Antioxidant activity and phenolic content of phenolic rich fractions obtained from black cumin (Nigella sativa) seedcake

Abstract

The antioxidant activities of crude methanolic extract (CME) and its fractions using ethyl acetate (EAF), hexane (HF) and water (WF) of black cumin seedcake were investigated. DPPH radical scavenging activity, β-carotene–linoleate bleaching, and inhibition of corn oil oxidation were used to evaluate the antioxidant capacity. The total phenolics were found to be 78.8, 27.8, 32.1 and 12.1 mg gallic acid equivalents (GAE)/g in EAF, CME, WF and HF, respectively. The CME and EAF exhibited the highest DPPH followed by WF and HF. The extract/fractions showed high effect on reducing the oxidation of β-carotene. The effect of extract/fractions on the oxidative stability of corn oil at 70 °C was tested in the dark and compared with butylated hydroxyanisole (BHA). The oil peroxide and anisidine values were generally lower with addition of PRFs in comparison to a control. The predominant phenolic compounds identified by HPLC–DAD in CME and WF of black cumin seedcake were hydroxybenzoic, syringic and p-cumaric acids.

Full text

Antioxidant activity and phenolic content of phenolic rich fractions obtained
from black cumin (Nigella sativa) seedcake
Abdalbasit Adam Mariod
a,c
, Ramlah Mohamad Ibrahim
a,b
, Maznah Ismail
a,b,*
, Norsharina Ismail
a,b
a
Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia
b
Department of Nutrition and Dietetic, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia
c
Department of Food Science and Technology, Sudan University of Science and Technology, P.O. Box 71, Khartoum North, Sudan
article info
Article history:
Received 1 November 2008
Received in revised form 29 January 2009
Accepted 17 February 2009
Keywords:
Antioxidant activity
b-carotene–linoleic acid assay
1,1-Diphenyl-2-picrylhydrazyl (DPPH)
Nigella sativa
Phenolic rich fractions
Oil stability
abstract
The antioxidant activities of crude methanolic extract (CME) and its fractions using ethyl acetate (EAF),
hexane (HF) and water (WF) of black cumin seedcake were investigated. DPPH radical scavenging activ-
ity, b-carotene–linoleate bleaching, and inhibition of corn oil oxidation were used to evaluate the antiox-
idant capacity. The total phenolics were found to be 78.8, 27.8, 32.1 and 12.1 mg gallic acid equivalents
(GAE)/g in EAF, CME, WF and HF, respectively. The CME and EAF exhibited the highest DPPH followed by
WF and HF. The extract/fractions showed high effect on reducing the oxidation of b-carotene. The effect of
extract/fractions on the oxidative stability of corn oil at 70 °C was tested in the dark and compared with
butylated hydroxyanisole (BHA). The oil peroxide and anisidine values were generally lower with addi-
tion of PRFs in comparison to a control. The predominant phenolic compounds identified by HPLC–DAD in
CME and WF of black cumin seedcake were hydroxybenzoic, syringic and p-cumaric acids.
Ó2009 Elsevier Ltd. All rights reserved.
1. Introduction
Epidemiological and in vitro studies strongly suggest that foods
containing phytochemicals such as phenolic compounds have po-
tential protective effects against many diseases. Therefore, they
may be used as antimutagenic, antibacterial, antiviral and anti-
inflammatory agents (Senevirathne et al., 2006). There is increas-
ing evidence that consumption of a variety of phenolic compounds
present in natural foods may lower the risk of serious health disor-
ders because of the antioxidant activity of these compounds (Her-
tog, Feskens, Hollman, Katan, & Kromhout, 1993; Surh, 2002; Surh
et al., 1999). When added to foods, antioxidants minimize rancid-
ity, retard the formation of toxic oxidation products, maintain
nutritional quality and increase shelf life (Jadhav, Nimbalkar, Kulk-
arni, & Madhavi, 1996). The antioxidant activity of extracts of sev-
eral plants including their leaves, bark and roots (Kubola &
Siriamornpun, 2008; Mariod, Matthaus, & Hussein, 2008), fruits
and seeds (Liyana-Pathirana, Shahidi, & Alasalvar, 2006; Malencic,
Maksimovic, Popovic, & Miladinovic, 2008), tree nuts oils (Mira-
liakbari & Shahidi, 2008) and seedcake (Mariod, Matthaüs, Eichner,
& Hussein, 2006; Matthaüs, 2002) has been extensively studied.
Seeds of black cumin (Nigella sativa L.) are used as a spice in
cooking and in a wide traditional medicinal uses, the seed volatile
oil and its main active constituent, thymoquinone, are extensively
reported to exhibited protective effect against many diseases
depending on its high antioxidant activity (El-Dakhakhny, Barakat,
El-Halim, & Aly, 2000; Nagi & Mansour, 2000). Peroxide value (PV)
is often used as an indicator for the initial stages of oxidation (Gray,
1978). The anisidine value is a more meaningful test for the assess-
ment of the oils quality during heating than the peroxide value.
Measurement of the content of conjugated dienes (at 234 nm)
and trienes (at 268 nm) is employed to assess the oxidative stabil-
ity of vegetable oils (St Angelo, Ory, & Brown, 1975).
High performance liquid chromatography (HPLC) with diode ar-
ray detection (DAD) is an indispensable tool for the provisional
identification of the main phenolic structures present in foods
(Chirinos et al., 2009).
