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This Article Appeared in a Journal Published by Elsevier. The This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Journal of Ethnopharmacology 121 (2009) 268–273 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm Cardioprotection by Guanxin II in rats with acute myocardial infarction is related to its three compounds Xi Huang a,d,∗, Feng Qin a, Hong-Min Zhang b, Hong-Bin Xiao c, Long-Xin Wang c, Xue-Ya Zhang d, Ping Ren a a Laboratory of Ethnopharmacology and Institute of Integrated Traditional Medicine and Western Medicine, Xiangya Hospital, Central South University, Changsha 410008, China b Henan Institute of Ophthalmology, and Department of Ophthalmology of Henan Provincial People’s Hospital, Zhengzhou 450003, China c Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116012, China d Laboratory of Ethnopharmacology, West China Hospital, Sichuan University, Chengdu 610041, China article info abstract Article history: Aim: We tested the hypothesis that cardioprotection afforded by traditional Chinese Guanxin II (GXII) Received 6 August 2008 formula is related to absorbed bioactive compounds (ABCs). Received in revised form 2 October 2008 Methods: Sprague–Dawley rats with acute myocardial infarction (AMI) were induced by coronary occlu- Accepted 29 October 2008 sion. ABCs including ferulic acid (F), hydroxyl safflor yellow A (A), tanshinol (T), protocatechualdehyde Available online 8 November 2008 (P) and paeoniflorin (E) were measured in blood after oral GXII. The effects of GXII and FATPE, alone and in combination, and of some components of FATPE on infarct size, myocardial apoptosis and caspase-3 Keywords: activity were determined. Myocardial blood flow (MBF) in AMI rat was detected 2 h after oral GXII and Traditional Chinese medicine Absorbed bioactive compounds FAT. Myocardial infarction Results: FATPE was found in rat blood. FAT was similar to FATPE and GXII in decreasing infarct size, Guanxin II myocardial apoptosis and caspase-3 activity of AMI. Both FAT and GXII were similar in increasing of MBF. Apoptosis Conclusion: GXII and FAT protect the heart from ischemic injury by increasing MBF, and decrease infarct size by inhibiting myocardial apoptosis and caspase-3 activity. These findings provide a potential cardio- protective cocktail. © 2008 Elsevier Ireland Ltd. All rights reserved. 1. Introduction miltiorrhiza Bge., Carthamus tinctorius L., Paeonia lactiflora Pall., Ligusticum chuanxiong Hort. and Dalbergia odorifera T. Chen in a Ischemic heart disease (IHD) is the number one killer in the ratio of 2:1:1:1:1 (Ye et al., 2003). Our previous study indicated that Western world (Venardos et al., 2007), and its main cause of death GXII has cardioprotection in ischemia–reperfusion injury (Zhao is acute myocardial infarction (AMI) (Kloner, 2006). Numerous et al., 2007). However, its absorbed bioactive compounds (ABCs) studies have indicated that the efficacy of pharmacological agents and mechanism of action are still largely unclear. We postulated (Tissier et al., 2008) and reperfusion (Abrams and Thadani, 2005) that the formula’s effects are related to its ABCs (Huang et al., is unsatisfactory in the treatment of AMI. So, as the cardioprotec- 1991). tive target during and after AMI, the importance of apoptosis is GXII is composed of five herbs. As shown in Fig. 1, its main com- sufficiently emphasized (Webster, 2007). pounds are ferulic acid (F), hydroxyl safflor yellow A (A), tanshinol Traditional Chinese Guanxin II (GXII), which is also called Guan- (T), protocatechualdehyde (P) and paeoniflorin (E). The content of Xin-Er-Hao (GXEH), formula has anti-anginal effects, attenuates these water-soluble FATPE components in GXII is relatively high. ST-segment depression, lowers total cholesterol and low-density FATE are all effective against myocardial ischemia (Huang et al., lipoprotein cholesterol (Chen, 1981; Xu et al., 2001; Huang, 2002a) 1996; Zhao et al., 1996; Liu et al., 2006; Han et al., 2008; Liu et and increases coronary flow velocity (Zhao et al., 2007; Zeng et al., al., 2008; Nizamutdinova et al., 2008). FATPE of GXII were used 2008) and antioxidative (Qin et al., 2008). GXII contained Salvia to test the ABC hypothesis via bioethnopharmaceutical analytical pharmacology (BAP) (Huang, 2002a). In brief, BAP is a strategy for elucidating ABCs of the formulae directed by ethnopharmaceutical ∗ and ethnopharmacokinetic analytics. Corresponding author at: Institute of Integrated Traditional Medicine and West- We aimed to evaluate whether GXII and FATPE induced simi- ern Medicine, Xiangya Hospital, Central South University, Changsha 