Abstract
S-Allylcysteine (SAC) is an extensively studied natural product which has been proven to confer cardioprotection. This potentiates SAC into many clinical relevance possibilities, hence, the use of it ought to be optimally elucidated. To further confirm this, an ischemia/reperfusion model has been used to determine SAC at 10 mM and 50 mM on cardiac function, cardiac marker, and mitochondrial permeability. Using Langendorff setup, 24 adult male Wistar rats，hearts were isolated to be perfused with Kreb-Henseleit buffer throughout the ischemia/ reperfusion method. After 20 min of stabilization, global ischemia was induced by turning off the perfusion genetic evaluation for 35 min followed by 60 min of reperfusion with either Kreb-Henseleit buffer or SAC with the dose of 10 mM or 50 mM. The cardiac function was assessed and coronary effluent was collected at different timepoints throughout the experiment for lactate dehydrogenase (LDH) measurement. The harvested hearts were then used to measure glutathione while isolated mitochondria for mPTP analysis. SAC-reperfused hearts were shown to prevent the aggravation of cardiac function after I/R induction. It also dose-dependently upregulated glutathione reductase and glutathione level and these were also accompanied by significant reduction of LDH leakage and preserved mitochondrial permeability. Altogether, SAC dose-dependently was able to recover the post-ischemic cardiac function deterioration alongside with improvement of glutathione metabolism and mitochondrial preservation. These findings highly suggest that SAC when sufficiently supplied to the heart would be able to prevent the deleterious complications after the ischemic insult.
1. Introduction
Cardiovascular diseases are the main non-communicable disease [1] as it has been the principal cause of death globally for many years [2]. It is a multifactorial pathological condition as an individual is susceptible to it by having sedentary lifestyle, smoking habit, and eating unhealthy diet. Insufficient blood supply instigated by blockage inside coronary artery eventually causes ischemia; a condition of imbalance between oxygen supply and demand, consequently diminishing cardiac function and its viability.Clinically, re-establishing blood flow to the surviving cardiomyocytes after an insult is a therapeutic efficient strategy to salvage ischemic myocardium from inevitable necrosis, subsequently limiting the damage and reduce the cardiac dysfunction risk [3]. However, it also comes with the disadvantage that may be damaging to myocardium if the symptoms and causes are not controlled and targeted precisely. -A reperfusion injury which manifests in form of myocardial stunning [4], fatal arrhythmias [5], no-reflow phenomenon [6] and lethal reperfusion injury [3] after re-establishing blood flow, is a complex clinical condition caused by interaction of various mechanisms such as oxidative stress, mitochondrial dysfunction, mitochondrial permeability transition pore (MPTP opening), calcium (Ca2+) overload, inflammation and microvascular injury [3]. Nevertheless, to date, the drugs prescribed to the post-ischemic patients such as Angiotensin-Converting Enzyme (ACE) inhibitor comes with side effects, hence a safer adjunctive therapy seems to be more promising. Natural antioxidant supplement as an alternative in combatting free radical damage after an ischemic injury could be a promising strategy. Among all other emerging antioxidants, S-Allylcysteine (SAC); an aged-garlic compound has demonstrated cardioprotective effects. In fact, it has been reported that SAC was able to limit cardiac remodelling by reducing oxidative stress, increasing antioxidant level, and attenuating fibrosis [7,8]. Interestingly, it has been proposed to confer cardioprotection also by stimulation of cystathionine-γ-lyase/hydrogen sulphide axis that ultimately increases antioxidant levels [9]. Hydrogen sulphide is an endogenous gas that therapeutically plays many roles in bodily functions particularly on the cardiovascular [10]. SAC is also a prominent thiol donor that is able to regulate the redox homeostasis, by donating electron to the free radicals. Thiol functional group (-SH) in the chemical structure of SAC [11] also enhances sulfhydration of some proteins that has been proven beneficial elsewhere [12]. The compound which has been reviewed to have multifaceted roles in protecting the heart has yet to be tested on its possibility of exerting direct effect on the heart function. With current ambiguous clinical strategy in re-establishing blood flow to the patients, thus this study is aimed to investigate the effect of SAC on the cardiac function, cardiac injury, antioxidant status, and mitochondrial permeability after ischemia/reperfusion induction.
2. Materials and methods
2.1. Materials
SAC (>98%; Certificate of analysis included in Supplementary Fig. 1). All other chemicals used in this study were of analytical grade unless otherwise mentioned.
