Protective roles of puerarin and Danshensu on acute ischemic myocardial injury in rats

ARTICLE IN PRESS

Phytomedicine 14 (2007) 652–658 www.elsevier.de/phymed

Protective roles of puerarin and Danshensu on acute ischemic myocardial injury in rats L. Wua,, H. Qiaoa, Y. Lib, L. Lia,b a

College of Pharmaceutical Sciences, Zhejiang University, Zijingang Campus of Zhejiang University, Hangzhou 310058, China Xiyuan Hospital, China Academy of Traditional Chinese Medical Sciences, Beijing 100091, China

b

Abstract Ischemic heart diseases have been the leading cause of death in both developed and developing countries over the past decades. The aim of this study was to investigate the cardioprotective effects of the complex preparation (called Shenge), made of puerarin (isolated from Pueraria lobata Ohwi., also called Kudzu) and Danshensu (isolated from the Chinese herb, Salvia miltiorrhiza), on acute ischemic myocardial injury in rats and its underlying mechanisms. The left anterior descending (LAD) coronary artery was occluded to induce myocardial ischemia in the hearts of SD rats. Shenge was injected into the tail vein 15 min after occlusion at doses of 0, 30, 60, or 120 mg/kg body wt. ST elevation was then measured at 60, 120, and 240 min after Shenge administration. The ischemic size, serum levels of creatine kinase isoenzyme-MB (CK-MB), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA), and ST elevation were measured after the rats were sacrificed. Shenge decreased ST elevation induced by acute myocardial ischemia, reduced ischemic size, serum levels of CK-MB, LDH and MDA, and increased serum activity of SOD in a dose-dependent manner. The combined use of puerarin and Danshensu at a ratio of 1:1 showed the most effective activity. In conclusion, Shenge exerts significant cardioprotective effects against acute ischemic myocardial injury in rats, likely through its antioxidant and anti-lipid peroxidation properties, and thus may be an effective and promising medicine for both prophylaxis and treatment of ischemic heart disease. r 2007 Elsevier GmbH. All rights reserved. Keywords: Acute myocardial ischemia; Shenge; Puerarin; Danshensu

Introduction Heart ischemia is one of the main causes related to sudden death in the world. In the past decades, there has been a great increase in the use of complementary treatments such as herbal remedies in the treatment of diseases. Many traditional plants have been claimed to be useful for the control of problems due to ischemia and associated pathologies. Corresponding author.

E-mail address: [email protected] (L. Wu). 0944-7113/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2007.07.060

Danshen (Salvia miltiorrhiza Bunge.) and Gegen (Pueraria lobata Ohwi., also called Kudzu) are two traditional Chinese medicinal herbs used widely in China, Japan, Korea, and Taiwan for the treatment of angina pectoris, myocardial infarction, and other cardiac symptoms (Gordon and Weng, 1992). Danshen and Gegen are commonly used in combination for their putative cardioprotective and anti-atherosclerotic effects. It has been reported that the combined preparation suppresses acetylated low-density lipoproteins uptake by human macrophages, enhances expression of intercellular adhesion molecules, and increases adhesion of monocytes to endothelial cells in vitro in a

ARTICLE IN PRESS L. Wu et al. / Phytomedicine 14 (2007) 652–658

O

653

OH

O

HO H H OH OH H

O CH OH 2

COOH

HO

H

H OH

A

HO

OH

B

Fig. 1. Chemical structures of puerarin (A) and Danshensu (B).

dose-dependent manner (Overstreet et al., 2002). In order to evaluate the efficacy and to understand the mechanisms of Danshen and Gegen, two representative active principles of the two herbs, puerarin from Puerariae radix and Danshensu (3,4-dihydroxyphenyllactic acid) from Salvia miltiorrhiza, were selected for study. Puerarin (40 -7-dihydroxy-8-b-D-glucosylisoflavone), the chemical structure of which is shown in Fig. 1A, is a C-glycoside compound extracted from Gegen. A number of studies have revealed that puerarin possesses many physiological activities, including improvement of blood circulation (Pan et al., 2005), prevention of cardiovascular diseases (Ma et al., 2002; Liu et al., 2000), and improvement of arrhythmia (Yan et al., 2004). According to several phytochemical studies, Danshensu is abundant and structurally representative of the water-soluble active components of Danshen (Luo et al., 2001). Its chemical structure is D(+)b-(3,4-dihydroxyphenyl) lactic acid, shown in Fig. 1B. It has been demonstrated that Danshensu reduces lipid peroxidation on mitochondrial membrane by scavenging free radicals, and inhibits permeability and transmission of mitochondrial membrane by reducing thiol oxidation. However, there is little information on the cardioprotective effects of the puerarin and Danshensu combination in vivo. The aim of this study was to evaluate the cardioprotective effects of the puerarin and Danshensu combination and to understand their underlying mechanism.

