LWT - Food Science and Technology 78 (2017) 198e207

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LWT - Food Science and Technology

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Genetic diversity in fatty acid composition and antioxidant capacity of

Nigella sativa L. genotypes
S.N. Saxena b, *, S.S. Rathore b, Y. Diwakar a, R.K. Kakani b, K. Kant b, P.N. Dubey b,
R.K. Solanki b, L.K. Sharma b, D. Agarwal b, S. John b
a
Central Plantation Crops Research Institute, Kidu, Bilinede Village, Puttur Taluk, Dakshina Kannad, Karnataka 574243, India
b
ICAR-National Research Centre on Seed Spices, Tabiji, Ajmer, Rajasthan 305206, India

a r t i c l e i n f o a b s t r a c t

Article history: A study was conducted to explore the suitability of Nigella sativa L. oil for human consumption on the
Received 23 July 2016 basis of fatty acid (FA) composition in twenty three selected genotypes. Total oil content ranged from 147
Received in revised form to 270 ml/kg. Eleven fatty acids including palmitic, oleic, and linoleic acids were found during fatty acid
14 December 2016
methyl esters (FAME) analysis. Linoleic acid was major contributor in the range of 608.9 ml/l (AN-15) to
Accepted 17 December 2016
Available online 23 December 2016
713.9 ml/l (AN-8). Saturated fatty acids ranged from 121 to 181 ml/l in genotype AN-23 and AN-3,
respectively. Signicant genetic variation was observed with respect to mono-unsaturated fatty acids.
Poly-unsaturated fatty acid being dominant existed in the range of 651e771 ml/l. Metabolic capacity to
Keywords:
Fatty acids ratio
inter-conversion of fatty acids and nutritive value of Nigella oil is described on the basis of various fatty
Nigella oil acid ratios. Multivariate analysis revealed that, oil content has positive correlation with linoleic acid. The
PUFA Euclidean based clustering revealed, that genotypes AN-23, AN-5, AN-8, AN-9, AN-10, AN-11, AN-19, AN-
MUFA 21 and AN-24 are suitable for trait specic breeding programme for higher oil content, increased lino-
Edible oil lenic and reduced palmitic acid with higher nutritive value. Genotype AN-4 exhibited good combination
Oxidative stability of higher polyunsaturated fatty acids (PUFA) as well as oxidative stability.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Plants seed are major sources of oils that have nutritional, in-
dustrial and pharmaceutical importance. Fatty acids (FAs) compo-
sition is an important indicator for suitability of oil for a particular
purpose. Interest in the specic fatty acids composition for ideal
edible oils has been emerging with the growing scientic views,
that all fats are not equivalent with regard to consumer health. Till
date seed oil of several thousand plant species have been chemi-
cally analysed and a few of them are taken into cultivation as an oil
crop. The variation in fatty acids composition within a species also
proved to be useful in chemotaxonomy and phylogenetic studies
(Velasco & Goffman, 2000). Exploitation of lesser known crops is a
possible solution for growing and diversied nutritional needs of
growing population. Black cumin (Nigella sativa L.) commonly
known as Nigella, belongs to the family Ranunculaceae, is a well
known herb cultivated from the Southern and Eastern-rim of the
* Corresponding author.
E-mail address: shail.nrcss@gmail.com (S.N. Saxena).
Mediterranean basin to Iran, Pakistan, and India. Nigella seeds are
rich in nutritional values and are used as seasoning and avoring of
food, bread, pickles and bakery products. It is important for both oil
and bioactive compounds. Studies have shown a wide spectrum of
properties curing various diseases (Dubey, Singh, Mishra, Kant, &
Solanki, 2016; Iqbal et al., 2011). The seeds as whole or their ex-
tracts have antitumor (Khan, Sharma, & Sultana, 2009), antidia-
betics (Fararh, Atoji, Shimizu, & Takewaki, 2008), antibacterial,
antiviral (Edris, 2007; Mashhadian & Rakhshandeh, 2005), anti-
oxidant, galactagogue, carminative, laxative and antiparasitic
properties (Kanter et al., 2003). The oil of Nigella possesses
phenolic, avonoids and other related compounds which increased
its antioxidant activity (Badary, Taha, Ayman, El-Din, & Abdel-
Wahab, 2003; Kruk, Michalska, & Klanda, 2000) and is consid-
ered highly prized nutritive oil. Inspite of the above facts, Nigella
seed oil does not really have a signicant economic market share
(Padhye, Banerjee, Ahmad, Mohammad, & Sarkar, 2008).
Oil seed crops rich in essential polyunsaturated fatty acids
(PUFA) can be more economic alternatives compared to traditional
crops. Linoleic acid (LA) is one of the important PUFA, referred as
essential fatty acid (EFA) and recognized as essential biochemical

http://dx.doi.org/10.1016/j.lwt.2016.12.033
0023-6438/ 2016 Elsevier Ltd. All rights reserved.
 S.N. Saxena et al. / LWT - Food Science and Technology 78 (2017) 198e207 199

