Neuroprotective effects of chloroform and petroleum ether extracts of Nigella sativa seeds in stroke model of rat.

PURPOSE: Stroke still remains a challenge for the researchers and scientists for developing ideal drug. Several new drugs are being evaluated showing excellent results in preclinical studies but when tested in clinical trials, they failed. Many herbal drugs in different indigenous system of medicine claim to have beneficial effects but not extensively evaluated for stroke (cerebral ischemia). AIM: The present study was undertaken to evaluate chloroform and petroleum ether extract of Nigella sativa seeds administered at a dose of 400 mg/kg, per orally for seven days in middle cerebral artery occluded (MCAO) rats for its neuroprotective role in cerebral ischemia.
MATERIALS AND METHODS: Focal cerebral ischemia was induced by middle cerebral artery occlusion for two hours followed by reperfusion for 22 hours. After 24 hours, grip strength, locomotor activity tests were performed in different treatment groups of rats. After completing behavioral tests, animals were sacrificed; brains were removed for the measurement of infarct volume followed by the estimation of markers of oxidative stress.
RESULTS: Both chloroform and petroleum ether extracts-pretreated rats showed improvement in locomotor activity and grip strength, reduced infarct volume when compared with MCAO rats. MCA occlusion resulted in the elevation of levels of thiobarbituric acid reactive substance (TBARS), while a reduction in the levels of glutathione (GSH) and antioxidant enzymes viz. superoxide dismutase (SOD) and catalase levels were observed. Pre-treatment of both extracts of Nigella sativa showed reduction in TBARS, elevation in glutathione, SOD, and catalase levels when compared with MCAO rats.
CONCLUSION: The chloroform and petroleum ether extract of Nigella sativa showed the protective effects in cerebral ischemia. The present study confirms the antioxidant, free radical scavenging, and anti-inflammatory properties of Nigella sativa already reported.

Cerebral ischemia or stroke still remains a challenge for the researcher and scientist for developing ideal drug for this disorder as it is the third commonest cause of mortality and the fourth leading cause of disease burden.[12] Several new drugs are being evaluated showing excellent results in pre-clinical studies but when tested in clinical trials, they failed. Many herbal drugs from Indian system of traditional medicines and also in Chinese system of medicine are being evaluated and showed beneficial effects in stroke.[2] These drugs not only improve the blood flow to the dying neurons but also have beneficial effect on the biochemical mechanisms involved in the development of stroke and thus, will prove to be more useful in the treatment of cerebral ischemia.[23]

Nigella sativa L. (Ranunculaceae) is an annual herbaceous flowering plant. It tastes slightly bitter and peppery with a crunchy texture. Seeds are angular, generally small size, dark grey or black color. Nigella sativa seeds are used for edible and medicinal purposes in many countries, including Egypt, Syria, Iran, and to a slight extent in Tunisia. They are used as a condiment in bread and other dishes.[4] Nigella sativa is widely studied for so many pharmacological activities including analgesic,[5] anti-inflammatory,[6] antioxidant and anti-eicosanoid,[78] anti-bacterial,[9] calcium channel blocking effects,[10] decreasing effect on intracellular calcium in the mast cells, immunomodulatory,[11] protective action against ischemia/reperfusion-induced gastric mucosal lesions.[12] Hypotensive,[1314] and hepato-protective[15] effects have also been reported. Since old times, Nigella sativa is considered an important medicinal herb as natural remedy for wide range of diseases. Thymoquinone, an active constituent of the Nigella sativa, is reported to possess a strong antioxidant property.[8] Thymoquinone and co-constituent of Nigella sativa protects organs against oxidative damage induced by a variety of free radical-generating agents including doxorubicin-induced cardio-toxicity, carbon tetrachloride-induced hepato-toxicity,[16] nephropathy produced by cisplatin,[17] autoimmune as well as allergic encephalomyelitis.[1819]

The present study was conducted to evaluate neuroprotective effects of chloroform and petroleum ether extracts of Nigella sativa seeds in middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia in rats.

Structure of thymoquinone

Materials and Methods

Plant material

The dried seeds of Nigella sativa were procured from the Khari Bawli, Delhi. The seeds were identified, authenticated; extracts were standardized by one of the co-author from Department of Pharmacognosy and Phytochemistry, Jamia Hamdard, New Delhi before the commencement of the study.

