Journal of Chemistry

Volume 2014 (2014), Article ID 693573, 6 pages

http://dx.doi.org/10.1155/2014/693573

Research Article

Protective Effects of Elaeagnus angustifolia Leaf Extract against Myocardial Ischemia/Reperfusion Injury in Isolated Rat Heart

Binsheng Wang,¹ Hengyi Qu,² Jun Ma,³ Xiling Sun,¹ Dong Wang,⁴ Qiusheng Zheng,¹ and Du Xing⁵

¹Binzhou Medical University, Yantai 264000, China

²Affiliated Hospital of Binzhou Medical University, Yantai 264000, China

³Yantai University, Yantai 264005, China

⁴Shandong Provincial Qianfoshan Hospital, Jinan, Shandong 250014, China

⁵Yuhuangding Hospital of Yantai City, Yantai 264000, China

Received 10 April 2014; Accepted 18 June 2014; Published 8 July 2014

Academic Editor: Wang Chunming

Copyright © 2014 Binsheng Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The purpose of this study is to clarify the cardioprotective property of the aqueous extract of Elaeagnus angustifolia L. leaf (EA) against myocardial ischemia/reperfusion injury in isolated rat heart. The myocardial ischemia/reperfusion (I/R) injury model of isolated rat heart was set up by the use of improved Langendorff retrograde perfusion technology. Compared with the ischemia/reperfusion (I/R) group, the aqueous extract of Elaeagnus angustifolia L. leaf (0.5 mg/mL, 1.0 mg/mL) pretreatment markedly improved the coronary flow (CF) and raised left ventricular developed pressure (LVDP) and maximum rise/down velocity (max). The infarct size of the EA-treated hearts was smaller than that of I/R group. After treatment with EA, the superoxide dismutase (SOD) activity increased; malondialdehyde (MDA) and protein carbonyl content reduced more obviously () than that of I/R injury myocardial tissue. Conclusion. Results from the present study showed that the aqueous extract of Elaeagnus angustifolia L. leaf has obvious protective effects on myocardial I/R injury, which may be related to the improvement of myocardial oxidative stress states.

1. Introduction

Cardiovascular disease is one of the main causes of mortality of people all over the world [1]; acute myocardial infarction (MI) is one of the leading causes of death in human cardiovascular disease, which is caused by thrombotic occlusion of the coronary artery induced by the death of myocardial cells. Quick return to normal blood supply is the only effective way to reduce heart injury. Paradoxically, reperfusion can cause myocardial injury and further lead to cardiac dysfunction, referred to as “reperfusion injury” [2–4]. Therefore, mitigating myocardial ischemia-reperfusion (I/R) injury is a very important way for the ischemic heart disease treatment [5].

I/R injury is a very complicated process which involves a lot of kinds of possible mechanisms. Oxidative stress damage plays an important role in the progression of I/R injury, which is one of the most important mechanisms involved in I/R injury [6, 7]. Some studies have shown that increased expression of antioxidant enzymes will protect against I/R injury. A lot of antioxidants, such as vitamin E, catalase (CAT), melatonin, and superoxide dismutase (SOD), have been reported to protect the heart from I/R injury; all the evidences confirm that reduced oxidative stress could decrease the injury induced by I/R [8, 9].

Elaeagnus angustifolia, used as a food ingredient or herbal drug for its reputed medicinal properties, commonly called wild olive, silver berry, Russian olive, or oleaster, is a species of Elaeagnus, native to western and central Asia, from southern Russia and Kazakhstan to Turkey and Iran [10]. It has been found that Elaeagnus angustifolia is used in traditional Turkish medicine as antipyretic, diuretic, tonic, and antidiarrheal and as a medication against kidney disorders (inflammatory or kidney stone) [11, 12] and in Iranian folk medicine for its anti-inflammatory, antinociceptive, and analgesic effects. Also, decoction and infusion of its fruits are considered to be a good remedy for fever, jaundice, asthma, tetanus, and rheumatoid arthritis [13]; the aqueous and ethanolic extracts of its fruits induced a muscle relaxant effect in a dose dependent manner [14]. In the present study, we investigated the effects of aqueous extract from Elaeagnus angustifolia L. leaf on protecting rat heart against I/R injury.

