylation reaction
with acetic anhydride in pyridine. The ORTEP representation of the
X-ray structure on 1 is presented in Figure 4.9 Consequently, the
total structure of hydracyanoside A (1) was determined as shown.
Hydracyanoside B (2),10
½
a 2D6
ꢀ
ꢁ26.0 (MeOH), was isolated as a
Acknowledgments
white powder. The IR spectrum of 2 showed absorption bands at
3400, 2365, and 1080 cmꢁ1 ascribable to hydroxyl, cyano, and
ether functions. The molecular formula C21H29NO13 was deter-
mined from the positive-ion FABMS at m/z 526 (M+Na)+ and by
This research was supported by the 21st COE program and Aca-
demic Frontier Project from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.
HRFABMS measurement. Acid hydrolysis of 2 liberated D-glucose,
which was identified by HPLC analysis using an optical rotation
detector.6 The proton and carbon signals of 2 in the 1H and 13C
NMR spectra were superimposable on those of 1, except for the sig-
References and notes
nals due to the 6-positon of the 2-O-b-D-glucopyranoside moiety
1. (a) Zhang, H.; Matsuda, H.; Yamashita, C.; Nakamura, S.; Yoshikawa, M.
Eur. J. Pharmacol. 2009, 606, 255–261; (b) Kurume, A.; Kamata, Y.;
Yamashita, M.; Wang, Q.; Matsuda, H.; Yoshikawa, M.; Kawasaki, I.;
Ohta, S. Chem. Pharm. Bull. 2008, 56, 1264–1269; (c) Matsuda, H.; Wang,
Q.; Matsuhira, K.; Nakamura, S.; Yuan, D.; Yoshikawa, M. Phytomedicine
2008, 15, 177–184; (d) Zhang, H.; Matsuda, H.; Kumahara, A.; Ito, Y.;
Nakamura, S.; Yoshikawa, M. Bioorg. Med. Chem. Lett. 2007, 17, 4972–4976;
(e) Wang, Q.; Matsuda, H.; Matsuhira, K.; Nakamura, S.; Yuan, D.;
Yoshikawa, M. Biol. Pharm. Bull. 2007, 30, 388–392.
on 2. The planar structure of 2 was characterized by means of
DQF COSY and HMBC experiments,7 which showed long-range cor-
relations between the following protons and carbons: H-100 and
C-2; H-1000 and C-600 (Fig. 2). On the basis of the above-mentioned
evidence, the structure of 2 was characterized to be a cyanogenic
diglycoside with the same aglycon as 1. Finally, by comparison of
the 2- and 100-proton signals of 2 with those of known cyanogenic
glycosides on the 1H NMR (CD3OD) spectrum as that used to char-
acterize the configuration at the C-2 position of 1, the C-2 orienta-
tion of 2 was determined to be R (Table 2). Consequently, the
structure of hydracyanoside B (2) was determined as shown.
2. Towers, G. H. N.; McInnes, A. G.; Neish, A. C. Tetrahedron 1964, 20, 71–77.
3. Seigler, D. S.; Pauli, G. F.; Fröhlich, R.; Wegelius, E.; Nahrstedt, A.; Glander, K. E.;
Ebinger, J. E. Phytochemistry 2005, 66, 1567–1580.
4. The fresh leaves and stems of H. macrophylla, which were cultivated in Sichuan
province of China, were collected in 2008. A voucher of the plant is on file in
our laboratory (Phamacognosy-2008-HM).
5. Hydracyanoside A (1): A white powder, ½a D25
ꢁ67.4 (c 0.02, MeOH). HRFABMS:
ꢀ
Hydracyanoside C (3),11
½
a 2D5
ꢀ
ꢁ16.7 (MeOH), was isolated as a
Calcd for C15H19NO8Na (M+Na)+: 364.1008. Found: 364.1012. UV (MeOH) kmax
(log
e
) nm 282 (3.68), 237 (3.96), 209 (4.54). IR (KBr) kmax cmꢁ1 3380, 2943,
white powder. The IR spectrum of 2 showed absorption bands
ascribable to hydroxyl, cyano, and ether functions. The molecular
formula C20H28NO12 was determined from the positive-ion FABMS
at m/z 496 (M+Na)+ and by HRFABMS measurement. Acid hydroly-
2365, 1618, 1516, 1080 cmꢁ1
.
