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Phytomedicine 14 (2007) 621627 www.elsevier.de/phymed

Effect of thymoquinone and Nigella sativa seeds oil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampus
Hossein Hosseinzadeha,, Siavash Parvardehb, Marjan Nassiri Aslb, Hamid R. Sadeghniab, Toktam Ziaeec
Pharmaceutical Research Center, Faculty of Pharmacy, Mashhad University of Medical Sciences, P.O. Box 1365-91775, Mashhad, I.R. Iran b Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Science, Mashhad, I.R. Iran c Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, I.R. Iran Received 22 March 2004; accepted 1 September 2006
a

Abstract
It has been previously reported that Nigella sativa oil (NSO) and thymoquinone (TQ), active constituent of N. sativa seeds oil, may prevent oxidative injury in various models. Therefore, we considered the possible effect of TQ and NSO on lipid peroxidation level following cerebral ischemia-reperfusion injury (IRI) in rat hippocampus. Male NMRI rats were divided into nine groups, namely, sham, control, ischemia and ischemia treated with NSO or TQ. TQ (2.5, 5 and 10 mg/kg), NSO (0.048, 0.192 and 0.384 mg/kg), phenytoin (50 mg/kg, as positive control) and saline (10 ml/kg, as negative control) were injected intraperitoneally immediately after reperfusion and the administration was continued every 24 h for 72 h after induction of ischemia. The transient global cerebral ischemia was induced using four-vessel-occlusion method for 20 min. Lipid peroxidation level in hippocampus portion was measured as malondialdehyde (MDA) based on its reaction with thiobarbituric acid (TBA) following ischemic insult. The transient global cerebral ischemia induced a signicant increase in TBA reactive substances (TBARS) level (po0.001), in comparison with sham-operated animal. Pretreatment with TQ and NSO were resulted a signicant decrease in MDA level as compared with ischemic group (66.971.5 vs. 29772.5 nmol/g tissue for TQ, 10 mg/kg; po0.001 and 153.571.3 nmol/g tissue for NSO, 0.384 mg/kg; po0.001). Using a reversed-phase HPLC system, the amount of TQ in NSO was also quantied and was 0.58% w/w. These results suggest that TQ and NSO may have protective effects on lipid peroxidation process during IRI in rat hippocampus. r 2006 Elsevier GmbH. All rights reserved.
Keywords: Nigella sativa; thymoquinone; lipid peroxidation; ischemia-reperfusion; rat

Introduction
Nigella sativa L., commonly known as black seed or black cumin, is used in folk medicine as a natural
Corresponding author. Tel.: +985118823252; fax: +985118823251. E-mail address: hosseinzadehh@mums.ac.ir (H. Hosseinzadeh).

remedy for a number of disease and condition such as asthma, hypertension, diabetes, inammation, cough, bronchitis, headache, eczema, fever, dizziness and gastrointestinal disturbances (Ali and Blunden, 2003). Furthermore, modern pharmacological and toxicological studies have demonstrated that crude extracts of the seeds and some of its active constituents (volatile oil, thymoquinoneTQ) might have protective effect

