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Journal of Ethnopharmacology 121 (2009) 268–273

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Journal of Ethnopharmacology

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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 cardioprotective 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

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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. Supernatant (0.8 ml) was transferred into a 5-ml centrifuge tube 2.3. Determination of FATPE in Guanxin II and evaporated to dryness under a stream of nitrogen at 45 ◦C. The residue was dissolved in 400 ␮l mobile phase and 30 ␮l of the solu- FATPE contents were detected by a high-performance liquid tion was injected into an HPLC column. Absorption of FATPE in the chromatography (HPLC) method developed in our previous study supernatant was identified by HPLC coupling two stages of mass (Zhao et al., 2007). F served as the reference compound for Ligus- analysis (MS/MS). ticum chuanxiong Hort.; T and P served as the reference compounds The HPLC system of Waters 2690 included a gradient controller, for Salvia miltiorrhiza Bge.; E served as the reference compound for an automatic sample injector and a 996-photodiode array detector. Author's personal copy

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Separation was performed on a CAPCELL PAK C18 ACR (2.0 × 50 mm) 3. Infarct size determination from Japanese. The mobile phase was methanol/1% aqueous acetic acid with gradient elution (0.01 min, 5:95; 0.3 min, 5:95; 2 min, The heart was excised immediately at 24 h after coronary artery 100:0; 3 min, 100:0) and the flow rate was 0.8 ml/min. The col- ligation. The area at risk (AAR) was delineated after 1 ml 2% umn temperature was set at 40 ◦C. The injection volume was 40 ␮l. Evans blue dye was injected into the left ventricular cavity. The MS was performed on a Finnigan TSQ (San Jose, CA, USA) mass excised heart was frozen in −20 ◦C and sliced into 1-mm thick spectrometer equipped with an APCI interface. Mass spectrometric sections, which were incubated in a 1% solution of 2,3,5-triphenyl- conditions were optimized in order to achieve maximum sensitiv- tetrazolium chloride (TTC) in phosphate buffer, at pH 7.4 and 37 ◦C ity. The APCI conditions were as follows: corona discharge voltage, for 10 min. The TTC-stained area (red staining), TTC-stained nega- 4.5 kV; heated capillary temperature, 330 ◦C; nebulization tem- tive area (myocardial infarction) and Evan’s-blue-stained area (area perature, 450 ◦C; nitrogen was used both as sheath gas (70 psi, not at risk) were digitally captured and determined using Image- 1 psi = 6894.76 Pa) and auxiliary gas (25 a.u.). Argon was used as ProPlus. The infarct size was expressed as the percentage of the the collision gas with collision energy of 35 V. AAR. The AAR was isolated and the cardiac tissue was processed using the procedures described below for immunohistological and biochemical assays. 2.5. Series II: Effects of GXII and FATPE alone versus in combination, and some of FATPE on infarct size 3.1. Series III: Effects of GXII, FATPE and components of FATPE on myocardial apoptosis and caspase-3 activity This series was to ascertain whether GXII had efficacy on infarct size and which compounds were related to this efficacy. Groups of Rats were divided into the following five groups of 14 rats: (1) seven rats each were: (1) AMI + vehicle rats were orally given vehi- sham-operated (rats underwent an identical surgical procedure cle (0.9% NaCl); (2) AMI + GXII: rats were orally given GXII extract except that the coronary artery was not ligated); (2) AMI + vehicle; (30 g/kg) 30 min before ischemia; (3) AMI + F: rats were orally given (3) AMI + GXII; (4) AMI + FATPE; and (5) AMI + some components of sodium ferulic (5.64 mg/kg, equivalently 5.07 mg/kg of ferulic acid) FATPE. The method of administration was the same as Series II. All 30 min before ischemia; (4) AMI + T: rats were orally given tanshi- volumes of vehicle in Series II–IV were identical to those of the drug nol (28.08 mg/kg) 30 min before ischemia; (5) AMI + A: rats were groups. orally given hydroxysafflor yellow A (77.73 mg/kg) 30 min before ischemia; (6) AMI + P: rats were orally given protocatechualde- 3.2. Apoptosis detection hyde (0.54 mg/kg) 30 min before ischemia; (7) AMI + E: rats were orally given peoniflorin (111.45 mg/kg) 30 min before ischemia; (8) Apoptotic samples in border area of myocardial infarction were AMI + FATPE: rats were orally given FATPE 30 min before ischemia; taken from excised heart immediately 3 h after the coronary occlu- and if possible, (9) AMI + some of FATPE: rats were orally given some sion. Apoptotic cardiomyocytes were quantitatively detected by of FATPE 30 min before ischemia; Vehicle and all drug groups in terminal deoxy-nucleotidyl transferase mediated dUTP nick end- Series III and IV were gavaged 30 min before coronary occlusion. labeling (TUNEL) assay using a Cell Death Detection Kit (Roche) The doses of FATPE were identical to its content in GXII. according to the manufacturer’s instructions.