2.2. Authentication analysis
The purity of SAC was validated by LC-MS, 1H and 13C NMR and specific optical rotation analyses. The direct infusion Orbitrap analysis was performed on LTQ Orbitrap mass spectrometer (Thermo Scientific, San Jose, USA). The spectral m/z from 50 to 500 was recorded in positive mode. The electrospray ionisation conditions were as follows: Source accelerating voltage, 4.0 kV; capillary temperature, 280 ◦ C; sheath gas flow, 40 arb; auxiliary gas, 20 arb. The1H and 13C-APT NMR analyses were performed on the Bruker Avance III 600 MHz FT-NMR spectrometer (in CD3OD) using residual solvent peak to reference the spectra. 10 mg of SAC was dissolved in deionized water and specific optical rotation analysis was performed on Autopol V Automatic Polarimeter (Rudolph Research Analytical, New Jersey, USA).
2.3. Animals
A total of 24 male Wistar Rats (250–350 g, 10–12 weeks old) were used in this study. The rats were acclimatized in the laboratory condition for 1 week with ad libitum access to water and normal rat diet before carrying out the experiment. Throughout the experiment, all rats were housed under the same laboratory conditions of ambient room temperature and lighting (12 h light-dark cycle). All the procedures involving animal handling were subjected to approval from Universiti Kebangsaan Malaysian Animal Ethic Committee (FSK/2016/SATIRAH/ 23-NOV./812-NOV.-2016-NOV.-2017).
2.4. Ischemia/reperfusion via Langendorff isolated perfused heart system
The rats were randomly allotted to four different groups during the Langendorff Isolated Perfused Heart System which are Sham, I/R Control, 10 mM SAC, and 50 mM SAC. After anesthetized by the mixture of ketamine-xylazine (KTX), rats thoracic cavity were dissected open and hearts were isolated for ischemia/reperfusion protocol on Langendorff apparatus as previously described [13,14]. Briefly, hearts were cannulated via aorta to the Langendorff perfusion system (AD Instruments, Australia) and retrogradely perfused with warm Krebs-Henseleit buffer (pH 7.4, containing 118 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 1.18 mM MgSO4⋅7H2O 1.2 mM KH2PO4, 11 mM Glucose, and 1.8 mM CaCl2; continuously bubbled with 95% O2 and 5% CO2 at 37 ◦ C). Water-filled latex balloon was then inserted into left ventricle to detect LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP) and the first derivatives of LV pressure (LV ± dP/dt). LV developed pressure was calculated by subtracting LVEDP from LVSP. For I/R induction, the hearts were first allowed to stabilize for 20 min and baseline readings for LV markers, heart rate and coronary flow were all recorded on LabChart Pro Software [15]. Global ischemia was then induced by turning off perfusion to the heart for 35 min. The hearts temperature was maintained by immersing the hearts into the warm Kreb-Henseleit perfusion buffer. After 35 min of global ischemia, the hearts were reperfused with either Krebs-Henseleit buffer alone (IR Control) or with SAC (10 mM or 50 mM) for 60 min to monitor post-ischemic recovery of LV function. Recovery of LV markers were expressed as % relative to the baseline values and represented as mean ± SEM for all animals. Coronary flow volume per minute was measured in 1 min. While the coronary effluents were collected at different timepoint (0, 1, 2, 5, 10, 15, 30, 45, 60th minute) after reperfusion to measure lactate dehydrogenase activity and total lactate dehydrogenase release. After reperfusion ended, the hearts were harvested for further analysis.
2.5. Coronary effluent lactate dehydrogenase kinetic activity
Lactate dehydrogenase activity was assayed as previously described with slight modifications [16]. Briefly, 10 μL of coronary effluent collected from the perfused hearts procedure were added into microplate wells followed by 240 μL of PBS.Then 10 μL of nicotinamide adenine dinucleotide (NADH) solution (2.5 mg/mL) were added and the plate was incubated at room temperature for 20 min. Next, 10 μL of sodium pyruvate solution (2.5 mg/mL) was added. Activity of LDH was measured five times at 340 nm for 5 min with 1 min interval.