2. Material and Methods

2.1. Plant Material

The mature leaves of Elaeagnus angustifolia L. were collected during the month of October from Shihezi, Xinjiang, China, and the authenticity of the material was verified by one of the authors and later confirmed by a botanist.

2.2. Chemicals

Triphenyltetrazolium chloride (TTC) was obtained from the Beijing Biodee Biotechnology Co., Ltd. (Beijing, China). The malondialdehyde (MDA), super oxide dismutase (SOD), and protein carbonyl analyzing reagent kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All of the other chemicals used in the study for the biochemical estimation were of analytical grade obtained commercially.

2.3. Preparation of Extract

Fresh Elaeagnus angustifolia L. leaves were washed in distilled water and air-dried in the shade. Thirty grams of leaves was cut into small pieces and extracted with 70°C distilled water (DW) (DW: material, 10 : 1, v/w) thrice in an incubator at 85°C for 3 h. The water extract was filtered through a 2 μm pore sterile filter paper. The combined filtrates were concentrated in a vacuum at 60°C, and the resulting filtrates were 50 mL (amount to 6 × 10² mg/mL crude drug).

2.4. Experimental Groups and Drug Delivery

Male Wistar rats (250–280 g) were obtained from Xinjiang Medicine University Medical Laboratory Animal Center (SDXK 2011-004). All experimental procedures complied with the Institutional Animal Care and Use Committee of National Institute Pharmaceutical Education and Research.

The rats were divided into four groups randomly: control group (Sham), I/R group, low concentration of aqueous extract of Elaeagnus angustifolia L. leaves (EA) treatment group, and high concentration of EA treatment group.

2.5. Preparation for Isolated Heart

The male Wistar rats (250–280 g) were anesthetized by an intraperitoneal injection of 60 mmol/L chloral hydrate (0.35 g/kg). To anticoagulate, 250 U/kg of heparin was sublingually venously injected into the rats. Thoracic surgery was performed to remove the heart [15], hearts were excised and mounted on Langendorff’s apparatus quickly, and then the hearts were immersed in ice-cold Krebs-Henseleit buffer (1.2 mM KH₂PO₄, 1.2 mM CaCl₂, 25 mM sodium acetate, 120 mM NaCl, 1.2 mM MgSO₄, and 11 mM glucose; pH 7.4) piped with a gas mixture comprised of 95% O₂/5% CO₂; then the hearts were maintained in 37°C in a water-jacketed organ chamber [16]. The pressure recording was established by a water-filled latex balloon, coupled to a pressure transducer (Statham), which was inserted into the left ventricular cavity via the left auricle.

Control group hearts were stabilized by perfusion for 95 min and the I/R group hearts were stabilized for 30 min, and then, after 20 min global ischemia, the reperfusion was established for 45 min. Hearts in EA treatment group were stabilized for 20 min, instead of K-H buffer with EA (0.5 mg/mL, 1 mg/mL) for 10 min, and then global ischemia for 20 min and reperfusion for 45 min were established.

2.6. Measurement of Heart Hemodynamic Parameters

A computer-based data acquisition system was used to continuously monitor the hemodynamic parameters including the following functional parameters: left ventricular developed pressure (LVDP, LVDP = LVSP − LVEDP), left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), maximum rise/down velocity of left intraventricular pressure (max), and heart rate (HR) were monitored continuously using 4S AD Instruments biology polygraph (Powerlab, Australia). The coronary flow (CF) was measured using a flowmeter with an in-line probe.

2.7. Evaluation of Myocardial Infarct Size

Frozen hearts were cut into 2 mm thick cross-sectional slices for evaluation of myocardial size. The slices were stained by putting them into the 1% triphenyltetrazolium chloride (TTC) solution for 10 minutes at 37°C. The stained slices were transferred to a formalin solution for 10 min after TTC staining; then we placed them into phosphate buffer (pH 7.4) [17]. The heart slices were then digitally imaged using a digital camera. The ischemic (white) and nonischemic (red) area were measured digitally using Image Pro Plus software. The infarct size was represented as percentage of the ischemic area.