Materials and methods Medicines The Puerariea radix and Salvia miltiorrhiza were purchased from a local market in China. An authenticated voucher specimen was deposited in the herbarium of the College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China. The isolation and determination of purity of puerarin were performed as

Fig. 2. A typical chromatogram of the Shenge preparation from Shunfeng Pharmaceutics: (1) Danshensu, (2) puerarin, and (3) salvianolic acid B.

reported previously (He et al., 2004). The purity of puerarin (Fig. 1A) was more than 97% based on highperformance liquid chromatographic (HPLC) analysis (Yu et al., 2002). The isolation and determination of purity of Danshensu (3,4-dihydroxyphenyl-lactic acid) (Fig. 1B) were performed as reported previously (Gu et al., 2004). The purity of Danshensu was more than 90% based on HPLC analysis (Fig. 2). Salvianolic acid B is a minor constituent of S. miltiorrhiza. All experiments in this study were performed with a Waters 2695 HPLC system. The analytical column was a Zorbax SB-C18 (5 mm, 4.6  150 mm) with a Zorbax SB-C18 (5 mm, 4.6  45 mm) guard column (both from Agilent Technologies). Shenge, which was prepared as a frozen dried powder and contained in a vial with 50 mg puerarin and 50 mg Danshensu, was provided by Shunfeng Pharmaceutics Co., Ltd, Foshan, China. Diltiazem hydrochloride injection (10 mg/vial), used as a positive control, was purchased from Tanabe Seiyaku Co., Ltd, Tianjin, China.

Surgical preparation of animals All protocols were performed in accordance with the guidelines established by the Guide for the Care and Use of Laboratory Animals of Zhejiang University, and approved by the local Ethics Committee. We used male Sprague–Dawley rats (Zhejiang Experimental Centre, China) weighing 180–215 g each. A myocardial infarction model was produced by ligation of the left coronary artery, as described previously (Stanton et al., 2000). Briefly, we anesthetized rats with sodium pentobarbital (30 mg/kg body wt.) and ventilated them with a volumeregulated respirator. We exposed hearts by left thoracotomy, and ligated the left coronary artery 2–3 mm

ARTICLE IN PRESS 654

L. Wu et al. / Phytomedicine 14 (2007) 652–658

from its origin between the pulmonary artery conus and the left atrium with a 6–0 Prolene suture. The sham group underwent thoracotomy and cardiac exposure without coronary ligation. The surviving rats were maintained on standard rat chow.

Experimental protocol Experiment I: determination of the proper ratio of puerarin to Danshensu The experiment was designed to determine the proportion of puerarin to Danshensu using the rat model of heart ischemia. Male albino rats (Sprague– Dawley) weighing 180–220 g each were randomly selected and placed into six experimental groups of ten rats each. Group I served as control (heart ischemia control). Groups II–VI were administered the test compound, the mixture of puerarin and Danshensu, at a dose of 100 mg/kg body wt. The proportion of puerarin to Danshensu in group II was 1:0, that in group III was 1:1, that in group IV was 1:2, that in groupV was 2:1, and that in group VI was 0:1. All experimental rats were sacrificed 4 h after occlusion. The hearts were then removed and cut into 5 pieces. Experiment II: determination of the dose of the mixture compound Rats were divided randomly into six groups: control group (n ¼ 10); diltiazem hydrochloride 1 mg/kg body wt. group (n ¼ 10); Shenge 30 mg/kg body wt. group (n ¼ 10); Shenge 60 mg/kg body wt. group (n ¼ 10); Shenge 120 mg/kg body wt. group (n ¼ 10); and the sham-operated group (n ¼ 10). Shenge or diltiazem hydrochloride (a calcium-antagonist) was injected into the tail vein of each of the experimental rats 15 min after left anterior descending (LAD) occlusion. Electrocardiograms were recorded before LAD occlusion, 15 min after LAD occlusion, and 60, 120 and 240 min after drug administration. All experimental rats were sacrificed 4 h after occlusion. Blood (3 ml) was taken from the abdominal aorta and centrifuged at 2500g for 10 min for the preparation of serum. The heart tissues were removed and dissected free of the atria and the right ventricle.

