Iridoid glycosides from the roots of Scrophularia ningpoensis Hemsl.

Article abstract of DOI:10.1016/j.cclet.2014.05.007

Two new iridoid glycosides, named scrophularianoids A (1) and B (2), were isolated from the roots of Scrophularia ningpoensis. The chemical structures were established on the basis of extensive analyses of spectroscopic data. Compounds 1 and 2 were inactive in our preliminary in vitro myocardial protective bioassay.

Full text of DOI:10.1016/j.cclet.2014.05.007

G Model
CCLET 2985 1–3
Chinese Chemical Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
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Original article
3
Iridoid glycosides from the roots of Scrophularia ningpoensis Hemsl.
Q1 Ling-Juan Zhu a,b, Cheng Qiao a,b, Xiu-Yu Shen a,b, Xue Zhang a,b, , Xin-Sheng Yao
*
a,b,c,**
4
5
6
7
a College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
b Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
c Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Two new iridoid glycosides, named scrophularianoids A (1) and B (2), were isolated from the roots of
Scrophularia ningpoensis. The chemical structures were established on the basis of extensive analyses of
spectroscopic data. Compounds 1 and 2 were inactive in our preliminary in vitro myocardial protective
bioassay.
Received 3 March 2014
Received in revised form 4 April 2014
Accepted 16 April 2014
Available online xxx
ß 2014 Xue Zhang and Xin-Sheng Yao. Published by Elsevier B.V. on behalf of Chinese Chemical
Society. All rights reserved.
Keywords:
Scrophularia ningpoensis
Iridoid glycosides
Scrophularianoid A
Scrophularianoid B
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9
1. Introduction
2. Experimental
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30
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15
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The common figwort Scrophularia ningpoensis Hemsl. (Scro-
phulariaceae) is one of over 300 known species of the genus
Scrophularia, which is well known for its variety of iridoids [1]. Its
dried roots are used as anti-inflammatory agents for the
treatment of fever, swelling, sore throat, and constipation
2.1. General
The dried roots of S. ningpoensis Hemsl. were collected from 31
Pan’an, Zhejiang province, China, in September 2011, and 32
authenticated by Zeng-Xi Guo, director pharmacist of TCM, at 33
Zhejiang Institute for Food and Drug Control, Zhejiang Province, 34
China. A voucher specimen was deposited at College of Traditional 35
Chinese Materia Medica, Shenyang Pharmaceutical University, 36
[2,3].
A series of chemical constituents including iridoids,
phenylethanoid glycosides, triterpene saponins, flavones, and
volatile oils have been isolated from S. ningpoensis [4–6]. Our
previous study on this plant led to the isolation of monoterpene
pyridine alkaloids, iridoid glycosides, phenylpropanoid glyco-
sides and phenolic acids, some of which showed inhibitory
activity against KCl induced [Ca2+]i increase in rat cardiomyo-
cytes and cardio-protective effects against the apoptosis induced
by H2O2 [7,8]. Further investigations on this plant have resulted
in the isolation of two new iridoid glycosides, named scrophu-
larianoids A (1) and B (2) (Fig. 1). In this paper, the isolation and
structural elucidation of the two new compounds and their
activity in an in vitro myocardial protective bioassay are
reported.
China.
37
2.2. Extraction and isolation
38
The dried roots of S. ningpoensis (25 kg) were chopped into 39
small pieces and extracted with 70% EtOH under reflux twice for 40
2 h each time. The combined extracts were concentrated by 41
evaporation to yield a residue of 5.6 kg, which was chromato- 42
graphed on a D101 column eluted with EtOH/H2O gradiently. The 43
50% EtOH eluted fraction (250.0 g) was then separated using silica 44
gel column chromatography (CC) by gradient elution with CH2Cl2/ 45
MeOH (100:0 ! 0:100) to afford 10 fractions. Fraction 8 (91.7 g) 46
was fractionated on silica gel CC eluted with CH2Cl2/MeOH 47
(95:5 ! 0:100) to give 10 subfractions. Subfraction 5 (10.2 g) 48
was applied to an ODS column using stepwise gradient mixtures of 49
MeOH/H2O (10:90 ! 100:0) system. Subfraction 5.5 (1.7 g) was 50
further passed over an HW-40 column with MeOH/H2O 51
(10:90 ! 80:20) gradiently. Purification of the eluate of 10% 52
*
Corresponding author at: College of Traditional Chinese Materia Medica,
Shenyang Pharmaceutical University, Shenyang 110016, China.
