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International Journal of Pharmacology & Toxicology / 5(1), 2015, 53-61.

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ANTI-ISCHEMIC PROPERTIES OF NIGELLA SATIVA AGAINST CARDIAC AND NON-CARDIOVASCULAR ISCHEMIA Ahmed A. A. Bader Department of Pharmacology and Toxicology, Unaizah College of Pharmacy, Qassim University, Al-Qassim 51452, Kingdom of Saudi Arabia.

ABSTRACT Ischemic Cardiovascular Diseases (ICVD) may be broadly classified into ischemic coronary heart disease, renal hypertension, ischemic cerebrovascular disease and peripheral vascular disease. The black seed, Nigella sativa (NS), a member of the family of ranunculaceae, is commonly used as a natural food additive. NS and its active constituents have been documented to exhibit antioxidant, hypotensive, calcium channel blockade and diuretic properties which may contribute to reduce blood pressure and exerting anti-ischemic effects via reduction in sympatheic overactivity and oxidative stress; suggesting a potential role of NS in the management of cardiovascular diseases as hypertension and ischemic coronary heart diseases and other non-cardiovascular ischemia. This study aiming for exploring the anti-ischemic properties of NS that protecting he heart and other vital organs against ischemia and decreasing incidence of myocardial infraction and ischemicreperfusion injuries on different body organs in experimental animal models and from clinical trials as found in ischemic heart patients received NS. The results provide a clear evidence that both the NS oil and its active ingredients, in particular thymoquinone (TQ), possessing reproducible anti-oxidant effects through enhancing the oxidant scavenger system against several ischemic insults and suggesting that the regular use of NS seed is recommended for prevention of ischemic cardiac and non-cardiovascular diseases. Keywords: NS: Nigella sativa, TQ: thymoquinone, ICVD: Ischemic Cardiovascular Diseases, Anti-oxidant. INTRODUCTION Ischemic cardiovascular diseases (CVD) may be broadly classified into ischemic coronary heart disease, ischemic renal hypertension, ischemic cerebrovascular disease and ischemic peripheral vascular disease, in which the blood supplies to the heart, the brain and the peripheral vasculature, respectively, are compromised [1]. Various of vascular ischemia implicates oxygen-derived free radicals (especially superoxide and hydroxyl radical) and highenergy oxidants (such as peroxynitrite) are as mediators of inflammation, shock, and ischemia/reperfusion injury [2]. The oxidantinjury can potentially occur during ischemia and reperfusion due to an excess production of oxygen free radicals, a decrease in antioxidant defenses, or both. Because antioxidants function by removing the toxicoxygen metabolites; they are generally highly

effective in reducing ischemia-reperfusion injury activity [3]. MATERIALS AND METHODS NS is one of the members of Ranunculaceae family, commonly grows in the Middle East, Eastern Europe and Eastern and Middle Asia. NS and its oil are being used as food additives as well as natural remedies for many ailments forover thousands of years. Many active ingredients have been isolated from NS, including: thymoquinone, thymo-hydroquinone, dithymoquinone, thymol, carvacrol, nigellicine and alphahedrin. In addition, many pharmacological effects of NS and its active principles have been identified, such as immune stimulation, anti-inflammatory, anti-cancer and antimicrobial activity [4].

Corresponding Author:-Ahmed Abdul-Sabour Ahmed Bader

Email:[email protected]