Fig. 2. Representative chromatogram of FATPE in plasma sample at 30 min after oral administration of GXII to acute myocardial infarction rat. (T) Tanshinol, (P) protocatechualdehyde, (E) peoniﬂorin, (F) ferulic acid and (A) hydroxysafﬂor yellow A. Author's personal copy

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3.3. Measurement of myocardial caspase-3 activity

A colorimetric assay kit (Chemicon International, Temecula, CA, USA) was used to detect myocardial caspase-3 activity. The frozen myocardial left ventricular samples from 3 h after coronary artery ligation were homogenized in ice-cold lysis buffer. The Bradford method was used to measure protein concentrations. Supernatant that contained 100 ␮g protein was loaded and incubated with 20 ␮g caspase-3 substrate Ac-DEVD-pNA for 90 min. The free pNA was quantified using a microplate spectrophotometer at microplate spectrophotometer at 405 nm. The results were calculated using Fig. 3. Effects of GXII, F, A, T, P, E, FATPE, FAT, FA, FT and AT on infarct size in rats. The a standard curve (3.906–500 ␮M pNA) and expressed as ␮mol area of necrosis (AN) was expressed as the percent of the area at risk (AN/AAR). (GXII) Guanxin II, (F) ferulic acid, (A) hydroxysafflor yellow A, (T) tanshinol, (P) pNA/mg protein. protocatechualdehyde and (E) peoniflorin. n =7,**p < 0.01 vs. vehicle.

3.4. Series IV: MBF measurement in AMI rats As illustrated in Fig. 4, there were very few TUNEL-positive cells Fluorescent microspheres 10 ␮m in diameter (FluoSpheres®, in myocardial tissue in the sham-operated rats. In contrast, numer- Invitrogen) were used to measure MBF in the area at risk (RA, ous apoptotic cardiomyocytes were detected in the border zone of the ischemic zone) and the remote control area (CA, the nonis- the infarct tissue in AMI rat. Three hours after coronary occlusion, chemic zone) of heart of AMI 2 h after oral administration of GXII GXII, FATPE and FAT all dramatically reduced the number of TUNEL- (30 g/kg, n = 11), FAT in combination (n = 10, the dose is the same as positive cells to 31.31–38.83% (p < 0.01) in the border area, when Series II and III), vehicle (n = 9) and sham-operated groups (n = 9), compared with the vehicle group. This suggests that FAT actually as described previously (Thibault et al., 2005). The animals were contributed to the anti-apoptotic effect of GXII. killed and the heart was excised and two different areas of the Myocardial caspase-3 activity, a final common pathway in left ventricle were examined separately: (1) RA in the anterior wall caspase-dependent apoptosis, was measured to quantitatively and and (2) CA in the posterior wall. The GXII, FAT and vehicle groups specially provide more evidence for the anti-apoptotic mech- received fluorescent microspheres injections 2 h after the start of anism of GXII and its ABC. As illustrated in Fig. 5, compared the experiment. Using the fluorescence data, MBF for each sample with sham-operated controls, 3 h following ligation of coronary was calculated according to the formula:

(FI /FI ) × 0.39 ml/ min MBF = tissue blood W where FI is ﬂuorescent intensity and W is wet tissue weight (g).Statistical analysis Data are expressed as means ± S.D. All data were analyzed by SPSS 10.0 software. Comparisons between multiple groups were carried out using analysis of variance (ANOVA) and t Tukey’s post hoc test (Sonne et al., 2008). Comparisons between two groups were carried out using Student’s t test. The values of p < 0.05 were considered to be statistically signiﬁcant.