2.6. Left ventricle mitochondrial isolation
Left ventricle tissues were homogenized with sucrose buffer as previously described [17] and centrifuged at 600g for 5 min to allow removal cell debris from the homogenate. Supernatant was transferred into clean microcentrifuge tubes and centrifuged at 12,000g for 8 minto separate mitochondria pellet from cytosol. After the discard of supernatant, pellet was re-suspended in 1 mL isolation buffer to purify mitochondria. The suspension was then re-centrifuged at 12,000g for 8 min. Supernatant was discarded and the mitochondrion was resuspended in 200 μL of ice-cold sucrose buffer.
2.7. In vitro MPTP opening assay
Briefly, 0.2 mg rat heart mitochondria were suspended in 180 μL of swelling assay buffer (containing 10 mM Tris, 120 mM KCl, 5 mM KH2PO4 and 5 mM of succinic acid). Baseline absorbance at 520 nm was recorded using MultiSkan™ microplate reader. Then, 200 μM CaCl2 stock was quickly added into sample wells. Absorbance of samples at 520 nm wavelength was continuously measured in kinetics mode for 10 min. Absorbance transmediastinal esophagectomy data were normalized to baseline absorbance (absorbance change from 520 nm; ΔA520), and data were expressed as area above the curve [18].
2.8. Biochemical analysis
A small portion of cut left ventricle was weighed and homogenized according to its weight using phosphate buffered saline of pH 7.4. Homogenized samples were centrifuged at the speed of 10,000 rpm for 20 min. Supernatant was collected for reduced glutathione (GSH), oxidized glutathione (GSSG) and glutathione reductase (GR) level measurement. Briefly, 40 μL of PBS of pH 8 was added into the microplate wells. 50 μL of sample were added and immediately 10 μL of 5,5′ -Dithiobis 2′ Nitrobenzoic were added into the wells. Prior to the GSH level measurement absorbance of wavelength of 415 nm, the reaction was incubated at room temperature for 15 min.
2.9. Statistical analysis
All data arepresented as mean ± standard error of mean (SEM). Twoway analysis of variance (ANOVA) followed by a Tukey，s post-hoc test was used to analyse differences between groups using GraphPad Prism 6 (GraphPad Inc., United States). Statistical significance was considered at p < 0.05.
Fig. 1. Spectrum of S-allylcysteine (Tokyo Chemical Industry Co., Ltd). (A) Full-scan production spectra of [M + H]+ for SAC. (B) 600 MHz 1H NMR spectrum of SAC in CD3OD. (C) 150 MHz APT-13C NMR spectrum of SAC in CD3OD.
3. Results
3.1. Authenticity of SAC
SAC is a water-soluble organosulfur compound found abundant (up to 0.3 mg/kg) in aged garlic. Structurally, SAC is the sulfur-derivative of L-amino acid cysteine in which an allyl group replaces the proton on the sulfur. In this study, the reference SAC standard (>95%) was purchased from Tokyo Chemical Industry Co. Ltd. as white crystalline powder with optical purity of 100% (Supplementary Fig. 1). The authentication of the SAC standard was validated by means of high resolution mass spectrometry, 1H and 13C NMR analyses and specific optical rotation measurement.The ESI-MS [M + H]+ ion peak was observed atm/z 162.0582 corresponding to C6H11NO2S. In addition, the fragmentation patterns illustrated in Fig. 1A were in agreement to SAC [19]. The specific optical rotation [α]D(20) -16.489 (c 1.0, H2O) justified high enantiomeric excess of the standard purchased [20] (Supplementary Fig. 2).
The1H NMR spectrum (Fig. 1B) revealed three doublet of doublets at 。3.68 (J = 8.4, 4.2 Hz), 。3.12 (J = 14.4, 4.2 Hz) and 。2.86 (J = 14.4, 8.4 Hz) corresponding to H-α and H2β
respectively which in accordance to cysteine. The presence of S-allyl chain was represented by two doublet of doublets at 。5.24 (J = 17.4, 1.8 Hz) and 5.12 (J = 9.6, 0.6 Hz) of the terminal olefinic protons while multiplet centred at 。5.83 and 。3.23 revealed the vinylic methine and allylic thio-methylene protons.Six signals were observed in the 13C-APT NMR spectrum (Fig. 1C) consists of carbonyl (δ 172.8), olefinic methine (δ 135.2), olefinic methylene (δ 118.4), allylicthio methylene (δ 35.5), C-α (δ 55.3), and Cβ (δ 33.0) of cysteine (Fig. 3). All data were in accordance with those reported previously [21].
