Anti-ischemic effect of Aurantii Fructus on contractile dysfunction of ischemic and reperfused rat heart

Journal of Ethnopharmacology 111 (2007) 584–591

Anti-ischemic effect of Aurantii Fructus on contractile dysfunction of ischemic and reperfused rat heart Moonkyu Kang a,1 , Jong-Hoon Kim b,1 , Chongwoon Cho a , Hwan-Suck Chung a , Chang-Woon Kang b , Yangseok Kim c , Minkyu Shin c , Moochang Hong c , Hyunsu Bae a,c,∗ b

a Purimed R&D Institute, Kyung-Hee University, #1 Hoegi-Dong, Dongdaemun-Ku, Seoul 130-701, Republic of Korea Department of Veterinary Physiology, College of Veterinary Medicine, Chonbuk National University, 664-14, 1GA, Duckjin-Dong, Duckjin-Gu, Jeonju City, Jeollabuk-Do 561-756, Republic of Korea c Department of Physiology, College of Oriental Medicine, Kyung-Hee University, #1 Hoegi-Dong, Dongdaemun-Ku, Seoul 130-701, Republic of Korea

Received 11 May 2006; received in revised form 26 December 2006; accepted 9 January 2007 Available online 12 January 2007

Abstract Aurantii Fructus (AF) is one of the most well-known traditional herbal medicines frequently used for the treatment of cardiovascular symptoms in Korea. The anti-ischemic effects of AF on ischemia-induced isolated rat heart were investigated through analyses of changes in perfusion pressure, aortic flow, coronary flow, and cardiac output. The subjects in this study were divided into two groups: an ischemia-induced group without any treatment, and an ischemia-induced group with AF treatment. There were no significant differences in perfusion pressure, aortic flow, coronary flow, and cardiac output between them before ischemia was induced. The supply of oxygen and buffer was stopped for 10 min to induce ischemia in isolated rat hearts, and AF was administered during ischemia induction. AF treatment significantly prevented decreases in perfusion pressure, aortic flow, coronary flow, and cardiac output under ischemic conditions (p < 0.01). These results suggest that AF has distinct anti-ischemic effects through recovery of contractile dysfunction in ischemic heart. © 2007 Published by Elsevier Ireland Ltd. Keywords: Aurantii Fructus; Traditional herbal medicine; Anti-ischemia effect

1. Introduction Cardiac ischemia is a condition in which blood flow and oxygen supply are insufficient to the heart muscle. The main cause of cardiac ischemia is narrowed coronary arteries. When arteries are narrowed, less blood and oxygen reach the heart muscle. Cardiac ischemia leads to coronary heart disease, angina pectoris, myocardial infarction, heart failure and ultimately heart attack (Shirai, 2004). Aspirin, beta-blockers, angiotensin-converting enzyme inhibitors, and lipid-lowering agents are currently the backbone of pharmacologic therapy for this disorder (Mehta et al., 2000). Because of the adverse effects associated with these anti-ischemia drugs, many trials have been recently per∗

Corresponding author at: Department of Physiology, College of Oriental Medicine, Kyung-Hee University, #1 Hoegi-Dong, Dongdaemun-Ku, Seoul 130-701, Republic of Korea. Tel.: +82 2 961 9316; fax: +82 2 967 2080. E-mail address: [email protected] (H. Bae). 1 Two first authors contributed equally to this work. 0378-8741/$ – see front matter © 2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2007.01.007

formed to find and develop new anti-ischemic drugs through herbal medicines that would minimize side effects. Numerous animal and clinical studies of various herbal medicines have been performed, and some have reported significant improvements in controlling ischemic symptoms without any noticeable adverse effects (Sun et al., 2002). Aurantii Fructus is one of the best-known traditional herbal medicines frequently used to treat cardiovascular symptoms in Korea. AF has been used clinically as a traditional oriental medicine against shock, indigestion, and accumulation and obstruction of phlegm and stifling in the chest. Aurantii Fructus is reported to reduce portal pressure in portal hypertensive rats through increase of mean arterial pressure, prevent infective shock by increase of cardiac output and inhibit blood coagulation by anti-fibrinolytic effect (Huang et al., 1995). Other potential therapeutic effects of AF extracts, such as hypocholesterolemic actions through HMG-CoA reductase inhibition (Liu et al., 2002) and hypolipidemic actions (Kurowska and Manthey, 2004) and anti-asthma action through inhibition of Ig E production (Kim et al., 1999)