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NS as a natural remedy has been documented to possess numerous therapeutic values, including diabetes, tumors, hypercholesterolemia, hypertension, inflammation, and gastrointestinal disorders [5-10]. This study aiming for exploring the anti-ischemic properties of NS and its constituents against oxidative stress and ischemic reperfusion injury inducing vascular dysfunction and in addition to outline the possible mechanisms of actions underlying these beneficial effects that protecting heart and other vital organs against ischemia and decreasing incidence of myocardial infraction and ischemic-reperfusion injuries on different body organs as proved from experimental animal models and from clinical trials in ischemic patients received NS. Nigella sativa (NS) The seed oil of NS was found to be rich in polyphenols and tocopherols. The seeds contain 36–38% fixed oils, 0.4–2.5% essential (volatile) oil, proteins, alkaloids, and saponins [11-13]. Thymoquinone (TQ) is the most pharmacologically active ingredient found abundantly (30– 48%) in the black seeds, together with its derivatives such as dithymoquinone, thymohydroquinone, and thymol [14]. Antioxidant Property of NS The seed oil of NS is well known for its strong antioxidant properties [15]. Previous studies have documented that pretreatment with TQ, the main active constituents in seed oil, protected organs against oxidative damage induced by a variety of free radical generating agents, such as carbon tetrachloride and including the alkylating agents, cisplatin, and the cardiotoxic anticancer drug doxorubicin [16-17]. The free radical scavenging effects of TQ, dithymoquinone, and thymol were tested against several reactive oxygen species (ROS), and all the tested compounds from NS exerted strong antioxidant effects; thymol acted as singlet oxygen quencher, while TQ and dithymoquinone showed superoxide dismutase (SOD)-like activity [18]. Pathogenesis of Vascular ischemia 1. Role of Oxidative Stress in atherosclerosis and vascular dysfunction. The most atherogenic type of plasma lipids is LDLc component of total serum cholesterol; that become oxidized by a process of low level oxidation when bound to the LDL receptor, internalized, and transported through the endothelium [19]. Oxidation of the serum LDL may induce a decreased uptake of L-arginine that may derange the eNOS, leading to over production of super-oxide free radical ; that induce more vascular damage due to defect in biosynthesis of NO[20]. Free radicals possess one or more unpaired electrons in their outer electronic orbits. Free radicals and ROS are formed continuously in normal physiological process. ROS are highly reactive and unstable by nature;

hence, they can damage various cellular components including lipid membranes [21]. Excessive production leads to oxidative stress with an increase in the formation of nitrogen-oxygen derivative free radicals as well as a decrease in antioxidant capacity [22]. Oxidative stress occurs when there is an imbalance between production of ROS and antioxidant defence system in favour of the former [23-24]. Lipid peroxides are derived from polyunsaturated fatty acid (PUFA) oxidation and are capable to initiate lipid peroxidation via free radicals chain reaction. Molinaldehyde acid (MDA) is a major end product of PUFA peroxidation and is often used as an indicator of cell injury. Increase in the production of MDA may be due to the formation of reactive oxidants. Lipid peroxidation leads to structural changes of the lipid molecules, and the changes are more severe as lipids are the main constituent of biological membranes. Generally, lipid peroxides carrying a risk factor for atherosclerotic complications strongly producing vascular ischemia . NO is synthesized predominantly in the vascular endothelium. Endothelial nitric oxide synthase (eNOS) is required for the synthesis of NO from amino acid Larginine. Reduced NO bioavailability, that is, a reduction in NO production by free radicals or an increase in deactivation of NO due to imbalance between antioxidant and oxidant levels may be the mechanism underlying endothelium dysfunction that producing vascular ischemia. Generation of ROS such as superoxide anions may cause vascular and cellular injury by oxidizing membrane lipids, proteins, and nucleic acids. Furthermore, superoxide anions may reduce NO bioavailability by binding to the gaseous molecule and forming peroxynitrite which itself is a free radical [25]. 2. Evidences of Oxidative Stress Involved in vascular dysfunction and ischemia. There is a growth evidence suggesting that vascular dysfunction and ischemia may be attributable to the increased production of ROS [26-27]. Oxidative stress may play a vital role in the development of vascular dysfunction and ischemia via the following mechanisms: enhanced sequestration of NO by ROS [28], formation of lipid peroxidation products [29], and depletion of NOS cofactor (tetrahydrobiopterin) [30] . Lastly, it may cause functional and structural changes in vascular wall and blood vessel [31] . These vascular alterations may be mediated by several ways, including direct injury to endothelial and vascular smooth muscle cells, effects on endothelial cell eicosanoid metabolism, altered redox state, increases in intracellular free calcium concentration, stimulation of inflammatory, and growth signalling events [32-33]. Oxidative stress promotes proliferation of vascular smooth muscle cells as well as collagen deposition which cause thickening of tunica media and narrowing of the vascular lumen, which eventually leads to an increase in the total peripheral resistance and vascular dysfunction and ischemia [34].