Determination of ST elevation Electrocardiograms were recorded and ST elevation, which can be observed after successful induction of ischemia, was measured.

Determination of the infarct size The heart tissue was washed with PBS three times. Sections were sliced and then incubated for 10 min in

0.025% nitrotetrazolium blue chloride (NBT) for pathological examinations. Photographs were taken and infarction size was measured with an Image C Morphology analysis system (Chansan, Shanghai, China). Ischemic areas and left ventricular areas were, respectively, added up by five slices of the same heart tissue. The ischemic risk area ratio was defined as the percentage of total ischemic risk area over the total left ventricle area measured (Hangaishi et al., 2001).

Determination of serum creatine kinase isoenzymeMB (CK-MB), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA) activity Blood was sampled from abdominal aorta and serum obtained after centrifugation at 2500g for 10 min. CKMB and LDH levels were determined spectrophotometrically at 340/660 nm and 340 nm, respectively, using diagnostic kits (Olympus Diagnostics, Beijing, China) and a clinical chemistry analyzer (AU400, Olympus, Hamburg, Germany). SOD and MDA levels were measured spectrophotometrically using diagnostic kits (Jiancheng, Nanjing, China), according to the manufacturer’s instructions.

Statistical analysis All data were expressed as means7standard deviation (SD). A database was set up with the SPSS 11.0 software package (SPSS Inc., Chicago, IL, USA). A Dunett t-test was used to compare the data obtained before and after occlusion. Differences among groups were analyzed by one-way ANOVA. A probability of less than 0.05 was considered to be statistically significant.

Results Effect of the ratio of puerarin to Danshensu on the infarct size As shown in Fig. 3, after ligation of the left descending coronary artery, the infarct area in the heart of the control rats reached 35.54%. Although puerarin or Danshensu alone could not apparently reduce the infarct size, all mixtures of puerarin and Danshensu could decrease the infarct area significantly. When the ratio of puerarin to Danshensu was 1:1, the infarct area was found to be more effectively decreased (only 21.45%, po0.01). The results indicated that the ratio of 1:1 should be a proper proportion for the treatment of heart ischemia.

ARTICLE IN PRESS L. Wu et al. / Phytomedicine 14 (2007) 652–658

Effect of Shenge on the infarct size

655

Effect of Shenge on the serum levels of CK-MB, SOD, LDH, and MDA

The ischemic risk area ratio was 31.9778.23% in the control group. Treatment of Shenge at doses of 30, 60, and 120 mg/kg body wt. resulted in dose-dependent reductions in the infarct size of 22.8075.88%, 22.3075.20%, and 19.9779.26%, respectively. There were significant differences in the ratios between the control rats and those treated with Shenge at 60 and 120 mg/kg body wt. (Table 1). Treatment with diltiazem hydrochloride also reduced ischemic risk ratio in rats with acute myocardial ischemia.

Effect of Shenge on ST elevation Shenge and diltiazem hydrochloride significantly decreased ST elevation at doses of 60 and 120 mg/kg body wt., compared with the controls (all po0.01) (Fig. 4).

The serum levels of CK-MB, LDH, and MDA were increased significantly (633.16776.8, 248.7739.6, and 13.5071.90 U/ml, respectively), while the serum SOD level was significantly decreased (220.48738.51 U/ml), as compared with the normal controls and shamoperated rats (Fig. 5). Shenge at doses of 30, 60, and 120 mg/kg body wt. reduced the serum level of CK-MB significantly (to 420.31767.13, 385.59770.4, and 68.20740.9 U/ml, respectively), and that of LDH (to 237.2722.0, 204.7726.0, and 195.9731.1 U/ml, respectively) (all po0.05, Figs. 5A and 5B). In addition, Shenge at a dose of 120 mg/kg body wt. decreased the serum MDA level (to 11.2671.16 U/ml, po0.05), but increased the serum SOD level (to 261.85742.75 U/ml, po0.05) (Figs. 5C and 5D).

Discussion 40

35.54

infarct to total ratio (%)

30.69

31.85

35 30

25.55 *

26.85 *

IV

V

21.45 **

25 20 15 10 5 0 I

II

III

VI

Fig. 3. Cardioprotective effects of different ratios of puerarin to Danshensu on myocardial infarct size, after occlusion of the left anterior descending coronary artery. Group I served as control. The ratios of puerarin to Danshensu were 1:0 in group II, 1:1 in group III, 1:2 in group IV, 2:1 in group V, and 0:1 in group VI, respectively. **, po0.01, * po0.05 vs. control (group I).