Preparation of extracts

Chloroform and petroleum ether were used as solvents for the preparation of extracts. Nigella sativa seeds were crushed to coarse powder, and then 100 g of the prepared powder was mixed with 500 ml of respective solvent in the soxhlet apparatus by heating at their boiling point, and the solutions were filtered and evaporated under reduced pressure to obtain chloroform and petroleum extracts.[20]

Animals

The study was carried out under controlled conditions using adult Wistar albino rats of 250-300 g, procured from Central Animal House Facility of Hamdard University, New Delhi, selected and acclimatized accordingly. All animals were housed in cages kept at a temperature of 23-30°C with a natural light - dark cycle. They had free access to standard pellet diet (Amrut Laboratory rat and mice feed, Nav Maharastra Chakan oil mills Ltd., Pune) and tap water. The experimental protocol was approved by an Institutional Animal Ethics Committee CPCSEA Jamia Hamdard New Delhi. (173/CPCSEA 28 JAN-2008, Project no. 575). Ethical norms were strictly followed during all experimental procedures.

Drugs and administration

Animals were divided into six groups, each group consisting of six rats each receiving different treatments per orally for seven days. GP I - Normal control, GP II - Sham-operated, GP III - MCAO only, GP IV - Aspirin + MCAO, GP V - Chloroform extract + MCAO, GP VI - Petroleum ether extract + MCAO. All the different extracts were made accordingly and administered at a dose of 400 mg/kg per orally daily for seven days; care was taken to ensure the same proportion of each extract to be dosed to the each animal for the comparisons. The last dose was administered three hours before inducing cerebral ischemia in rats. Normal control group and sham-operated animals received distilled water. One hundred mg of aspirin was suspended in 5 ml of the 0.5% CMC suspension, and 0.5 ml of this suspension was administered orally at 100 mg/kg for the same days as above.

Ischemia was induced for two hours followed by reperfusion for 22 hours. After 24 hours, behavioral parameters were assessed, and the animals were then immediately sacrificed for infarct volume and oxidative stress parameters in brains.

Induction of cerebral ischemia

Rats were anesthetized with chloral hydrate (dissolved in distilled water) at a dose of 400 mg/kg i.p. A middle incision was made, and the right common carotid artery, external carotid artery, and internal carotid artery was exposed under an operative magnifying glass. A 4.0 monofilament Nylon thread (40-3033 Pk 10, Doccol Corporation Pennsylvania Ave, Red lands, CA-USA) with its tip rounded by heating quickly near a flame was advanced from the external carotid artery into the lumen of the internal carotid artery until resistance was felt, which ensures the occlusion of the origin of the middle cerebral artery. The nylon filament was allowed to remain in place for two hours. After two hours, the filament was retracted so as to allow the reperfusion of ischemic region. Sham-operated rats had the same surgical procedures, except that the occluding monofilament was not inserted. Adequate precautions were taken to prevent the infection.[21]

After 24 hours, the animals were studied for locomotor activity, grip strength test. Immediately after behavioral tests, the animals were sacrificed, their brain removed, infarct volume was measured, homogenate was prepared from the brain slices, and biochemical estimations were carried out.

Behavioral tests

Locomotor activity (closed field activity monitoring)

Locomotor activity was measured using a digital photoactometer. Each animal was observed for a period of 10 min in a square closed arena equipped with infrared light-sensitive photocells. The apparatus was housed in a darkened light and sound attenuated ventilated testing room. During activity testing, only one animal was tested at a time.[22]

Grip test

Grip strength meter was used for recording the grip strength of the animal. The animal's front paws were placed on the grid of grip strength meter and was moved down until its front paws grasping the grid was released. The force achieved by animal was then displayed on the screen and was recorded as kg unit.[23]

Estimation of oxidative stress markers

Following the behavioral testing, the animals were decapitated, and the brains were quickly removed, and necrotic part of the brains were taken for estimation, weighed, and homogenized in ice-cold KCl phosphate buffer (0.1 M pH 7.4) 10 times (w/v) and centrifuged at 2000 rpm for five min at 4°C. The supernatant containing crude membrane were used for the estimation of Thiobarbituric acid reactive substance(TBARS) and replace glutathione(GSH). The remaining supernatant was again centrifuged at 10,000 rpm at 4°C for 20 min. The post-mitochondrial supernatant was used for the study of antioxidant enzyme activities and protein estimation. Catalase and super oxide dismutase activities were determined immediately after sample preparation. Protein concentrations were determined according to Lowry et al.[24] using purified bovine serum albumin as standard.