1H NMR (600 MHz, CD3OD) d 3.74 (1H, dd, J = 2.0,
12.0 Hz, Ha-600), 3.94 (1H, dd, J = 6.2, 12.0 Hz, Hb-600), 3.85 (3H, s, OCH3), 4.27
(1H, d, J = 7.6 Hz, H-100), 5.77 (1H, s, H-2), 6.94 (1H, d, J = 8.2 Hz, H-50), 7.02 (1H,
dd, J = 2.0, 8.2 Hz, H-60), 7.05 (1H, d, J = 2.0 Hz, H-20). 13C NMR data (150 MHz,
CD3OD) dC: given in Table 1. FABMS: m/z 364 [M]+.
sis of 3 liberated D
-glucose.6 The 1H (CD3OD) and 13C NMR (Table 1)
6. (a) Sugimoto, S.; Nakamura, S.; Yamamoto, S.; Yamashita, C.; Oda, Y.; Matsuda,
H.; Yoshikawa, M. Chem. Pharm. Bull. 2009, 57, 257–261; (b) Nakamura, S.;
Hongo, M.; Sugimoto, S.; Matsuda, H.; Yoshikawa, M. Phytochemistry 2008, 69,
1565–1572.
spectra7 of 3 indicated the presence of a methine bearing an oxy-
gen function [d 5.79 (1H, s, H-2)], an A2B2-type aromatic ring
[d 6.84 (2H, d, J = 8.2 Hz, H-30,50), 7.39 (2H, d, J = 8.2 Hz, H-20,60)],
7. The 1H and 13C NMR spectra of 1–3 were assigned with the aid of distortionless
enhancement by polarization transfer (DEPT), double quantum filter
correlation spectroscopy (DQF COSY), heteronuclear multiple quantum
coherence (HMQC), and heteronuclear multiple bond connectivity (HMBC)
experiments.
two b-D
-glucopyranosyl moieties [d 4.23 (1H, d, J = 7.6 Hz, H-100),
4.51 (1H, d, J = 8.2 Hz, H-1000)], and a cyano group (dc 119.6, C-1).
The planar structure of 3 was characterized by means of DQF COSY
and HMBC experiments, which showed long-range correlations be-
tween the following proton and carbon: H-100 and C-2; H-1000 and C-
300 (Fig. 2). Finally, by using a similar NMR method as that used to
characterize the configuration at the C-2 position of 2, the C-2 ori-
entation of 3 was determined to be R (Table 2). Consequently, the
structure of hydracyanoside B (2) was determined as shown.
8. Seigler, D. S.; Pauli, G. F.; Nahrstedt, A.; Leen, R. Phytochemistry 2002, 60, 873–
882.
9. Crystal data for acyl derivative (1a) of hydracyanoside A (1). C25H29NO13, MW
551.50, T = 294 K, k = 1.54187 Å, monoclinic, space group P212121 (#19),
a = 8.24352(15) Å, b = 16.7081(3) Å, c = 20.7844(4) Å, V = 2862.71(9) Å3, Z = 4,
Dcalcd = 1.155 mg/m3,
l
(Cu K
a
) = 8.960 cmꢁ1, F(0 0 0) = 1160.00, crystal size,
0.35 ꢂ 0.08 ꢂ 0.07 mm. No. of reflections measured: total, 33343; unique, 5184

4642
S. Nakamura et al. / Tetrahedron Letters 50 (2009) 4639–4642
H-20). 13C NMR data (150 MHz, CD3OD) dC: given in Table 1. FABMS: m/z 526
(Rint = 0.033). Refinement method: full-matrix least-squares on F2, goodness of
fit indicator 1.011, final R1[I > 2.00
r
(I)] = 0.0509 maximum peak in final Diff.
[M]+.
Map and minimum peak 0.49 eꢁ/Å3 and ꢁ0.45 eꢁ/Å3.
11. Hydracyanoside C (3): A white powder, ½a D25
ꢁ16.7 (c 0.15, MeOH). HRFABMS:
ꢀ
10. Hydracyanoside B (2): A white powder, ½a D26
ꢀ
ꢁ26.0 (c 0.30, MeOH). HRFABMS:
Calcd for C20H28NO12Na (M+Na)+: 496.1431. Found: 496.1435. UV (MeOH) kmax
Calcd for C21H29NO13Na (M+Na)+: 526.1537. Found: 526.1542. UV (MeOH) kmax
(log e
) nm 275 (3.41), 232 (4.02), 203 (4.23). IR (KBr) kmax cmꢁ1 3400, 2924,
(log
e
) nm 282 (3.62), 236 (3.87), 207 (4.51). IR (KBr) kmax cmꢁ1 3400, 2926,
2363, 1616, 1518, 1080. 1H NMR (600 MHz, CD3OD) d 4.23 (1H, d, J = 7.6 Hz, H-
100), 4.51 (1H, d, J = 8.2 Hz, H-1000), 5.79 (1H, s, H-2), 6.84 (2H, d, J = 8.2 Hz, H-
30,50), 7.39 (2H, dd, J = 8.2 Hz, H-20,60). 13C NMR data (150 MHz, CD3OD) dC:
given in Table 1. FABMS: m/z 496 [M]+.
2365, 1618, 1516, 1080. 1H NMR (600 MHz, CD3OD) d 3.87 (3H, s, OCH3), 4.29
(1H, d, J = 7.6 Hz, H-100), 4.51 (1H, d, J = 7.6 Hz, H-1000), 5.73 (1H, s, H-2), 6.96
(1H, d, J = 8.8 Hz, H-50), 7.03 (1H, dd, J = 2.0, 8.8 Hz, H-60), 7.03 (1H, d, J = 2.0 Hz,