410008, China. Tel.: +86 731 4327222 fax: +86 731 4328386. lar cardioprotection in rats with acute infarction different from our E-mail address: [email protected] (X. Huang). previous rats with ischemia–reperfusion injury. If so, we further 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.10.029 Author's personal copy X. Huang et al. / Journal of Ethnopharmacology 121 (2009) 268–273 269 Fig. 1. Chemical structures of five compounds derived from Guanxin II (GXII). determined which ABCs were related to efficacy and the mecha- Paeonia lactiflora P. and A served as the reference compound for nisms involved. Carthamus tinctorius L. The purity of all reference compounds pur- chased from National Institute for Control of Pharmaceutical and 2. Materials and methods Biological Products (Beijing, China) was >99%. The yield of lyophilized powder of GXII was about 22.86% 2.1. Preparation of Guanxin II decoction (w/w). According to the HPLC method used, the contents (mg/g) of FATPE in GXII were as follows: T, 0.936 ± 0.013; P, 0.018 ± 0.001, The ratio of Salvia miltiorrhiza Bge., Ligusticum chuanxiong Hort., E, 3.715 ± 0.123, A, 2.591 ± 0.021; and F, 0.169 ± 0.004. The over- Paeonia lactiflora P., Carthamus tinctorius L. and Dalbergia odorifera all intra- and inter-day variations were less than 5% for all five T. Chen from GXII was 2:1:1:1:1. They were bought in the Phar- analytes. These results demonstrated that the developed method macy of Xiangya Hospital. They were also authenticated by the is reproducible with good precision. The accuracy tests were car- herbal medicine botanist Professor Hu ZH, Department of Botanical ried out using a recovery test. Recovery of all five tested bioactive Anatomy of Northwest University in China. The voucher specimen compounds were >90% was deposited in Laboratory of Ethnopharmacology, Xiangya Hos- pital, Central South University. GXII was boiled twice in distilled 2.4. Series I: Identification of FATPE in serum of rats with acute water (1:12, w/v) for 30 min (Zhao et al., 2007). The blended super- myocardial infarction natants were then lyophilized. The rat dose of GXII (30 g/kg) in the present study was converted 2.2. Experimental design according to our previous study of human (GXII, 4.5 g/kg) (Zhao et al., 2007). That is, the rat is 6.7 times the doses of human. It is con- The study included: (I) FATPE content determination to calcu- sistent with literature (Pinkel, 1958). The administration volume in late the dose of each component in the following experiments; (II) Series I–IV was 1.5 ml/100 g. ABCs detection to provide direct evidence for ABCs of GXII according Midline sternotomy was performed after intubation and artifi- to BAP strategy (Huang, 2002a,b); (III) evaluating the effect of GXII cial ventilation, following anesthesia with sodium pentobarbitone and FATPE alone and in combination, and some of the FATPE compo- (40 mg/kg i.p.) The heart was rapidly exteriorized and the left ante- nents, on infarct size, to establish which components were related rior descending coronary artery was ligated ∼2 mm from its origin to this activity; (IV) measurement of myocardial apoptosis and with a 6–0 prolene suture. AMI was confirmed by the presence caspase-3 activity to elucidate the cardioprotective mechanism, of regional cyanosis, and ST elevation by electrocardiography. In and (V) myocardial blood flow (MBF) determination to elucidate this series of seven rats, 30 g/kg of GXII was orally administered the mechanism of acute action of GXII and its components. 10 min before the start of the surgical procedure. The operation All experiments in male Sprague–Dawley rats (200–240 g) from time was ∼10 min, and the mortality was ∼9%. Whole blood used SLAC (Shanghai, China) conformed to the Regulations for the for the determination of absorbed compounds was obtained by Administration of Affairs Concerning Experimental Animals (1988), decapitation 10 min after the end of the surgical procedure. which were approved by the Animal Experimental Center for Cen- Whole blood (1.2 ml) was centrifuged at 3000 × g for 20 min. tral South University (Changsha, China). Animals were housed in Serum (0.5 ml) was recovered and kept at −76 ◦C until analysis. The a temperature-controlled facility with a 12-h light/dark cycle, and serum sample was thawed and transferred to a 5-ml centrifuge had unlimited access to food and water for 7 days. The rats were tube, then mixed with 1.2 ml 80% ethanol for 12 h. After vortex- fasted for 12 h with free access to water before drug administration. ing, the resulting mixture was centrifuged at 12,000 × g for 10 min.
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