3.2. SAC reversed cardiac function exacerbation post-ischemia
Changes of cardiac function after ischemia were observed and recovery of the left ventricular function was recorded relative to baseline to determine the effects of SAC administration on postischemic cardiac dysfunction (Fig. 1). Compared to sham, I/R controls exhibited significantly (p < 0.05) reduced cardiac function as shown by the drop in recovery % of left ventricular systolic pressure (LVSP) (Fig. 2A), left ventricular developed pressure (LVDP) (Fig. 2B),maximum contraction rate in left ventricular pressure (LV + dP/dt) (Fig. 2C) and minimum relaxation rate (LV-dP/dt) (Fig. 2D). SAC administration however dosedependently improved the recovery of LVSP, LVDP, LV + dP/dt, LV-dP/ dt. Meanwhile, for the relaxation rate of left ventricular (Fig. 2E), I/R control hearts showed a significant (p < 0.05) increase in left ventricle end-diastolic pressure (LVEDP) as compared to Sham while SACperfused hearts showed a trend of decrease (p = 0.06) in LVEDP as compared to the I/R Control. LVEDP elevation during I/R reflects the impairment in ventricular relaxation capacity. In this study, coronary flow recovery rate (Fig. 2F) was also significantly lowered (p < 0.05) in I/R controls compared to Sham. Conversely, rat hearts administered with 50 mM SAC exhibited significant (p < 0.05) improvement in coronary flow recovery rate compared to the I/R controls. Rate pressure product (RPP) that was obtained by multiplying heart rate with LVDP was also measured. All I/R groups had a significantly (p < 0.05) lower RPP compared to Sham on 1st, 2nd, 5th, 10th, 15th, 30th, 45th, and 60th minute after reperfusion. Hearts that were supplied with SAC 50 mM however manifested a significant (p < 0.05) increase in RPP compared to I/R alone. 3.3. SAC reduced lactate dehydrogenase release and activity Lactate dehydrogenase release was used as a marker to measure the degree of injury after I/R. Lactate dehydrogenase release peaked significantly higher (p < 0.05) in I/R Control compared to Sham, SAC 10 mM, and SAC 50 mM for the first 2 min after onset of reperfusion (Fig. 3A). Total lactate dehydrogenase release was also significantly higher in I/R Control hearts coronary effluent. 50 Mm of SAC significantly reversed the total LDH release compared to I/R Control (Fig. 3B). 3.4. SAC enhanced glutathione ratio and glutathione reductase activity In determining whether SAC could increase antioxidant capacity in the heart in the context of I/R, level of endogenous glutathione (in ratio of GSH-to-GSSG) and activity of glutathione reductase were measured in the rat hearts after I/R. I/R Control hearts had significantly (p < 0.05) lowered level of reduced glutathione (GSH) (Fig. 4A), reduced glutathione/oxidized glutathione (GSH/GSSG) ratio (Fig. 4C), and glutathione reductase activity (Fig. 4D) as compared to Sham hearts. SAC administration however managed to reverse the reduced glutathione level back to baseline, dose-dependently. A trend of increment (p = 0.07–0.08) was shown by 50 mM SAC-perfused hearts as compared to I/ R Control in both GSH (Fig. 4A) and GSH/GSSG ratio (Fig. 4C). 3.5. SAC prevented MPTP opening in cardiac mitochondria MPTP opening is a crucial event that occurs during I/Rand it is a key determinant of the lethal reperfusion injury. In this study, in vitro mitochondria experiments were conducted in addition to the Langendorff study to determine whether SAC possess the ability to prevent MPTP opening in cardiac mitochondria. Isolated rat heart mitochondria were pre-incubated with DMSO vehicle alone, cyclosporine A (CsA, 10 μM) or SAC (10 mM or 50 mM) prior to the addition of Ca2+ bolus that induces MPTP opening. MPTP opening was reflected by the decrease in absorbance monitored at 520 nm as shown in Fig. 5A. Area above the curve analysis for the decrease in absorbance is shown in Fig. 5B. Compared to the vehicle, CsA which blocks MPTP by binding to cyclophilin D effectively attenuated the Ca2+-induced MPTP opening. SAC also exhibited dose-dependent potential to significantly prevent MPTP opening as compared to the vehicle treatment (Fig. 5A and B). 