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have been previously reported. It is also known that several flavonoids are included in AF (see Table 3). Among those flavonoids, naringin is known for its anti-ischemic action (Singh and Chopra, 2004), while synephrine is known for its anti-shock action (Huang et al., 2001). Also, naringin and hesperidin are known for their anti-diabetic (Jung et al.,2004, 2006), antioxidant (Chen et al., 1990) and cholesterol-lowering actions (Kurowska and Manthey, 2004). Other potential therapeutic effect of hesperidin, such as vasorelaxing effect has been previously reported (Calderone et al., 2004). These functions of Aurantii Fructus suggest that Aurantii Fructus can serve as an effective anti-ischemia agent to improve bad blood circulation induced by abnormal blood coagulation, adjust blood flow to normal, and finally recover dysfunction of heart induced by ischemia. However, it is not reported for any anti-ischemic effect of Aurantii Fructus. Therefore, the purpose of this study is to evaluate the protective effects of Aurantii Fructus on postischemic contractile dysfunction in isolated rat hearts. 2. Materials and methods 2.1. Preparation of Aurantii Fructus (AF) We obtained the spray-dried AF extract from Sunten Pharmaceutical Co. (Taipei, Taiwan, lot number: 263901). AF was deposited in the Department of Physiology, College of Oriental Medicine, Kyung-Hee University. One gram of AF powder contained 0.66 g of AF extract and 0.34 g of starch. We dissolved 3 mg of AF powder in 0.66 ml of KH buffer, centrifuged at 15,000 rpm for 10 min, transferred the supernatant to another tube and filtered through a 0.2 ␮m syringe filter. Then, 1.02 mg of starch was removed in final AF solution preparation and the AF concentration of that solution was finally 3 mg/ml. The resultant solution was administered into the aortic line for 5 min to observe the effects of AF on an ischemia-induced heart with a 65 mmHg perfusion pressure to see its anti-ischemic effects. 2.2. The HPLC analysis of standard material of Aurantii Fructus One thousand milligrams of the commercial sprayed dried water extract including 34% starch was accurately weighed, placed in test tubes, and dissolved in 10 ml of chloroform (HPLC reagent, J.T. Baker Co. Ltd., U.S.A.). This was filtered using a 0.45 ␮m syringe filter (PVDF, Waters, U.S.A.). The marker substance (standard material) used for the quantitative analysis was hesperidin (Sigma Chemicals, U.S.A). Ten milligrams of the standard material was dissolved in a solution. This solution was then diluted at 0.1, 0.5, 1.0, 1.5 and 2.0 mg/ml. In order to obtain a standard HPLC chromatogram, each standard solution was again diluted at 0.1, 0.5, 1.0, 1.5, 2.0 mg/ml. The relationship between the concentration and the peak-area was measured by the minimum square method (R2 value). The HPLC apparatus used was a Waters Breeze System (717+ Autosampler, 2487 dual ␭ absorbance detector, 1525 binary HPLC Pump, Waters Co., Milford, USA). Another Waters Breeze System (Ver. 5.00,