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RESULTS AND DISCUSSION Possible Pharmacological Actions of NS against vascular ischemia. The therapeutic properties of NS have been carried out by various previous experimental and clinical trials. A wide spectrum of pharmacological actions of NS have been explored which may include antidiabetic, anticancer, immunomodulator, analgesic, antimicrobial, anti-inflammatory, spasmolytic, bronchodilator, hepato-protective, renal protective, gastroprotective, antioxidant properties [47-52]. NS exerting pleiotropic vascular properties effects including cardiac depressant effect, calcium channels blocking property, and diuretic effect [53]. In addition, NS was significantly ameliorating the inflammatory changes in vital organs (liver, kidney and lungs) and preventing multi-organ dysfunctions in aged rats and recommending that the dietary supplement of NS for elderly people can improve their prognosis on exposure to sepsis [54]. Thymoquinone, the active constituent of Nigella sativa seeds, is a pharmacologically active quinone, which possesses several properties including analgesic and anti-inflammatory actions [55]. It has been reported that thymoquinone prevents oxidative injury in various in vitro and in vivo studies in rats [5657]. It has been suggested that thymoquinone may act as an antioxidant agent and prevents membrane lipid peroxidation in tissues [58]. The mechanism of action is still largely unknown. But, it seems these effects may be related to inhibition of eicosanoid generation, namely thromboxane B2 and leucotrienes B4 (by inhibiting cyclooxygenase and 5-lipooxygenase, respectively), and inhibition of membrane lipid peroxidation [59]. Moreover, it has been demonstrated that NS can significantly prevent hepatotoxicity [60]. Furthermore, the results obtained some other experimental studies suggest that the NS seed extract decreases cerebral ischemia reperfusion injuryinduced pathological stress in the rat model [36]. Although, the exact mechanism of action of anti-ischemic activities is not clear; NS seed has a variety of antiischemic activities including antioxidant [61, 62],antieicosanoid [63], calcium channel blocker effects [64] and a decreasing effect on intracellular calcium in mast cells [65]. Myocardial infarction (MI) is a deleterious enigma which is the principal cause of death in both developed and developing countries as a result of cardiovascular diseases, and has been the object of intense investigation by clinicians and basic medical scientists [66]. MI results from the prolonged myocardial ischemia with necrosis of myocytes due to interruption of blood supply to an area of heart [67]. The myocardial protective effect of NS (black cumin) could be due to its protective activity as result of the presence of phytoconstituents such as thymoquinone, dithymoquinone, thymohydro-quinone, thymol, carvacrol, tanethole and 4-terpineol [68].

Furthermore, the previous results indicated that the prior administration of the black cumin attenuates isoproterenol induced MI. The cardioprotective effect of the black cumin is probably related to its ability to strengthen the myocardial membrane by its membrane stabilizing action, with a decrease in activity of Creatinine Kinase-MB (CK-MB) isoenzyme in the serum; suggesting a protective effect of NS against myocardial injury [37]. Recently, it has been shown that NS oil had a marked protective action against ischemia/ reperfusioninduced gastric mucosal lesions, an effect that was associated with suppression in the levels of lipid peroxide (LPX) and lactate dehydrogenase (LDH) and an increase in those of glutathione (GSH) and superoxide dismutase (SOD) [69]. Ischemic stroke results from a transient or permanent reduction in the cerebral blood flow, that is restricted to the territory of a major brain artery [70]. Three major approaches of possible mechanisms had been investigated to ameliorate ischemia-induced brain damage by (i) interfering with the excitatory action of glutamate; (ii) preventing intracellular accumulation of Calcium and (iii) preventing the destructive actions of reactive oxygen species [71]. Vascular ischemia can cause an increase in the lipid peroxidation reaction and elevation in production of free radicals, which contributes to a secondary damage of the nervous tissues [72-73]. Diabetes is associated with marked increase in ischemic heart disease [74]. The form of dyslipidemia, that is most characteristic of the diabetes has increased triglyceride and decreased HDL-Cholesterol level [75-76]. Atherosclerosis is a multifactorial progressive disease characterized by inflammatory, athero-thrombotic and fibroproliferative changes leading to macro and microvascular complications with different clinical sequelae particularly in diabetic persons [77]. Hypercholesterolemia is the major risk factor associated with the increased incidence of atherosclerosis [78]. It had been recorded that NS having a favorable impact on total cholesterol, LDLc, triglycerides and fasting blood sugar, that was observed in the intervention group with NS oil capsules in comparison to the control group [39, 79]. There are various mechanisms were proposed for the lowering of cholesterol by NS seed, the seed may either inhibit de novo cholesterol synthesis or stimulate bile acid excretion and both effects would lead to a decrease in serum cholesterol [80]. The mechanism may be due to stabilization of atherosclerotic plaques due to anti-inflammatory effect of NS or its immunomodulatory effect [81]. It was reported in a previous clinical study that there was an effective reduction of serum cholesterol by braka oil (Oil of NS) in hypercholesterolemic patients [81]. Moreover, it was observed that the influence of thymoquinoe on doxorubicin-induced hyperlipidemic nephropathy in rats, that the rats treated with thymoquinone (10mg/kg/day) for five days significantly had lowered serum urea, triglycerides and total cholesterol

