Cardioprotective effect of 'methylamine irisolidone', a new compound, in hypoxia/reoxygenation injury in cultured rat cardiac myocytes

Article abstract of DOI:10.1002/cbdv.200800314

'Methylamine irisolidone' (=5,7-dihydroxy-6-methoxy-3-(4-methoxyphenyl)-8- [(methylamino)-methyl]-4H-[1]benzopyran-4-one), a new compound, is a structurally modified kakkalide with good water solubility. In this study, we investigated its effect on hypoxia/reoxygenation (H/R) injury in cultured rat cardiac myocytes. The results showed that methylamine irisolidone could significantly inhibit lactate dehydrogenase (LDH) release, enhance the mitochondrial membrane potential, decrease intracellular calcium (Ca ²⁺ ) associated with the attenuation of reactive oxygen species (ROS) generation, reduce contents of malondialdehyde (MDA), and increase the activity of superoxide dismutase (SOD) after H/R in a dose-dependent manner. The present study demonstrated that methylamine irisolidone can directly protect cardiomyocytes against H/R injury, primarily as a result of reduction of the intracellular Ca²⁺ overload coincident with an attenuation of ROS generation and ROSmediated lipid peroxidation, which may contribute to the preservation of mitochondrion function and antioxidant against H/R injury.

Full text of DOI:10.1002/cbdv.200800314

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Cardioprotective Effect of ꢀMethylamine Irisolidoneꢁ, a New Compound, in
Hypoxia/Reoxygenation Injury in Cultured Rat Cardiac Myocytes
by Yan-Ling Mua)b), Yan-Ying Xieb), Ling Zhoub), Ying Zhongb), Lu Liub), Hong Baib),
Yuan-Shu Wangb), and Xiu-Mei Zhang*a)
a) Department of Pharmacology, School of Medicine, Shandong University, 44# W. Wenhua Road,
Jinan 250012, P. R. China (phone/fax: þ86-531-88383146; e-mail: zhangxiumei0909@126.com)
b) Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan 250062, P. R. China
ꢀMethylamine irisolidoneꢁ (¼ 5,7-dihydroxy-6-methoxy-3-(4-methoxyphenyl)-8-[(methylamino)-
methyl]-4H-[1]benzopyran-4-one), a new compound, is a structurally modified kakkalide with good
water solubility. In this study, we investigated its effect on hypoxia/reoxygenation (H/R) injury in
cultured rat cardiac myocytes. The results showed that methylamine irisolidone could significantly inhibit
lactate dehydrogenase (LDH) release, enhance the mitochondrial membrane potential, decrease
intracellular calcium (Ca2þ ) associated with the attenuation of reactive oxygen species (ROS)
generation, reduce contents of malondialdehyde (MDA), and increase the activity of superoxide
dismutase (SOD) after H/R in a dose-dependent manner. The present study demonstrated that
methylamine irisolidone can directly protect cardiomyocytes against H/R injury, primarily as a result of
reduction of the intracellular Ca2þ overload coincident with an attenuation of ROS generation and ROS-
mediated lipid peroxidation, which may contribute to the preservation of mitochondrion function and
antioxidant against H/R injury.
Introduction. – A series of isoflavones, including kakkalide, tectoridin, and glycitin,
isolated from Puerariae flos (PF), are used in traditional Chinese medicine for the
treatment of liver injury, fever, diabetes mellitus, and menopause [1]. It was reported
that kakkalide (¼ 5-hydroxy-6,4’-dimethoxyisoflavone-7-b-d-xylopyranosyl-(1!6)-O-
b-d-glucopyranoside) was transformed into irisolidone (¼ 5,7-dihydroxy-6-methoxy-3-
(4-methoxyphenyl)-4H-[1]benzopyran-4-one) by human intestinal bacteria (Scheme)
and then displayed the pharmacological activity [2][3]. In other words, kakkalide is a
prodrug, and irisolidone is the active metabolite. In general, hydrophilic compounds
with sugar moieties can not easily permeate into the cell membrane [4], and hence
glycosides are not easily absorbed from the gastrointestinal tract. Several studies
indicated that the irisolidone form has more biologically active properties compared to
the kakkalide form [1][2][5–12]. However, irisolidone must be dissolved by
hydroxypropyl-b-cyclodextrin or carboxymethylcellulose (CMC). Therefore, in our
study, ꢀmethylamine irisolidoneꢁ (¼ 5,7-dihydroxy-6-methoxy-3-(4-methoxyphenyl)-8-
[(methylaminomethyl)]-4H-[1]benzopyran-4-one), a new compound possessing good
water solubility compared with irisolidone and its glycoside, was synthesized (Scheme)
[13]. The effect of methylamine irisolidone on cardioprotection associated with the
alleviation of calcium (Ca2þ ) overload and oxidative stress, as well as stabilization of
the membrane, and preservation of mitochondrial functions was reported.
ꢂ 2009 Verlag Helvetica Chimica Acta AG, Zꢃrich

