BLACK CUMIN SEED (NIGELLA SATIVA LINN.) OIL AND ITS FRACTIONS PROTECT AGAINST BETA AMYLOID PEPTIDE-INDUCED TOXICITY IN PRIMARY CEREBELLAR GRANULE NEURONS NORSHARINA ISMAIL1,2, MAZNAH ISMAIL1,2,5, LATIFFAH A. LATIFF1, MUSALMAH MAZLAN3 and ABDALBASIT A. MARIOD2,4 1 Faculty of Medicine and Health Sciences Universiti Putra Malaysia 43400 UPM Serdang, Selangor Malaysia 2 Laboratory of Molecular BioMedicine Institute of Bioscience 43400 UPM Serdang, Selangor Malaysia 3 Department of Biochemistry Faculty of Medicine Universiti Kebangsaan Malaysia 50300 Kuala Lumpur Malaysia 4 Department of Food Science & Technology Sudan University of Science & Technology PO Box 71 Khartoum North Sudan Submitted for Publication July 31, 2008 Revised Received and Accepted September 17, 2008 ABSTRACT The neuroprotective effect of Nigella sativa oil (NSO) and its fractions against beta amyloid (Ab)-induced cell death in primary rat cerebellar granule neurons was investigated. Primary cultures were pretreated for 5 h with NSO and its fractions – hexane fraction (HF), ethyl acetate fraction (EAF) and water fraction (WF) – before incubating with 10 mM Ab1-40 for 24 h. Cell viability was investigated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) 5 Corresponding author. TEL: 03-89472115; FAX: 03-89472116; EMAIL: maznah@medic.upm. edu.my; myhome.e@mail.com Journal of Food Lipids 15 (2008) 519–533. All Rights Reserved. © 2008, The Author(s) Journal compilation © 2008, Wiley Periodicals, Inc. 519 520 N. ISMAIL ET AL. and lactate dehydrogenase (LDH) assays. Results of the MTS assay showed that the WF and NSO were significantly protective against cell death induced by 10 mM Ab1-40 at 1, 10 and 100 mg/mL probably because of antioxidant properties as determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical trapping method. HF and EAF had low DPPH scavenging effect and were only effective at 100 mg/mL. However, NSO and its fractions were weakly protective against cell membrane damage in the LDH assay. NSO and its fractions, especially the WF, may play a role in the prevention of Ab-induced cell death. PRACTICAL APPLICATIONS Neurodegeneration in Alzheimer’s disease (AD) has been associated with the toxic effects of beta amyloid (Ab), a by-product of amyloid precursor protein. Natural products that are able to reduce Ab-induced neurotoxicity are candidates for preventing as well as for treating this neurodegenerative condition. Thus, the neuroprotective effects of Nigella sativa against Ab toxicity may play a potential role in preventing AD progression. INTRODUCTION The black seeds of Nigella sativa Linn., belonging to the family Ranunculaceae, provide a highly nutritional product that has been used extensively as a supplement to help maintain good health and well-being (Cheikh-Rouhou et al. 2008). Several pharmacological properties of N. sativa have been reported, including antioxidant, anti-inflammatory, antimicrobial, antitumor, immunomodulatory, antihypertension, antinociceptive, uricosuric, choleretic, antifertility, antidiabetic and antihistaminic (Ali and Blunden 2003). In addition, N. sativa L. is also used to enhance memory. N. sativa oil (NSO) is a rich source of quinones, unsaturated fatty acids, traces of alkaloids and terpenoids (Gali-Muhtasib et al. 2006). The saponifiable and unsaponifiable lipids in NSO may act synergistically to contribute to its various health benefits. However, its use in preventing neuronal cell death induced by various neurotoxins has not been investigated. Therefore, this study was undertaken to elucidate the potential of NSO and its fractions in protecting against neurodegeneration. One of the most prevalent neurodegenerative diseases is Alzheimer’s disease (AD). It is the commonest cause of dementia affecting the elderly worldwide. According to the amyloid cascade mechanism, beta amyloid (Ab) peptide is the prime suspect in the neurodegeneration occurring in the brain of an Alzheimer’s disease patient. Therefore, the best treatment for cognitive NEUROPROTECTIVE EFFECT OF N. SATIVA OIL 521 decline is to prevent the toxic effects of the Ab peptide (Shen et al. 2006). Ab is a by-product of Amyloid Precursor Protein (APP), a plasma membrane protein normally enriched in neurons (Jung et al. 1996). The