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[82]. It has been observed that NS is very useful in treatment of thrombosis due to their lipid reducing, blood diluting and anti-oxidant property [44]. Oxygen free radicals, produced on reperfusion, play a critical role in the vascular injury caused by ischemia-reperfusion [83]. It has been shown that NS has protective effect against ischemia reperfusion injury to various organs [59, 69, 84]. Many studies have suggested

the beneficial antioxidant effects of antioxidant nutrients such as NS, vitamin E, green tea extract, ginkgo biloba extract, resveratrol and niacin for reducing or preventing cerebral or muscle injury during ischemia-reperfusion [85, 86]. Furthermore, NS and thymoquinone possessing strong antioxidant activity that had been shown to decrease ischemia-reperfusion (I/R) injury in rat hippocampus [87] or gastric mucosal tissues [69].

Table 1. Significant anti-ischemic effects of NS and its constituents against different cardio and non-cardiac vascular ischemia in experimental and clinical trials Reference Study model Constituents Laboratory Findings Conclusion and Recommendations [35] Experimental study of NS oil (i.p) 0.2 ↓ SBP, tissue MDA, luminol, NS seed oil could have a renovascular ischemic mL/kg and lucigenin CL therapeutic antihypertensive hypertension in rats ↑ tissue Na+ and K+-ATPase effect against renovascular and ↑ plasma NO hypertension in ischemic ↓ plasma CK, LDH, and ADMA hypertensive rat. [36] Experimental study of NS aqueous (1 g / Significant reduction in NS seed extracts could have four-vessel occlusion kg) and Ethanolic neuronal cell injuries a therapeutic protective model in rats (1.6 g / kg) extracts of rat hippocampus effects against cerebral ischemia. [37] Experimental study of Black cumin (150 Significant ↓ in serum AST, Pretreatment with black isoproterenol-induced mg/kg body weight) ALT, LDH, CK, and tissue lipid cumin offered a protective myocardial infarction intragastrically for a profile of TG, cholesterol, free effects against isoproterenol in rats period of 15 days fatty acids in NS treated rat induced myocardial groups infarction in rats. [38] Experimental study of NS seed extract Significant ↓ in arterial BP, Anti-ischemic effect could L-NAME-induced (p.o) 400mg/kg SBP, DBP, and serum LDH; ↑ be related to thee significant hypertension in rats serum NO decrease in blood lipids and the increase in serum NO [39] Experimental study of NS (5 g/kg/day) p.o NS showed a promising blood NS recommended as a Experimentally lipid lowering effects when dietary supplement for long induced compared to placebo and also term use without toxic hypercholesterolemia has a favourable significant effects for primary in rabbits increase in serum HDL prevention of hypercholesterolemia and atherosclerosis and also has therapeutic potential actions for ischemic patients with coronary artery disease. [40] Experimental study of NS oil (0.2 mL/kg) Significant ↓ in serum AST, Suggested that NS oil hepatic ischemia / intraperitoneally, ALT, and LDH in treated rats in treatment can protect the rat reperfusion injury in before ischemia and comparison with non-treated liver against ischemiarats before reperfusion rats reperfusion injury [41] Experimental study of The aqueous (1, 1.5 Extracts of NS had suppressed Concluded that NS extracts ischemic / reperfusion and 2 g/kg) and the increase of MDA levels in having some protective injury of skeletal ethanolic extracts the rat skeletal muscle and effects against skeletal muscle in rats (1.6, 2.4 and 3.2 therefore inhibiting lipid muscle tissue injury on g/kg) of NS peroxidation following exposure to ischemiaischemia-reperfusion injury. reperfusion and the antiischemic effects could be due to antioxidant properties and free radical scavengering of NS