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Scheme. Synthesis of Methylamine Irisolidone
Results and Discussion. – Effects of Methylamine Irisolidone on the Survival of
Cultured Cardiomyocytes. Prior to investigation of the effect of methylamine
irisolidone on cardiomyocyte damage induced by hypoxia/reoxygenation, an experi-
ment was performed to determine whether methylamine irisolidone was toxic to
cardiomyocytes. After 12 h of incubation in the absence (control) or presence of
different concentrations (10ꢀ7, 10ꢀ6, and 10ꢀ5 m) of methylamine irisolidone, cell
viability was quantified by the MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-
tetrazolium hydrobromide) assay. At indicated concentrations of methylamine
irisolidone, the viability of exposed cardiomyocytes did not differ from control cells
(data not shown). Thus, methylamine irisolidone was used at concentrations of 10ꢀ7
–
10ꢀ5 m in the following experiments. Exposure of cardiomyocytes to hypoxia for 3 h,
followed by 6 h reoxygenation in vitro led to 45.2ꢁ3.8% attenuation of MTTreduction.
The cell viability of cardiomyocytes treated with methylamine irisolidone during H/R
significantly increased to 65.2ꢁ4.5, 75.2ꢁ18.9, and 87.1ꢁ8.2% at 10ꢀ7, 10ꢀ6, and 10ꢀ5
m, respectively, in comparison to methylamine irisolidone-untreated cells (Fig. 1,a).
Similar results were obtained for LDH release. In cultures exposed to H/R alone, LDH
release was 46.2ꢁ3.8% of total content. However, in cultures treated with methyl-
amine irisolidone, LDH release decreased to 36.1ꢁ2.9, 28.9ꢁ1.5, and 21.3ꢁ2.4% of
control at 10ꢀ7, 10ꢀ6, and 10ꢀ5 m, respectively (Fig. 1,b). These data indicated that
adding methylamine irisolidone prior to H/R could reduce cell death and elevate cell
viability.
Measurement of Reactive Oxygen Species (ROS), Malondialdehyde (MDA), and
Superoxide Dismutase (SOD) Activity. It is well accepted that the generation of ROS is
a major mechanism of cell injury after ischemia/reperfusion. The failure of oxygen
radical scavengers to consistently prevent the physiological effects of ROS-mediated

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Fig. 1. Effect of methylamine irisolidon on cell viability (a) and LDH release (b) of cultured rat
cardiomyocytes. Cardiomyocytes were exposed to hypoxia for 3 h, followed by 6 h reoxygenation in the
absence or presence of methylamine irisolidon (10ꢀ7, 10ꢀ6, and 10ꢀ5 m). Data are shown as meanꢁS.D.
(n¼6). Significant differences compared to control group: p<0.01 (##); significant differences
compared to H/R model group: p<0.05 (*), p<0.01 (**).
injury has, to some extent, challenged the importance of ROS in ischemia/reperfusion
injury, and a robust burst of ROS has been reported after reoxygenation [13]. The
attenuated ROS generation was also associated with lower contents of MDA as a
product of lipid peroxidation. SOD is the primary line of defense against tissue and
cellular damage induced by ROS. Therefore, we further investigated whether the
decreased ROS production is associated with increased activities of SOD after
methylamine irisolidone treatment. As shown in Fig. 2,a and b, cardiomyocytes
exposure to hypoxia/reoxygenation markedly increased the generation of ROS and the
content of MDA to 450.2ꢁ39.8 and 390.2ꢁ35.3%, respectively, of control. Methyl-
amine irisolidone significantly attenuated the generation of ROS induced by hypoxia/
reoxygenation. Results indicated that the levels of ROS decreased to 361.3ꢁ35. 8,
246.0ꢁ28.4, and 180.3ꢁ21.4% of control at 10ꢀ7, 10ꢀ6, and 10ꢀ5 m of methylamine
irisolidone, respectively. Moreover, the levels of MDA also significantly decreased to
330.8ꢁ29.8, 226.6ꢁ32.1, and 150.3ꢁ15.5% by 10ꢀ7, 10ꢀ6, and 10ꢀ5 m of methylamine
irisolidone, respectively (Fig. 2,b). We also measured the antioxidant SOD activity.
After hypoxia/reoxygenation, the activities of SOD decreased to 56.3ꢁ5.4% of
control. Methylamine irisolidone significantly recovered the activities of SOD. They
increased to 64.3ꢁ7.1, 72.6ꢁ6.6, and 84.3ꢁ9.5% at 10ꢀ7, 10ꢀ6, and 10ꢀ5 m of
methylamine irisolidone, respectively, under hypoxia/reoxygenation conditions
(Fig. 2,c).
Mitochondrial Membrane Potential (MMP) Changes in Cultured Cardiomyocytes.
Among ROS-producing systems, mitochondria are considered as one of the most
important intracellular sources of ROS. Mitochondrial dysfunction leads to cell death
by reducing ATP production, increasing production of ROS, and releasing death
regulatory and signaling molecules from the intermembrane space [14]. Therefore, we
studied the effect of methylamine irisolidone on the regulation of the MMP in cultured
cardiomyocytes. The traditional mitochondrial probe Rhodamine 123 (Rho123) has