components of human diet (Gomez, Bermejo, & Kohen, 2011). transferred to amber coloured glass vials and stored at 20
C until
Though, sufcient work has been done on medicinal properties of further analysis.
Nigella seeds, there are meagre reports on extent of genotypic
variability in cultivated genotypes of Nigella in India with reference 2.4. FAME analysis
to fatty acid composition. To formulate an effective breeding pro-
gramme for desirable fatty acid composition, information regarding Fatty Acid Methyl Esters (FAME) were prepared according to
heritability, genetic advance and correlation among fatty acids is a AOCS Method CE 1e62. Diluted FAME were separated on an Agilent
pre requisite. Hence, the present study has been conducted to Series GC-MS (Agilent Technologies, Santa Clara, California, USA;
explore the genetic variation in selected genotypes of N. sativa GC -7820 A, MS-5975) equipped with an HP5 (Universal column)
based on analysis of oil composition, antioxidant potential and its (30 m  0.325 mm x 0.25 mm); Agilent J&W GC column with an
suitability for human consumption by blending with other food auto sampler. A sample of 1 ml was used in split mode (20:1) with
oils. an auto sampler. Helium was used as the carrier gas at a ow rate of
1.0 ml/min. The column temperature was programmed from 50
C
2. Materials and methods to 280
C with equilibrium time of 3 min, held for 30 min. Injector
temperatures were set at 250
C. The fatty acids were identied by a
2.1. Plant material comparison of their retention indices and their identication was
conrmed by computer matching of their mass spectral fragmen-
Seeds of twenty three genotypes of Nigella were collected from tation patterns of compounds in the NIST-MS library and published
different Nigella growing parts of India. Crop has been raised in mass spectra with the help of Chemtation software (Agilent Tech-
farm of ICAR-NRCSS, Ajmer (26
270 000 N, 74
380 2400 E), India nologies, Santa Clara, California, USA).
during rabi season (winter season which commences from
November rst week up to February) of year 2012e13 and 2013e14 2.5. Estimation of phenolics and antioxidant capacity
under standard package and practices. A detailed description of the
materials used in this study is shown in Table 1. Total phenol concentrations were determined using a Folin-
Ciocalteu assay, as described by Amin, Norazaidah, and Hainida
2.2. Chemicals (2006). An aliquot of 0.1 ml from extracted oil was taken in a test
tube and made the volume 1 ml by adding n-hexane solvent. Three
All reagents and fatty acid standards were procured from Sigma- ml of 100 g/l sodium carbonate was added. Previously 10-fold
Aldrich,St. Louis, Missouri, USA and were analytical or HPLC grade. diluted Folin-Ciocalteu reagent was added to the mixture. The
mixture was allowed to stand at room temperature for 90 min and
then absorbance was measured at 710 nm. Gallic acid was used as
2.3. Oil extraction
the standard phenol. The amount of phenolic content was calcu-
lated by using the standard curve of Gallic acid having R2 (a sta-
The Nigella seeds were ground in a grinder before oil extraction.
tistical measure of how close the data are to the tted regression
Twenty gram ground seeds were homogenized and oil was
line) value ranged from 0.96 to 0.99 and was expressed as mg Gallic
extracted with 150 ml of n-hexane following the procedure of
Acid Equivalents/ml oil (GAE/ml).
Soxhlet extraction. Six extraction cycles were performed for
Total avonoid concentration was determined by using previ-
maximum recovery of oil. Each cycle took 1 h, thus total duration of
ously reported method by Chang, Yang, Wen, and Chern (2002). An
extraction was 6 h. The oil extract was concentrated under vacuum
aliquot of 0.1 ml from extracted oil was taken in a test tube and
in a rotary evaporator (JSGW, Amballa Cantt, India) at 35
C. Solvent
100 ml aluminium chloride (1 mol/l) solution was added carefully
free oil was weighed to determine the oil content and then
from the side wall of the test tube followed by addition of 100 ml
potassium acetate. The total volume was made 4 ml by adding
Table 1 2.8 ml of n-hexane solvent in the test tube. After 30 min incubation
List of Nigella genotypes used in this study. of reaction mixture at room temperature, stable yellow colour was
Genotype Location developed. Absorbance was measured at 415 nm. Quercetin was
used as the standard avonoids. The amount of avonoid was
AN-1 NRCSS, Tabiji, Ajmer, Rajasthan
AN-2 Beawar, Ajmer, Rajasthan
calculated by using the standard curve of quercetin having R2 value
AN-3 Kekri, Ajmer, Rajasthan ranging from 0.96 to 0.99 and was expressed as mg Quercetin
AN-4 Pisangan, Ajmer, Rajasthan Equivalents/ml (QE/ml) extracted oil.
AN-5 Pushkar, Ajmer, Rajasthan The antioxidant activity of oil extract was evaluated on the basis
AN-6 Digod, Kota, Rajasthan
of its activity in scavenging the stable DPPH (2, 2-Diphenyl-1-
AN-7 Anta, Kota, Rajasthan
AN-8 Local market, Jaipur, Rajasthan Picrylhydrazyl) radical using the method described by Shimada,
AN-9 Local market, Nagaur, Rajasthan Fujikawa, Yahara, and Nakamura (1992). Oil extract was diluted in
AN-10 Phalodi, Jodhpur, Rajasthan methanol to give at least 5 different concentrations. An aliquot (1,
AN-11 Masodha, Faizabad, Uttar Pradesh 1.5, 2, 2.5 ml) of the oil extract of each concentration was mixed
AN-13 Kumargang, Faizabad, Uttar Pradesh
AN-14 Kanpur, Uttar Pradesh
with 1 ml of 1 mol/l DPPH solution. The mixture was then ho-
AN-15 Chaubepur, Kanpur, Uttar Pradesh mogenized and left to stand for 30 min in the dark. The absorbance
AN-16 Ludhiana, Punjab was measured at 517 nm against a blank of methanol using a
AN-17 Mandsaur, Madhya Pradesh spectrophotometer. DPPH solution plus methanol was used as
AN-18 Neemuch, Madhya Pradesh
control and Butyl hydroxyl toluene (BHT) was used as a standard
AN-19 Ratlam, Madhya Pradesh
AN-20 Sangod, Kota reference synthetic antioxidant with R2 value ranging from 0.95 to
AN-21 Digod, Kota 0.99. Results were expressed as mg Butyl hydroxyl toluene (BHT)
AN-22 Banaskantha, Gujarat Equivalent/ml oil. Results were expressed as a mean standard
AN-23 Pushkar, Ajmer, Rajasthan deviation from three replicate measurements. The percent scav-
AN-24 Ranakpur, Gujarat
enging effect (capacity of scavenging DPPH free radicals) was
200 S.N. Saxena et al. / LWT - Food Science and Technology 78 (2017) 198e207
calculated as follows:
A517 of Control  A517 of Extract
Scavenging capacity %
A517 of Control
 100
2.6. Fatty acid ratio
Due to inter-correlation of different fatty acids present in a
particular genotype, it is difcult to evaluate individual genotype
