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
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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
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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
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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
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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.
ACKNOWLEDGMENTS
We thank Then Sue Mian and Maizatul Akma (Universiti Kebangsaan,
Kuala Lumpur, Malaysia) for technical support with tissue culture. This study
was funded by Intensifying Research Priority Areas grants provided by the
Government of Malaysia and Universiti Putra Malaysia.
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