Several studies on black cumin seeds (Nagi & Mansour, 2000)
and shoots and roots (Bourgou et al., 2008) have been reported re-
cently but there are no relevant studies on antioxidant activity of
black cumin seedcake. Therefore, the purpose of the present study
was to investigate phenolic compounds of black cumin seedcake
extract/fractions and to evaluate their antioxidant activity (AOA)
by using different in vitro methods. The different extracts were dis-
solved in few amount of methanol and applied to corn oil at levels
of 0.25% and 0.5% to examine their antioxidative activity; the
development of the peroxide value (PV), anisidine value (AV) dur-
ing oxidation of corn oil was evaluated at 70 °C for 72 h.
0308-8146/$ - see front matter Ó2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.02.051
*Corresponding author. Address: Laboratory of Molecular Biomedicine, Institute
of Bioscience, University Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia.
Tel.: +60 3 8947 2115; fax: +60 3 8947 2116.
E-mail addresses: maznah@medic.upm.edu.my,myhome.e@gmail.com (M. Ismail).
Food Chemistry 116 (2009) 306–312
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

2. Materials and methods
2.1. Materials
All solvents used were of analytical grade. Methanol, ethyl ace-
tate, hexane, chloroform, butylated hydroxyanisole (BHA), b-caro-
tene, linoleic acid and Folin–Ciocalteau reagent as well as
polyoxyethylene sorbitan monopalmitate (Tween 40) were ob-
tained from Merck (Merck, Darmstadt, Germany).
Black cumin (N. sativa) seeds, a product of Iran, were purchased
from a local herbal medicine store in Kuala Lumpur, Malaysia.
Black cumin seeds were cleaned under running tap water for
10 min, rinsed twice with distilled water and air-dried in an oven
at 40 °C overnight. The seeds were ground to a powder using an
electric grinder (National, Model MX-915, Kadoma, Osaka, Japan)
for 10 min and then passed through a 35 mm (42 mesh) sieve.
The oil was extracted from the ground seeds by extraction with
n-hexane (b.p. 50–60 °C) using Soxhlet apparatus for 6 h. following
the AOCS (1993) method Aa 4-38. Drying the residue under vac-
uum at 40 °C for 30 min led to a fine and homogenous fat-free
powder which was weighed and was used to extract phenolic
compounds.
2.1.1. Corn oil
The edible corn oil which was produced by Lam Soon edible oil
Co. Ltd. Shah Alam, Selangor, Malaysia was obtained from local
store Serdang Malaysia; the oil was free of any synthetic
antioxidant.
2.2. Extraction of phenolic compounds
Twenty grams of the dried ground black cumin seedcake were
extracted successively with 80% methanol (3 200 ml) by sonica-
tion (Hwasin Technology, Seoul, Korea) to obtain crude methanolic
extract (CME) with solid to solvent ratio of 1:10 (w/v) at room tem-
perature for 1 h; then combined and concentrated by removing
methanol by rotary evaporator (Buchi, Flawil, Switzerland). The
obtained CME (4.4 g) was fractionated by using hexane, ethyl ace-
tate and water (3 100 ml) individually, where the residue from
each fractionation step was used to obtain the subsequent fraction.
Each extraction process involved homogenisation of CME and its
fractions and solvent at 13,000 rpm for 15 min followed by sonica-
tion at constant temperature of 30 °C for 1 h. The CME and its frac-
tions (hexane fraction HF, ethyl acetate fraction EAF, water fraction
WF) were filtered through filter paper Whatman no. 1. Then sol-
vents were removed by using rotary evaporator (Buchi, Flawil,
Switzerland). The yield of each extract and its fractions was mea-
sured before kept in 80 °C for further analysis.
2.3. Determination of total phenolics in black cumin seedcake
The total phenolic (TPC) in the CME and its fractions was
determined with the Folin–Ciocalteu reagent following the method
of Kaur, Arora, and Singh (2008) using gallic acid as standard.
Absorbance was measured at 760 nm using spectrophotometer
(Shimazu, Co., Ltd., Kyoto, Japan). TPC of sample was expressed
as gallic acid in terms of mg gallic acid equivalents (GAE)/100 g
dried samples.
2.4. Antioxidant activities (AOA) measurement
2.4.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
activity test
The antioxidant activity of phenolic extract/fractions from black
cumin seedcake was measured following Gordon, Paiva-Martins,
and Almeida (2001) using the stable radical 1,1-diphenyl-2-pic-
rylhydrazyl (DPPH). A methanolic solution (100
l
L) of the phenolic
compounds extracted from the seedcakes was placed in a cuvette
and 0.5 mL of a methanolic solution of DPPH (50 mg DPPH/
100 mL MeOH) was added. After 30 min incubation in darkness
and at ambient temperature (23 °C), the resultant absorbance
was recorded at 515 nm. The decrease in absorbance at 515 nm
was determined using a spectrophotometer (Shimadzu Co., Ltd.,
Kyoto, Japan). The absorbance of the DPPH radical without antiox-
idant, i.e. the control was measured. The data is commonly re-
ported as IC
50
, which is the concentration of antioxidant required
for 50% scavenging of DPPH radicals in the specified time period.
All determinations were performed in triplicate.