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Waters Co., Milford, U.S.A.) was used for data acquisition and integration. The quantity of standard material solution added to each herbal extract was calculated using the following formula: amount (mg) of standard materials = {quantitative amount (mg) of standard materials × AT/AS}/n (n = 3), where AT is the peakarea of the test samples containing the standard materials and AS is the peak-area of standard materials). From the results of the standard calibration curve, the R2 values of all marker substances range between 0.991 and 0.999. The standard material used for the quantitative analysis of Aurantii Fructus is hesperidin (M.W. 610.56) and its content in Aurantii Fructus is 5.13 ± 0.15 mg/g (0.51 ± 0.02%). 2.3. Heart preparation and perfusion apparatus Male Sprague–Dawley rats weighing 250–300 g were supplied by Taconic Korea (Taconic Korea, Seoul, Korea). The rats were housed and allowed free access to food and tap water under strictly controlled pathogen-free conditions (room temperature: 23 ± 1 ◦ C; relative humidity: 50 ± 10%; light cycle: 07:00–19:00). The rats were fed a standard rodent pellet chow and acclimatized to their environment for 2 weeks before commencement of the experiments. Next, the rats were randomly divided into two groups (n = 10), an ischemia-induced group and ischemia-induced group with AF treatment. The rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg). Heparin (1000 U/kg) was injected through a femoral vein to prevent blood coagulation. The hearts were rapidly excised and placed in ice-cold (4 ◦ C) Krebs–Henseleit (KH) bicarbonate buffer (NaCl 120.0 mM, NaHCO3 25 mM, KCl 4.8 mM, KH2 PO4 1.2 mM, CaCl2 1.25 mM, MgSO4 1.2 mM, and glucose 11.0 mM), which immediately stopped the contractile activity of the heart. Aorta and left atrium cannulation was performed rapidly, and the hearts were perfused in Langendorff mode at a pressure of 100 cm H2 O with KH buffer. The buffer was saturated with 95% O2 /5% CO2 at pH 7.4 and thermostatically kept at a constant temperature of 37 ◦ C. Global ischemia was achieved by clamping both the aortic and atrial lines for 10 min. The Langendorff heart preparation involves the cannulation of the aorta, which is then attached to a reservoir containing oxygenated perfusion fluid. This fluid is then delivered in a retrograde direction down the aorta either at a constant flow rate delivered by an infusion or roller pump or a constant hydrostatic pressure usually in the range of 60–100 mmHg (Sutherland and Hearse, 2000). In Langendorff perfusion (non-working heart model), perfusion fluid entered the heart via the aorta retrograde from the aortic reservoir located 100 cm above the heart. The aortic reservoir, which was the thermostatically maintained oxygenator, carried out a perfusate to the aorta at a 100 cm H2 O hydrostatic pressure maintained with the use of a constant head device (CHD). This system maintains the function of the heart, but does not maintain circulation of perfusate to the ventricle. Such a system is used to recover and maintain heart function for 15 min after isolation and ischemia induction. The working heart preparation is a more complex preparation with ventricular filling via the left atrium and ejection in the normal direction via the aorta. This preparation offers the advantage of an ability to mea-

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sure pump function with different filling pressures and afterloads (Sutherland and Hearse, 2000). In the working heart model, the left atrium cannula and aortic cannula were open and perfusion fluid entered the heart via the left atrium from an atrial bubble trap located 20 cm above the heart. The left ventricle ejected perfusate via the aorta and elasticity chamber (aortic pressure chamber) against a 20 cm H2 O hydrostatic pressure to the aortic bubble trap. The same system is used to maintain heart function 20 min before induction of heart ischemia, and to recover heart function for 60 min after ischemia surgery using the Langendorff system (non-working heart model). The system makes it possible to compare the recovery of heart function before and after induction of heart ischemia. Aortic and coronary perfusates were not recirculated in the present study. The entire apparatus was thermostatically maintained by a water jacket and coil heat chamber. Aortic flow and coronary flow were measured by timed collection of perfusate from the aortic and pulmonary trunk cannula, respectively. Cardiac output was calculated by summing the aortic and coronary flows. Heart rate was obtained by an ECG monitoring system (S&W Medico Teknik A/S, Denmark) with three electrodes attached to the epicardium. Systolic and diastolic aortic pressures were measured throughout the working heart model perfusion periods in the aortic outflow line with a hemodynamic monitoring system (S&W Medico Teknik A/S, Denmark). 2.4. Ischemia induction of isolated-perfused rat heart Male Sprague–Dawley rats weighing 250–300 g were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg). The hearts were rapidly excised and mounted on a Langendorff apparatus (IPH-W, Labo Support, Osaka, Japan) via the aorta, and then perfused at a constant pressure of 65 mmHg with KH buffer. The heart was constantly warmed by a circulating water jacket at 37 ◦ C. The buffer was gassed with 95% O2 /5% CO2 at pH 7.4. To measure left ventricular pressure, a pressure transducer was connected to the aortic cannula. Heart rate was monitored from left ventricular pressure. Coronary flow was measured by coronary flow volume (ml/min). After stabilization (non-working system) to 100 cm H2 O (100 mmHg) for 15 min via the aortic cannula, the perfusion pressure was reduced to 20 cm H2 O (20 mmHg) for 20 min at the LA cannula (working system), and then ischemia was induced for 10 min accompanied by AF injection for 5 min. When ischemia was started, AF extract (50 ml of 3 mg/ml AF) was dissolved in KH buffer, centrifuged at 15,000 rpm for 10 min, transferred the supernatant to another tube and filtered through a 0.2 ␮m syringe filter, and the resultant AF solution injected into the aortic line for 5 min to observe the effects of AF on an ischemia-induced heart with a 65 mmHg perfusion pressure. Ischemic conditions were maintained for 5 additional min. In the control group, equal volumes of KH buffer were injected into the aortic line for 5 min. Those hearts were retrograde perfused for 15 min according to the Langendorff method as described by Li et al. (1996) to recover heart function after ischemia induction as previously described in Section 2.3. Then, the heart was perfused again through the working heart system for 60 min. The functional recovery rates between

