300
C.-J. He et al. / Phytochemistry Letters 6 (2013) 299–301
Table 1
2006), (Z)-p-coumaric acid 4-O-
(E)-feruloyl-4- -glucoside and (Z)-feruloyl-4-
and Tomohiko, 2011).
b
-glucoside (Iorizzi et al., 2001),
1H and 13C NMR Data (400 and 100 MHz, resp., MeOH-d4) of 1 and 2 with J values (in
b
b
-glucoside (Park
Hz) in parentheses.
No.
1
2
The protective activities of the compounds against cardiomyo-
cyte damage induced by pentobarbital sodium in primary cultured
neonatal rat cardiomyocytes were investigated by the MTT
method. The results showed that pentobarbital sodium induced
a significant inhibition of MTT reduction. At concentrations of
dH
dC
dH
dC
1
–
125.6
127.6
147.0
150.5
119.5
123.4
168.4
125.9
150.1
120.3
121.0
154.5
117.5
166.9
104.9
74.7
–
121.7
132.7
117.6
155.1
121.7
125.6
169.2
127.3
149.4
120.1
120.7
154.2
117.2
166.6
104.1
74.6
2
–
–
3
–
7.41 d (2.8)
4
–
–
10
mM, 1 mM, 0.1 mM, and 0.01 mM, compound 1 increased the cell
5
7.00 d (8.8)
7.04 dd (8.8, 2.8)
viability from 39.7% to 50.8%, 50.9%, 48.8%, and 46.9%, respectively,
and recovered beating rhythm when examined under a micro-
scope. Meanwhile, compound 2 increased the cell viability to 49.0%
6
7.21 d (8.8)
8.21 d (8.8)
7
–
–
10
–
–
20
–
–
at a concentration of 10
M, 0.1 M, and 0.01
concentration of 10 M, were inactive in the assay against
m
M, but was inactive at concentrations of
30
7.31 d (8.8)
6.95 dd (8.8, 3.2)
–
7.25 d (8.8)
6.94 dd (8.8, 2.8)
–
1
m
m
m
M. The known compounds, at
a
40
m
50
60
7.33 d (3.2)
–
7.29 d (2.8)
–
cardiomyocyte damage in primary cultured neonatal rat cardio-
myocytes.
70
100
4.93 d (8.0)
3.62 dd (9.2, 8.0)
3.47 m
4.93 d (8.0)
3.58 dd (8.8, 8.0)
3.47 m
Although several dianthramide compounds and their gluco-
200
´
sides were isolated from Aconitum and Delphinium genus (Dıaz
300
78.1
77.8
et al., 2005; Kosuge and Yokota, 1976; Sun et al., 2009), the
protective effects on myocardial cell have not been investigated.
This study discovered two new dianthramide glucosides from A.
carmichaelii and first reported the activity of dianthramide
compounds against cardiomyocyte damage. It can be seen that
dianthramide glucosides were bioactive components in A. carmi-
chaelii which has been used as cardiac drug for thousands of years
in East Asia. However, it is very difficult to attribute the biological
activities of a plant to one or a few active principles.
400
3.39 m
71.3
3.36 m
71.2
500
3.43 m
78.6
3.42 m
78.2
600a
600b
MeO–3
MeO–7
3.92 dd (12.0, 2.0)
3.70 dd (12.0, 6.0)
3.87 s
62.6
3.87 dd (12.0, 1.6)
3.66 dd (12.0, 5.6)
–
62.6
61.8
53.1
–
3.88 s
3.90 s
53.1
atom and the chemical shift for the carbonyl group, in combination
with the IR absorption band at 1646 cmꢀ1, implied that two
aromatic rings were linked by an amide (Sun et al., 2009). All the
above-mentioned spectroscopic data suggested that 1 was a
dianthramide glucoside containing methoxy and hydroxy func-
tional groups (Dı´az et al., 2005).