2.8. Assay of Oxidative Stress

At the end of the perfusions, the hearts were kept at −70°C for later oxidative stress analysis. Liquid nitrogen-chilled tissue pulverizer was used to crush the frozen hearts to a powder. For tissue analyses, weighed amounts of the frozen tissues were homogenized in appropriate buffer using microcentrifuge tube homogenizer. Then, the SOD activity, malondialdehyde (MDA), and protein carbonyl content were analyzed spectrophotometrically according to the instruction of the assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.9. Statistical Analysis

Data were presented as mean ± standard deviation from at least six independent experiments. Differences in hemodynamic parameters, infarct size, MDA level, SOD activity, and carbonyl content level were analyzed using the one-way ANOVA with Student’s -test. Statistical significance was considered at the probability value of less than 0.05. Analyses were carried out using the Origin 8.0 software (Origin Lab Corporation, Northampton, MA, USA).

3. Result

3.1. The Aqueous Extract of Elaeagnus angustifolia L. Leaves (EA) Enhanced Recovery of I/R Induced Changes in Cardiac Function

The cardiac function was monitored continuously using 4S AD Instruments biology polygraph. Compared to the sham I/R group, I/R injury caused a significant decrease in left ventricular developed pressure (LVDP), maximum rise/down velocity of left intraventricular pressure (max), and coronary flow (CF). As shown in Figure 1, the EA administration significantly blunted the reduction of LVDP (Figure 1(a)), max (Figures 1(b) and 1(c)), and CF (Figure 1(d)) caused by I/R injury. However, there were no significant changes in HR (Figure 1(e)) among all of the four groups.

(a)

(b)

(c)

(d)

(e)

Figure 1: Effects of the aqueous extract of Elaeagnus angustifolia L. leaves upon hemodynamics in a model of ischemia-reperfusion using isolated rat heart. Data are the mean ± SD. Values marked with and are significantly different from control group. Values marked with and are significantly different from I/R group.

3.2. The EA Reduced Myocardial Infarct Size following I/R Injury

At the end of reperfusion, myocardial infarct size was assessed using the TTC staining method. As illustrated in Figure 2, ischemia for 20 min followed by 45 min of reperfusion resulted in development of substantial myocardial infarcts, while the 0.5 mg/mL and 1.0 mg/mL EA preconditioning substantially decreased I/R induced percentage of myocardial infarct size.

(a)

(b)

Figure 2: Effects of the aqueous extract of Elaeagnus angustifolia L. leaves upon myocardial infarct size. Data are the mean ± SD. Values marked with and are significantly different from I/R group.

3.3. The EA Improved Oxidative Stress State Induced by I/R

To identify the possible mechanisms of EA on cardioprotection, the SOD activity and MDA level were determined in myocardial tissue. The SOD activity significantly increased (Figure 3(a)), while MDA level (Figure 3(b)) was significantly decreased in EA pretreatment groups compared with that of I/R group. Protein carbonyls are formed through oxidation of proteins by a variety of mechanisms which are the sensitive markers of oxidative injury. As shown in Figure 2, the hearts subjected to global myocardial ischemia for 20 min followed by 45 min of reperfusion showed a significant increase of level of carbonyl content, while the EA preconditioning substantially decreased I/R induced rise of level of carbonyl content.

(a)

(b)

(c)

Figure 3: Effects of EA on SOD activity (a), MDA level (b), and carbonyl content level (c). Values are means with their standard deviation (); compared with control group; compared with I/R group.

4. Discussion

The present work was aimed at studying the cardioprotective activity of the aqueous extract of Elaeagnus angustifolia L. leaf (EA) in ischemia/reperfusion (I/R) induced cardiotoxicity in isolated rat heart. The results of this study revealed that EA at the doses of 0.5 mg/mL and 1.0 mg/mL dependently and significantly ameliorated the cardiotoxicity by restoring cardiac function and myocardial biochemical parameters towards the normal values.