Fig. 1. Determination of maximum AF dose that results in maximum antiischemic effect. Cardiac output was calculated by summing the aortic and coronary flows. Aortic flow and coronary flow were measured by timed collection of perfusate from the aortic and pulmonary trunk cannula, respectively, throughout the working heart model with a hemodynamic monitoring system in both groups to detect the maximal anti-ischemia effect according to AF dose (0–30 mg/ml). White histograms represent the mean ± S.E.M. from 10 rats per control group without any treatment under pre ischemia conditions. Striped histograms represent the mean ± S.E.M. from 10 rats per AF treatment under pre ischemia conditions. Web histograms represent the mean ± S.E.M. from 10 rats per AF treatment group under post-ischemia conditions according to AF dose (0–30 mg/ml).

the ischemia-induced group and ischemia-induced group with AF treatment after ischemia induction were compared through changes in perfusion pressure, aortic flow, coronary flow, and cardiac output to observe the anti-ischemia effect of AF. 2.5. Statistical analysis The results are presented as the mean ± S.E.M. Statistical significance was compared between the treatment and control groups by Student’s t-test. Results with a p < 0.05 were considered statistically significant. 3. Results 3.1. Determination of maximal effective dose of AF on ischemia-induced isolated rat heart The maximum effective amount of AF on ischemia-induced isolated rat heart was assessed by measuring cardiac output, the direct parameter of heart pump function, with and without AF treatment after induction of ischemia while increasing the AF dose from 0.1 to 30 mg/ml. As seen in Fig. 1, there was no difference between groups with and without 3 mg/ml of AF treatment under pre-ischemic conditions [89.8 ± 2.8 (99%) versus 90.6 ± 2.4 (100%)]. This result suggests that AF itself does not influence cardiac output in normal conditions. The maximum recovery effect for cardiac output after ischemia was obtained with 30.0 mg/ml of AF. The recovery effect of AF on cardiac output after ischemia increased

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Table 1 Heart rate in ischemia-induced isolated rat heart Group

Control AF treatment

Pre-ischemia (beats/min)

Post-ischemia (beats/min)

15 min

10 min

30 min

60 min

284.1 ± 22.7 (100) 277.5 ± 16.1 (100)

271.5 ± 15.8 (96.8) 263.7 ± 16.3 (96.2)

268.9 ± 19.3 (95.0) 266.7 ± 18.9 (96.4)

275.6 ± 23.3 (96.9) 268.8 ± 19.5 (95.2)

Each number represents the mean ± S.E.M. from 10 rats per group. The numbers in brackets of each low are percentage comparison values when the heart rates of the control and AF treatment groups under pre-ischemic conditions were described as 100%, respectively. Values in parenthesis are in percent.

continuously with doses of 30.0 mg/ml (63.5 ± 3.2 ml/min with 3 mg/ml, 65.8 ± 4.8 ml/min with 10 mg/ml and 68.9 ± 4.1 ml/min with 30 mg/ml). However, 10 mg/ml and 30 mg/ml of AF, the concentrations of maximum recovery effect for cardiac output, are very high concentrations for application. Also, the recovery for cardiac output after ischemia between treatment of 3 mg/ml and 30.0 mg/ml AF was not significantly different (63.5 ± 3.2 ml/min with 3 mg/ml, 65.8 ± 4.8 ml/min with 10 mg/ml and 68.9 ± 4.1 ml/min with 30 mg/ml). Thus, 3.0 mg/ml was determined to be the appropriate AF dose to optimize the anti-ischemic effect on ischemia-induced isolated rat heart. 3.2. Heart rate in ischemia-induced isolated rat heart Since it is well-known that heart rate does not significantly change under ischemic conditions (Yu et al., 2001; Galagudza et al., 2004), the heart rate of ischemia-induced isolated rat heart was assessed. As shown in Table 1, the heart rate between pre-ischemic and post-ischemic conditions was not significantly different [284.1 ± 22.7 (100%) versus 275.6 ± 23.3 (96.9%)]. Also, the heart rate between the control and AF treatment groups under post-ischemic conditions was not significantly different [275.6 ± 23.3 (96.9%) versus 268.8 ± 19.5 (95.2%)]. These results indicate that heart rate does not change in ischemiainduced isolated rat heart regardless of AF treatment.