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Fig. 2. Effect of methylamine irisolidone on ROS generation (a), MDA level (b), and SOD activity (c) of
cultured rat cardiomyocytes. Cardiomyocytes were exposed to hypoxia for 3 h, followed by 6 h
reoxygenation in the absence or presence of methylamine irisolidon (10ꢀ7, 10ꢀ6, and 10ꢀ5 m). Data are
expressed as percentage of control (meanꢁS.D., n¼6). Significant differences compared to control
group: p<0.01 (##); significant differences compared to H/R model group: p<0.05 (*), p<0.01 (**).
been widely used to evaluate the MMP in a variety of cell types [15–17]. Our results
indicated that H/R induced a decrease in the MMP of cultured cardiomyocytes, and this

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
change was prevented by the addition of methylamine irisolidone (10ꢀ7, 10ꢀ6, and 10ꢀ5
m) in a dose-dependent manner (Fig. 3).
Fig. 3. Effect of methylamine irisolidone on the mitochondrial membrane potential in cultured rat
cardiomyocytes. Cardiomyocytes were exposed to hypoxia for 3 h, followed by 6 h reoxygenation in the
absence or presence of methylamine irisolidon (10ꢀ7, 10ꢀ6, and 10ꢀ5 m). Data are expressed as
percentage of Rho123 fluorescence intensity (FI) of the control (meanꢁS.D., n¼3). Significant
differences compared to control group: p<0.01 (##); significant differences compared to H/R model
group: p<0.05 (*), p<0.01 (**).
Intracellular Ca2þ Content. The present results indicated that, after H/R, the
intracellular Ca2þ content was increased to 64.3ꢁ7.2% compared with the control (ca.
40%). Pretreatment with various concentrations (10ꢀ7, 10ꢀ6, and 10ꢀ5 m) of methyl-
amine irisolidon significantly decreased the level of Ca2þ in the cultured cardiomyo-
cytes to 60.1ꢁ6.6, 52.3ꢁ8.1 and 45.7ꢁ6.6%, respectively (Fig. 4). Based on the above
results, we suggest that alleviating Ca2þ accumulation during myocardial ischemia and
reperfusion may be one of the mechanisms of the cardioprotective effect of
methylamine irisolidone.
This project was financially supported by National Natural Science Foundation of China (No.
30701022) and the National Natural Science Foundation of Shandong Province (No. Y2007C156). All
work has been conducted in the Key Laboratory for Modern Medicine and Technology of Shandong
Province.
Experimental Part
General. Rhodamine 123 (Rho123) and reactive oxygen species (ROS) detection kits were
purchased from Beyotime Company (Jiangsu, China). Malondialdehyde (MDA) and superoxide
dismutase (SOD) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing,
China). Dulbeccoꢁs Modified Eagle Medium (DMEM) was from GIBCOꢀ Invitrogen Corporation
(USA). Fluo-3/AM ester was obtained from Biotium (USA). 3-(4,5-Dimethylthiazole-2-yl)-2,5-
diphenyl-2H-tetrazolium hydrobromide (MTT) was from Sigma. Other reagents were from standard
suppliers or as indicated in the text. Stock solns. of Fluo-3/AM ester (2 mm) were made in DMSO.
Rho123 (2 mm) was dissolved in DMF. Stock solns. were stored in small aliquots at ꢀ208 and diluted in
exper. medium immediately before use.
Synthesis and Structural Proofs of Methylamine Irisolidone. ꢀMethylamine irisolidonꢁ (¼ 5,7-
dihydroxy-6-methoxy-3-(4-methoxyphenyl)-8-[(methylamino)methyl]-4H-[1]benzopyran-4-one) was