Measurement of lipid peroxidation

TBARS, a measure of lipid peroxidation, was measured as described by Ohkawa, et al.[25] Briefly, 1 ml of suspension medium was taken from the 10% tissue homogenate. 0.5 ml of 30% trichloroacetic acid (TCA) was added to it, followed by 0.5 ml of 0.8% thiobarbituric acid (TBA) reagent. The tubes were covered with aluminum foil and kept in shaking water bath for 30 minutes at 80°C. After 30 minutes, tubes were taken out and kept in ice-cold water for 30 minutes. These were centrifuged at 3000 rpm for 15 minutes.

The absorbance of the supernatant was read at 540 nm at room temperature against appropriate blank. Blank consist of 1 ml distilled water, 0.5 ml of 30% TCA, and 0.5 ml of 0.8% TBA. TBARS values were expressed as n moles malonaldehyde (MDA)/mg protein.

Measurement of reduced GSH

Glutathione was measured according to the method of Ellman.[26] The equal quantity of homogenate (w/v) and 10% TCA were mixed and centrifuged to separate the proteins. To 0.01 ml of this supernatant, 2 ml of phosphate buffer (pH 7.4), 0.5 ml 5, 5’-dithiobisnitro benzoic acid (DTNB), and 0.4 ml of double distilled water was added. The mixture was vortexed, and the absorbance was read at 412 nm within 15 minutes. GSH values were expressed as μ moles GSH mg protein.

Measurement of catalase

Catalase activity was measured by the method of Claiborn.[27] A total of 0.1 ml of supernatant was added to cuvette containing 1.9 ml of 50 mM phosphate buffer (pH 7). The reaction was started by the addition of 1 ml freshly prepared 30 mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically at 240 nm. Catalase values were expressed as n moles H2O2 consumed/min/mg protein.

Measurement of superoxide dismutase

Superoxide dismutase activity was measured by the method of Kagiyama, et al.[28] The supernatant was assayed for SOD activity by following the inhibition of pyrogallol auto-oxidation. One hundred μl of cytosolic supernatant was added to Tris HCl buffer (pH 8.5). The final volume of 3 ml was adjusted with the same buffer. At least 25 μl of pyrogallol was added, and changes in absorbance at 420 nm were recorded at 1 minute interval for three minutes. The increase in absorbance at 420 nm after the addition of pyrogallol was inhibited by the presence of SOD.

Photomicrograph

Rats were sacrificed, and their brains were quickly removed and sectioned coronally into six slices each with a 2 mm thickness. The six brain slices were immersed in 2% triphenyltetrazolium chloride (TTC) for 30 min at 37°C and then fixed with formalin. Infracted areas were identified as regions lacking the brick red staining of normal brain tissue. These brain slices were scanned with the help of scanner, and their images were taken and saved as bitmap images. These bitmap images were identified with the help of scion image version 4.0.3.2 software program, by which we measured the infarct volume of brain slices. It was expressed as mm3.

Statistical analysis

All the data were expressed as the mean ± SEM. For a statistical analysis of the data, group means were compared by one-way analysis of variance followed by Dunnett's t’ test. P < 0.05 was considered significant. It is carried out with graph pad in Stat 3 software.

Results

Effect of chloroform and petroleum extract of Nigella sativa seeds on locomotor activity in MCAO rats

Spontaneous locomotor activity was observed over a period of 10 min for each rat in each group. In the MCA occluded rats, significant reduction in locomotor count was observed (P < 0.00l). There was significant improvement in locomotor counts observed with both extracts as compared to the MCAO rats (P < 0.001) [Figure 1].

Figure 1

Effect of chloroform and petroleum ether extracts of Nigella sativa seeds on locomotor activity in middle cerebral artery occluded rats MCAO = Middle cerebral artery occlusion, No of animals = 6, Data Represented as mean ± SEM; ***P < 0.001 vs. Vehicle control; +++P < 0.001 vs. Sham-operated ###P < 0.001, vs. MCAO Significance by one way ANOVA followed by Dunnett's t’ test

Effect of chloroform and petroleum extract of Nigella sativa seeds on grip strength in MCAO rats

MCAO group showed a significant decrease in grip strength as compared to the normal rats (P < 0.01). Pre-treatment of different extracts showed improvement in grip strength when compared with MCAO rats (P < 0.001) [Figure 2].