Ab peptide is also found at low concentrations as a normal constituent of biological fluids, where it is known as soluble Ab (sAb) (Busciglio et al. 1993). sAb is predominantly Ab1-40, although shorter and longer sequences exist, including Ab1-28 and Ab1-42 (Vigo-Pelfrey et al. 1993). The most abundant fragment is Ab1-40 corresponding to about 90% of the total Ab peptide. Around 10% is represented by the more amyloidogenic fragment Ab1-42. Ab1-39 and Ab1-43 are minor species naturally occurring in senile plaques. Brain sAb appears to be elevated in AD, and the elevation precedes amyloid plaque formation in the brain of Down syndrome patients. Moreover, Casoli et al. (2008) found that Ab1-40 might be dangerous to neurons, and exogenous Ab may cause neuronal degeneration in primary cultured neurons (Yankner 1996). The AD protein of APP was found to be associated in cerebellar and hippocampal synapses during postnatal development and aging of rats (Ribaut-Barassin et al. 2003). The cerebral cortex and hippocampus are known to contribute early in AD-related memory and cognitive deficits, whereas the cerebellum is often observed in more advanced stages (Kobayashi et al. 1998). The cerebellum is not only responsible for motoric function but is also engaged in cognition and learning (Wegiel et al. 1999). Neuronal cell dysfunction and oxidative cell death caused by the Ab contribute to the pathogenesis of AD. Antioxidants that prevent the detrimental consequences of reactive oxygen species (ROS) are considered to be a promising approach to neuroprotection (Behl and Moosmann 2002). Alzheimer’s patients with moderate impairment taking high doses of AT display some beneficial effect with respect to the rate of deterioration of cognitive functions (Sano et al. 1997). In the present study, we examined the protective effects of NSO and its fractions – hexane fraction (HF), ethyl acetate fraction (EAF) and water fraction (WF) – against Ab1-40-induced cell death in primary rat cerebellar granule neuron (CGN) cultures, by examining cell viability using inner salt (MTS) and lactate dehydrogenase (LDH) assays. The antioxidant properties of these fractions were determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method. MATERIALS AND METHODS Sample Preparation N. sativa, a product of Iran, was purchased from a local herbal medicine store in Kuala Lumpur, Malaysia. The seeds were cleaned under running tap 522 N. ISMAIL ET AL. water for 10 min, rinsed twice with distilled water and air-dried overnight in an oven at 40C. The seeds were ground to a powder using an electric grinder (model MX-915, National, Tokyo, Japan) for 10 min and then were passed through a 35-mm (42-mesh) sieve. a-Tocopherol was prepared as stock solution in dimethyl sulfoxide (DMSO). Chemicals and Reagents Fetal bovine serum (FBS) was purchased from GIBCO BRL Life Technologies (Grand Island, NY); CellTiter 96 AQueous One Solution Cell Proliferation Assay and CytoTox 96 Nonradioactive Cytotoxicity Assay were purchased from Promega (Southampton, U.K.). Ab1-40, Ab40-1, a-tocopherol, DPPH and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Oil Extraction from N. sativa Seeds One hundred grams of dry-powdered N. sativa seeds was dissolved in methanol. The extraction was carried out in a flask while stirring overnight with a magnetic stirrer and was repeated in the dark three times at room temperature. The combined extracts were filtered through a Whatman No. 4 filter paper (Fisher Scientific Co Ltd., Ottawa, Canada) to obtain a clear supernatant and the solvent was evaporated in a vacuum rotary evaporator (Buchi, Flawil, Switzerland) at 40C. Half of the extracted oil was further fractionated to obtain HF, EAF and WF. The first two fractions were later concentrated under vacuum and WF was freeze-dried. The oil and its fractions were kept at -20C in amber dark bottles for further analysis within 2 months. DPPH Free Radical-scavenging Activity This assay was performed as described by Yoshida et al. (1989) with slight modifications. Stock solutions of NSO and its fractions were prepared as 1 mg/mL and were dissolved in ethanol. The solutions were diluted to different concentrations with twofold serial dilutions (500–2 mg/mL) in a 96-well microtiter