2.4.2. The b-carotene–linoleic acid assay
The antioxidant activity (AOA) of the different extract/fractions
was evaluated using the b-carotene–linoleic acid assay following
the method of Amarowicz, Karamac, and Shahidi (2003). In brief
a solution of b-carotene was prepared by dissolving 2 mg of b-car-
otene in 10 ml of chloroform. Two millilitres of this solution were
pipetted into a 100 ml round-bottom flask. After chloroform was
removed under vacuum, using a rotary evaporator at 40 °C,
40 mg of purified linoleic acid, 400 mg of Tween 40 as an emulsifier
and 100 ml of aerated distilled water were added to the flask with
vigorous shaking. Aliquots (4.8 ml) of this emulsion were trans-
ferred into a series of tubes containing 200
l
l of the extract
(200 ppm in methanol). The total volume of the systems was ad-
justed to 5 ml with methanol. As soon as the emulsion was added
to each tube, the zero time absorbance was measured at 470 nm
with a Shimadzu spectrophotometer (Shimadzu Co., Ltd., Kyoto, Ja-
pan). Sub-sequent absorbance readings were recorded over a 2 h
period at 20 min intervals by keeping the samples in a water bath
at 50 °C. Blank samples, devoid of b-carotene, were prepared for
background subtraction.
2.4.3. Stability of corn oil as affected by the addition of black cumin
phenolic rich fractions (PRFs)
The collected different black cumin seedcake PRFs were applied
to 100 g commercial edible corn oil obtained from local market
(free of any antioxidant) at levels of 0.25% and 0.5% to examine
their antioxidative activity. BHA at a level of 0.02% was used as a
standard. A control sample was prepared by using the same
amount of methanol used to dissolve the antioxidant and the ex-
tracts (Moure et al., 2000). The corn oil with added antioxidants
were heated at 70 °C for 72 h. Samples (5 g) were removed period-
ically every 4, 8, 24, 32, 48 and 72 h for analysis. The absorbance at
234 and 270 nm, peroxide value (PV) and p-anisidine value (AV)
were determined by the AOCS (1993) methods.
2.5. HPLC–DAD system for analysis of phenolic compounds
HPLC analysis was performed using Agilent G1310A pumps
(Agilent, Stevens Creek Blvd Santa Clara, USA), with diode array
detector and chromatographic separations were performed on a
LUNA C-18 column (5
l
m, 250 4.6 mm) (Phenomenex, Torrance,
CA, USA). The composition of solvents and used gradient elution
conditions were described previously by Chirinos et al. (2009) with
some modifications. The mobile phase was composed of solvent
(A) water–acetic acid (94:6, v/v, pH 2.27) and solvent (B) acetoni-
trile. The solvent gradient was as follows: 0–15% B in 40 min, 15–
45% B in 40 min and 45–100% B in 10 min. A flow rate of 0.5 ml/
min was used and 20
l
l of sample were injected. Samples and mo-
bile phases were filtered through a 0.22
l
m Millipore filter, type
GV (Millipore, Bedford, MA) prior to HPLC injection. Each fraction
was analyzed in duplicate. Phenolic compounds were identified
and quantified by comparing their retention time and UV–Vis spec-
A.A. Mariod et al. / Food Chemistry 116 (2009) 306–312 307

tral data to known previously injected standards (Chirinos et al.,
2009).
2.6. Statistical analyses
Statistical analyses were conducted using SPSS (Statistical Pro-
gram for Social Sciences, SPSS Corporation, Chicago, IL) version
12.0 for Windows. Analysis of variance (ANOVA) and Pearson’s cor-
relation coefficients were performed to compare the data. All
determinations were done at least in triplicate and all were aver-
aged. The confidence limits used in this study were based on 95%
(p< 0.05).
3. Results and discussion
3.1. Amount of extractable compounds vs extractable phenolic
compounds
The results of using different solvents for the extraction/frac-
tionation of phenolic compounds are given in Table 1. From this ta-
ble it was evident that black cumin seedcake contained noticeable
amounts of extractable compounds. It is clear that the different sol-
vents used for the extraction and fractionation of black cumin
seedcake, had different abilities to extract substances from this
seedcake. In general, the amount of total extractable compounds
decreased with decreasing polarity of the solvent in the order of
water, ethyl acetate, methanol and hexane.
The extraction of extractable substances from black cumin
seedcake with water was found most effective. With this solvent,
the highest amount of total extractable compounds (TEC) was ex-
tracted and found to be 642.0 mg/g followed by EAF 229.0 and CME
218.0 mg/g in black cumin seedcake, respectively. These findings
are in good agreement with that of Matthaüs (2002) who studied
the antioxidant activity of extracts obtained from seedcake of dif-
ferent oilseeds and he found that extraction with water gave the
highest total extractable compounds. So the extraction of black cu-
min seedcake with water resulted in the highest amount of TEC.
The extraction with hexane showed the lowest TEC (125.0 mg/g)
in comparison with that of the other solvents.