for breeding purposes. Hence, different ratios viz., elongation ratio
(ER), desaturation ratio (DR), oleic desaturation ratio (ODR), and
linoleic desaturation ratio (LDR) were estimated. The ER estimates
the relative weight of the elongation pathway from oleic (C18:1) to
eicosenoic (C20:1) and higher chain fatty acids, while the DR esti-
mates the relative weight of the desaturation pathway from oleic to
linoleic (C18:2) and linolenic acid (C18:3) within the overall fatty
acid biosynthetic system. The ER and DR calculation was done as
per the formula suggested by Velasco, Goffman, and Becker (1998),
while ODR (efciency of the desaturation from oleic to linoleic acid)
and LDR (from linoleic to linolenic acid) were calculated as per
Pleines and Friedt (1988).
ER (% C20:1)/(%C20:1 %C18:1 %C18:2 %C18:3)
DR (% C18:2 %C18:3)/(%C 20:1 %C18:1 %C18:2 %C18:3)
ODR (% C18:2 %C18:3)/(%C18:1 %C18:2 %C18:3)
LDR (% C18:3)/(%C18:2 %C18:3)
Ratio of monounsaturated fatty acids (MUFA)/Saturated fatty
acids (SFA) (M/S), polyunsaturated fatty acids PUFA/Saturated fatty
acids (SFA) (P/S), and (PUFA MUFA)/SFA (M P/S) have been
worked out to analyze individual genotype for nutritional impor-
tance (Badr, Arzoo, & Bakeet, 2014).
2.7. Statistical analysis
Obtained data were analysed in Completely Randomized Design
while phenotypic and genotypic correlation coefcients for fatty
acid were calculated as per Panse and Sukhatme (1978). Experi-
ment was conducted in triplicate. Fatty acid diversity for fatty acids
among 23 genotypes was compared by an Euclidean distance ma-
trix and by preparing dendrogram using XLSTAT software version
2015. The same software was used to perform principal component
analysis (PCA). Genetic distance calculations was analysed by
matrices for all pairs of genotypes constructed from the interval
and ratio of lipid data using the Euclidean distance method (Jerry,
2001). Cluster analysis was performed based on the genetic dis-
tance matrices generated by the Euclidean distance method to
reveal the patterns of genetic relationships among genotypes.
3. Results and discussion
Table 2 showed genotypic variation in total oil, individual fatty
acid methyl esters, different fatty acid ratios, phenolic, avonoid
content, antioxidant content and free radical scavenging percent-
age in 23 nigella genotypes. Analysis of variance showed signicant
genotypic differences for total oil, FAME and all characteristics
except Saturated Fatty Acids (SFA) in different oils.
3.1. Total oil and fatty acids
Total oil content in twenty three Nigella genotypes ranged from
a minimum of 147 ml/kg (AN-16) to a maximum of 270 ml/kg (AN-
24) with an average of 205 ml/kg (Table 3). The oil yield is lower
than reported by many researchers. Nigella seeds contains more
than 30% oil and possibly up to 40% (Akram Khan, 1999; Matthaus &
Ozcan, 2011;; Cheikh-Rouhou et al., 2007). However, phenological
events as water-stress or saline conditions, and eventually cool
temperatures can reduce this yield to 13e23% (Al-Kayssi, Shihab, &
Mustafa, 2011; Atta, 2003; Bourgou, Bettaieb, Saidani, & Marzouk,
2010; Iqbal, Ghafoor, Ullah, & Ahmad, 2014). The agro-ecological
conditions prevailing at research farm from where seeds of Ni-
gella genotypes were collected is similar to the above conditions,
hence yielded less oil. Among FAME, linoleic acid (18:2 n-6), pal-
mitic acid (16:0), oleic acid (18:1 n-9) and stearic acid (18:0) were
the major fatty acids present in Nigella seed oil. Presence of these
fatty acids was also reported by Ramadan and Morsel (2003). Apart
from these other fatty acids viz., myristic acid (14:0), Heptadecanoic
acid (17:0), Arachidic (20:0), 9-Hexadecenoic acid (16:1 n-7), 10-
heptadecenoic acid (17:1 n-7), Linolenic acid (18:3 n-3) and 11,14-
Eicosenoic acid (20:2 n-6) were present in lesser amount. Pres-
ence of these minor fatty acid in Nigella seed oil is also reported by
Atta (2003) & Hamrouni-Sellami, Kchouk, and Marzouk (2003).
Amount of SFA, MUFA, PUFA, EFA, fatty acids presents in 23
Nigella genotypes are shown in Table 4. SFA ranged from a mini-
mum of 121 ml/l in AN-23 to maximum of 181 ml/l in AN-3 ge-
notype with an average availability of 159 ml/l. Among SFA,
palmitic acid was the major contributor which recorded up to
93 ml/l in AN-23 to 151 ml/l in AN-3 and was followed by steric acid
(20.2 ml/l in AN-15 to 39.1 ml/l in AN-6) (Table 3). Myristic acid
(14:0), Heptadecanoic acid and Arachidic (20:0) were present
below 1% in all genotypes. Palmitic acid is a major contributor for
elevating the levels of total blood cholesterol, especially LDL that
can lead a greater risk to cardiovascular diseases (Wardlaw, 2003,
pp. 143e159). Similar to the present investigation Gharby et al.
(2015) also reported 11.9e13.1% palmitic acid and 2.3e3.25% stea-
ric acid in solvent extracted or cold press extracted oil of Nigella
seeds. However, Cheikh-Rouhou et al. (2007) reported higher pal-
mitic acid content in Tunisian and Iranian origin Nigella seeds.
There existed genetic variation in total SFA among studied geno-
types. The MUFA content were ranged from 80 ml/l in genotype AN-
7 to 199 ml/l in AN-15. Among MUFA, oleic acid, a member of n-9
group was dominant fatty acid, ranging from a minimum of 68 ml/l
(AN-7) to a maximum of 190 ml/l in AN-15, while other MUFA
found below 10.0 ml/l (Table 3). Presence of MUFA in present study
is similar as reported by Iqbal et al. (2014). The intake of high
content of monounsaturated fatty acids (MUFAs) especially oleic
acid (18:1) is associated with decrease in total cholesterol and low-
density lipoprotein cholesterol (Denny et al., 2006), thus its con-
sumption reduces the incidence of coronary heart disease (CHD).
Similarly, PUFA were the dominant fatty acids in Nigella seed oil
and their contribution was ranging from 651 ml/l in genotype AN-
15 to 771 ml/l in genotype AN-23. Among PUFA, level of Linoleic
acid (n-6) was maximum (714 ml/l) in AN-8, while minimum level
was observed in AN-15 (609 ml/l) with an average of 681 ml/l.
Other n-6 member 11, 14-Eicosadienoic acid (20:2 n-6) was ranging
between 29.8 ml/l (AN-4) to 80.9 ml/l (AN-23). It is reported that
linoleic acid is one of the most signicant PUFA in human diet due
to its ability of preventing heart and vascular diseases. However,
there are reports that n-6 fatty acids are precursors of pro-
inammatory compounds in the body. Fritsche (2008) in a review
examined the existing evidence that consuming a diet rich in
linoleic acid (LA), an essential PUFA of the n-6 family, promotes
inappropriate or excessive inammatory responses. He found that
 S.N. Saxena et al. / LWT - Food Science and Technology 78 (2017) 198e207 201