plate. The reaction mixtures containing an ethanolic solution of 200 mM DPPH (100 mL), freshly prepared and stored in a dark chamber, were added to each as well. The plate containing the mixtures was shaken gently and incubated in the dark at room temperature for 30 min. Absorbance was read at 517 nm by microplate reader (Opsys MR, Thermo Labsystems, Franklin, MA). In this assay, the synthetic antioxidant butylated hydroxytoluene (BHT) was used as positive control. All antioxidant assays were carried in triplicate and the readings were averaged. Percentage of inhibition of DPPH radical was calculated using the formula NEUROPROTECTIVE EFFECT OF N. SATIVA OIL Percent inhibition ( I%) = 523 OD ( DPPH ) − OD (DPPH + sample ) × 1000 OD ( DPPH ) where optical density (OD) is the absorbance at 517 nm. Primary CGN Culture Preparation All experiments were performed according to the requirement of the Universiti Putra Malaysia Use and Care of Animal Ethics Committee. Primary neuronal cultures were prepared from the cerebellar tissue of 7-day old newborn Sprague–Dawley rats. The tissue was dissected out in phosphate buffered saline (pH 7.2), and the meninges and blood vessels were cleaned. The cerebella were then chopped with a scalpel and were transferred to a trypsin solution at 37C for 7 min, with gentle swirling. The trituration steps were carried out using a fire-polished glass pipette in Dulbecco’s modified Eagle’s medium (DMEM) containing 19 mM NaHCO3, 26.2 mM KCl, 7 mM p-aminobenzoic acid, 100 mU/L insulin and 50 mg/mL gentamicin, and the supernatant was taken. After centrifugation, the suspended cells were distributed in a 96-well poly-L-lysine-coated plate. The cells were grown in DMEM supplemented with heat-inactivated 10% FBS and were maintained at 37C in a humidified 5% CO2 and 95% air. After 48 h, non-neuronal cell division was inhibited by a 24-h exposure to 10 mM cytosine arabinofuranoside. The experiment was performed using matured cultures at 4 days in vitro. Preparation of Aggregated Ab The stock solutions of Ab1-40 and Ab40-1 (Sigma-Aldrich) were prepared by dissolving the peptide with sterile distilled water at a concentration of 1 mg/mL. The stock solutions were then stored at 37C and were used for the aged Ab condition after 4 days of incubation, followed by dilution in culture medium at final concentration of 10 mM for cell culture experiments. MTS Reduction Assay The CGNs were placed in 96-well plates at a density of 1 ¥ 105 cells/well and were allowed to attach to the plate and to mature for 96 h before treatment. The Ab1-40 and its reverse sequence Ab40-1 were exposed to the cells with various concentrations (1, 5 and 10 mM) for 24 h in the absence of any extracts. This is to determine the IC50 (the concentration of Ab peptide that provide 50% inhibition or cells viability) of both Ab peptides that induced toxicity to the CGNs. NSO and its fractions (HF, EAF and WF), and a-tocopherol with various concentrations were added separately and were pretreated with CGNs 524 N. ISMAIL ET AL. for 5 h before being exposed to the IC50 of Ab peptide. After 24 h of incubation, 50 mL of the sample media was transferred to a new 96-well plate to assess LDH release because of cell death. The mitochondrial function of cultured CGNs was measured by MTS conversion assay, which served as a general indicator of cell viability. Briefly, the CellTiter 96 AQueous One Solution Reagent (Promega) was thawed in a water bath at 37C for 10 min. Twenty microliters of MTS reagent was pipetted into each well containing the samples in 100 mL of culture medium. The plates were then incubated for 4 h at 37C in a humidified 5% CO2 atmosphere. The optical density (OD) of the wells was determined using a microplate reader (Opsys MR, Thermo Labsystems) at 490-nm wavelength. LDH Release Assay LDH released into the media was determined using CytoTox 96 nonradioactive cytotoxicity assay (Promega). In this colorimetric assay, LDH converts lactate and nicotinamide adenine dinucleotide (NAD+) to pyruvate and NADH, respectively. This initial reaction is coupled to a second reaction where diaphorase converts iodonitrotetrazolium salt and NADH to a redcolored formazan compound and NAD+, respectively. Briefly, 50 mL of assay buffer was added to