**
Q2
Corresponding author at: Key Laboratory of Structure-Based Drug Design &
Discovery of Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, China.
E-mail addresses: syzxalice@163.com (X. Zhang), tyaoxs@jnu.edu.cn (X.-S. Yao).
http://dx.doi.org/10.1016/j.cclet.2014.05.007
1001-8417/ß 2014 Xue Zhang and Xin-Sheng Yao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Please cite this article in press as: L.-J. Zhu, et al., Iridoid glycosides from the roots of Scrophularia ningpoensis Hemsl., Chin. Chem. Lett.
(2014), http://dx.doi.org/10.1016/j.cclet.2014.05.007

G Model
CCLET 2985 1–3
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L.-J. Zhu et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
The mixture was evaporated to dryness in vacuo, and then the
residue was dissolved in H2O and extracted with CHCl3. The
aqueous layer was collected. After drying in vacuo, the residue was
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
dissolved in pyridine (1 mL) containing
(1 mg) (Sigma, USA) and the mixture was heated at 60 8C for 1 h.
Then, o-tolyl isothiocyanate (5 L) (Alfa Aesar, UK) was added to
L-cysteine methyl ester
m
the mixture, which was heated at 60 8C for 1 h. The reaction
mixture was directly analyzed by HPLC. Analytical HPLC was
performed on a reversed-phase C18 column (250 mm ꢂ 4.6 mm
i.d., 5
mm, Phenomenex, Gemini) at 35 8C with isocratic elution of
Fig. 1. Chemical structures of compounds 1 and 2.
25% CH3CN containing 0.1% formic acid for 40 min at a flow rate
0.8 mL/min. Peaks were detected by a UV detector at 250 nm.
The derivatives of 1 and 2 both gave one peak at tR 21.6 min. The
53
54
MeOH by semi-preparative HPLC with 44% MeOH gave 1 (7.0 mg)
and 2 (5.4 mg).
derivatives of
to the same method. The peaks were recorded at tR 20.2 (
and 21.6 ( -glucose) min, respectively.
D
-glucose and
L
-glucose (Sigma, USA) were subjected
L-glucose)
55
2.3. Scrophularianoid A
D
56
57
58
59
60
Yellowish amorphous powder; ½a D25
ꢀ
ꢁ 40:8 (c 0.5, MeOH). UV
3. Results and discussion
Compound 1 was isolated as a yellowish amorphous powder,
87
(MeOH) lmax (log e ) nmax
): 232 (3.16), 279 (4.26) nm; IR (KBr, cmꢁ1
3366, 2936, 1711, 1637, 1577, 1496; HR-ESI-MS m/z 533.1637
[M+Na]+, calcd. 533.1635 for C24H30O12Na; 1H and 13C NMR data,
see Table 1.
88
89
90
91
92
93
94
95
96
97
98
99
½
a 2D5
ꢀ
ꢁ 40:8 (c 0.5, MeOH). The molecular formula C24H30O12 was
determined on the basis of HR-ESI-MS (m/z 533.1637 [M+Na]+). Its
UV spectrum exhibited the characteristic cinnamoyl chromophore
absorption at 279 nm. Its IR spectrum displayed absorption bands
at 1711 cmꢁ1 for conjugated ester carbonyl group(s), 1637, 1577,
and 1496 cmꢁ1 for aromatic ring(s), and 3366 cmꢁ1 for OH
group(s). The 1H NMR spectrum of 1 showed resonances for a
61
2.4. Scrophularianoid B
62
63
64
65
66
Yellowish amorphous powder; ½a D25
ꢀ
ꢁ 50:0 (c 0.5, MeOH). UV
(MeOH)
l
max (log
e
): 232 (3.41), 279 (4.26) nm; IR (KBr, cmꢁ1
)
nmax
3370, 2926, 1691, 1635, 1578, 1514; HR-ESI-MS m/z 565.1904
[M+Na]+, calcd. 565.1897 for C25H34O13Na; 1H and 13C NMR data,
see Table 1.