3.2. Total phenolic compounds (TPC)
The content of the total phenolic compounds of CME and its dif-
ferent fractions (HF, EAF and WF) from black cumin seedcake
determined using Folin–Ciocalteau method expressed as gallic acid
equivalents is shown in Table 1. Results in this table show that EAF
in black cumin samples contained the highest amount of total phe-
nolic compounds followed by WF, CME and HF, respectively. The
estimation of phenolic content amongst different fractions of black
cumin seedcake, surprisingly revealed that the ethyl acetate
fraction exhibited higher phenol content of 78.8 ± 0.08 mg/g GAE
followed by the water fraction (32.1 ± 0.003 mg/g) > crude
methanolic extract (27.8 ± 0.011 mg/g) > hexane fraction (12.1 ±
0.003 mg/g GAE). Ethyl acetate is often used as an extraction sol-
vent with a significant selectivity in the extraction of low-molecu-
lar-weight phenolic compounds and high-molecular-weight
polyphenols (Scholz & Rimpler, 1989). It was observed that the
crude extract had a lower phenolic content as compared to the
water and ethyl acetate fractions.
The relationship between the total extractable materials and its
content of phenolic compounds was represented in percentages of
TPC/TEC. The total phenolics found in the total extractable com-
pounds was low in all fractions. The ratio of total phenolic com-
pounds to the total extractable compounds ranged from 5.0% to
34.4% (Table 1). From these results it can be understood that in
CME, HF, EAF and WF of black cumin seedcake more than 87.3%,
90.3%, 65.6% and 95.0% of the extractable materials, respectively,
were compounds other than phenolic compounds (Table 1).
3.3. DPPH scavenging activity test
DPPH and b-carotene/linoleic acid methods were used to evalu-
ate the antioxidant activity of black cumin seedcake. The antioxi-
dant activity of the extracts in corn oil was assessed using
peroxide and anisidine values in comparison with a synthetic anti-
oxidant. The DPPH radical has been widely used to test the free
radical scavenging ability of different seedcakes and fat-free resi-
dues of the oilseeds (Mariod et al., 2006; Matthaüs, 2002; Peschel,
Diekmann, Sonnenschein, & Plescher, 2007). The DPPH scavenging
activities of different extract/fractions of seedcakes of black cumin
are shown in Table 1.
The DPPH values for investigated extract/fractions were ex-
pressed as IC
50
; the IC
50
values for different PRFs from black cumin
seedcake were 2.26, 2.65, 1.89 and 2.17 for CME, HF, EAF and WF,
respectively. From this table the CME and different fractions of
black cumin seedcake showed potent free radical scavenging activ-
ity on DPPH. The EAF showed the highest DPPH radical scavenging
activity followed by WF, CME and HF. From Table 1, a correlation
was found between the TPC and IC
50
when the TPC level was high;
the IC
50
was low which indicates high antioxidant activity. This is
due to the high amount of polyphenolic constituents present in the
PRFs from black cumin seedcake that act as free radical scavengers.
It was clear that the antioxidant potential of CME and its frac-
tions in DPPH assay was linearly correlated to its total phenolic
compounds. The antioxidant activity increased proportionally to
the polyphenol content, and a positive linear relationship between
IC
50
values and total phenolic compounds was found. Malencic
et al. (2008) found that antioxidant activity of soya bean seed ex-
tracts increased proportionally to the polyphenol content with a
linear relationship between DPPH values and total polyphenols.
In the same manner Chew, Lima, Omara, and Khoob (2008) found
a correlation between the TPC and IC
50
in edible seaweeds extracts
and they mentioned that, high level TPC gives low IC
50
and results
in high level of antioxidant capacity due to the high amount of
polyphenolic constituents
3.4. b-carotene bleaching (BCB) assay
In the BCB assay, the oxidation of linoleic acid generates peroxyl
free radicals due to the abstraction of hydrogen atom from diallylic
methylene groups of linoleic acid (Kumaran & Karunakaran, 2006).
The free radical then will oxidize the highly unsaturated b-caro-
tene. The presence of antioxidants in the extract will minimize
the oxidation of b-carotene by hydroperoxides. Hydroperoxides
formed in this system will be neutralized by the antioxidants from
the extracts. Thus, the degradation rate of b-carotene depends on
the antioxidant activity of the extracts.
Effect of black cumin seedcake extract/fractions (CME, HF, WF
and EAF) on oxidation of b-carotene/linoleic acid at 50 °C is shown
in Fig. 1. It was clear that the presence of antioxidants in the black
Table 1
Total extractable compounds (TEC), Total phenolic compounds (TPC), DPPH IC
50
(mg/
ml) of different extract/fractions obtained from black cumin seedcake.
Extraction TEC (mg/g)
a
TPC
a
TPC/TEC (%) DPPH IC
50
(mg/ml)
CME 218.0 27.8 ± 0.11 12.7 2.26 ± 0.21
HF 125.0 12.1 ± 0.03 9.7 2.65 ± 0.32
EAF 229.0 78.8 ± 0.08 34.4 1.89 ± 0.12
WF 642.0 32.1 ± 0.03 5.0 2.17 ± 0.41
a
Results are mean ± SD (n=3), results are given in mg/g extract.
308 A.A. Mariod et al. / Food Chemistry 116 (2009) 306–312

cumin seedcake extract/fractions reduced the oxidation of b-caro-
tene by hydroperoxides from these extract/fractions. The control
sample, without addition of extract solution, oxidized most rap-
idly. There were significant differences (p< 0.05) between the dif-
ferent extract/fractions, control and BHA. Thus, the degradation
rate of b-carotene depends on the antioxidant activity of the ex-
tracts. The phenolic compound rich fractions of black cumin seed-
cake exhibited antioxidant activity in a b-carotene–linoleate model
system. From Fig. 1 the effect of hexane and water fractions on the
coupled oxidation of linoleic acid and b-carotene was the highest,
and that, the antioxidant activity of N. sativa PRFs followed the or-
der: hexane > water > crude methanol extract > ethyl acetate, and
there was a significant difference (p< 0.05) between the antioxi-
dant activities of these fractions.