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against nephrotoxicity and hepatotoxicity induced by either disease or chemicals (Ali and Blunden, 2003). Nigella sativa oil (NSO) has also antipyretic, analgesic, anti-inammatory, antimicrobial, and antineoplastic activity (Ali and Blunden, 2003). TQ, active constituent of N. sativa seeds, is a pharmacologically active quinone, which possesses several properties including analgesic and anti-inammatory actions (Abdel-Fattah et al., 2000; Houghton et al., 1995), protection against chemical induced carcinogenesis (Hassan and El-Dakhakhny, 1992; Worthen et al., 1998), and the inhibition of eicosanoids generation (Houghton et al., 1995). Moreover, it has been reported that TQ prevents oxidative injury in hepatocytes induced by carbon tetrachloride or tert-butyl hydroperoxide in various in vitro (Daba and Abdel-Rahman, 1998) and in vivo (Mansour et al., 2001; Nagi et al., 1999) hepatotoxicity models, as well as acetic acid-induced colitis in rats (Mahgoub, 2003). It has been suggested that TQ may act as an antioxidant agent and prevent the membrane lipid peroxidation in hepatocytes (Mansour et al., 2002). Compelling evidence implicates free radicals as major contributors to ischemic tissue injury in the CNS. It has been well documented that ischemia increases lipid peroxidation reactions and reactive oxygen species (ROS), which cause secondary neural tissue damage (Dubinsky et al., 1995; Patel et al., 1996). Lipid peroxidation is an autocatalytic mechanism leading to oxidative destruction of cellular membranes (Cheeseman, 1993). Their destruction can lead to cell death and to production of toxic and reactive aldehyde metabolites called free radicals. Among these free radicals, malondialdehyde (MDA) is the most important (Paradis et al., 1997). Therefore, the use of antioxidants, free radical scavengers or trapping agents may be rational in cerebral ischemia-reperfusion injury (IRI) (White et al., 2000; Streck et al., 2003; Gilgun-Sherki et al., 2002). Recently, there is overwhelming attention to plant products and natural agents that can limit free radicalmediated injuries, for better therapeutic management of IRI. The aim of this study was to investigate the effect of NSO and TQ, active constituent of NSO, on lipid peroxidation level following global cerebral IRI in rat hippocampus.

in groups of ve in standard laboratory conditions. They were kept at constant room temperature (2172 1C) under a 12 h light/dark cycle at least 10 days prior to testing. Commercial food pellets and tap water were freely available. The animals were divided into nine groups, each of which contained 10 rats. Group 1 was the sham group in which only surgery was done without induction of ischemia; groups 2 and 3 were the control groups in which saline solution plus 0.8% Tween 80 (10 ml/kg, as negative control) or phenytoin (50 mg/kg, as positive control) was given intraperitoneally. In groups 49, TQ (2.5, 5 and 10 mg/kg, i.p.) or NSO (0.048, 0.192 and 0.384 mg/kg, i.p.) was administrated immediately after reperfusion and the administration was continued every 24 h for 72 h after the induction of ischemia. All animal experiments were carried out in accordance with Mashhad University of Medical Sciences, Ethical Committee acts.

Chemicals
Thymoquinone, 2-isopropyl-5-methyl-1, 4-benzoquinone, was purchased from Aldrich Chemical Co. TBA (2-thiobarbituric acid), n-butanol, phosphoric acid, potassium chloride and tetramethoxypropane (TMP) were obtained from Merck. The HPLC grade methanol and 2-propanol were purchased from Caledon. The other drugs used in this investigation were xylazine (Loughrea Co., Galway, Ireland), ketamine (Rotexmedica, GmbH, Germany) and phenytoin (IPDIC, Rasht, I.R. Iran). TQ was suspended in 0.8% Tween 80 (in normal saline). N. sativa seeds were authenticated by Pharmacognosy Department, School of Pharmacy, MUMS, Iran. The seeds were washed, dried, and crushed to a powder. Twenty grams of the powdered seeds were added to 400 ml of distilled water and the extraction was carried out by steam distillation. The yield was equal to 0.4%.

Induction of global cerebral ischemia


Transient global cerebral ischemia was produced using the four-vessel occlusion (4-VO) rat model as modied for complete occlusion of the vertebral arteries and control of collateral circulation (Pulsinelli and Brierley, 1979; Pulsinelli and Buchan, 1988). Briey, under intraperitoneal ketamine/xylazine anesthesia (60 and 6 mg/kg, respectively), the alar foramina of rst cervical vertebrae were exposed and the vertebral arteries were electrocauterized permanently. On the next day and under brief anesthesia, the common carotid arteries (CCAs) were dissected from surrounding tissues and temporarily ligated using the microvascular clamps

Materials and methods


Animals
Male NMRI rats weighing 200250 g were obtained from the animal house of Pharmaceutical Research Center, Bu-Ali Research Institute of Mashhad University of Medical Sciences (Mashhad, Iran) and housed

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for 20 min. At the end of the ischemic period, the ligature was removed and reperfusion was supplied. After maintaining animals in suitable situation for 72 h, the animals were decapitated and the hippocampus portion was homogenized in 1.5% cold KCl solution to give a 10% homogeny suspension and used for MDA assay.