Table 2
ANOVA for Complete Randomized Design analysis of total oil, fatty acids, fatty acid ratio, phenolic, avonoid, antioxidant content, and DPPH (2, 2-Diphenyl-1-Picrylhydrazyl)
scavenging capacity in Nigella genotypes.

Variables Abbreviation SS MSS (22c) F value

Total oil e 814.27 37.012b 14.41

Myristic acid (14:0) 0.13 0.006b 14.13
Palmitic acid (16:0) 57.63 2.619b 2.40
9-Hexadecenoic acid (16:1 n-7) 18.02 0.819b 30.74
Margaric acid (17:0) 1.54 0.070b 8.56
10-Heptadecenoic acid (17:1) 2.17 0.098b 12.04
Stearic acid (18:0) 3.06 0.13b 3.68
Oleic acid (18:1 n-9) 517.99 23.54b 12.66
Linoleic acid LA (18:2 n-6) 677.09 30.77a 1.95
a-Linolenic acid ALA (18:3 n-3) 2.81 0.12b 18.35
Arachidic acid (20:0) 0.10 0.004b 6.03
11,14-Eicosadienoic acid (20:2 n-6) 57.68 2.62b 17.31
Saturated Fatty acids SFA 78.42 3.56 1.32
Mono-Unsaturated Fatty Acids MUFA 482.72 21.94b 12.31
Poly-Unsaturated Fatty Acids PUFA 794.66 36.12a 2.22
Essential Fatty Acids EFA 706.36 32.10a 2.01
Elongation ratio ER 0.0067 0.0003b 12.70
Desaturation ratio DR 0.078 0.003b 12.65
Oleic desaturation ratio ODR 0.081 0.003b 12.82
Linoleic desaturation ratio LDR 0.00054 0.00b 17.66
Mono-Unsaturated Fatty Acids/Saturated Fatty acids M/S 2.30 0.10b 9.50
Poly-Unsaturated Fatty Acids/Saturated Fatty Acids P/S 18.96 0.86a 1.96
(Mono-Unsaturated Fatty Acids Poly-Unsaturated Fatty Acids)/Saturated Fatty acids (M P)/S 21.12 0.96a 1.87
Phenolic Content PC 25,226.55 1146.66b 24.13
Flavonoid Content FC 14 557.14 661.68b 4.09
Antioxidant Content AC 640.96 29.13b 25.24
Scavenging capacity SC 5734.80 260.67b 10.78

SS: Sum of Square MSS: Mean Sum of Square.

a
Signicant at 5% level of Signicance.
b
Signicant at 1% level of Signicance.
c
Values in parenthesis is degree of freedom for treatments.