4. Discussion We herein present our novel findings on the cardioprotective effect of SAC in ischemic injury model. Ischemia/reperfusion protocol on isolated perfused hearts system was chosen as a tool to evaluate its effect directly on the heart. SAC is the most abundant organosulfur compound in aged garlic. During the aging process, γ-glutamylcysteines is hydrolysed completely to SAC (Fig. 6), where it remains unaltered for up to 2 years [22]. Oral administered SAC is rapidly absorbed by GIT with almost 100% bioavailability. SAC is metabolised to N-acetyl-SAC with half-life of 10 h and 30 h excretion time [23].Re establishing blood flow to the ischemic region has been the clinically relevant alternative in reducing the cardiovascular damage or dysfunction risk after ischemia. Even if reperfusion is a physiological adaptation of the heart itself in salvaging the ischemic tissue, it also contribute to myocardial cellular injury that accelerates necrosis due to cell swelling, disruption of myofibril, formation of contraction bands, and deposition of intra-mitochondrial calcium phosphate granules [24]. However, a cardioprotective compound addition with the blood flow reestablishment would attenuate the post-ischemic condition better. In the present study, SAC; a natural compound derived from aged garlic extract dose-dependently reversed the cardiac dysfunction after ischemia near to the normal baseline. Other than being a prominent anti-oxidant,SAC is also known to release the cardioprotective hydrogen sulphide gas [9]. Theoretically, possession of cysteine favours it to be a substrate to an endogenous enzyme called cystathionine-γ-lyase, which subsequently release hydrogen sulphide as a product. Therefore, it is important to consider a future research investigating into the possible CSE/H2S pathway initiated by SAC treatment with inhibitors such as β-Cyanoalanine (BCA) and propargylglycine (PAG) [9,25]. Fig. 2. SAC direct administration to the heart attenuated post-ischemic left ventricular dysfunction. (A) LVSP Recovery (%), (B) LVDP Recovery (%), (C) LV + dP/dt Recovery (%), (D) LV-dP/dt Recovery (%), (E) LVEDP, (F) coronary flow recovery, and (G) rate pressure product. Data arepresented as mean ± SEM for n = 5-6 per group. $p < 0.05 for sham vs control I/R group, I/R + SAC 10 mM, and I/R + SAC 50 mM using two-way ANOVA; *p < 0.05 for sham vs. control I/R group; #p < 0.05 for I/R Control vs I/R + SAC 50 mM groups using one-way ANOVA with Tukey post-hoc test. Fig. 3. SAC direct administration to the heart prevented post-ischemic deleterious impairment in cardiomyocyte injury. Time-course and area-under-curve (AUC) analysis for changes in (A) and total LDH release in (B) from Langendorff-perfused rat hearts isolated from 35 min of ischemia and 60 min of reperfusion. All values are given as mean ± SEM for n = 6 per group. *p < 0.05 for sham vs. control I/R group; and #p < 0.05 for I/R Control vs I/R + SAC 10 mM and 50 mM groups using one-way ANOVA with Tukey post-hoc test and two-way ANOVA. Fig. 4. SAC direct administration to the heart ameliorated post-ischemic cardiac glutathione level and metabolism. (A) GSH, (B) GSSG, (C) ratio of GSH:GSSG, and (D) glutathione reductase. All values are given as mean ± SEM for n = 6 per group. *p < 0.05 for sham vs. control I/R group; and #p < 0.05 for I/R Control vs I/R + SAC 50 mM groups using one-way ANOVA with Tukey post-hoc test. The result of this study demonstrated that I/R Control deteriorated significantly in cardiac function when compared to Sham. However, this present study suggests that SAC administration was able to restore the deleterious cardiac function effect after ischemic injury. Free radicalmediated mechanism might be the key factor of I/R [26] injury but the prominent antioxidant property of SAC might have not been the only sole mechanism that modulates the cardiac function after ischemia. It could be possible that the heart function recovery might be due to the hydrogen sulphide release, as evidenced in various previous studies of experimental models [27–29]. It is noteworthy that the result of SAC being able to recover cardiac function might subsequently favour in reducing the injury marker, improving antioxidant level, and preventing excessive mitochondrial opening altogether. Fig. 5. SAC prevented mitochondrial permeability transition pore opening in isolated cardiac mitochondria. (A) Change in absorbance monitored at 520 nm after addition of 200 μM Ca2+ bolus in isolated rat heart mitochondria pre-treated with CsA and SAC and (B) the area above the curve determined for the change in absorbance. *p < 0.05 vs. vehicle treatment using one-way ANOVA with Tukey post-hoc test for three independent experiments. Fig. 6. Catabolism of γ-glutamylcysteines to SAC via hydrolysis. The significant increase in LDH leakage following I/R demonstrated that myocardial injury occurred as result of ischemia. During ischemia, cardiomyocytes undergo necrosis by which cellular and subcellular compartments disintegrates and releases