It is clear that HF, WF fractions performed better in their effect
on reducing the oxidation of b-carotene than CME and EAF frac-
tions, and that their degradation rate of b-carotene dose not de-
pends on their antioxidant activity. There was no correlation
between the degradation rate and the bleaching of b-carotene, by
other words, no correlation between TPC, and BCB. This is due to
the different types of antioxidants that are assayed by the two
methods, where TPC gives an indication of the levels of both lipo-
philic and hydrophilic compounds. BCB in contrast, only gives an
indication of the levels of lipohilic compounds (Chew et al.,
2008). Most studies showed there was no correlation between
TPC and BCB (Mariod et al., 2006; Matthaüs, 2002).
3.5. Stability of corn oil as affected by the addition of black cumin
phenolic rich fractions (PRF)
From the above results of TPC, DPPH and BCB it is clear that the
phenolic compounds of seedcakes from black cumin contains effec-
tive antioxidants, as phenolics are present in seeds mainly within
the hulls to protect the seeds from invasive diseases development
and from consumption by insects (Liyana-Pathirana et al., 2006).
Synthetic antioxidants e.g. tartery-butylhydroquinone (TBHQ),
butylated hydroxytoluene (BHT) and butylated hydroxyanisole
(BHA) are added to fats and oils to retard oxidation of unsaturated
fatty acids and to decrease the development of rancidity, natural
phenolic antioxidants inhibit oxidation reactions when added to
oils by acting as a hydrogen donor and afford relatively stable free
radicals and/or non-radical products (Wanasundara & Shahidi,
1994).
The effect of black cumin phenolic rich fractions (PRFs) and BHA
on corn oil oxidation at 250 and 500 mg/100 g oil is shown in Figs.
2 and 3, respectively. The development of PV during the oxidation
of corn oil was evaluated at 70 °C. This temperature was ideal, be-
cause at higher temperatures the peroxides will decompose very
fast (Duh & Yen, 1997). In Fig. 2 the PV of corn oil (control) with
and without black cumin extract/fractions (CME, HF, WF and
EAF) or BHA showed a gradual increase. As demonstrated in this
figure, a maximum PV of 17.2 meq O
2
/kg was reached after 72 h
of storage in the control without addition of extract or BHA. Signif-
icant differences (p< 0.05) were found between the control and dif-
ferent PRFs or BHA, which decreased and slowed down the rate of
peroxide formation, resulting in lower PVs after 72 h of storage at
70 °C. The PVs of corn oil containing CME, and EAF fractions were
found to be more effective than BHA while WF and HF fractions
were found to be less effective than the synthetic antioxidant.
In Fig. 3. The PV of corn oil (control) with and without black cu-
min extract/fractions (CME, HF, WF and EAF) or BHA showed a
gradual increase. As demonstrated in this figure, a maximum PV
of 17.5 meq O
2
/kg was reached after 72 h of storage in the control
without addition of extract or BHA. Significant differences
(p< 0.05) were found between the control and different PRFs or
BHA, which decreased and slowed down the rate of peroxide for-
mation, resulting in lower PVs after 72 h of storage at 70 °C. The
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 20 40 60 80 100 120
Time (min)
Absorbance at 470 nm
BHA HF WF CME EAF Control
Fig. 1. Effect of Nigella sativa extract/fractions (HF, WF, CME and EAF) on oxidation
of b-carotene/linoleic acid at 50 °C.
0
2
4
6
8
10
12
14
16
18
0 1020304050607080
Time (hr)
PV (meq O2 kg oil)
Contro l BHA WF250 HF250 CME250 EAF250
Fig. 2. Oxidation of corn oil treated with Nigella sativa CME and its fractions
250 mg/100 g oil during storage at 70 °C.
0
2
4
6
8
10
12
14
16
18
0 1020304050607080
Time (hr)
PV (meq O
2
kg oil)
Control BHA WF500 HF500 CME500 EAF 500
Fig. 3. Oxidation of corn oil treated with Nigella sativa CME and its fractions
500 mg/100 g oil during storage at 70 °C.
A.A. Mariod et al. / Food Chemistry 116 (2009) 306–312 309

PVs of corn oil containing CME, and WF fractions were found to be
more effective than EAF and HF.
It can be concluded that black cumin seedcake PRFs at concen-
trations of 0.25% and 0.5% were effective in stabilizing corn oil dur-
ing storage at 70 °C, and addition of 0.25% PRFs from black cumin
as natural antioxidant was found to be better in inhibition corn
oil oxidation than using 0.5% and this concentration will be prefer-
able from economic point of view. The PRFs from black cumin seed
cake possessed good antioxidant activity and extended the induc-
tion period and decreased the formation of peroxides in corn oil
more effectively than BHA at rate of 250 mg/100 g oil.