Table 3
Total oil content and fatty acid composition of Nigella genotypes.

Genotypes Oil (ml/kg) Fatty Acids (ml/l)

14:0 16:0 16:1 n-7 17:0 17:1 n-7 18:0 18:1 n-9 18:2 n-6 18:3 n-3 20:0 20:2 n-6

AN-1 158 (17) 1.4 (0.2) 119 (10) 2.3 (0.2) 3.7 (0.2) 2.4 (0.2) 28.9 (1.2) 125 (18.7) 666.4 (45.6) 8.0 (1.4) 2.36 (0.2) 38.2 (3.3)
AN-2 184 (14) 1.5 (0.2) 112 (14) 2.5 (0.9) 6.6 (0.9) 2.1 (0.2) 37.0 (1.7) 145 (18.1) 637.4 (48) 6.0 (0.9) 2.07 (0.2) 39.7 (2.3)
AN-3 163 (11) 2.1 (0.2) 151 (39) 16.6 (0.2) 2.1 (0.2) 4.4 (0.4) 23.7 (2.4) 106 (18.2) 646.2 (43.2) 5.6 (0.4) 1.64 (0.2) 39.8 (2.6)
AN-4 217 (11) 2.4 (0.1) 129 (14) 15.6 (0.5) 3.7 (0.5) 6.5 (0.6) 26.2 (3.4) 80 (11.7) 696 (39.4) 10.1 (10.8) 1.05 (0.2) 29.8 (2.6))
AN-5 237 (17) 1.4 (0.2) 118 (6.7) 2.3 (0.2) 2.6 (0.2) 5.9 (0.6) 25.0 (2.6) 85 (14.2) 712.4 (41.3) 4.0 (0.4) 0.9 (0.2) 41.2 (1.6)
AN-6 162 (14) 1.2 (0.1) 111 (16) 2.4 (0.3) 2.2 (0.3) 3.7 (0.4) 39.1 (3.7) 84 (7.2) 713.4 (28.6) 4.0 (0.4) 1.97 (0.4) 36.8 (2.4)
AN-7 217 (11) 1.8 (0.2) 136 (13) 3.2 (0.9) 7.9 (0.9) 8.8 (1) 32.0 (2.6) 68 (8.7) 699 (49.1) 3.6 (0.4) 2.21 (0.3) 37 (1.2)
AN-8 245 (11) 1.0 (0.3) 122 (20) 2.4 (0.4) 6.5 (0.4) 5.4 (0.6) 27.2 (2.8) 75 (7) 714 (39.8) 5.0 (0.4) 1.84 (0.3) 39.4 (2.1)
AN-9 219 (22) 0.9 (0.3) 117 (7.8) 2.4 (0.3) 3.2 (0.3) 3.3 (0.4) 26.5 (1.5) 86 (6.2) 704.3 (38.1) 9.0 (1) 2.29 (0.3) 43.8 (2.4)
AN-10 216 (13) 1.8 (0.3) 134 (11) 2.5 (0.4) 2.6 (0.4) 4.5 (0.4) 27.6 (2) 78 (6.9) 704 (46.7) 4.0 (0.9) 2.29 (0.3) 39.1 (2.6)
AN-11 219 (6) 1.7 (0.2) 127 (12) 2.6 (0.3) 3.7 (0.3) 5.9 (0.7) 27.6 (3.2) 98 (8.2) 688.4 (54.9) 3.8 (0.2) 2.30 (0.2) 38.5 (2.6)
AN-13 175 (17) 1.2 (0.2) 122 (10) 4.0 (0.5) 4.6 (0.5) 3.3 (0.3) 28.8 (4.3) 125 (18.3) 668.4 (36.2) 4.0 (0.9) 1.98 (0.2) 35.8 (1.9)
AN-14 195 (28) 0.9 (0.2) 125 (16) 14.1 (0.3) 3.7 (0.3) 4.6 (0.5) 25.5 (3) 105 (7.0) 676.8 (64) 3.8 (0.2) 1.19 (0.2) 38.5 (4)
AN-15 217 (29) 1.1 (0.1) 123 (1.7) 2.4 (0.2) 2.6 (0.2) 6.9 (0.7) 20.2 (0.7) 190 (17.5) 609 (45.5) 2.8 (0.3) 2.38 (0.2) 39.4 (3.1)
AN-16 147 (19) 1.0 (0.2) 122 (11) 2.3 (0.6) 4.3 (0.6) 3.7 (0.3) 26.7 (5.2) 125 (14.7) 667.6 (48.1) 2.9 (0.3) 2.03 (0.3) 42 (2.6)
AN-17 154 (6) 1.1 (0.3) 125 (14) 2.4 (0.4) 2.6 (0.4) 4.5 (0.5) 26.3 (3.8) 128 (4) 664.5 (33.2) 3 (0.2) 1.92 (0.2) 39.4 (2.5)
AN-18 167 (31) 1.2 (0.3) 120 (18) 10.8 (0.7) 4.1 (0.7) 4 (0.2) 26.2 (1.4) 105 (17.6) 684.2 (40) 3.4 (0.4) 2.05 (0.4) 38.3 (2.1)
AN-19 193 (17) 0.3 (0.1) 124 (12) 2.2 (0.4) 4.6 (0.4) 5.2 (0.5) 23.6 (4.1) 98 (10.6) 687.8 (24.4) 8 (0.4) 2.01 (0.3) 43.1 (2.7)
AN-20 223 (17) 0.8 (0.1) 141 (12) 15.9 (0.2) 3.3 (0.2) 2.4 (0.3) 24.9 (2.5) 89 (11.4) 684.6 (35.2) 4 (0.4) 1.62 (0.2) 31.6 (5.8)
AN-21 251 (20) 1.1 (0.2) 121 (18) 2.2 (0.3) 5.8 (0.3) 9.0 (1) 28.4 (4.1) 79 (9.8) 709.4 (45.2) 3.1 (0.3) 2.05 (0.2) 38.4 (3.2)
AN-22 231 (18) 1.8 (0.1) 139 (22) 9.5 (0.5) 4.7 (0.5) 4.1 (0.5) 21.0 (3.6) 126 (14) 648.1 (35.4) 3.4 (0.4) 1.66 (0.3) 39.7 (3)
AN-23 243 (14) 1.0 (0.1) 93 (16) 7.8 (0.6) 3.3 (0.6) 4.6 (0.5) 22.2 (2) 95 (16.1) 684.2 (33.9) 6 (0.6) 2.17 (0.3) 80.9 (8)
AN-24 270 (14) 1.3 (0.2) 125 (20) 2.3 (0.2) 2.5 (0.2) 3.6 (0.4) 29.1 (5.1) 102 (19.5) 686.1 (36.6) 5 (0.5) 2.22 (0.2) 40.3 (2)