3.4. Recovery effect of AF on decreased perfusion pressure and aortic flow of ischemia-induced isolated rat heart Perfusion pressure was substantially decreased by ischemia induction to an average of 64.9 ± 2.3% of control under preischemic conditions (Table 2 and Fig. 2). However, such decreases were recovered by AF treatment to an average of 79.9 ± 3.3% of control before ischemia was induced (p < 0.01, Table 2 and Fig. 2). These anti-ischemic effects of AF on perfusion pressure (mmHg) were continuously observed for 10–60 min during the post-ischemic period (Fig. 3). However, any effects of AF on perfusion pressure under normal conditions were not observed for 5–20 min during the pre-ischemic period and 10–60 min during the post-ischemic period (control versus AF, p > 0.05, Fig. 3). Taken together, these results suggest that AF does not influence perfusion pressure and does recover decreased perfusion pressure induced by ischemia specifically. Similarly, AF treatment successfully recovered the aortic flow reduced by ischemia to 66.6 ± 3.5% of the control value (p < 0.01, Table 2 and Fig. 2). In the working heart model,

3.3. Overall anti-ischemic effects of AF on ischemia-induced isolated rat heart The degree of ischemic injury was assessed by measuring the extent of perfusion pressure, aortic flow, coronary flow, and cardiac output, all of which are basic assessments of cardiac function. All four parameters were substantially decreased by induction of ischemia to an average of 64.9 ± 2.3%, 48.7 ± 3.0%, 58.4 ± 1.8%, and 51.2 ± 2.8%, respectively (100% being pre-ischemic values, Table 2 and Fig. 2). However, AF treatment recovered such decreases to an average of 79.9 ± 3.3%, 66.6 ± 3.5%, 80.7 ± 2.1%, and 70.2 ± 3.2%, respectively, compared to pre-ischemic conditions (p < 0.01, Table 2 and Fig. 2). These recovery rates correspond to average increases of 23% (perfusion pressure), 37% (aortic flow), 38% (coronary flow), and 37% (cardiac output) compared to control under post-ischemic conditions (p < 0.01, Fig. 2). These results indicate that AF treatment significantly recovered heart dysfunction induced by ischemia.

Fig. 2. Overall anti-ischemic effects of AF on ischemia-induced isolated rat heart. Perfusion pressure (PP), aortic flow (AF), coronary flow (CF), and cardiac output (CO) were measured by timed collection of perfusate from the aortic and pulmonary trunk cannula of both groups to detect an anti-ischemia effect. Each histogram represents the mean ± S.E.M. from 10 rats per group, the control group without any treatment (first histogram, N/C), the AF treatment group (second histogram (+) AF) under normal conditions, the control group without any treatment (third histogram, Ischemia), and the AF treatment group (fourth histogram (+) AF + ischemia) under ischemic conditions. ** Significantly different from control group (p < 0.01) based on Student’s t-test.

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Table 2 Overall anti-ischemic effects of AF on ischemia-induced isolated rat heart Time (min)