The effect of black cumin PRFs and BHA on corn oil oxidation
(measured by p-anisidine value) is shown in Table 2. The succes-
sive heating of corn oil (at 70 °C for 72 h) mixed with PRFs leads
to autoxidation and formation of primary products that decom-
posed readily and formed aldehydes, ketones and alcohols as sec-
ondary products. As anisidine value is a more meaningful test for
the assessment of the heating oils quality during heating than
the peroxide value, so it was used here because it measures the
secondary products of oxidation reactions. Using PRFs extracted
from black cumin seedcake as natural antioxidant, at the ratio of
250, 500 mg/kg oil, inhibited the formation of the secondary prod-
ucts in comparison with the control, the amount of secondary
products formed seem to be less than that formed in the control
samples. These results indicated that almost addition of CME and
WF gave better effect than HF and EAF as shown in Table 2. The
Table 2
Comparative inhibition of edible corn oil oxidation by Nigella sativa seedcake phenolic rich fractions at 250 and 500 mg/100 g oil measured by p-anisidine value.
Time (h) Control BHA HF250 HF500 EAF250 EAF500 CME250 CME500 WF250 WF500
0 0.11 ± 0.1
a
0.11 ± 0.1
a
0.12 ± 0.1
a
0.12 ± 0.1
a
0.12 ± 0.1
a
0.12 ± 0.1
a
0.11 ± 0.1
a
0.12 ± 0.1
a
0.11 ± 0.1
a
0.13 ± 0.1
a
4 3.86 ± 0.2
a
1.80 ± 0.2
b
2.95 ± 0.1
c
1.49 ± 0.1
b
2.76 ± 0.2
c
2.02 ± 0.2
d
2.80 ± 0.3
c
2.62 ± 0.2
c
2.39 ± 0.2
d
2.57 ± 0.3
c
8 4.47 ± 0.3
a
2.71 ± 0.1
b
3.51 ± 0.2
c
3.37 ± 0.2
c
3.96 ± 0.1
d
3.81 ± 0.2
d
3.21 ± 0.1
c
2.92 ± 0.1
b
3.34 ± 0.2
c
3.50 ± 0.2
c
24 4.91 ± 0.2
a
3.14 ± 0.1
b
4.50 ± 0.2
c
3.99 ± 0.1
d
4.51 ± 0.3
c
3.95 ± 0.2
d
3.52 ± 0.1
e
3.50 ± 0.1
e
3.90 ± 0.2
d
4.50 ± 0.1
c
32 5.32 ± 0.3
a
3.52 ± 0.1
b
4.91 ± 0.2
c
4.58 ± 0.2
d
4.81 ± 0.3
c
4.41 ± 0.2
d
3.90 ± 0.1
b
3.80 ± 0.1
b
4.52 ± 0.2
d
4.50 ± 0.1
d
48 5.79 ± 0.2
a
3.87 ± 0.1
b
5.52 ± 0.2
c
5.42 ± 0.1
c
5.40 ± 0.2
c
5.01 ± 0.1
d
4.40 ± 0.1
e
4.20 ± 0.1
e
4.80 ± 0.1
d
4.80 ± 0.1
d
72 6.94 ± 0.2
a
4.10 ± 0.1
b
6.20 ± 0.2
c
6.40 ± 0.2
d
5.70 ± 0.1
e
6.02 ± 0.1
e
4.80 ± 0.1
f
4.60 ± 0.1
f
5.10 ± 0.1
e
5.20 ± 0.1
e
Means in every row without a common superscript differ significantly at p< 0.05. BHA, butylated hydroxyanisol; HF, hexane fraction; WF, water fraction; CME, crude
methanolic extract; EAF, ethyl acetate fraction.
Fig. 4. HPLC/DAD chromatogram of phenoilc compounds in black cumin (A) CME. Detection was at 280 nm. Peak: (4) hydroxybenzoic acid, (5) syringic acid, (6) p-cumeric (B)
WF. Detection was at 280 nm. Peak: (4) hydroxybenzoic acid, (5) syringic acid, (6) p-cumeric (C) Standards of phenolic acids recorded at 280 nm. Peak: (1) gallic acid, (2) (+)-
catechin, (3) chlorogenic acid, (4) hydroxybenzoic acid, (5) syringic acid, (6) p-cumaric, (7) vanillin, (8) ferulic acid, (9) quercetin.
310 A.A. Mariod et al. / Food Chemistry 116 (2009) 306–312

phenolic rich fractions (PRFs) obtained from black cumin seedcake,
seem to be less effective in inhibition of secondary products than
BHA. From Table 2 it was clear that using 250 mg from these nat-
ural antioxidants gave the same effect of using 500 mg so using
250 mg will be more preferable from economical point of view.
3.6. Identification of phenolic compounds using HPLC–DAD
To know what is/are the responsible active ingredient(s) in
black cumin seedcake PRFs, HPLC–DAD was used. CME and WF
fractions were used to identify their important phenolic com-
pounds. Fig. 4 shows a representative chromatogram of the (A)
CME, (B) WF of black cumin seedcake PRFs and standard (C) mon-
itored at 280 nm.
It shows that the crude methanolic extracts and water fraction
of black cumin seedcake contains hydroxybenzoic, syringic and p-
cumaric acids, with high area in p-cumaric acid in both two frac-
tions. These compounds have been identified according to their
retention time and the spectral characteristics of their peaks com-
pared to those of standards in Fig. 4C, as well as by spiking the
sample with standards. p-cumaric acid was detected to be the ma-
jor phenolic component in the two fractions (CME and WF), con-
tributing about 66.8% and 72.1% to the total amount,
respectively, and showing the levels of 0.631 and 3.83 mg/100 g
dry weight (DW) in CME and WF, respectively (Table 3). Hydroxy-
benzoic and syringic acids were also predominant, but slightly
higher in WF (0.989 and 0.496 mg/100 g DW) than in CME (0.188
and 0.125 mg/100 g DW), respectively. Results demonstrated that
differences in CME and WF phenolic composition were signifi-
cantly more quantitative than qualitative, where water fraction
showed higher amount than methanol crude extract. Black cumin
seedcake CME and WF possess similar composition. The amounts
of the detected p-cumaric acid in both fractions is much higher
than (0.36 mg/100 g dry sample) that reported by Bourgou et al.