Values are reported as means standard deviation (SD) of three replicate analyses (n 3).

high LA in the diet or circulation does not associate with higher
in vivo or ex vivo pro-inammatory responses. Another important
member of PUFA is ALA (n-3 fatty acid), amount of which was
ranging from a minimum of 2.8 ml/l in genotype AN-15 to a
maximum of 10.1 ml/l in AN-4 with an average of 4.9 ml/l. The
maximum level of EFA (LA ALA) was observed in AN-5 (743 ml/l)
and minimum in AN-15 (641 ml/l).
In the present study, linolenic acid (C18:3) was found to be
present in minor quantity as compare to earlier reports. Studies
showed that oleic and linoleic acid are more desirable for cooking
oils as compared to linolenic acid because the later is unstable and
readily oxidized. Linoleic acid is constituent of EFA which is more
202 S.N. Saxena et al. / LWT - Food Science and Technology 78 (2017) 198e207

Table 4
Fatty acid groups, phenol, avonoids, antioxidant activity and scavenging capacity in oil of Nigella genotypes.

Genotypes SFA (ml/l) MUFA (ml/l) PUFA (ml/l) EFA (ml/l) Phenolics (mg GA E/ml) Flavonoids (mg QE/ml) *AC (mg BHT E/ml) **SC (%)

AN-1 158 (8.3) 130 (19.1) 713 (49.4) 703 (11.7) 170 (6.9) 769 (19.9) 9.7 (0.4) 33 (2.7)
AN-2 167 (14.5) 150 (18.4) 683 (47.6) 671 (0.9) 171 (5.2) 780 (20.2) 10.7 (2.0) 31 (4.8)
AN-3 181 (37.7) 127 (19.1) 692 (41.2) 681 (20.8) 212 (5.6) 781 (15.0) 12.7 (1.0) 32 (2.8)
AN-4 162 (17.7) 102 (12.3) 736 (38.2) 731 (11.3) 181 (8.9) 818 (12.4) 14.8 (0.7) 48 (4.0)
AN-5 148 (9.2) 93 (14.9) 758 (40.1) 743 (10.7) 171 (5.1) 778 (18.3) 9.5 (1.5) 42 (4.6)
AN-6 156 (18.8) 90 (7.7) 754 (27.4) 736 (10.1) 166 (6.8) 785 (19.6) 2.4 (1.4) 11 (4.3)
AN-7 180 (16.0) 80 (9.3) 740 (48.2) 735 (17.2) 152 (8.2) 785 (21.8) 10.2 (0.7) 34 (2.1)
AN-8 158 (22.1) 83 (7.5) 758 (39.8) 741 (2.3) 158 (3.9) 783 (13.8) 8.4 (1.1) 28 (3.5)
AN-9 150 (9.0) 92 (6.7) 757 (37.1) 739 (19.8) 193 (8.5) 792 (11.7) 8.3 (0.6) 11 (6.4)
AN-10 168 (12.4) 85 (7.4) 747 (45.6) 739 (23.7) 167 (4.1) 769 (16.3) 4.6 (0.7) 17 (2.1)
AN-11 162 (9.5) 107 (7.8) 731 (53.0) 724 (1.3) 154 (7.6) 762 (20.7) 4.1 (1.0) 18 (7.3)
AN-13 159 (6.1) 132 (18.2) 708 (34.7) 696 (10.0) 190 (5.0) 785 (20.1) 6.6 (1.2) 23 (3.7)
AN-14 156 (12.9) 124 (8.3) 719 (61.0) 723 (44.8) 164 (6.8) 769 (9.2) 3.0 (0.6) 15 (6.0)
AN-15 150 (2.2) 199 (7.6) 651 (44.3) 641 (16.4) 178 (4.4) 770 (23.9) 3.9 (0.8) 17 (8.8)
AN-16 157 (10.9) 131 (15.1) 713 (50.6) 682 (35.8) 171 (3.9) 779 (17.2) 4.8 (1.2) 17 (3.6)
AN-17 157 (12.1) 135 (4.2) 707 (31.0) 689 (25.7) 175 (4.7) 762 (21.6) 6.4 (0.7) 22 (2.0)
AN-18 154 (16.5) 120 (18.1) 726 (38.3) 714 (24.2) 157 (8.7) 739 (8.9) 5.2 (0.8) 21 (6.3)
AN-19 155 (10.8) 105 (11.3) 739 (22.1) 710 (25.3) 152 (8.8) 758 (14.3) 6.5 (1.6) 22 (4.4)
AN-20 172 (9.7) 107 (12.0) 720 (34.9) 708 (36.8) 144 (7.0) 772 (7.8) 10.7 (1.0) 36 (3.2)
AN-21 159 (18.6) 90 (10.6) 751 (42.3 741 (9.0) 129 (3.8) 783 (18.0) 6.9 (1.1) 22 (5.4)
AN-22 168 (17.7) 140 (14.9) 691 (34.3) 664 (40.6) 150 (9.8) 782 (9.0) 7.5 (1.2) 24 (5.5)
AN-23 121 (14.3) 107 (16.8) 771 (32.8) 704 (38.8) 143 (8.4) 785 (17.8) 6.6 (0.4) 27 (7.5)
AN-24 160 (14.7) 108 (20.1) 731 (34.7) 715 (18.3) 131 (9.7) 769 (22.7) 9.7 (1.4) 25 (4.3)