Figure 2

Effect of chloroform and petroleum ether extracts of Nigella sativa seeds on grip strength in middle cerebral artery occluded rats MCAO = Middle cerebral artery occlusion, No of animals = 6, Data Represented as mean ± SEM; ***P < 0.001 vs. Vehicle control; +++P < 0.001 vs. Sham-operated ###P < 0.001, vs. MCAO Significance by one way ANOVA followed by Dunnett's t’ test

Effect of chloroform and petroleum extract of Nigella sativa seeds on TBARS in MCAO rats

The TBARS levels measured after 24 hour of middle cerebral artery occlusion were found to be significantly increased in the MCAO rats than in the normal rats. Both extract produced significantly reduction in TBARS levels when compared to that of MCAO (P < 0.001) [Figure 3].

Figure 3

Effect of chloroform and petroleum ether extracts of Nigella sativa seeds on TBARS in middle cerebral artery occluded rats MCAO = Middle cerebral artery occlusion, No of animals = 6, Data Represented as mean ± SEM; ***P < 0.001 vs. Vehicle control; +++P < 0.001 vs. Sham-operated ###P < 0.001, vs. MCAO Significance by one way ANOVA followed by Dunnett's t’ test

Effect of chloroform and petroleum extract of Nigella sativa seeds on glutathione in MCAO rats

The brain glutathione levels were estimated in all the groups. Levels of reduced glutathione in MCAO rats were significantly reduced when compared to the sham-operated rats. In both extracts pretreated rats, the glutathione levels were significantly elevated when compared with MCAO group (P < 0.001) [Figure 4].

Figure 4

Effect of chloroform and petroleum ether extracts of Nigella sativa seeds on GSH in middle cerebral artery occluded rats MCAO = Middle cerebral artery occlusion, No of animals = 6, Data Represented as mean ± SEM; ***P < 0.001 vs. Vehicle control; +++P < 0.001 vs. Sham-operated ###P < 0.001, vs. MCAO Significance by one way ANOVA followed by Dunnett's t’ test

Effect of chloroform and petroleum extract of Nigella sativa seeds on SOD in MCAO rats

The levels of SOD after 24 hours in MCA occluded group were significantly reduced as compared to the normal rats. In both chloroform and petroleum ether pretreated group, the levels of SOD were significantly increased as compared to the MCAO group (P < 0.001) [Figure 5].

Figure 5

Effect of chloroform and petroleum ether extracts of Nigella sativa seeds on SOD in middle cerebral artery occluded rats MCAO = Middle cerebral artery occlusion, No of animals = 6, Data Represented as mean ± SEM; ***P < 0.001 vs. Vehicle control; +++P < 0.001 vs. Sham-operated ###P < 0.001, vs. MCAO Significance by one way ANOVA followed by Dunnett's t’ test

Effect of chloroform and petroleum extract of Nigella sativa seeds on catalase in MCAO rats

The levels of catalase were reduced in MCA occluded group (P < 0.001) as compared to the normal rats. Both extracts pretreated rats showed elevation in the levels as compared to the MCAO group (P < 0.001) [Figure 6].

Figure 6

Effect of chloroform and petroleum ether extracts of Nigella sativa seeds on catalse in middle cerebral artery occluded rats MCAO = Middle cerebral artery occlusion, No of animals = 6, Data Represented as mean ± SEM; ***P < 0.001 vs. Vehicle control; +++P < 0.001 vs. Sham-operated ###P < 0.001, vs. MCAO Significance by one way ANOVA followed by Dunnett's t’ test

Effect of chloroform and petroleum extract of Nigella sativa seeds on infarct volume in MCAO rats

TTC dye staining of brains of all extracts pretreated animals showed significant improvement in the infarct volume aspirin, chloroform and petroleum extracts pretreated animals showed a highly significant reduction in infarct volume as compared with MCAO rats (P < 0.001) [Figures 7 and 8].