3. Experimental
3.1. General experimental procedures
Detailed analysis of 2D NMR data verified the location of the
substitutents. In the HMBC spectrum, the correlations (Fig. 1) of H-
5 with C-1, C-3, and C-4; H-6 with C-1, C-2, C-4, C-5, and C-7; MeO-
3 with C-3; MeO-7 with C-7, suggested the presence of a 1,2,3,4-
tetrasubstituted methyl benzoate moiety, containing a methoxyl
and a hydroxyl group located at C-3 and C-4, respectively; the
correlations of H-30 with C-10, C-20, and C-50; H-40 with C-20, C-50,
and C-60; H-60 with C-10, C-20, C-40, C-50, and C-70; H-100 with C-20,
indicated that the glycosyl and the hydroxyl groups were attached
at C-20 and C-50, respectively, in the 1,2,5-trisubstituted salicylate
NMR spectra were recorded on a Bruker-AV-400 spectrometer.
HRESIMS were measured with Waters Synapt G2 HDMS. IR spectra
were recorded on a Vector 22 FT-IR spectrometer. UV spectra were
obtained on a Shimadzu UV-260 spectrophotometer. Optical
rotations were measured with a Perkin-Elmer 341 plus. Column
chromatography was performed with silica gel (200–300 mesh,
Jiangyou Silical Gel Development Co., Yantai, China), MCI gel
CHP20P (75–150
Sephadex LH-20 (40–70
m
m, Mitsubishi Chemical, Co., Japan), and
m, Amersham Pharmacia Biotech AB,
m
Uppsala, Sweden). Preparative TLC (0.4–0.5 mm) was conducted
with glass plates precoated silica gel GF254 (Yantai).
moiety. Therefore, compound 1 was established to be N-(20-
b-D-
glucopyranosyl-50-hydroxysalicyl)-4-hydroxy-3-methoxyanthra-
nilic acid methyl ester.
3.2. Plant material
The NMR, IR, and UV spectra of 2 were very similar to those of 1,
indicating that 2 was also a dianthramide glucoside. The molecular
formula was determined as C21H23NO11 by HR-ESI-MS. Compari-
son of the 1H NMR data of 1 and 2 (Table 1) showed that the
resonances for the 1,2,3,4-tetrasubstituted phenyl in 1 were
The lateral root of A. carmichaelii was collected in July of 2010
from the culture field in Jiangyou, Sichuan Province, China. Plant
identity was verified by Prof. Min Li (Chengdu University of TCM,
Sichuan, China). A voucher specimen (SFZ-0710) was deposited at
the School of Pharmacy, Chengdu University of TCM, Chengdu,
China.
replaced by those of a 1,2,4-trisubstituted phenyl in 2 [dH 8.21 (1H,
d, J = 8.8 Hz, H-6), 7.41 (1H, d, J = 2.8 Hz, H-3), 7.04 (1H, dd, J = 8.8,
2.8 Hz, H-5)], while the resonance of the methoxyl group was
absence in 2. These spectroscopic data, in combination with the
molecular composition, indicated that 2 was a 3-demethoxy
analogue of 1. The suggestion was verified by analysis of the 2D
NMR data that amended the assignments of resonances in the NMR
spectra of 2. Particularly, HMBC correlations (Fig. 1) between H-3
with C-1, C-2, C-3, and C-4; H-5 with C-1, C-3, and C-4; H-6 with C-
1, C-2, C-4, C-5, and C-7, confirmed the hydroxyl group at C-4. Thus,
b-D
2 was assigned to be N-(20- -glucopyranosyl-50-hydroxysalicyl)-
4-hydroxyanthranilic acid methyl ester.
The known glycosides were identified by comparison of
spectroscopic data with those reported in the literature as
adenosine (Sang et al., 2002), isomaltol-glucoside (Ota et al.,
3.3. Extraction, isolation and characterization of compounds
The air-dried lateral roots of A. carmichaelii (5 kg) was
powdered and extracted three times with 95% EtOH (30 l) for
2 h under reflux. The EtOH extract was concentrated to yield a
semi-solid (620 g), which was suspended in water and then
successively partitioned with petroleum ether, EtOAc and n-BuOH
(5 ꢃ 2.5 l). The n-BuOH extract (85 g) was subjected to CC over
silica gel with a gradient of increasing MeOH in CHCl3 to afford
eleven fractions A–K. Adenosine (250 mg) was crystallizated from
the fraction H and recrystallizated with MeOH. Fraction I (9.5 g),
eluted with CHCl3–MeOH (4:1, v/v), was resubjected to CC over