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Fig. 4. Flow cytometric analysis of intracellular Ca2þ concentration by Fluo-3/AM staining. H/R
increased the intracellular Ca2þ content, and pretreatment with methylamine irisolidon (10ꢀ7, 10ꢀ6, and
10ꢀ5 m) reduced intracellular Ca2þ content compared to the H/R model group. Intracellular Ca2þ
fluorescence intensity is presented as percentage of the total analyzed cells (meanꢁS.D., n¼3).
Significant differences compared to control group: p<0.01 (##); significant differences compared to H/R
model group: p<0.05 (*), p<0.01 (**).
synthesized by the New Drug Center of Shandong Academy of Medical Sciences (Scheme) [13]. Briefly,
dried flowers of Puerariae flos (PF) were extracted with EtOH. Evaporation of the solvent under
reduced pressure gave kakkalide. Hydrolysis of kakkalide gave irisolidone. Formaldehyde (1 ml) and
dimethylamine (2 ml) were added to the soln. of irisolidone (1 g) in EtOH (30 ml). After being stirred at
708 for 4 h, the mixture was cooled and filtered to give methylamine irisolidone (1.3 g). Pale yellowish
amorphous powder. M.p. 185–1908. 1H-NMR ((D6)DMSO, 300 MHz): 13.05 (s, NH); 8.05 (s, HꢀC(2));
7.45 (d, J¼8.8, HꢀC(2’), HꢀC(6’)); 6.97 (d, J¼8.8, HꢀC(3’), HꢀC(5’)); 4.03 (s, CH2); 3.78 (s,
MeOꢀC(4’)); 3.70 (s, MeOꢀC(6)), 2.53 (s, MeN). 13C-NMR ((D6)DMSO, 150 MHz): 177.8 (C(4));
170.3 (C(7)); 158.7 (C(4’)); 152.4 (C(2)); 152.1 (C(5)); 151.0 (C(9)); 133.5 (C(6)); 130.0 (C(2’), C(6’));
123.8 (C(3)); 120.6 (C(1’)); 113.5 (C(3’), C(5’)); 98.7 (C(10)); 97.1 (C(8)); 58.6 (MeOꢀC(6)); 55.0
(MeOꢀC(4’)); 43.4 (CH2); 32.0 (MeN) [18].
Culture of Neonatal Rat Cardiac Myocytes. Primary cultures of neonatal rat cardiac myocytes were
prepared as described in [19][20] with some modifications. In brief, hearts were removed from 1- to 3-
day-old Wistar rats anesthetized by Et2O under aseptic conditions and placed in ice cold Ca2þ- and Mg2þ
-
free Phosphate Buffered Saline (PBS). The ventricles were minced with scissors into 1-mm3 fragments,
and they were then enzymatically digested with 0.25% trypsinogen. The detached cells were collected by
centrifugation at 300g and incubated in 100-ml tissue culture flasks for 60 min at 378 in a humidified
incubator with 5% CO2. The nonadjacent cardiac myocytes were harvested and seeded into 100-ml tissue
culture flasks and incubated in DMEM supplemented with 20% fetal bovine serum (FBS). 5-Bromo-29-
deoxyuridine (BrdU; 100 mm) was added during the first 48 h to inhibit proliferation of nonmyocytes. By
this method, contractile cardiomyocyte-rich cultures with 90–95% myocytes were obtained routinely.
The cardiomyocytes were then incubated in DMEM containing 20% FBS without BrdU, and all assays
were performed within 48–72 h after incubation.
Exper. Protocol of H/R Injury. Before hypoxic exposure, cell medium was replaced by modified
Tyrodeꢁs soln. (in mm: NaCl 136.9, KCl 2.68, Na2HPO4 ·12 H2O 8.1, KH2PO4 1.47, CaCl2 0.9, MgCl2 ·
6 H2O 0.49; pH 7.2). The cardiac myocytes were transferred to and kept in an anaerobic incubator
(CIMO, Shanghai) flushed with the anaerobic gas mixture N2/H2/CO2 85 :10 :5 at 378 for 3 h, and were
then removed to reoxygenate for another 6 h with normal DMEM. To examine the role of methylamine
irisolidone in H/R injury, the myocytes were treated as follows: i) cardiomyocytes were cultured in
normal DMEM for a total of 21 h under normoxic conditions (control); ii) after 12 h incubation in