intracellular enzyme like LDH [30]. Among several mechanisms explained, free radicals have been shown to initiate lipid peroxidation resulting in altered membrane integrity [31]. Thus, LDH makes a good indication in showing the extent of the injury and predictor for pathological changes after ischemia [32]. This study demonstrated that SAC was able to significantly lower the LDH release on the first and second minute after reperfusion even though there was no significant difference on the fifth and tenth minute after reperfusion. This indicates that SAC did not wholly prevent the LDH release but delayed the injury instead. Consistent with this result, in vivo study on SAC treatment was able to reverse collagen deposition compared to the vehicle group in myocardial injury model, indicating a reduced injury [7]. In addition to that, SAC’s effect in lowering the total LDH release showed that it also conferred cardiomyocyte survival from excessive injury. This clearly suggests that SAC could reduce the cardiomyocyte necrosis and injury. SAC is likely to reduce the cardiomyocyte disintegration because of its ability in tackling the free radical damage-mediated injury by inducing endogenous antioxidant [9]. Although the mechanism underlying it is yet to be fully elucidated, restoration of myocardial injury marker clearly suggests the cardioprotective property of SAC. In any pathological condition, oxidative stress status plays an important role in either aggravating or combatting the disease. Hence, oxidative stress monitoring is essential. Glutathione is a natural endogenous antioxidant enzyme that is involved in quenching electrophilic substance such as hydrogen peroxide [33]. Recovery after ischemia causes reactive oxygen species attack within the cells and further alleviate cell injury. In our study, I/R Control group was observed to have significantly decreased reduced glutathione, glutathione reductase, and the ratio of reduced glutathione and oxidized glutathione. This clearly depicts that reperfusion might have disrupted the glutathione metabolism and availability, making it more susceptible to I/R injury. SAC administration exhibited recovery of GSH level and GSH/GSSG ratio although not in a significant manner. Glutathione reductase; the enzyme that reduce the oxidized state of glutathione significantly increased in SAC 50 Mm group compared to I/R, indicating that there was an increased activity of oxidized conversion to reduced state of glutathione to neutralize the free radicals. We speculated that SAC dose-dependently has selleck compound an antioxidant action on the ischemia/ reperfusion injury by preventing glutathione depletion through its thiol group [11].
In this study, we also determined whether SAC has potential to prevent MPTP opening in isolated rat heart mitochondria. MPTP opening is thought to mediate lethal reperfusion injury during I/R. Several studies have shown that pharmacological agents targeting MPTP reduced the extent of cardiomyocyte injury and improved cardiac function recovery after I/R in animal models [18,34]. It is interesting to note that SAC could potentially prevent MPTP opening in dosedependent manner. Currently it is unclear how SAC interacts with MPTP protein components; however, it is possible that thiol group in SAC might regulate redox state of these proteins through sulfhydration. It is well-documented that redox state and reactive oxygen species production increases susceptibility to Ca2+-induced MPTP opening [35]. Nonetheless, this data showed that SAC might prevent MPTP opening in the cardiomyocytes during I/R to limit tissue injury. Regardless, further studies are warranted to characterize the interaction between SAC with MPTP components during I/R in vivo and to elucidate the exact mechanism involved in
cardioprotective mechanism.
5. Conclusion
The findings of SAC cardioprotective property from this study by alleviating the cardiac function after I/R is noteworthy. SAC could improve the heart function after such an ischemic insult, although the underlying mechanism is still unclear. However, it could be possible that the increasing antioxidant availability via the thiol-containing structure improved the oxidative stress. It is also possible that these antioxidants were able to sustain at mitochondrial level from necrosis evidenced by reduced LDH injury marker, and eventually reversed the ischemic injuries. Altogether, at experimental level, SAC is able to demonstrate prevention of the injurious effects of ischemia to the cardiac function and injury, as well as the glutathione metabolism.