4. Results

4.1. Determination of FATPE in serum

The chromatographic conditions were optimized to obtain good separation of the target compounds and avoid the interference of endogenous substances. Following oral GXII (30 g/kg) administration to AMI rats, the retention times and the main product ion of T, P, A, E and F were 0.68, 1.22, 1.78, 1.96 and 2.08 min, and 197, 137, 611, 539 and 193 m/z, respectively, as shown in Fig. 2. These parameters were consistent with reference substances of FAPTE.

4.2. Effect of GXII and FATPE (alone and in combination) and FAT on infarct size, apoptosis and caspase-3 activity

According to the content of FATPE multiplied by 30 g/kg, the Fig. 4. Representative photomicrographs of the in situ detection of DNA fragments in doses were 5.07 (F), 77.73 (A), 28.08 (T), 0.54 (P) and 111.45 (E) heart tissue from rats subjected to sham ischemia or 3 h of ischemia receiving vehicle mg/kg, respectively. As shown in Fig. 3, the area of necrosis (AN) or GXII. Cardial myocytes are identified by anti-a-actinin antibody, total nuclei were was expressed as a percentage of the area at risk (AN/AAR). Among labeled with DAPI (blue), and apoptotic nuclei were detected by TUNEL staining the components of FATPE, FAT alone had slight and moderate effects (green). (B) Summary of percent TUNEL-positive myocytes. Sham (sham-operated and PE alone did not, FA, FT and AT in combination only had also control), vehicle (orally given vehicle, n = 7), GXII (orally given Guanxin II), FATPE (orally given ferulic acid, hydroxyl safflor yellow A, tanshinol, protocatechualdehyde moderate effects. However, FAT in combination had a similar effect and paeoniflorin) and FAT (orally given tanshinol, ferulic acid and hydroxyl safflor to GXII or FATPE in combination in reducing infarct size. yellow A). n =7,**p < 0.01 vs. vehicle. Author's personal copy