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[42]

Experimental study of adrenaline-induced dyslipidemia and left ventricular hypertrophy in rats

Injection of NS with different doses (0.5, 1 and1.5 mg/kg body weight, BW)

NS showed significant antidyslipidemic effects with significantly increase in free radical scavenging activity and with attenuation of ischemic cardiomyocyte damage

[43]

Clinical study on patients with coronary artery disease

NS seed oil capsules with the dose (900 mg/ oral per day) for 8 weeks.

Significant decreased in serum levels of LDL, TG, VLDL and total cholesterol after 8 weeks of treatment, with significant increase in serum HDL of treated patients compared with pre-treated group

[44]

Clinical study on patients with stable coronary artery disease

NS seed powder 500 mg/daily orally

[45]

Clinical study on patients with mild hypertension

NS seed 100mg/kg 200mg/kg, per day

Significant decrease in serum total cholesterol, LDL,VLDL, TG after the treatment, with significant increase in serum HDL cholesterol after six months of treatment Significant ↓ SBP and DBP (dose dependent) ↓ total and LDL cholesterol

[46]

Clinical study on patients with ischemic heart disease

NS seeds in a dose of 200 mg twice daily; for 3 months

extract and orally

NS exerted beneficial reductive effects on blood glucose levels especially in diabetic patients, with a significant increase in serum HDL and a decrease in serum TG after regular intake of NS

Suggested that NS having antidyslipidemic, antioxidative and cardioprotectiving effects on heart and NS might be used as a therapeutic alternative or as a combination with drug in the treatment of dyslipidemia that increase risk of cardiac ischemia and LVH. NS seed oil can be usefull in the treatment of mild dyslipidemia due to its potential hypocholesterolemic and hypolipidemic effects NS has ability to reduce lipid profile which is a major risk factor for ischemic coronary artery disease in cardiac patients. NS seed extract could be exerting antihypertensive and hypolipidemic effects; which may be beneficial for ischemic hypertensive diseases by decreasing serum total cholesterol and LDL cholesterol NS could have beneficial anti-ischemic effects on patients with ischemic heart disease after regular intake for 3 months

Abbreviation NS: Nigella sativa; L-NAME: L-NG-nitroarginine methyl ester; i.p: intraperitoneal; i.v: intravenous; p.o: per os; TQ: thymoquinone; De-TQ: de-thymoquinonated; SBP: systolic blood pressure; DBP: diastolic blood pressure; MDA: malondialdehyde; CL: chemiluminescence; CK: creatine kinase; LDH: lactate dehydrogenase; ADMA: asymmetric dimethylarginine; NO: nitric oxide; GFR: glomerular filtration rate; GSH: glutathione; LDL: low-density lipoprotein; AST and ALT ; aspartate and alanine transaminase; LDH; lactate dehydrogenase; CK; Creatine kinase; VLDL; very low density lipoprotein ; LDL ; Low density lipoprotein; HDL ; high density lipoprotein; TG; triglycerides; NO; nitric oxide ; LVH; left ventricular hypertrophy CONCLUSION The protective anti-ischemic effects of NS against different vascular ischemia could be possibly contributed to their multiple pleiotropic effects including cardiac depressant and/ or calcium channel blockade actions that are very helpful for decreasing myocardial oxygen demands and for strengthening myocardial

membrane by its membrane stabilizing action in ischemic patients, and in addition to this it could be attributed largely to hypolipidemic and antioxidant properties of NS. NS is a promising medicinal plant with many therapeutic properties and this effect could be related to the presence of active alkaloid components and/or other constituents. Further studies should be carried out on human to confirm

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its efficacy. It is an important area for further research and development to combine NS with other anti-ischemic drugs to investigate their possible synergistic effects and

preferable pharmacological properties against different vascular dysfunction and ischemic insults.

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