Control

AF treatment

PP

AF

CF

CO

PP

AF

CF

CO

Pre-ischemia 5 10 15 20

93.7 93.5 93.8 92.3

± ± ± ±

1.00 0.87 1.14 1.18

70.3 67 66.7 66.2

± ± ± ±

1.17 1.32 0.94 1.22

22.9 23.3 22 22.8

± ± ± ±

0.82 0.75 0.97 0.39

92.2 90.3 88.7 89

± ± ± ±

1.47 1.51 1.28 1.26

90.3 89.4 87.6 87.6

± ± ± ±

1.01 1.63 2.09 1.80

69 67.33 65 67.03

± ± ± ±

1.15 2.40 3.21 3.79

22.05 22.67 21.8 23.06

± ± ± ±

1.15 1.20 0.90 1.30

91.05 ± 1.15 89.67 ± 2.38 86.8 ± 3.19 90.09 ± 3.77

Post-ischemia 10 20 30 40 50 60

64.3 63.7 59 58.6 54.1 51.6

± ± ± ± ± ±

1.11 1.16 1.30 1.46 1.12 1.01

34.7 34.9 32.4 31.7 32.4 31.7

± ± ± ± ± ±

1.01 1.15 1.38 1.12 1.40 1.64

13.8 13.9 12.7 13.4 12.9 13.8

± ± ± ± ± ±

0.53 0.81 0.72 1.14 0.67 0.88

48.5 48.8 45.1 45.1 45.3 45.5

± ± ± ± ± ±

1.11 1.31 1.64 1.68 1.61 1.55

73.3 73.60 72.60 71.80 71.50 69.70

± ± ± ± ± ±

2.91** 3.32** 3.31** 2.53** 2.13** 2.32**

45.67 47.67 42.00 43.33 44.02 51.03

± ± ± ± ± ±

3.38** 2.96** 3.06** 2.73** 1.30** 2.65**

17.33 19.00 19.87 18.58 17.90 18.80

± ± ± ± ± ±

1.86** 1.76** 0.80** 1.20** 1.30** 1.70**

62.67 66.67 61.87 61.91 61.92 69.83

± ± ± ± ± ±

3.35** 2.93** 3.01** 2.71** 1.30** 2.62**

Each number represents the mean ± S.E.M. from 10 rats per group. ** Significantly different from control group (p < 0.01) based on Student’s t-test. PP, perfusion pressure; AF, aortic flow; CF, coronary flow and CO, cardiac output.

AF treatment continuously recovered decreases in aortic flow 10–60 min after ischemia was induced (Fig. 4).

from 10 to 60 min in the working heart model after ischemia was induced (Fig. 5).

3.5. Recovery effect of AF on decreased coronary flow of ischemia-induced isolated rat heart

3.6. Recovery effect of AF on decreased cardiac output of ischemia-induced isolated rat heart

Induction of ischemia elicits a substantial decrease in coronary flow up to 58.4 ± 1.8% compared to control (Table 2 and Fig. 2). However, AF treatment dramatically recovered coronary flow to 80.7 ± 2.1% of control values under pre-ischemic conditions (p < 0.01, Table 2 and Fig. 2). Such recovery continued

Cardiac output was substantially decreased by induction of ischemia to an average of 51.2 ± 2.8% of control (see Table 2 and Fig. 2). However, such decreases were recovered by AF treatment to an average of 70.2 ± 3.2% of control under pre-ischemic conditions (p < 0.01, see Table 2 and Fig. 2). Also, such decreases

Fig. 3. Recovery effect of AF on decreased perfusion pressure (PP) of ischemiainduced isolated rat heart. Perfusion pressure was measured throughout the working heart model perfusion periods in the aortic outflow line with a hemodynamic monitoring system in the control and AF treatment groups to detect an anti-ischemia effect. Each symbol represents the mean ± S.E.M. from 10 rats per group with denoting (䊉) the control group without any treatment, () the AF treatment group under normal conditions, () the control group without any treatment, and () the AF treatment group under ischemic conditions. ** Significantly different from the control group without any treatment under ischemic conditions (p < 0.01) compared to the AF treatment group under ischemic conditions based on Student’s t-test.

Fig. 4. Recovery effect of AF on the decreased aortic flow (AF) of ischemiainduced isolated rat heart. Aortic flow (AF) was measured by timed collection of perfusate from the aortic and pulmonary trunk cannula in the control and AF treatment groups to detect an anti-ischemia effect. Each symbol represents the mean ± S.E.M. from 10 rats per group with denoting (䊉) the control group without any treatment, () the AF treatment group under normal conditions, () the control group without any treatment, and () the AF treatment group under ischemic conditions. ** Significantly different from the control group without any treatment under ischemic conditions (p < 0.01) compared to the AF treatment group under ischemic conditions based on Student’s t-test.

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4. Discussion

Fig. 5. Recovery effect of AF on decreased coronary flow (CF) of ischemiainduced isolated rat heart. Coronary flow (CF) was measured by timed collection of perfusate from the aortic and pulmonary trunk cannula in the control and AF treatment groups to detect an anti-ischemia effect. Each symbol represents the mean ± S.E.M. from 10 rats per group with denoting (䊉) the control group without any treatment, () the AF treatment group under normal conditions, () the control group without any treatment, and () the AF treatment group under ischemic conditions. ** Significantly different from the control group without any treatment under ischemic conditions (p < 0.01) compared to the AF treatment group under ischemic conditions based on Student’s t-test.

were significantly increased by AF treatment to an average of 37% of control under post-ischemic conditions (p < 0.01, see Table 2 and Fig. 2). In the working heart model during the postischemic period, AF treatment significantly recovered decreases in cardiac output (Fig. 6).

Fig. 6. Recovery effect of AF on decreased cardiac output (CO) of ischemiainduced isolated rat heart. Cardiac output (CO) was calculated by summing the aortic and coronary flows (CO = CF + AF). Each symbol represents the mean ± S.E.M. from 10 rats per group with denoting (䊉) the control group without any treatment, () the AF treatment group under normal conditions, () the control group without any treatment, and () the AF treatment group under ischemic conditions. ** Significantly different from the control group without any treatment under ischemic conditions (p < 0.01) compared to the AF treatment group under ischemic conditions based on Student’s t-test.