each 50 mL sample of media removed from each well prior to MTS analysis. Samples were then incubated in the dark for 30 min at room temperature, and then 50 mL of a stop solution was added into each well and the absorbance was measured using a microplate reader (Opsys MR, Thermo Labsystems) at 490 nm. Statistical Analysis All assays were carried out in three separate experiments with triplicate samples. Each individual experiment was performed in a single 96-well plate containing appropriate negative and positive untreated controls. Statistical analysis was conducted by one-way analyses of variance, Tukey’s multiple comparison and Student’s t-test using Statistical Package for the Social Sciences (SPSS Inc., Chicago, Illinois, USA) version 13.0. P < 0.01 was considered as statistically significantly different. RESULTS AND DISCUSSION NSO obtained by methanolic extraction yielded 36.3 g oil, which is about 36% of 100-g powdered N. sativa seeds. Half of the oil was further fractionated with hexane, ethyl acetate and water. The yielded fractions were 23.2, 6.6 and 3.6 g for HF, EAF and WF, respectively (Fig. 1). As a step toward iden- NEUROPROTECTIVE EFFECT OF N. SATIVA OIL 525 Nigella sativa oil and fraction yield (g) 45 40 35 30 25 20 15 10 5 0 NSO HF EAF WF FIG. 1. YIELD OF NIGELLA SATIVA OIL (NSO) AND ITS FRACTIONS NSO was obtained by methanolic extraction and was further fractionated to hexane fraction (HF), ethyl acetate fraction (EAF) and water fraction (WF). tifying the active principle(s) of N. sativa, the oil obtained from the methanolic extract was fractionated successively using different organic solvents on the basis of polarity. Methanol is a solvent that is able to extract both polar and nonpolar compounds. On the other hand, waxes, fats and fixed oil were obtained by HF. Alkaloids were extracted with ethyl acetate, while glycosides are a class of phytochemicals that can be extracted with water. The results in Fig. 2 show that WF and NSO render the best scavenging activity among all fractions, with concentration in the range of 1.6–200.0 mg/ mL, but were still lower than that of the synthetic antioxidant BHT. The sequence of the DPPH scavenging effect of extracts tested was as follows: BHT > WF > NSO > HF > EAF at 200 mg/mL. At the lowest concentration tested, 1.6 mg/mL, the DPPH radical-scavenging activity was 46.0, 43.2, 42.4 and 41.6% of WF, NSO, HF and EAF, respectively. At 12.5 mg/mL concentration, the corresponding values were 45.8, 44.1, 42.0 and 40.1%, respectively; at 100 mg/mL concentration, these were 56.1, 50.6, 46.8 and 41.8%, respectively. There were significant differences (P < 0.01) between the WF and EAF at all concentrations. The EAF showed a lower scavenging activity than WF, NSO and HF for the whole range of concentrations analyzed. In another study, Khattak et al. (2008) reported that the DPPH radical-scavenging activity of N. sativa at 5 mg/mL was 92.0 and 79.4% for methanol and water extracts, respectively. Meanwhile, Erkan et al. (2008) found that the DPPH IC50 value for black seed essential oil was 515.0 ⫾ 20.1 mg/mL. In this study, WF and NSO were hypothesized to have a better protective effect against Ab-induced toxicity in CGNs, as they were found to have higher free radicalscavenging activities compared with other fractions. 526 N. ISMAIL ET AL. 100 90 Inhibition (%) 80 70 60 50 40 30 20 10 0 0 50 100 150 200 Concentration (µg/mL) BHT WF NSO HF EAF FIG. 2. 1,1-DIPHENYL-2-PICRYLHYDRAZYL (DPPH) RADICAL-SCAVENGING ACTIVITY OF NIGELLA SATIVA OIL (NSO) AND ITS FRACTIONS The DPPH radical-scavenging activity (inhibition %) of NSO and its fractions: water fraction (WF), hexane fraction (HF), ethyl acetate fraction (EAF) and synthetic antioxidant butylated hydrotoluene (BHT). Cell viability (% MTS reduction) 120 100 80 60 Aβ 40-1 40 Aβ 1-40 20 0 0 2 4 6 8 10 Concentration (µM) FIG. 3. INNER SALT (MTS) ASSAY OF Ab1-40 (BETA AMYLOID) AND Ab40-1 TOXICITY The cerebellar granule neurons were treated with various concentrations (1, 5 and 10 mM) of Ab1-40 and its reverse sequence Ab40-1 for 24 h. The percentage of dimethyl sulfoxide was at 0.01% (v/v). Levels of cell viability were measured using the MTS assay. The viability of untreated control cells was defined as 100%. Values