After reperfusion of the ischemic myocardium, significant myocardial dysfunction, including LVDP, max, CF and HR, and myocardial infarct, was induced by I/R. Reperfusion is a key role among a number of events leading to myocardial dysfunction associated with I/R injury. In the present study, we found that EA improved recovery of I/R-altered hemodynamic parameters (LVDP, max, and CF and HR) and attenuated infarct size induced by I/R significantly.

The generation of reactive oxygen species (ROS) plays an important role in the I/R induced myocardial injury [18, 19]. The ROS can be diminished by antioxidant systems that include antioxidant enzymes, such as SOD in the normal conditions [20]. However, when the amount of ROS is beyond the capacity of those enzymes and cannot be diminished during reperfusion, oxidative stress occurs. More and more evidence suggested that cardiomyocyte death and myocardial injury occurred during ischemia and reperfusion accompanied with oxidative stress [21–23]. Therefore, reduction of oxidative stress is one of the favorable strategies to alleviate myocardial injury induced by I/R. MDA is a maker of the peroxidation of cell membrane lipids caused by ROS. SOD is one of the most significant antioxidant enzymes, functioning as a superoxide anion scavenger, protein carbonyl is a sensitive biomarker of oxidative injury of proteins, and carbonyls are formed through oxidation of proteins. To further investigate the mechanism of cardioprotective effect of EA, an experiment was performed to examine whether EA affected the levels of MDA, protein carbonyls, and SOD activity induced by I/R. The present results illuminated that EA protected against myocardial I/R induced injury, accompanied by the attenuation of MDA production and protein carbonyls content and enhancement of SOD activity indicating that one of the mechanisms of the cardioprotection of EA was associated with its antioxidant effects.

In the present investigation, administration of EA significantly enhanced the recovery of I/R-altered cardiac function by blunting the reduction of left ventricular developed pressure (LVDP), maximum rise/down velocity of left intraventricular pressure (max), and coronary flow (CF) decreased by I/R injury. Also, EA treatment resulted in significant modulation of cardioprotection content, the SOD activity, and MDA level. Therefore, it can be concluded that the aqueous extract of Elaeagnus angustifolia L. leaf has obvious protective effects on myocardial I/R injury, which may be related to the improvement of myocardial oxidative stress states.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contribution

Binsheng Wang and Hengyi Qu contribute equally to this work.

Acknowledgments

This study was supported by the Natural Science Foundation of Shandong Province (no. ZR2011HL013) to Dong Wang, the Xinjiang Production and Construction Corps Fund for Distinguished Young Scientists (2011CD006), and International Cooperation Project 2012BC001 to Qiusheng Zheng.

References

G. Martínez-Sánchez, L. Delgado-Roche, A. Díaz-Batista, et al., “Effects of ozone therapy on haemostatic and oxidative stress index in coronary artery disease,” European Journal of Pharmacology, vol. 691, no. 1–3, pp. 156–162, 2012.

P. A. Grace, “Ischaemia-reperfusion injury,” British Journal of Surgery, vol. 81, no. 5, pp. 637–647, 1994.

H. Inafuku, Y. Kuniyoshi, S. Yamashiro et al., “Determination of oxidative stress and cardiac dysfunction after ischemia/reperfusion injury in isolated rat hearts,” Annals of Thoracic and Cardiovascular Surgery, vol. 19, no. 3, pp. 186–194, 2013.

J. J. Ley, R. Prado, J. Q. Wei, N. H. Bishopric, D. A. Becker, and M. D. Ginsberg, “Neuroprotective antioxidant STAZN protects against myocardial ischemia/reperfusion injury,” Biochemical Pharmacology, vol. 75, no. 2, pp. 448–456, 2008.

E. Gao, Y. H. Lei, X. Shang et al., “A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse,” Circulation Research, vol. 107, no. 12, pp. 1445–1453, 2010.

M. Khan, S. Varadharaj, L. P. Ganesan et al., “C-phycocyanin protects against ischemia-reperfusion injury of heart through involvement of p38 MAPK and ERK signaling,” The American Journal of Physiology—Heart and Circulatory Physiology, vol. 290, no. 5, pp. H2136–H2145, 2006.