Table 1.

Danshen and Gegen, officially listed in the Chinese pharmacopoeia, have been reported to be effective in treating myocardial infarction in animal models and cardiovascular diseases in humans. A preparation of Danshen and Gegen has been demonstrated to modulate key early atherogenic events in vitro (Sieveking et al., 2005). There is, however, little information in the literature on the combined use of puerarin and Danshensu in vivo and the mechanism underlying the reciprocal synergy of the main active ingredients of the herbs. The results in this study demonstrated for the first time that the combined use of puerarin and Danshensu had similar cardioprotective effects as diltiazem, causing smaller infarct size compared with vehicle. Moreover, we showed that Shenge dose-dependently reduced the size of infarct caused by ischemic injuries, which is the most reliable indicator of myocardial protection. Pharmacological augmentation of endogenous myocardial antioxidants has been identified as a promising therapeutic approach in diseases associated with increased oxidative stress (Jin et al., 2003). In the present

Effect of Shenge on the myocardial infarct size after occlusion of the left anterior descending coronary artery

Group

Infarct size (cm2)

Normal size (cm2)

Infarct-to-total ratio (%)

Control (n ¼ 10) Diltiazem hydrochloride (n ¼ 10) Shenge (30 mg/kg body wt., n ¼ 10) Shenge (60 mg/kg body wt., n ¼ 10) Shenge (120 mg/kg body wt., n ¼ 10) Sham-operated (n ¼ 10)

1.1370.30 0.6170.10 0.7970.20 0.7870.18 0.7770.32 0

2.4070.29 3.1070.41 2.6970.29 2.7270.28 3.1570.59 3.5070.37

31.9778.23 16.3772.14** 22.8075.88 22.3075.20* 19.9779.26* 0

**po0.01, * po0.05 vs. control. (group I).

ARTICLE IN PRESS 656

L. Wu et al. / Phytomedicine 14 (2007) 652–658

0.6 0.5

ST elevation (mv)

0.4

Control Diltiazem hydrochloride

0.3

Shenge(120 mg/kg) Shenge(60 mg/kg)

0.2

Shenge(30 mg/kg) Sham

0.1 0 before ischemia

-0.1

after ischemia

60 min after

120 min after

240 min after

) am Sh

)

kg

m 0

Sh

en

ge

(6

g/

)

kg

kg

g/

0

m

g/ m

20 (1

ge en

Sh

ge en Sh

(3

m

l

ze

ro

ia

nt Co

D

0 (3 ge

en

*

*

*

*

ilt

)

Sh

kg

)

g/ m

g/

kg

m

g/

Sh

en Sh

en

ge

ge

(1

(6

20

0

m

ia ilt D Sh

kg

)

m ze

ro nt Co

A

B *

C

*

*

am

350 300 250 200 150 100 5 0

Co nt ro l Sh D en ilt ia ge ze (1 m 20 Sh m en g/ ge kg (6 ) Sh 0 m en g ge /k g) (3 0 m g/ kg )

m Sh a

) kg g/

Sh e

ng

e(

30

m

m

g/

kg g/ e( ng Sh e

ng

e(

12

0

60

m

ilt D Sh e

kg

)

m ia

ze

ro nt Co

)

SOD (U/ml)

**

Sh

18 16 14 12 10 8 6 4 2 0 l

MDA (U/ml)

350 300 250 200 150 100 50 0

am

LDH (U/ml)

800 700 600 500 400 300 200 100 0 l

CK-MB (U/ml)

Fig. 4. Effect of Shenge at different doses and diltiazem hydrochloride on ST elevation.