represent means ⫾ standard error of the mean. Figure 3 shows that the viability of CGNs exposed to 10 mM Ab1-40 for 24 h was significantly (P < 0.01) lower than the control group. It was found that incubation for 24 h of 10 mM Ab1-40 was able to kill almost 50% of the cells (P < 0.01) compared to the control cells. Therefore, 10 mM Ab1-40 with NEUROPROTECTIVE EFFECT OF N. SATIVA OIL 527 24-h incubation was chosen to induce cell death on CGNs for subsequent experiments. The reverse sequence of Ab40-1 did not have any effect on cell survival with no significant difference compared to the control cells. Therefore, only Ab1-40 was used for further neuroprotective assays. Although there are different lengths of Ab peptide that can contribute to neuronal cell death in the brains of AD patients, Ab1-40 has been found to be more abundant than Ab1-42. The Ab1-42 fragment is more amyloidogenic and showed potent toxicity than Ab1-40; however, there is still limited study on the CGNs against Ab1-40 toxicity. Our data show that Ab1-40 at 10 mM was cytotoxic as it decreased the number of cell viability. Heo and Lee (2005) indicated that Ab1-40 was directly toxic to neuronal cell cultures at high micromolar concentrations. This study has thus demonstrated the Ab1-40 toxicity on cerebellar neurons. On the other hand, stock peptide solutions (1 mg/mL in distilled water) were aged by incubation at 37C to lead to the formation of stable oligomeric peptide aggregates (Mattson et al. 1997). This aged Ab1-40 was found to be toxic to the neurons (Levine 1995). The mechanism by which sAb peptide converts into the insoluble form is also unclear, although environmental factors such as pH shift, metal ions, temperature, peptide concentration and ionic strength probably induce molten-globule states of Ab1-42 (Matsunaga et al. 2002a,b) In our study, the Ab1-40 was aged by incubation at 37C for 4 days. This aggregated Ab1-40 was shown to be toxic to the cerebellar neurons. We observed the formation of amyloid fibrils of the aged Ab1-40 by transmission electron microscope (data not shown). Therefore, we hypothesized that Ab1-40 also contributed into insoluble form because of environmental factors similar to Ab1-42. The neurotoxicity of the oil and its fractions was screened by 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, MTS assay in the dose range of 1–200 mg/mL. The dose given within this range did not show any toxicity but rather stimulated the growth of CGNs compared to the untreated control cells (Fig. 4). DMSO, which is the solvent for all of extracts, at 0.01% (v/v), had no effect on cell viability either. No previous studies on the screening of the toxicity of NSO and its fractions on primary culture CGNs or by using neuron cell lines were found in the existing literature. Figure 5 shows that the mitochondrial activity was decreased to 54% after CGNs were exposed to 10 mM Ab1-40 for 24 h. NSO and WF were significantly (P < 0.01) protected from cell death at 1, 10 and 100 mg/mL compared to HF and EAF, which were only effective at higher doses. In addition, after the exposure of CGNs with 10 mM Ab1-40 24 h, the LDH activity increased markedly to 117% of the control indicating cell death. Inhibition rates in LDH assay at various concentrations of NSO and its fractions induced by Ab1-40 are shown in Fig. 6. However, these fractions showed weak protection toward plasma N. ISMAIL ET AL. Cell viability (% MTS Reduction) 528 250 200 150 100 50 0 0 50 100 150 200 Concentration (µg/mL) NSO HF EAF WF AT FIG. 4. EFFECT OF NIGELLA SATIVA OIL (NSO) AND ITS FRACTION TOXICITY ON CEREBELLAR GRANULE NEURONS (CGNs) The CGNs were treated for 24 h with various concentrations (1, 10, 50, 100, 200 mg/mL) of NSO and its fractions; hexane fraction (HF), ethyl acetate fraction (EAF) and water fraction (WF) and a-tocopherol (AT). The percentage of dimethyl sulfoxide was at 0.01% (v/v). Levels of cell viability were measured using the inner salt (MTS) assay. The viability of untreated control cells was defined as 100%. Values represent means ⫾ standard error of the mean. FIG. 5. NEUROPROTECTIVE EFFECT OF NIGELLA SATIVA OIL (NSO) AND ITS FRACTIONS AGAINST Ab1-40 The cerebellar granule neurons were pretreated for 5 h with NSO, hexane fraction (HF), ethyl