E. Murphy and C. Steenbergen, “Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury,” Physiological Reviews, vol. 88, no. 2, pp. 581–609, 2008.

D. K. Das and N. Maulik, “Antioxidant effectiveness in ischemia-reperfusion tissue injury,” Methods in Enzymology, vol. 233, pp. 601–610, 1994.

Z. Chen, T. D. Oberley, Y. S. Ho et al., “Overexpression of CuZnSOD in coronary vascular cells attenuates myocardial ischemia/reperfusion injury,” Free Radical Biology and Medicine, vol. 29, no. 7, pp. 589–596, 2000.

L. H. Bailey and E. Z. Bailey, Hortus Third: A Concise Dictionary of Plants Cultivated in the United States and Canada, Macmillan, New York, NY, USA, 1976.

F. A. Ayaz and E. Bertoft, “Sugar and phenolic acid composition of stored commercial oleaster fruits,” Journal of Food Composition and Analysis, vol. 14, no. 5, pp. 505–511, 2001.

I. Gürbüz, O. Üstün, E. Yesilada, E. Sezik, and O. Kutsal, “Anti-ulcerogenic activity of some plants used as folk remedy in Turkey,” Journal of Ethnopharmacology, vol. 88, no. 1, pp. 93–97, 2003.

A. Ahmadiani, J. Hosseiny, S. Semnanian et al., “Antinociceptive and anti-inflammatory effects of Elaeagnus angustifolia fruit extract,” Journal of Ethnopharmacology, vol. 72, no. 1-2, pp. 287–292, 2000.

H. Hosseinzadeh, M. Ramezani, and N. Namjo, “Muscle relaxant activity of Elaeagnus angustifolia L. fruit seeds in mice,” Journal of Ethnopharmacology, vol. 84, no. 2-3, pp. 275–278, 2003.

N. Khandoudi, M. Laville, and A. Bril, “Protective effect of the sodium/hydrogen exchange inhibitors during global low-flow ischemia,” Journal of Cardiovascular Pharmacology, vol. 28, no. 4, pp. 540–546, 1996.

J. L. Zweier, J. Fertmann, and G. Wei, “Nitric oxide and peroxynitrite in postischemic myocardium,” Antioxidants and Redox Signaling, vol. 3, no. 1, pp. 11–22, 2001.

D. J. Bester, K. Kupai, T. Csont et al., “Dietary red palm oil supplementation reduces myocardial infarct size in an isolated perfused rat heart model,” Lipids in Health and Disease, vol. 9, article 64, 2010.

H. P. Grill, J. L. Zweier, P. Kuppusamy, M. L. Weisfeldt, and J. T. Flaherty, “Direct measurement of myocardial free radical generation in an in vivo model: effects of postischemic reperfusion and treatment with human recombinant superoxide dismutase,” Journal of the American College of Cardiology, vol. 20, no. 7, pp. 1604–1611, 1992.

M. Shen, R. Wu, L. Zhao et al., “Resveratrol attenuates ischemia/reperfusion injury in neonatal cardiomyocytes and its underlying mechanism,” PLoS ONE, vol. 7, no. 12, Article ID e51223, 2012.

X. Yuan, B. Yu, Y. Wang et al., “Involvement of endoplasmic reticulum stress in isoliquiritigenin-induced SKOV-3 cell apoptosis,” Recent Patents on Anti-Cancer Drug Discovery, vol. 8, no. 2, pp. 191–199, 2013.

J. M. McCord, “Oxygen-derived free radicals in postischemic tissue injury,” The New England Journal of Medicine, vol. 312, no. 3, pp. 159–163, 1985.

J. A. Thompson and M. L. Hess, “The oxygen free radical system: a fundamental mechanism in the production of myocardial necrosis,” Progress in Cardiovascular Diseases, vol. 28, no. 6, pp. 449–462, 1986.

S. W. Werns, M. J. Shea, and B. R. Lucchesi, “Free radicals and myocardial injury: pharmacologic implications,” Circulation, vol. 74, no. 1, pp. 1–5, 1986.