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cardioprotection. This finding is different from the orthodox theory that ABCs of traditional Chinese formulae or single Western herbal remedies are too complex to be elucidated (Yuan and Lin, 2000; De Smet, 2002). The hypothesis was strongly supported by the similar efficacies of GXII and FAT (Figs. 3–5), and absorption of FATPE in blood (Fig. 2). Here, each dose of FAT was identical to its content in GXII. The BAP strategy for testing ABC hypothesis emphasized the ABC dosage identical to its content in formula, determination of ABC in blood and the active comparison of ABC and the parent formula (Huang, 2002b). As expected, we observed ischemia-induced apoptotic cardiomyocytes (Fig. 4). This was demonstrated by the presence of Fig. 5. Effects of GXII, FATPE and FAT on myocardial caspase-3 activity. Sham TUNEL-positive cells and an increase in caspase-3 activity (Fig. 5), (sham-operated control, n = 7), vehicle (orally given vehicle, n = 7), GXII (orally given a final common pathway in caspase-dependent apoptosis. These Guanxin II extract, n = 7), FATPE (orally given ferulic acid, hydroxyl safflor yellow A, tanshinol, protocatechualdehyde and paeoniflorin, n = 7) and FAT (orally given two changes were consistent with the widely accepted fundamen- tanshinol, ferulic acid and hydroxyl safflor yellow A). n =7,**p < 0.01 vs. Vehicle. tal pathogenetic mechanism that involves cardiomyocyte death during acute ischemia (Chen et al., 2008). This apoptosis led to AMI (Fig. 3). Acute oral administration of GXII (30 g/kg) reduced Table 1 the number of apoptotic cardiomyocytes by 32.31%; the per- Effects of GXII and FAT on myocardial blood flow of rats with acute myocardial infarction. centage of TUNEL-positive cardiomyocytes in vehicle control and GXII groups was 54.45 ± 14.96% and 36.83 ± 5.32%, respectively −1 MBF (ml/(min g )) (p < 0.01). Compared with the vehicle group, GXII-induced infarct Group Risk area Control area size was reduced (AN/AAR, 46.21 ± 14.68% versus 18.20 ± 4.96%, Sham, n = 9 3.68 ± 1.23 3.71 ± 1.30 p < 0.01, Fig. 3). As shown in Figs. 2–4, the percent of TUNEL- Vehicle, n = 9 3.42 ± 1.46 3.40 ± 1.52 positive cardiomyocytes and caspase-3 activity induced by FAT GXII, n = 11 6.91 ± 1.68 ** 7.12 ± 1.75 ** and FATPE all had the similar decreases compared with that of ± ± FAT, n = 10 6.87 1.91 ** 6.90 1.68 ** GXII. The above results indicated than FAT represent for the car- MBF: myocardial blood flow, GXII: Guanxin II, FAT: ferulic acid (F), hydroxyl safflor dioprotection of GXII, which is a potential native cardioprotective yellow A (A) and tanshinol (T). **p < 0.01 vs. vehicle. cocktail. Studies (Huang et al., 1996; Liu et al., 2006) indicated that ferulic acid was a nitrite scavenger, which had cardioprotection in artery (vehicle group) produced a 4.7-fold increase in caspase-3 ischemia–reperfusion injury and hemodynamics. Hydroxysafflor activity. GXII, FATPE and FAT apparently inhibited caspase-3 activ- yellow A protected the heart against ischaemia–reperfusion injury ity. by inhibiting mitochondrial permeability transition pore opening (Liu et al., 2008). Tanshinol possess the protection for myocardial 4.3. Effects of GXII and FAT on MBF mitochondrial membranes and microcirculatory disturbance from ischemia–reperfusion injury (Zhao et al., 1996; Han et al., 2008). As shown in Table 1, there were not differences of MBF between And Nizamutdinova et al. (2008) had also reported that paeoniflorin the ischemic and nonischemic zones and between sham-operated could protect the heart from myocardial ischemia/reperfusion and vehicle groups at acute ischemic stage of AMI, which is con- injury when high-dosage paeoniflorin was given by peritoneal sistent with literature (Boudina et al., 2002). After acute oral injection in rats. In the present study, FAT had slight or moder- GXII (n = 11) and FAT (n = 10), MBF of ischemic zone in AMI rat ate effect on infarct size and PE had no effect, when given alone was significantly increased compared with vehicle (n = 9) group by gavage to AMI in the dosage of GXII (Fig. 3). And the syner- (GXII, 6.91 ± 1.68 versus 3.42 ± 1.46, p < 0.01; FAT, 6.87 ± 1.91 versus gistic effect of FAT possesses the similar cardioprotection of GXII. − 3.42 ± 1.46, p < 0.01, ml/(min g 1)). To date, cardioprotection induced by FATPE or FAT in combination has not been reported. Our results showed that the synergisti- 5. Discussion cal interactions among FAT have happened when absorbed into blood. FATPE and FAT had similar cardioprotection as GXII against In addition, we consider that the acute anti-apoptotic effects infarct size and apoptosis, when gavaged acutely in AMI of high-dose GXII and FAT are induced via the indirect pathway (Figs. 2 and 3). As indicated in Section 1, this myocardiaol apopto- of increased MBF of ischemic zone, which had 1.02- and 1.01-fold sis induced by ischemic injury is different from that by reperfusion increases in MBF compared with that in the vehicle group. This indi- injury (Zhao et al., 2007). The crude extract of GXII is difficult rect pathway is also involved in the cardioprotection induced by E to be acutely gavaged during reperfusion, which is expediently via firstly improving hemodynamics and then protecting against gavaged before coronary ligation. Unexpectedly, compounds with ischemia–reperfusion injury (Nizamutdinova et al., 2008). This fewer than five of the components played the same role as GXII. indicates that GXII and FAT attenuate myocardial apoptosis via From this, we concluded that some of absorbed compounds have improvement of myocardial ischemia, which is the indirect action similar efficacy to the parent formula, further confirming the ABC on apoptotic cell. hypothesis. In conclusion, ABC of GXII is related to FAT. Via an indirect Our hypothesis (Huang et al., 1991) states the consistency of pathway, GXII and FAT protect the heart from ischemic injury by the number of absorbed and effective compounds. It was later increasing MBF of ischemic zone, and both GXII and FAT decrease supported by Homma et al. (1992). They have shown that the com- infarct size by inhibiting myocardial apoptosis. This suggests the pounds absorbed from the gut following oral administration are possibility of a novel cardioprotective cocktail in treating IHD, and actually related to the effects of the formula. In the present study, of a paradigm for elucidating the mechanism of action of ∼100,000 only three of the FATPE components played an important role in formulae. Author's personal copy

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