Tiobarbituric acid reactive species (TBARS) measurement


The lipid peroxidation level of the hippocampus portion was measured as MDA which is the end product of lipid peroxidation, and reacts with thiobarbituric acid (TBA) as a TBA reactive substance (TBARS) to produce a red colored complex which has peak absorbance at 532 nm (Fernandez et al., 1997). Three ml phosphoric acid (1%) and 1 ml TBA (0.6%) was added to 0.5 ml of homogenate in a centrifuge tube and the mixture was heated for 45 min in a boiling water bath. After cooling, 4 ml of n-butanol was added to the mixture and vortex-mixed for 1 min followed by centrifugation at 20,000 rpm for 20 min. The organic layer was transferred to a fresh tube and its absorbance was measured at 532 nm. 1,1,3,3-tetramethoxypropane was used as a standard of MDA.

MDA levels following IRI as compared with shamoperated animals (29772.5 vs. 17072.8 nmol/g tissue, po0.001) (Fig. 1). NSO and TQ pretreatment resulted in a signicant and dose-dependent reduction in the free radical-mediated lipid peroxidation as indicated by a decrease in the MDA levels, at various dose levels. In TQ-pretreated groups with doses 5 and 10 mg/kg, TBARS levels were 18270.9 (po0.001 as compared to vehicle-treated animal) and 66.971.5 nmol/g tissue (po0.001), respectively (Fig. 1). The TBARS levels in hippocampus portion of those pretreated with NSO 0.192 and 0.384 mg/kg were 220.70710.0 nmol/g tissue (po0.001 as compared to saline-treated animal) and 153.50713.2 nmol/g tissue (po0.001), respectively (Fig. 2).
MDA Conc. (nmol/g tissue)

350 300 250 200 150 100 50 0 *** ***

Ischemia-Reperfusion Sham Phenytoin (50 mg/kg) TQ 2.5 mg/kg TQ 5 mg/kg TQ 10 mg/kg

***

***

Quantication of TQ in N. sativa seeds oil


TQ quantication was carried out by HPLC on a reversed-phase Shim-pak C18 analytical column (250 4.6 mm, 4.6 mm particle size), using an isocratic mobile phase of water: methanol: 2-propanol (50:45:5% v/v) at a ow rate of 1 ml/min. UV monitoring was carried out at 254 nm. The chromatograms of a sample of N. sativa seed oil and standard TQ were showed in Figs. 3 and 4.

Fig. 1. Effect of thymoquinone (TQ) on lipid peroxidation following global cerebral ischemia. MDA levels were measured in 10% homogenates of hippocampus portion from rats subjected to 20 min of ischemia. All drugs were administrated immediately after reperfusion. Values are mean7SEM (n 10). **po0.001 as compared with vehicle (normal saline plus 0.8% Tween 80) treated animals (one-way ANOVA followed by TukeyKramer test).

MDA Conc. (nmol/g tissue)

350 300 250 200 150 100 50 0 *** ***

Statistical analysis
The data were expressed as mean7SEM statistical analysis was performed using one-way ANOVA followed by TukeyKramer post-hoc test for multiple comparisons. The p-values less than 0.05 were considered statistically signicant.

Ischemia-Reperfusion Sham Phenytoin (50 mg/kg) NSO 0.048 mg/kg NSO 0.192 mg/kg NSO 0.384 mg/kg

*** ***

Results
Effect of TQ and N. sativa oil on TBARS levels
The degree of free radical damage following IRI was assessed using lipid peroxidation, which was measured as MDA levels. There was an increase (64.2%) in the

Fig. 2. Effect of Nigella sativa oil (NSO) on lipid peroxidation following global cerebral ischemia. MDA levels were measured in 10% homogenates of hippocampus portion from rats subjected to 20 min of ischemia. All drugs were administrated immediately after reperfusion. Values are mean7SEM (n 810). ***po0.001 as compared with vehicle (normal saline plus 0.8% Tween 80) treated animals (one-way ANOVA followed by TukeyKramer test).