In the present study, the anti-ischemic effects of AF on ischemia-induced isolated rat heart were investigated through analyses of changes in perfusion pressure, aortic flow, coronary flow, and cardiac output. AF treatment significantly prevented decreases in perfusion pressure, aortic flow, coronary flow, and cardiac output under ischemic conditions. The onset of severe ischemia in the myocardium sets into motion a series of pathological events that continue until the tissues die. These changes begin seconds after ischemia and occur because the supply of oxygen is insufficient to support oxidative phosphorylation in the cardiac tissue (Bak et al., 2006). The isolated perfused small mammalian heart probably represents the optimal compromise in the conflict between the quantity and quality of data that can be acquired from an experimental model versus its clinical relevance-especially in relation to the modeling of ischemia (Hearse and Sutherland, 2000). At a practical level, the isolated heart, especially from small mammals, provides a highly reproducible preparation, which can be studied quickly and in large numbers at relatively low cost. It allows a broad spectrum of biochemical, physiological, morphological and pharmacological indices to be measured. These measurements can be made in the absence of the confounding effects of other organs, the systemic circulation and a host of peripheral complications such as circulating neurohormonal factors. This characteristic may be considered as an investigational advantage in that it allows the dissection of peripheral from cardiac responses. Certainly, the isolated perfused heart provides an excellent test-bed for undertaking carefully controlled dose–response studies of metabolic or pharmacological interventions. The preparation also readily allows the induction of whole heart or regional ischemia and this can be achieved at various levels of flow. Similarly, anoxia or hypoxia at various degrees of oxygen deprivation in the presence of normal flow can be easily imposed. The isolated heart preparation is amenable to reperfusion or reoxygenation at various rates and with various reperfusate compositions thus providing a powerful tool for assessing many aspects of ischemia- and reperfusioninduced injury. In practical terms, the rat heart is by far, the best characterized, it is also the heart most frequently used for more complex perfusion preparations such as working and blood perfused hearts. In terms of ease of handling, the rat has a great advantage over smaller hearts such as the mouse where intraventricular pressure recordings are more difficult. Although a number of other variants exist, isolated perfused heart preparations are largely based on adaptations of that originally described by Langendorff or the more complex working preparation described by Neely. The Langendorff heart preparation involves the cannulation of the aorta, which is then attached to a reservoir containing oxygenated perfusion fluid. This fluid is then delivered in a retrograde direction down the aorta either at a constant flow rate delivered by an infusion or roller pump or a constant hydrostatic pressure usually in the range of 60–100 mmHg. In both instances, the aortic valves are forced shut and the perfusion fluid is directed into the coronary ostia thereby perfusing the entire ventricular mass of the heart,

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Table 3 The known components of Aurantii Fructus Naringin Umbelliferone Limonin

Hesperidin d-Limonene Synephrine

This table was referred from http://www.tradimed.com.

draining into the right atrium via the coronary sinus. The working heart preparation is a more complex preparation with ventricular filling via the left atrium and ejection in the normal direction via the aorta. This preparation offers the advantage of an ability to measure pump function with different filling pressures and afterloads. Rat hearts are the most frequently used species for working heart preparations but all species can be used-even the dog or pig. Whereas the Langendorff preparation provides valuable information on left ventricular systolic and diastolic pressures and their derivatives, the working heart gives valuable data on cardiac pump function. Both isolated heart preparations are extremely valuable for assessing the direct cardiovascular effects of various therapeutic agents in terms of contractile function, electrical activity or metabolic function. Whether perfused with an asanguinous solution, washed red cells or blood in the Langendorff or the working mode, many investigators use the isolated heart for the study of regional or global ischaemia. Global whole heart zero-flow ischemia is readily induced in the Langendorff and the working heart simply by occluding the perfusion inflow lines. Graded whole heart ischemia at various degrees of flow can also be readily induced in the Langendorff preparation but is difficult to achieve in the working heart (as a consequence, investigators often switch the working heart temporarily back to the Langendorff mode for such treatment). Regional ischemia can also be induced in both preparations by ligating a coronary artery (usually the left main) and the size of the ischemic zone can be influenced by the positioning of the occlusion point (Sutherland and Hearse, 2000). Under ischemic conditions, myocardial oxidative metabolism is suppressed and glycolysis becomes an important source of ATP generation. The increased glycolytic rate in the face of impaired glucose oxidation leads to uncoupling of the two pathways and a buildup of lactate and H+ , a process that may continue during reperfusion. This accumulation of protons leads to downstream activation of pathways (Na+ /H+ exchanger, Na+ /Ca2+ exchanger) that result in Ca2+ overload, impaired contractile function, and/or cell death (Asano et al., 2003). It is known that AF contains several chemicals (Table 3), and of these components, naringin and hesperidin have been most widely recognized as having anti-ischemic effects that could potentially prevent Ca2+ overload or intake by the cell (Calderone et al., 2004; Singh and Chopra, 2004). In more detail, it has been reported that hesperidin and hesperetin, a major metabolite of hesperidin in vivo significantly activated vasorelaxation through potassium channel activation (Calderone et al., 2004), and naringenin, a major metabolite of naringin in vivo significantly inhibited Ca2+ uptake induced by phenylephrine in rat aorta (Orallo et al., 2005) and in voltage operated calcium channel in clonal rat pituitary GH C cells (Summanen et al., 2001). Reactive oxygen species and metabo-