Values are reported as means standard deviation (SD) of three replicate analyses (n 3); SFA: Saturated Fatty Acids; MUFA: Mono Unsaturated Fatty Acids; PUFA: Poly
Unsaturated Fatty Acids; EFA: Essentail Fatty Acids; GA E: Gallic Acid Equivalent; QE E: Quercetin Equivalent; BHT E: Butylated Hydroxy Toluene Equivalent; * Antioxidant
Content; **Scavenging Capacity of oil extract (%).

desirable for human health. Together, n-3 and n-6 fatty acids play
an essential role in brain function as well as normal growth and
development. Furthermore, they are important for reducing
cholesterol and heart disease (Silvers & Scott, 2002).
The results show an agreement with earlier reports by Iqbal
et al. (2014), except for the reduced level of oleic acid and
increased level of linoleic acid. This deviation might be due to a
higher biological conversion capacity of oleic to linoleic acid which
can be explained by higher desaturation ratio (DR) as well as higher
oleic desaturation ratio (ODR) value. Genetic factors, seed maturity,
seed production environment or lipid extraction methods might be
other reason. Unsaturated fatty acids (MUFA PUFA) which
amounted with an average of 841 ml/l are in agreement with those
reported by Iqbal et al. (2014) and Gharby et al. (2015).
Similar to the results obtained by Ramadan and Morsel (2003);
Atta (2003) and Hamrouni-Sellami et al. (2003), fatty acids like
myristic (14:0), palmitoleic (16:1), arachidic (C20:0) ecosadienoic
acid (20:2 n-6) were present as minor fatty acids in the present
study, however, Iqbal et al. (2014) did not observe the presence of
these fatty acids. The source of this variability might be genetic,
seed quality or seed production environment and the efcacy of the
analytical system applied.

Table 5
Various utility ratios of fatty acids for biochemical capacities and nutritive value in oil of Nigella genotypes.

Genotypes M/S P/S M P/S ER DR ODR LDR

AN-1 0.82 (0.08) 4.52 (0.13) 5.34 (0.20) 0.046 (0.002) 0.81 (0.01) 0.84 (0.01) 0.012 (0.001)
AN-2 0.90 (0.15) 4.12 (0.61) 5.03 (0.75) 0.048 (0.006) 0.78 (0.01) 0.82 (0.01) 0.009 (0.001)
AN-3 0.73 (0.25) 3.96 (0.98) 4.70 (1.22) 0.050 (0.007) 0.82 (0.01) 0.86 (0.02) 0.009 (0.001)
AN-4 0.64 (0.14) 4.59 (0.68) 5.23 (0.81) 0.037 (0.005) 0.87 (0.01) 0.90 (0.01) 0.014 (0.001)
AN-5 0.64 (0.14) 5.16 (0.56) 5.79 (0.70) 0.049 (0.005) 0.85 (0.01) 0.89 (0.01) 0.006 (0.001)
AN-6 0.59 (0.13) 4.91 (0.76) 5.50 (0.89) 0.044 (0.004) 0.86 (0.00) 0.90 (0.01) 0.006 (0.001)
AN-7 0.45 (0.09) 4.14 (0.63) 4.59 (0.72) 0.046 (0.005) 0.87 (0.00) 0.91 (0.01) 0.005 (0.001)
AN-8 0.53 (0.12) 4.87 (0.86) 5.40 (0.98) 0.047 (0.004) 0.86 (0.00) 0.91 (0.01) 0.007 (0.001)
AN-9 0.62 (0.08) 5.07 (0.53) 5.69 (0.61) 0.052 (0.005) 0.85 (0.00) 0.89 (0.00) 0.013 (0.001)
AN-10 0.51 (0.08) 4.48 (0.59) 4.99 (0.67) 0.048 (0.005) 0.86 (0.01) 0.90 (0.00) 0.006 (0.001)
AN-11 0.66 (0.06) 4.51 (0.11) 5.17 (0.06) 0.047 (0.006) 0.84 (0.02) 0.88 (0.01) 0.006 (0.001
AN-13 0.83 (0.10) 4.46 (0.12) 5.29 (0.06) 0.043 (0.004) 0.81 (0.02) 0.84 (0.02) 0.006 (0.001)
AN-14 0.80 (0.08) 4.62 (0.45) 5.41 (0.53) 0.047 (0.008) 0.83 (0.01) 0.87 (0.00) 0.006 (0.001)
AN-15 1.33 (0.12) 4.36 (0.35) 5.69 (0.41) 0.047 (0.006) 0.73 (0.02) 0.76 (0.02) 0.005 (0.001)
AN-16 0.84 (0.09) 4.55 (0.06) 5.39 (0.04) 0.050 (0.003) 0.80 (0.01) 0.84 (0.02) 0.004 (0.001)
AN-17 0.86 (0.07) 4.51 (0.27) 5.37 (0.34) 0.047 (0.005) 0.80 (0.01) 0.84 (0.01) 0.004 (0.001)
AN-18 0.78 (0.03) 4.74 (0.26) 5.51 (0.23) 0.046 (0.005) 0.83 (0.01) 0.87 (0.01) 0.005 (0.001)
AN-19 0.68 (0.03) 4.79 (0.22) 5.47 (0.21) 0.052 (0.005) 0.83 (0.00) 0.88 (0.01) 0.011 (0.001)
AN-20 0.62 (0.03) 4.20 (0.16) 4.82 (0.15) 0.039 (0.008) 0.85 (0.01) 0.89 (0.01) 0.006 (0.001)
AN-21 0.57 (0.06) 4.76 (0.33) 5.33 (0.38) 0.047 (0.007) 0.86 (0.01) 0.90 (0.01) 0.004 (0.001)
AN-22 0.83 (0.04) 4.16 (0.52) 4.99 (0.54) 0.049 (0.005) 0.80 (0.01) 0.84 (0.01) 0.005 (0.001)
AN-23 0.88 (0.04) 6.41 (0.62) 7.29 (0.58) 0.094 (0.012) 0.80 (0.00) 0.88 (0.02) 0.009 (0.001)
AN-24 0.67 (0.07) 4.59 (0.23) 5.26 (0.19) 0.049 (0.005) 0.830 (0.01) 0.87 (0.02) 0.007 (0.001)