(2008) in black cumin roots methanolic extract. While these
authors reported higher amount of hydroxybenzoic acid
(1.73 mg/100 g) in black cumin roots methanolic extract. Surpris-
ingly these authors did not identify any amount of syringic acid
in black cumin shoots or roots extracts. The levels of total phenolic
compounds in black cumin CME and WF determined by HPLC were
0.0094 and 0.053 mg/g DW, respectively, and thus lesser than (36.1
and 31.3 mg/g) the ones obtained by the Folin–Ciocalteu method.
This result is predictable due to the weak selectivity of the Folin–
Ciocalteu reagent, as it reacts positively with different antioxidant
compounds (phenolic and non-phenolic substances).
The EAF and HF fractions of black cumin seedcake also pre-
sented some phenolic compounds with less area (data not shown).
Previous studies reported that phenolic compounds such as
hydroxybenzoic, syringic, p-cumaric and vanillin containing signif-
icant antioxidant activities (Zhang, Liao, Moore, Wu, & Wang,
2009). The above mentioned HPLC–DAD results indicate that such
phenolic rich fractions from black cumin seedcake may inhibit the
oxidation of corn oil. Isolation and characterisation of such PRFs
may be useful in developing natural antioxidants.
4. Conclusions
This investigation indicated the presence of compounds pos-
sessing antioxidant activity in PRFs of black cumin seedcake. The
total phenolic content of the PRFs revealed that the EAF has a high-
er phenolic content 78.8 mg/g followed by the other fractions. The
PRFs showed a potential value as natural antioxidants and possibly
can be used to improve oxidative stability of corn oil. Some of the
black cumin PRFs were more effective than BHA in retarding the
formation of primary and secondary oxidation products of corn
oil. These PRFs act as free-radical terminators and chelating agents,
so the application of these natural antioxidants to stabilize edible
oils may be considered. Three phenolic compounds were identified
in CME and WF as hydroxybenzoic, syringic and p-cumaric acids
and quantified. Further more studies in isolation and quantification
of individual phenolic compounds, to elucidate their different anti-
oxidant mechanisms and the existence of possible synergism, if
any, amongst the compounds in black cumin seedcake, and effects
of these phenolics on antioxidant status in animal models are
needed to evaluate their potential benefits.
Acknowledgements
The first author is grateful to IBS/UPM for the senior research
fellowship granted during the period of this work. We thank Siti
Muskinah, Laboratory of Molecular Biomedicine IBS/UPM for anal-
ysis in HPLC–DAD equipment and excellent technical expertise
needed for this work. This study was funded by Intensifying Re-
search Priority Areas (IRPA) Grants provided by Government of
Malaysia and University Putra Malaysia.
References
Amarowicz, R., Karamac, M., & Shahidi, F. (2003). Antioxidant activity of phenolic
fractions of lentil (Lens culinaris). Journal of Food Lipids, 10, 1–10.
AOCS (1993). Official methods and recommended practices of the American oil
chemists’ society (4th ed.). Champaign, IL: AOCS Press.
Bourgou, S., Ksouri, R., Bellila, A., Skandrani, I., Falleh, H., & Marzouk, B. (2008).
Phenolic composition and biological activities of Tunisian Nigella sativa L. shoots
and roots. C. R. Biologies, 331, 48–55.
Chew, Y. L., Lima, Y. Y., Omara, M., & Khoob, K. S. (2008). Antioxidant activity of
three edible seaweeds from two areas in South East Asia. Lebensmittel-
Wissenschaft und – Technologie, 41, 1067–1072.
Chirinos, R., Betalleluz, I., Humána, A., Arbizub, C., Pedreschi, R., & Campos, C. (2009).
HPLC–DAD characterization of phenolic compounds from Andean oca (Oxalis
tuberose Mol.) tubers and their contribution to the antioxidant capacity. Food
Chemistry, 113, 1243–1251.
Duh, P. D., & Yen, Y. C. (1997). Antioxidant efficacy of methanolic extracts of peanut
hullsin soybeanand peanut oils.Journal ofAmerican Oil ChemistSociety,74, 745–748.
El-Dakhakhny, M., Barakat, M., El-Halim, M., & Aly, S. M. (2000). Effects of Nigella
sativa oil gastric secretion and ethanol-induced ulcer in rats. Journal of
Ethnopharmacology, 72, 299–304.
Gordon, M. H., Paiva-Martins, F., & Almeida, M. (2001). Antioxidant activity of
hydroxytyrosol acetate compared with that of other olive oil polyphenols.
Journal of Agricultural Food Chemistry, 49, 2480–2485.
Gray, J. I. (1978). Measurement of lipid oxidation, a review. Journal of the American
Oil Chemists’ Society, 55, 539–546.