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In this study, also phenytoin reduced the MDA level of hippocampus portion following IRI to 1217 1.82 nmol/g tissue (po0.001, as compared to the saline group) (Fig. 1).

TQ quantication
The chromatograms of a sample of N. sativa seed oil and standard TQ were showed in Figs. 3 and 4. Using the calibration curve, the quantication of TQ in a sample of commercial N. sativa seeds oil was achieved and was about 0.58% w/w.

Discussion
A great deal of effort has been directed toward searching for new compounds that can be used for protection of cerebral IRI. The results obtained in the
0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 0 2 4 6 8

present investigation suggest that NSO and TQ, active constituent of NSO, have an overall protective effect against lipid peroxidation during cerebral IRI in a rat model. A number of processes have been implicated in the pathogenesis of oxygen deprivation-induced cell injury. These include the disturbances of cell calcium homeostasis, depletion of adenine nucleotides, activation of enzymes like phospholipases with production of toxic lipid metabolites, proteases and endonucleases and generation of free radicals (ROS) that can cause oxidative damage to cellular macromolecules (Fisher, 2001). Generation of free radicals may be, at least partially, the basis of many neurological and neurodegenerative disorders such as ischemia-reperfusion, seizure, Parkinson and Alzheimers disease and antioxidant therapy have been well documented to protect against CNS injuries (Gilgun-Sherki et al., 2002; Love, 1999; Sun and Chen, 1998). The large numbers of polyunsaturated fatty acids (PUFAs) make

Detector A (254nm) pharmacology sample 1-100

Volts

10 12 14 16 18 20 22 24 26 28 30 32 34 Minutes

Fig. 3. A chromatogram of puried Nigella sativa seed oil sample. Detection was carried out at l254.
Detector A (254nm) pharmacology T5

0.007 0.006 0.005 Volts 0.004 0.003 0.002 0.001 0.000

0.0 2.5

5.0

7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 Minutes

Fig. 4. HPLC chromatogram of thymoquinone (TQ) dissolved in methanol (5 mg/ml).

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cell membranes particularly vulnerable to lipid peroxidation. The oxidation of PUFAs causes them to be more hydrophilic, thereby altering the structure of the membrane with resultant changes in uidity and permeability. Lipid peroxidation can also inhibit the function of membrane bound receptors and enzymes (Fisher, 2001; Love, 1999). We assessed the effect of NSO and TQ on lipid peroxidation, which was measured in terms of MDA, a stable metabolite of the free radical-mediated lipid peroxidation cascade. The MDA levels increased signicantly (po0.001) following cerebral IRI in hippocampus portion. NSO and TQ reversed the increase of MDA levels to a considerable extent, thereby conrming their antioxidant role in IRI. There are several reports about modulatory effect of phenytoin on lipid peroxidation following injuries like hypoxia/ischemia, CNS injuries, etc. (Kaptanoglu et al., 2005; Hsieh et al., 2000, 2001; Lampley et al., 1995; Wang et al., 1992). In agreement with these ndings, we also found that phenytoin signicantly reduced the TBARS levels in hippocampus portion following global cerebral ischemia. It has been shown that both the NSO and TQ inhibit non-enzymatic lipid peroxidation in liposomes (Houghton et al., 1995). Burits and Bucar showed that NSO as well as its compounds, especially TQ, have appreciable antioxidant and free radical scavenger properties but no pro-oxidant effect. The antioxidant action of N. sativa and/or TQ may explain the protective effect of these agents against various hepatotoxic and nephrotoxic models in vivo and in vitro (Badary et al., 1997; Daba and Abdel-Rahman, 1998; El-Dakhakhny et al., 2000; El Daly, 1998; Mansour et al., 2001; Nagi et al., 1999), as well as liver brosis and cirrhosis (Turkdogan et al., 2000). Al-Gharably et al. (1997) suggested that the protective effect of TQ against carbon tetrachloride-induced hepatotoxicity might be related to the ability of this agent to inhibit lipid peroxidation. Kanter et al. (2005) has also reported that NSO decreased the lipid peroxidation and liver enzymes, and increased the antioxidant defense system activity in the CCl4-treated rats. Same authors also showed that NSO treatment decreased tissue MDA and protein carbonyl levels and prevented inhibition of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) enzyme activities following experimental spinal cord injury in rats (Kanter et al., 2006). Recently, El-Abhar et al. (2003) showed that NSO and TQ have a marked protective action against ischaemia-reperfusion-induced gastric mucosal lesions. It has been reported that the aqueous extract of N. sativa seeds exhibits an inhibitory effect on nitric oxide production by murine macrophages and the active component(s) is/are non-protein in nature (Mahmood et al., 2003). TQ is of low toxicity (LD50 2.4 g/kg) and it