Figure 7

Effect of chloroform and petroleum ether extracts of Nigella sativa seeds on infarct volume in middle cerebral artery occluded rats MCAO = Middle cerebral artery occlusion, No of animals = 6, Data Represented as mean ± SEM; ***P < 0.001 vs. Vehicle control; +++P < 0.001 vs. Sham-operated ###P < 0.001, vs. MCAO Significance by one way ANOVA followed by Dunnett's t’ test

Figure 8

TTC dye staining of different brain sections following various treatments in rats Gp-1- Normal control, Gp-2- Sham operated, Gp-3- MCAO, Gp-4- Aspirin + MCAO, Gp-5- Chloroform extract + MCAO, Gp-6- Petroleum ether extract + MCAO

Discussion

Although a large number of therapeutic agents like thrombolytics, N-methyl D-aspartate receptor antagonists, calcium channel blockers, and antioxidants etc., have been used or being evaluated, there is still a large gap between the benefits by these agents and properties for an ideal drug for the treatment of stroke they offer.[29] The major approaches have been investigated to ameliorate ischemia-induced brain damage viz. interfering with the glutamate excitatory action, preventing intra-cellular accumulation of calcium ions, preventing the negative effects of reactive oxygen species.[30]

In recent years, much attention is being paid towards the exploration of herbal preparations to find out the appropriate and useful agents for the treatment and preventions of stroke and its consequences. Chloroform and petroleum extracts of Nigella sativa administered in the present study decreased TBARS levels as compared to the MCAO rats [Figure 3]. This was in agreement with our earlier reports, which showed that MCA occlusion followed by reperfusion increased TBARS formation in rats.[313233] It was already reported that Nigella sativa decreased lipid peroxidation and increased the antioxidant defense system activity.[34] The various chemical constituents such as flavonoids, fatty acids, sterols, and other volatile oils are responsible for its antioxidant effect. It has already been reported that petroleum extract contain saturated and unsaturated fatty acids. Linoleic acid, oleic acid, and palmitic acid were the major components in Nigella sativa seeds.[8] The volatile component of Nigella sativa seeds i.e., thymoquinone prevents membrane lipid peroxidation,[30] protected rats against transient forebrain ischemia-induced damage in the rat hippocampus,[31] work as a scavenger of various reactive oxygen species, including super oxide anion and hydroxyl radicals.[33343536] Nigella sativa is a herbal drug that phytochemically confirms to have antioxidant properties, which suppress reactive oxygen and nitrogen species formation;[30] this scavenging reactive oxygen and nitrogen species play a major role in protecting the antioxidant defense system.[3738]

The present study observed a significant decrease in GSH levels (P < 0.001) in MCA occluded rats as compared to pretreated groups. Similar observations were also reported elsewhere, in which the oxidative stress decreased GSH levels.[32333940] Pretreatment of chloroform and petroleum extracts of Nigella sativa showed elevation of GSH levels as compared to MCA occluded rats, thus confirming its antioxidant and free radical scavenging properties.[33343536]

Pretreatment with Nigella sativa extracts for seven days followed by MCA occlusion showed the return pattern towards normal in the reduction of GSH level when compared with MCA occluded rats, thus corroborate its antioxidant properties. In cerebral ischemia particularly after reperfusion free radicals production is elevated disrupting the endogenous antioxidant systems[4142] was observed in the present study and thus confirming the antioxidant properties of Nigella sativa.

Motor performance tests and grip strength test showed reduction in count and grip strength of animals. Our findings were in agreement with already reported observations and results.[323940] The mechanism for this reduction could be due to neuronal damage in the territory of the MCA.[29] The ischemic insult observed could be due to the alterations induce both biochemical (TBARS, GSH, SOD and catalase) and morphologically in rats brain. However, pretreatment groups with Nigella sativa for seven days showed improvement in the motor performance tests (P < 0.001). These finding suggested that the improvement in the motor performance test could be due to reduction in the ischemic lesion in the territory area.[29]

In the present study, we observed selective neuronal damage infarction as evident by TTC dye staining of rats brain [Figures 7 and 8]. Chloroform and petroleum extracts of Nigella sativa significantly reduced the infarct volume when compared to MCA occluded rats. Thus, pretreatment of both extracts of Nigella sativa showed neuroprotection (P < 0.001).

Conclusion

The present study utilized the chloroform and petroleum ether extracts of Nigella sativa seeds, showed reduction in TBARS levels, infarct volume, and elevation of GSH, SOD, and catalase levels. The neuroprotective effects observed in the present study by chloroform and petroleum ether extracts of Nigella sativa seeds thus confirms its antioxidant-free radical scavenger and anti-inflammatory properties already reported previously. However, further studies using multiple doses, with different models of stroke and isolation and characterization of chemical constituents present in the extracts, which are responsible for the protective effects, are required to establish the exact role of Nigella sativa as atherapeutic agent in stroke.