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normal DMEM, cardiomyocytes were transferred to modified Tyrodeꢁs soln., incubated for 3 h in an
anaerobic incubator (pO2 range in the incubator was maintained <1%), and, subsequently,
reoxygenated for 6 h in normal DMEM (H/R model); iii) cardiomyocytes were treated with
methylamine irisolidone (10ꢀ7, 10ꢀ6, and 10ꢀ5 m) for 12 h in normoxic DMEM supplemented with
20% FBS, transferred to Tyrodeꢁs soln., incubated for 3 h in an anaerobic incubator, and, subsequently,
reoxygenated for 6 h in normal DMEM (methylamine irisolidone-treated cells).
Assessment of Cell Viability and LDH Release. The survival of cardiomyocytes was determined by
the MTTassay and by the amount of LDH released into the culture medium [21]. Cells were seeded into
96-well plates (5ꢂ103 cells/well) and exposed to various doses of agent treatments for 12 h. Following
treatments, 10 ml of MTT soln. (5 mg/ml) was added into each well, and the cells were incubated for
another 4 h at 378. Afterwards, the media were discarded, and DMSO (150 ml) was added for the
dissolution of formazan crystals. The absorbance of each well was read at 570 nm with a Victor 1420
multilabel counter. Cell viability was expressed as a percentage of the control. After various treatments,
the amount of LDH released into the medium, and total cellular LDH activities were also determined
using a diagnostic kit (Jiancheng Bioengineering Institute, Nanjing, China). The total LDH release that
corresponded to complete astrocyte death was determined at the end of experiments, following repeated
freezing and thawing. Cell death was expressed as percentage of total LDH.
Measurement of ROS, MDA, and SOD. ROS were measured as described in [22] with some
modifications. In brief, cardiomyocytes were incubated with 6-carboxy-2’,7’-dichlorodihydrofluorescein
diacetate (DCFH-DA) at a final concentration of 10 mm for 20 min and washed three times with PBS
buffer. ROS Production in cardiomyocytes was measured by a Victor 1420 multilable counter with
excitation and emission settings at 485 and 525 nm, resp., and expressed as a percentage of the control.
The SOD activity was determined by the hydroxylamine assay (developed from xanthine oxidase assay),
and the MDA content in cardiomyocytes was determined by the thiobarbituric acid method [23]. All
groups of treated cells were washed twice, resuspended in PBS, sonicated for 30 s on ice, and centrifuged
at 1000g for 15 min. The supernatants were subjected to intracellular SOD activity and MDA content
assays according to the manufacturerꢁs instructions. Both SOD activity and MDA content are expressed
as percentage of the control.
MMP Changes in Cultured Cardiomyocytes. To measure the MMP [17][24], the fluorescent probe
Rho123 was used. Treated cells were collected and resuspended (1ꢂ106 cells/ml) in PBS, which
contained 1 mm Rho123 and incubated at 378 for 30 min. Samples were analyzed by FACScan flow
cytometer (Becton Dickinson, USA) with excitation and emission wavelengths of 488 and 525 nm, resp.
Fluorescent signal intensity was recorded and analyzed by CellQuest software (Becton Dickinson). The
cellular MMP was expressed as a percentage of control cells.
Myocardial Cell Intracellular Ca2þ Content. Myocardial cells were suspended in PBS buffer (1ꢂ106
cells/ml) and marked with the fluorescent probe Fluo-3/AM, previously dissolved in DMSO. The labeling
was effected as follows: 4 min of preincubation at 48, followed by incubation for 30 min at 378 (final probe
concentration 2.0 mm, final DMSO concentration 0.2%). This procedure is sufficient to inhibit Fluo-3/
AM uptake in endocytic vesicles [25]. Fluorescence measurement was carried out with the flow
cytometer (excitation and emission wavelengths of 488 and 525 nm, resp.). Intracellular Ca2þ content
was expressed as a percentage of control cells.
Statistical Analysis. Experiments were repeted at least three times unless otherwise indicated; figures
from one representative experiment are shown. Levels of statistical significance were determined using
ANOVA followed by Dunnettꢁs test. A p value below 0.05 was considered significant. SPSS11.0 Statistical
software was used for data analysis.
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Received October 21, 2008