lites are known to play important roles in the pathogenesis of ischemia/perfusion and anoxia/reoxygenation injury. The reduction of O2 results in the production of superoxides as well as hydrogen peroxide (H2 O2 ). H2 O2 is highly diffusible and induces cell damage. H2 O2 appears to affect not only lipids but also transmembrane proteins. The hydroxyl radical (OH) also participates in lipid hyperoxidation (Asano et al., 2003). Some chemicals in AF, including umbelliferone and d-limonene, are recognized as antioxidants (Table 3) capable of reducing reactive oxygen species (Baccard et al., 2000; Hakim et al., 2002). However, it has been reported that the amounts of those components in AF, except hesperidin and naringin, are very small (Li et al., 2004). Thus, it is thought that hesperidin and naringin in AF may work as anti-ischemic agents. 5. Conclusion The anti-ischemic effects of AF on ischemia-induced isolated rat heart were investigated through analyses of changes in perfusion pressure, aortic flow, coronary flow, and cardiac output. AF treatment significantly prevented decreases in perfusion pressure, aortic flow, coronary flow, and cardiac output under ischemic conditions. These results suggest that AF has distinct anti-ischemic effects. These results support the development of a novel anti-ischemia agent based on the pharmacological action of Aurantii Fructus. Acknowledgments This research was financially supported by a grant from the Ministry of Science and Technology of Korea (#2005-00198), a grant from the Korean Health 21 R&D Project, Ministry of Health & Welfare, Korea (A06-00044227), the Brain Korea 21 project in 2006 and research grant from Bio-Safety Research Institute, Chonbuk National University in 2006. References Asano, G., Takashi, E., Ishiwata, T., Onda, M., Yokoyama, M., Naito, Z., Ashraf, M., Sugisaki, Y., 2003. Pathogenesis and protection of ischemia and reperfusion injury in myocardium. Journal of Nippon Medical School 70, 384– 392. Baccard, N., Mechiche, H., Nazeyrollas, P., Manot, L., Lamiable, D., Devillier, P., Millart, H., 2000. Effects of 7-hydroxycoumarin (umbelliferone) on isolated perfused and ischemic-reperfused rat heart. Arzneimittelforschung 50, 890–896. Bak, I., Lekli, I., Juhasz, B., Nagy, N., Varga, E., Varadi, J., Gesztelyi, R., Szabo, G., Szendrei, L., Bacskay, I., Vecsernyes, M., Antal, M., Fesus, L., Boucher, F., de Leiris, J., Tosaki, A., 2006. Cardioprotective mechanisms of Prunus cerasus (Sour cherry) seed extract against ischemia-reperfusioninduced change in isolated rat hearts. American Journal of Physiology-Heart and Circulatory Physiology 291, H1329–H1336. Calderone, V., Chericoni, S., Martinelli, C., Testai, L., Nardi, A., Morelli, I., Breschi, M.C., Martinotti, E., 2004. Vasorelaxing effects of flavonoids: investigation on the possible involvement of potassium channels. NaunynSchmiedebergs Archives of Pharmacology 370, 290–298. Chen, Y.T., Zheng, R.L., Jia, Z.J., Ju, Y., 1990. Flavonoids as superoxide scavengers and antioxidants. Free Radical Biology and Medicine 9, 19–21. Galagudza, M., Kurapeev, D., Minasian, S., Valen, G., Vaage, J., 2004. Ischemic postconditioning: brief ischemia during reperfusion converts persistent ven-

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