cinnamoyl group at
d
7.62–7.65 (m, 2H, H-200, 600), 7.40–7.42 (m, 3H,
H-300, 400, 500), 7.75 (d, 1H, J = 16.0 Hz, H-700), and 6.59 (d, 1H,
J = 16.0 Hz, H-800), and a sugar moiety including an anomeric
proton signal at
proton signals at
(m, 2H, H-40, 50), 3.86 (br d, 1H, J = 11.6 Hz, H-6a0) and 3.66 (m, 1H,
H-6b0). Remaining resonances indicated a characteristic iridoid
d
4.58 (d, 1H, J = 8.0 Hz, H-10) together with five
d
67
2.5. Sugar analysis
3.22 (m, 1H, H-20), 3.38 (m, 1H, H-30), 3.30
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
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69
70
The absolute configuration of the sugar moiety was determined
by the method reported by Tanaka et al. [9]. Compounds 1 and 2
(2 mg, each) were heated in 2 mol/L aqueous HCl for 2 h at 90 8C.
skeleton at
d 5.95 (d, 1H, J = 0.8 Hz, H-1), 6.37 (d, 1H, J = 6.4 Hz,
H-3), 5.00 (dd, 1H, J = 6.4, 1.2 Hz, H-4), 3.80 (t, 1H, J = 4.0 Hz, H-6),
2.07 (dd, 1H, J = 14.0, 4.8 Hz, H-7a), 1.84 (dd, 1H, J = 14.0, 3.2 Hz,
H-7b), and 2.73 (br s, 1H, H-9), and two methylene protons at
(dd, 2H, J = 11.6, 6.8 Hz,). The 13C NMR spectrum of
d
1
4.22
in
Table 1
1H and 13C NMR data for compounds 1 and 2 (1H: 400 MHz, 13C: 100 MHz; in
combination with the DEPT and HSQC spectra indicated the
presence of 24 carbons including a cinnamoyl group at 168.6,
CD3OD).
d
No.
1
2
146.9, 135.9, 131.7, 130.2 (ꢂ2), 129.5 (ꢂ2), and 118.8, a sugar
dC
dH
dC
dH
moiety at
skeleton at
d
d
99.8, 78.4, 77.7, 74.7, 71.8, and 62.9, and an iridoid
143.1, 108.5, 92.7, 79.3, 78.0, 72.9, 69.5, 59.2, and 43.2.
1
3
4
5
6
7
92.7
5.95 d (0.8)
96.8
5.97 s
After acid hydrolysis and derivatization of 1 using the reported
method [9], an HPLC analysis of the derivatives revealed the
143.1
108.5
72.9
6.37 d (6.4)
98.2
71.2
82.9
73.1
47.2
4.99 d (8.0)
3.19 d (8.0)
5.00 dd (6.4, 1.2)
presence of
D-glucose. Additionally, the b-configuration was
78.0
3.80 t (4.0)
4.06 m
2.41 m
2.25 m
established based on the coupling constant (8.0 Hz) of the
anomeric proton.
According to the 1H and 13C NMR data, an iridoid glycoside
similar to harpagoside was proposed [2], except for the
absence of a methyl group and the presence of an oxygenated
43.2
2.07 dd (14.0, 4.8)
1.84 dd (14.0, 3.2)
8
79.3
59.2
69.5
88.1
57.0
23.1
57.5
100.3
74.8
78.1
72.2
78.2
63.4
9
2.73 br s
2.79 br s
1.62 s
10
OCH3
10
4.22 dd (11.6, 6.8)
3.54 s
methylene (
d 69.5) at C-8. HMBC correlation from d 2.73 (H-9) to
99.8
74.7
77.7
71.8
78.4
62.9
4.58 d (8.0)
3.22 m
4.68 d (7.6)
3.28 m
20
d
69.5 further confirmed the above deduction. The partial
30
3.38 m
3.41 m
structures of the iridoid, cinnamoyl, and glucose moiety were
assigned by detailed analyses of 1H –1H COSY, HSQC and HMBC
spectra. The glucose moiety was attached at C-1 of iridoid, based
on the HMBC correlations observed at H-1/C-10 and H-10/C-1.