Hertog, M. L., Feskens, E. M., Hollman, P. H., Katan, M. B., & Kromhout, D. (1993).
Dietary antioxidant flavonoids and risk of coronary heart-diseases the Zutphen
Elderly study. Lancet, 342, 1007–1011.
Jadhav, S. J., Nimbalkar, S. S., Kulkarni, A. D., & Madhavi, D. L. (1996). Lipid oxidation
in biological and food systems. In D. L. Madhavi, S. S. Deshpande, & D. K.
Salunkhe (Eds.), Food antioxidants (pp. 5–63). New York: Dekker Press.
Kaur, R., Arora, S., & Singh, B. (2008). Antioxidant activity of the phenol rich fractions of
leaves of Chukrasia tabularis A. Juss. Bioresource Technology, 99, 7692–7698.
Kubola, J., & Siriamornpun, S. (2008). Phenolic contents and antioxidant activities of
bitter gourd (Momordica charantia L.) leaf, stem and fruit fraction extracts
in vitro.Food Chemistry, 110, 881–890.
Kumaran, A., & Karunakaran, R. J. (2006). Antioxidant and free radical scavenging
activity of an aqueous extract of Coleus aromaticus.Food Chemistry, 97, 109–114.
Liyana-Pathirana, C. M., Shahidi, F., & Alasalvar, C. (2006). Antioxidant activity of
cherry laurel fruit (Laurocerasus officinalis Roem.) and its concentrated juice.
Food Chemistry, 99, 121–128.
Malencic, D., Maksimovic, Z., Popovic, M., & Miladinovic, J. (2008). Polyphenol
contents and antioxidant activity of soybean seed extracts. Bioresource
Technology, 99, 6688–6691.
Table 3
Phenolic compound content mg/100 g in black cumin methanolic extract and water
fraction.
Compounds Crude methanolic extract (CME) Water fraction (WF)
p-Cumeric acid 0.631 ± 0.32 3.83 ± 0.35
Hydroxybenzoic acid 0.188 ± 0.21 0.989 ± 0.42
Syringic acid 0.125 ± 0.12 0.496 ± 0.15
Values are means ± SD (n= 3), and they are given as mg/100 g dry weight of
investigated black cumin extract/fractions.
A.A. Mariod et al. / Food Chemistry 116 (2009) 306–312 311

Mariod, A. A., Matthaus, B., & Hussein, I. H. (2008). Antioxidant properties of
methanolic extracts from different parts of Sclerocarya birrea.International
Journal of Food Science and Technology, 43, 921–926.
Mariod, A. A., Matthaüs, B., Eichner, K., & Hussein, I. H. (2006). Antioxidant activity
of extracts from Sclerocarya birrea kernel oil cake. GRASAS Y ACEITES, 57,
361–366.
Matthaüs, B. (2002). Antioxidant activity of extracts obtained from residues of
different oilseeds. Journal of Agricultural and Food Chemistry, 50, 3444–3452.
Miraliakbari, H., & Shahidi, F. (2008). Antioxidant activity of minor components of
tree nut oils. Food Chemistry, 111, 421–427.
Moure, A., Franco, D., Sineiro, J., Dominguez, H., Nunez, M. J., & Lema, J. M. (2000).
Evaluation of extracts from Gevuina hulls as antioxidants. Journal of Agricultural
and Food Chemistry, 48, 3890–3897.
Nagi, M. N., & Mansour, M. A. (2000). Protective effect of thymoquinone against
doxorubicin induced cardiotoxicity in rats: A possible mechanism of protection.
Pharmacological Research, 41, 283–289.
Peschel, W., Diekmann, W., Sonnenschein, M., & Plescher, A. (2007). High
antioxidant potential of pressing residues from evening primrose in
comparison to other oilseed cakes. Food Chemistry, 97, 137–150.
Scholz, E., & Rimpler, H. (1989). Proanthocyanidins from Krameria triandra root.
Planta Medica, 55, 379–384.
Senevirathne, M., Kim, S., Siriwardhana, N., Ha, J., Lee, K., & Jeon, Y. (2006).
Antioxidant potential of Ecklonia cava on reactive oxygen species scavenging
metal chelating, reducing power and lipid peroxidation inhibition. Food Science
and Technology International, 12, 27–38.
St Angelo, A. J., Ory, R. L., & Brown, L. E. (1975). Comparison of methods for
determining peroxidation in processed whole peanut products. Journal of the
American Oil Chemists’ Society, 52, 34–35.
Surh, Y. J. (2002). Anti-tumor promoting potential of selected spice ingredients with
antioxidative and anti-inflammatory activities: A short review. Food and
Chemical Toxicology, 40, 1091–1097.
Wanasundara, U. N., & Shahidi, F. (1994). Stabilization of canola oil with flavonoids.
Food Chemistry, 50, 393–396.
Surh, Y. J., Hurh, Y. J., Kang, J. Y., Lee, E., Kong, G., & Lee, S. J. (1999). Resveratrol, an
antioxidant present in red wine, induces apoptosis in human promyelocytic
leukemia (HL-60) cells. Cancer Letters, 140(1–2), 1–10.
Zhang, Z., Liao, L., Moore, J., Wu, T., & Wang, Z. (2009). Antioxidant phenolic
compounds from walnut kernels (Juglans regia L.). Food Chemistry, 113, 160–165.
312 A.A. Mariod et al. / Food Chemistry 116 (2009) 306–312