acetate fraction (EAF), water fraction (WF) and a-tocopherol (AT) at various concentrations (1, 10 and 100 mg/mL) before being exposed to 10 mM Ab1-40 for 24 h and measured using inner salt (MTS) assay. The percentage of dimethyl sulfoxide was at 0.01% (v/v). Values represent means ⫾ standard error of the mean. * P < 0.01 versus Ab1-40, † P < 0.01 versus control. NEUROPROTECTIVE EFFECT OF N. SATIVA OIL 529 FIG. 6. NEUROPROTECTIVE EFFECT OF NIGELLA SATIVA OIL (NSO) AND ITS FRACTIONS AGAINST Ab1-40 The cerebellar granule neurons were pretreated for 5 h with of NSO, hexane fraction (HF), ethyl acetate fraction (EAF), water fraction (WF) and a-tocopherol (AT) at various concentrations (1, 10 and 100 mg/mL) before being exposed to 10 mM Ab1-40 for 24 h and measured using lactate dehydrogenase (LDH) assay. The percentage of dimethyl sulfoxide was at 0.01% (v/v). Values represent means ⫾ standard error of the mean. * P < 0.01 versus Ab1-40, † P < 0.01 versus control. membrane damage. Furthermore, NSO and WF at 10 and 100 mg/mL, HF at 10 mg/mL and EAF at all concentrations showed no significant difference with Ab1-40 10 mM alone (P < 0.01). Compared with NSO and its fractions, a-tocopherol at 0.1, 1.0 and 10.0 mM significantly (P < 0.01) protected both mitochondrial dysfunction (Fig. 5) and plasma membrane from damage (Fig. 6) by Ab1-40. The protective effect of a-tocopherol may be due to its antioxidant properties, which are well known as a natural antioxidant. In these findings, it is interesting to note that the neuroprotective activity of the NSO and its fractions especially WF appears to be similar to that of pure a-tocopherol at 1 mg/mL. Even at low concentrations, WF could still exert potent protective effect against Ab-induced cell death in neuron culture. Among the fractions, WF appeared to be most effective in protecting against Ab1-40-induced CGNs toxicity, implying that the active compounds are polar in nature with hydrophilic characteristics. However, the WF showed lower protective effect at 10 and 100 mg/mL 530 N. ISMAIL ET AL. compared with 1 mg/mL. This probably reflects the prooxidant activity of the WF at high concentrations. In the present study, WF has been found to protect against Ab1-40-induced CGNs toxicity. This result in agreement with findings of Irie and Keung (2003), Jeong et al. (2005), Yu et al. (2005) and Lai et al. (2006), who reported that water and aqueous extracts from different sources of herbal medicine are potent and protected against Ab1-40, Ab25-35 and Ab1-42 toxicity in cell line and primary cultures. Meanwhile, Huang et al. (2008) and Ban et al. (2006) found that ethanol and methanol extracts protected cells from Ab25-35-induced neurotoxicity. In this study, the methanolic extract of NSO also showed protective effect on Ab1-40-induced CGNs toxicity in dose-dependent manner on MTS assay. Plant crude extracts can offer many health beneficial properties as they are considered to be less toxic and more potent because of their complex nature and the possible synergistic effects of the components in the mixture. Black cumin seed oil has been part of a nutritious diet and its consumption is becoming increasingly popular all over the world (Cheikh-Rouhou et al. 2008). Although known to be beneficial to health, there is no reported study of its effect on the neurodegenerative disorder, particularly AD. This study is necessary to gain additional data on the health benefit of N. sativa and as an indication of the economic potential of these seeds as a new source of medicinal oils in future. Our in vivo models also showed the protective effect of N. sativa against the Ab and reduced the senile plaque formation (unpublished data). In conclusion, NSO and its fractions are able to prevent the toxicity of Ab toxin. NSO and its WF were found to possess better antioxidant properties than HF and EAFs, effectively trapping the DPPH-generated radicals, suggesting that these compounds may be protecting cells from Ab1-40 insult through the antioxidant pathway. Therefore, further studies are required to identify the bioactive compounds present in NSO and its WF and to examine their mechanisms of action contributing to this protective effect. 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