Furthermore, the linkage of the cinnamoyl group to iridoid
was established at C-8 by the HMBC long-range correlation at
H-10/C -900.
40
3.30 m
3.33 m
50
3.30 m
3.35 m
60
3.86 br d (11.6)
3.66 m
3.93 dd (12.0, 2.0)
3.70 m
100
200
300
400
500
600
700
800
900
135.9
129.5
130.2
131.7
130.2
129.5
146.9
118.8
168.6
136.0
129.4
130.2
131.7
130.2
129.4
146.3
120.2
168.7
7.62–7.65 m
7.40–7.42 m
7.40–7.42 m
7.40–7.42 m
7.62–7.65 m
7.75 d (16.0)
6.59 d (16.0)
7.58–7.61 m
7.39–7.41 m
7.39–7.41 m
7.39–7.41 m
7.58–7.61 m
7.66 d (16.0)
6.49 d (16.0)
The relative configuration of 1 was determined by comparison
with reported literature values. The chemical shift of C-1 at
indicated a cis-fused iridoid by comparing with the chemical shift
of C-1 ( 100–105) in trans-fused iridoids [10]. The C-1 shift
92.7) together with the difference between the C-3 and C-4 shifts
d 92.7
d
(
d
Please cite this article in press as: L.-J. Zhu, et al., Iridoid glycosides from the roots of Scrophularia ningpoensis Hemsl., Chin. Chem. Lett.
(2014), http://dx.doi.org/10.1016/j.cclet.2014.05.007

G Model
CCLET 2985 1–3
L.-J. Zhu et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
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168
169
170
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173
174
175
176
177
178
179
180
181
(
Dd 34.6) demonstrated a 6
b
-O-substituted iridoid compared to
4. Conclusion
182
the C-1 shift (
shifts (Dd > 47) for 6
basis of the above evidence, 1 is believed to be a new iridoid
glycoside, designated as scrophularianoid A.
d
> 99) and the difference between the C-3 and C-4
a
-O-substituted compounds [11]. On the
In conclusion, the 70% EtOH extract of the roots of S. ningpoensis 183
gave two new iridoid glycosides, named scrophularianoids A (1) 184
and B (2). The myocardial protective bioassay indicated that they 185
both had little cardioprotective effect against the apoptosis 186
Compound
2
was obtained as
a yellowish amorphous
powder, ½a 2D5
ꢀ
ꢁ 50:0 (c 0.5, MeOH). HR-ESI-MS gave a quasimo-
induced by H2O2.
187
lecular ion peak at m/z 565.1904 [M+Na]+, corresponding to the
molecular formula C25H34O13. Its UV absorption
lmax at 279 nm
Acknowledgments
188
was indicative of a cinnamoyl chromophore. Its IR spectrum
showed absorption bands of OH groups (3370 cm ꢁ1), conjugated
ester carbonyl group(s) (1691 cm ꢁ1) and aromatic ring(s) (1635,
1578, 1514 cm ꢁ1). The 1H and 13C NMR spectra of 2 were also
The authors thank Ming Zhu and Zeng-Xi Guo, director 189
pharmacists of TCM, at Zhejiang Institute for Food and Drug 190
Control, Zhejiang Province, China for the collection and identifica- 191
tion of the plant materials. We are grateful to Institute of 192
Traditional Chinese Medicine & Natural Products, College of 193
Pharmacy, Ji’nan University for collecting the optical rotation 194
similar to those0o0 f harpagoside [2]. Proton signals at
d 7.58–7.61
(m, 2H, H-200, 6 ), 7.39–7.41 (m, 3H, H-300, 400, 500), 7.66 (d, 1H,
J = 16.0 Hz, H -700), and 6.49 (1H, d, J = 16.0 Hz, H -800), together
with carbon signals at
129.4 (ꢂ2), and 120.2, suggested the presence of a cinnamoyl
moiety. Proton signals at
4.68 (d, 1H, J = 7.6 Hz, H-10), 3.28 (m,
1H, H-20), 3.41 (d, 1H, J = 8.8 Hz, H-30), 3.33 (m, 1H, H-40), 3.35
(m, 1H, H-50), 3.93 (dd, 1H, J = 12.0, 2.0 Hz, H-6a0) and 3.70 (m,
1H, H-6b0), together with carbon signals at
74.8, 72.2, and 63.4, belonged to a sugar moiety. Acid hydrolysis
followed by an HPLC analysis of the derivatives using an
authentic sample as reference [9] confirmed the presence of
glucose. Additionally, the configuration of the anomeric proton
was deduced to be based on the coupling constant (7.6 Hz) of
the anomeric proton.