D

Fig. 5. Effect of Shenge on the serum levels of creatine kinase-MB (CK-MB) (A), lactate dehydrogenase (LDH) (B), superoxide dismutase (SOD) (C), and malondialdehyde (MDA) (D). Bars represent standard deviations.

study, Shenge, when administrated at a dose of 120 mg/kg body wt., significantly increased the serum levels of SOD, indicating that Shenge possesses a potent antioxidant property. In addition, we observed that the level of LDH, a biochemical indicator of cellular damage, was also dose-dependently decreased. Previous studies have shown that acute myocardial ischemia generates numerous free radicals, causing damage to the cellular

membranes as a result of production of lipid peroxidation (Haiyun et al., 2004). The disturbance in the transport function of the myocardial cells causes the leakage of enzymes from cells due to altered permeability of membranes (Grech et al., 1996). MDA formed by the breakdown of lipid peroxides is often used to quantify the extent of lipid peroxidation, whereas serum CK-MB is a cardiac-specific marker of acute myocardial

ARTICLE IN PRESS L. Wu et al. / Phytomedicine 14 (2007) 652–658

infarction, or an indicator for myocardial tissue injury. The present study demonstrated that Shenge significantly restrained the increase of serum MDA and CKMB levels caused by acute ischemic injury, suggesting that Shenge may exert its protective effect against the myocardial ischemic injury by reducing lipid peroxidation. Rats treated with the mixture (Shenge) had high antioxidant activities but low MDA production in comparison with rats treated with saline or diltiazem, which implies that the mixture may affect the level of endogenous antioxidants or oxidative stress, or both. ST elevation in Q leads on the electrocardiogram is assumed to reflect the area of the myocardium at risk, which is a major determinant of final infarct size and clinical outcome. In the present study, we observed that Shenge suppressed ST elevation induced by ischemia until 240 min after administration. Xie et al. (2003) reported that, prior to percutaneous transluminal coronary angioplasty for patients with angina pectoris, treatment with intravenous puerarin attenuated the ST segment changes in the ECG. The suppression of ST elevation in the current study should be attributed to puerarin. Both puerarin and Danshensu are polyphenolic compounds, and their antioxidant properties are thought to be the common reason that they are pharmacologically useful against heart ischemia (Guerra et al., 2000). There are major differences between the mechanisms of actions of puerarin and Danshensu. Generally, puerarin protects the myocardium against ischemia injury via inhibiting mitochondrial permeability transition pore opening, blocking cardiac ion channels (Zhang et al., 2003), and opening the collateral circulation (Liu et al., 1999). Danshensu inhibits mitochondrial membrane permeability transmission by reducing protein thiol oxidation (Liu et al., 2001) and dilating the coronary artery. Taken together, the mechanisms of action of puerarin and Danshensu are distinct and complementary; therefore, their combined use might be synergistic and more effective. These mechanisms may partly explain the result that puerarin or Danshensu alone decreases the infarct myocardium only slightly, but significantly reduces the infarct size in hearts when administered in combination. In conclusion, the mixture of herbal active ingredients (Shenge) exerts significant cardioprotective effects against acute ischemic myocardial injury in rats, likely through its antioxidant and anti-lipid peroxidation properties, and thus may be used as an effective and promising medicine for both prophylaxis and treatment of ischemic heart disease.

Acknowledgements This project was partially supported by grants from the Science and Technology Department of Zhejiang Province,

657

China (2004C13022, 2005C13027), a grant from Zhejiang Department of Education (No. 20061328), Zhejiang Bureau of Traditional Chinese Medicine (No. 2006Z015), 973 Program (No.2005CB523405), PR China and a grant from the National Natural Science Foundation of China (30500661).