is generally well tolerated when given subchronically till 90 mg/kg/day for 90 days (Badary et al., 1998). N. sativa and/or TQ have also chemopreventive, anticarcinogenic and antimutagenic activity and reduce the toxic effects of standard antineoplastic agents (Nair et al., 1991; El Daly, 1998). It was rst reported that N. sativa extract inhibited the two-stage initiationpromotion of skin cancer by dimethylbenzo[a]anthracene-croton oil in mice (Salomi et al., 1991). Salomi et al. (1992) also showed that the crude extract of N. sativa seeds had a strong cytotoxic action on Erlich ascites carcinoma, Daltons ascites lymphoma and sarcoma 180 cells, while exhibiting minimal cytotoxicity to normal lymphocytes. The in vivo and in vitro inhibitory effects of TQ against 20-methylcholanthrene (MC)-induced brosarcoma (Badary and Gamal, 2001) and against benzo[a]pyrene-induced forestomach carcinogenesis (Badary et al., 1999) were reported in mice. It has been suggested that these effects may be related to interference with DNA synthesis, enhancement of the detoxication process and/or improving the oxidant status, as they were accompanied by restoration of the concentrations of reduced glutathione (GSH), lipid peroxides and the activities of some enzymes (Ali and Blunden, 2003). NSO and TQ have also anti-inammatory and analgesic actions (Khanna et al., 1993; Mutabagani and El-Mahdi, 1997; Abdel-Fattah et al., 2000; Al-ghamdi, 2001) and 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 membrane lipid peroxidation (Houghton et al., 1995). On the other hand and as mentioned before, Ca2+ overloading after traumatic or ischemic injury results in uncontrolled activation of neuronal protein kinases, phospholipases, proteases, endonucleases and NOS (Fisher, 2001; Love, 1999). The ability of TQ to interfere with the mobilization of extracellular Ca2+ required for muscular contraction has been demonstrated by our previous study (Parvardeh and Fatehi, 2003). In agreement with this view, it has been previously shown that TQ stimulates the k-opioid receptors and subsequently affects mostly Ca2+ channels, resulting in the blockade of Ca2+ entry (Abdel-Fattah et al., 2000; Werz and Macdonald, 1984, 1985). But the effect of TQ on ionic channels, especially calcium channels, is still unclear and needs to be more investigated. Eight fatty acids (99.5%) and 32 compounds (86.7%) have been identied in the xed and volatile oils of N. sativa L. seeds grown in Iran, respectively. The major compounds of the volatile oil are trans-anethole (38.3%), p-cymene (14.8%), limonene (4.3%), and carvone (4.0%) (Nickavar et al., 2003). We found that NSO contains 0.58% w/w TQ. It seems that this small amount of TQ is not involved into the anti-ischemia

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effect of NSO. We did not quantify other lipid soluble compounds, like thymol, dithymoquinone (DTQ) or thymohydroquinone (THQ), but suggest that these compounds in the oil act in a synergistic manner (Burits and Bucar, 2000). In conclusion, the results of the present study indicate that NSO and its active constituent TQ, have inhibitory effects against lipid peroxidation process during cerebral IRI in rat hippocampus.

Acknowledgment
The authors are thankful to the Vice Chancellor of Research, Mashhad University of Medical Sciences for nancial support.

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