d
168.7, 146.3, 136.0, 131.7, 130.2 (ꢂ2),
data and HR-ESI-MS spectra.
195
d
Appendix A. Supplementary data
196
d
100.3, 78.2, 78.1,
Supplementary data associated with this article can be found, in 197
the online version, at http://dx.doi.org/10.1016/j.cclet.2014.05.00- 198
7.
199
D-
References
200
b
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The remaining 1H and 13C NMR signals of 2 were similar to
2006, pp. 1060–1063.
[2] Y.M. Li, S.H. Jiang, W.Y. Gao, D.Y. Zhu, Iridoid glycosides from Scrophularia 204
ningpoensis, Phytochemistry 50 (1999) 101–104.
[3] A.T. Nguyen, J. Fontaine, H. Malonne, et al., A sugar ester and an iridoid glycoside 206
from Scrophularia ningpoensis, Phytochemistry 66 (2005) 1186–1191.
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Scrophularia ningpoensis Hemsl., Asian J. Tradit. Med. 2 (2007) 118–123.
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those of harpagoside, except for the absence of an olefinic bond at
C-3 and C-4, and the presence of two oxygenated methines (
71.2) and an additional methoxy group ( 57.5). HMBC correlation
from 3.54 (–OCH3) to 98.2 (C-3) indicated the attachment of the
methoxy group at C-3.
The relative configuration of 2 was determined by comparison
with 1 and further supported by NOESY analysis. The correlations
between H-1 and H-10, H-10 and H-4, and H-4 and H-6 indicated
d 98.2,
d
d
d
[7] L.J. Zhu, Y.L. Hou, X.Y. Shen, et al., Monoterpene pyridine alkaloids and phenolics 214
that C4-OH was in b-orientation. The a-configuration of C3-OCH3
from Scrophularia ningpoensis and their cardioprotective effect, Fitoterapia 88 215
was established based on the coupling constant (J3,4 = 8.0 Hz).
Thus, 2 is a new iridoid derivative and is named scrophularianoid B.
HR-ESI-MS, UV, IR, 1H NMR, 13C NMR, DEPT, 1H–1H COSY, HSQC,
HMBC and NOESY spectra of compound 1–2 are supplied in
Supporting information.
In our preliminary in vitro myocardial protective bioassay,
compounds 1 and 2 were evaluated by the MTT assay. However,
both of them showed little effect against H2O2-induced apoptosis
in cardiomyocytes.
(2013) 44–49.
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ningpoensis Hemsl. and their inhibition of [Ca2+]i increase induced by KCl, Chem. 218
Biodivers. 5 (2008) 1723–1735.
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mers by reversed-phase HPLC, Chem. Pharm. Bull. 55 (2007) 899–901.
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219
221
223
Phytochemistry 49 (1998) 2413–2415.
[11] S. Damtoft, S.R. Jensen, B.J. Nielsen, 13C and 1H NMR spectroscopy as a tool in 224
the configurational analysis of iridoid glucosides, Phytochemistry 20 (1981) 225
2717–2732.
226
Please cite this article in press as: L.-J. Zhu, et al., Iridoid glycosides from the roots of Scrophularia ningpoensis Hemsl., Chin. Chem. Lett.
(2014), http://dx.doi.org/10.1016/j.cclet.2014.05.007