References Gordon, M.H., Weng, X.C., 1992. Antioxidant properties of extracts from Danshen (Salvia miltiorrhiza Bunge). Food Chem. 44, 119–122. Grech, E.D., Dodd, N.J., Jackson, M.J., Morrison, W.L., Faragher, E.B., Ramsdale, D.R., 1996. Evidence for free radical generation after primary percutaneous transluminal coronary angioplasty recanalization in acute myocardial infarction. Am. J. Cardiol. 77, 122–127. Gu, M., Zhang, G., Su, Z., Ouyang, F., 2004. Identification of major active constituents in the fingerprint of Salvia miltiorrhiza Bunge developed by high-speed counter-current chromatography. J. Chromatogr. A 1041, 239–243. Guerra, M.C., Speroni, E., Broccoli, M., Cangini, M., Pasini, P., Minghetti, A., Crespi-Perellino, N., Mirasoli, M., Cantelli-Forti, G., Paolini, M., 2000. Comparison between Chinese medical herb Pueraria lobata crude extract and its main isoflavone puerarin: antioxidant properties and effects on rat liver CYP-catalysed drug metabolism. Life Sci. 67, 2997–3006. Haiyun, L., Yijia, L., Honggang, L., Honghai, W., 2004. Protective effect of total flavones from Elsholtzia blanda (TFEB) on myocardial ischemia induced by coronary occlusion in canines. J. Ethnopharmacol. 94, 101–107. Hangaishi, M., Nakajima, H., Taguchi, J., Igarashi, R., Hoshino, J., Kurokawa, K., Kimura, S., Nagai, R., Ohno, M., 2001. Lecithinized Cu, Zn-superoxide dismutase limits the infarct size following ischemia–reperfusion injury in rat hearts in vivo. Biochem. Biophys. Res. Commun. 285, 1220–1225. He, X., Tan, T., Xu, B., Janson, J.C., 2004. Separation and purification of puerarin using [beta]-cyclodextrin-coupled agarose gel media. J. Chromatogr. A 1022, 77–82. Jin, M., Qin, J., Wu, W., 2003. [Clinical study on Tianbaokang injection against oxidative injury of vascular endothelial function in ischemic apoplexy. Zhong. Yao Cai. 26, 148–151. Liu, Q., Wang, L., Lu, Z., Li, S., Xiong, Y., 1999. Effect of puerarin on coronary collateral circulation in dogs with experimental acute myocardial infarction. Zhongguo Zhong. Yao Za Zhi. 24:304–6, 320. Liu, Q., Lu, Z., Wang, L., 2000. Restrictive effect of puerarin on myocardial infarct area in dogs and its possible mechanism. J. Tongji Med. Univ. 20, 43–45. Liu, J., Shen, H.M., Ong, C.N., 2001. Role of intracellular thiol depletion, mitochondrial dysfunction and reactive oxygen species in Salvia miltiorrhiza-induced apoptosis in human hepatoma HepG2 cells. Life Sci. 69, 1833–1850. Luo, X., Bi, K., Zhou, S., Wei, Q., Zhang, R., 2001. Determination of Danshensu, a major active compound

ARTICLE IN PRESS 658

L. Wu et al. / Phytomedicine 14 (2007) 652–658

of Radix Salvia miltiorrhiza in dog plasma by HPLC with fluorescence detection. Biomed. Chromatogr. 15, 493–496. Ma, J., Cheng, G., Li, D., Liu, H., 2002. The effects of Radix Puerariae decoction on levels of antibody and interleukin-2 in mice. Zhong. Yao Cai. 25, 731–732. Overstreet, D.H., Ma, Z.Z., Lee, D.Y.W., 2002. The isoflavone puerarin reduces anxiety symptoms induced by acute 5-HT2C or inverse benzodiazepine agonists or alcohol withdrawal. Eur. Neuropsychopharmacol. 12, 361–362. Pan, H.P., Yang, J.Z., Mo, X.L., Li, L.L., Huang, Z.L., Ye, J., Huang, J., 2005. Protection of puerarin on the cerebral injury in the rats with acute local ischemia. Zhongguo Zhong. Yao Za Zhi. 30, 457–459. Sieveking, D.P., Woo, K.S., Fung, K.P., Lundman, P., Nakhla, S., Celermajer, D.S., 2005. Chinese herbs Danshen and Gegen modulate key early atherogenic events in vitro. Int. J. Cardiol. 105, 40–45. Stanton, L.W., Garrard, L.J., Damm, D., Garrick, B.L., Lam, A., Kapoun, A.M., Zheng, Q., Protter, A.A., Schreiner,

G.F., White, R.T., 2000. Altered patterns of gene expression in response to myocardial infarction [miscellaneous article]. Circ. Res. 86, 939–945. Xie, R.Q., Du, J., Hao, Y.M., 2003. Myocardial protection and mechanism of Puerarin injection on patients of coronary heart disease with ischemia/reperfusion. Zhongguo Zhong. Xi. Yi. Jie. He. Za Zhi. 23, 895–897. Yan, B., Wang, D.Y., Xing, D.M., Ding, Y., Wang, R.F., Lei, F., Du, L.J., 2004. The antidepressant effect of ethanol extract of radix puerariae in mice exposed to cerebral ischemia reperfusion. Pharmacol. Biochem. Behav. 78, 319–325. Yu, B.S., Yan, X.P., Zhen, G.B., Rao, Y.P., 2002. RP-HPLC determination of puerarin in Chinese traditional medicinal preparations containing pueraria. J. Pharm. Biomed. Anal. 30, 843–849. Zhang, G.Q., Hao, X.M., Dai, D.Z., Fu, Y., Zhou, P.A., Wu, C.H., 2003. Puerarin blocks Na+ current in rat ventricular myocytes. Acta Pharmacol. Sin. 24, 1212–1216.