Values are reported as means SD of three replicate analyses (n 3); M: Mono Unsaturated Fatty Acids; P: Poly Unsaturated Fatty Acids; S: Saturated Fatty Acids; ER:
Elongation Ratio; DR: Desaturation Ratio; ODR: Oleic Desaturation Ratio & LDR: Linoleic Desaturation Ratio.
 S.N. Saxena et al. / LWT - Food Science and Technology 78 (2017) 198e207 203

Table 6
Coefcient of correlation among oil, FAME and their group of fatty acids in genotypes of Nigella sativa L. germplasm.

Variables Oil (%) (14:0) (16:0) (16:1 n-7) (17:0) (17:1 n-7) (18:0) (18:1 n-9) (18:2 n-6) (18:3 n-3) (20:0) (20:2 n-6) SFA MUFA PUFA

Oil (%) 1 0.024 0.079 0.063 0.141 0.407 0.249 0.352 0.339 0.028 0.063 0.212 0.160 0.345 0.386
(14:0) 1 0.408 0.284 0.025 0.220 0.108 0.118 0.072 0.113 0.206 0.243 0.457a 0.052 0.137
(16:0) 1 0.433a 0.041 0.074 0.172 0.021 0.232 0.142 0.218 0.650a 0.899a 0.067 0.437a
(16:1 n-7) 1 0.195 0.121 0.354 0.117 0.150 0.143 0.586a 0.101 0.243 0.065 0.162
(17:0) 1 0.325 0.285 0.152 0.092 0.038 0.123 0.099 0.276 0.171 0.051
(17:1 n-7) 1 0.164 0.270 0.245 0.199 0.075 0.040 0.048 0.233 0.201
(18:0) 1 0.218 0.293 0.009 0.194 0.274 0.256 0.302 0.185
(18:1 n-9) 1 0.921a 0.215 0.248 0.003 0.111 0.981a 0.874a
(18:2 n-6) 1 0.156 0.169 0.003 0.113 0.954a 0.941
(18:3 n-3) 1 0.087 0.086 0.125 0.206 0.245
(20:0) 1 0.239 0.086 0.135 0.086
(20:2 n-6) 1 0.735a 0.026 0.327
SFA 1 0.064 0.353
MUFA 1 0.911a
PUFA 1

SFA: Saturated Fatty Acids; MUFA: Mono Unsaturated Fatty Acids; PUFA: Poly Unsaturated Fatty Acids.
a
Signicant at 0.01 and 0.05% probability.

3.2. Phenol, avonoid contents and antioxidant activity minimum of these ratio were noted in AN-3 (3.96) and AN-7 (4.59)
respectively (Table 5). The higher ratios of P/S and (M P)/S in AN-
All the genotypes showed appreciable amount of phenolic and 23 might be due to lower SFA and higher MUFA and PUFA content.
avonoid content in Nigella seed oil (Table 4). Total phenolic con- In a study Chang and Huang (1998) found that a low M/S ratio and
tent ranged from a minimum of 129 mg GAE/ml (AN-21) to a high P/M and (P M)/S ratios, not exceeding 2 are prerequisites for
maximum of 212 mg GAE/ml (AN-3) with an average of 164 mg GAE/ keeping plasma and liver cholesterol low. However, plasma total
ml while avanoid content was observed minimum in AN-18 cholesterol, and low density lipoprotein (LDL)eC concentrations
(739 mg QE/ml) to maximum of 818 mg QE/ml in genotype AN-4. were signicantly higher in rats fed with the (P M)/S ratio of 5.7
However, total antioxidant content was maximum in AN-4 compared with rats fed with the (P M)/S ratio of 1.5 or 2.0 (Chang,
(14.8 mg BHT E/ml) followed by AN-3 (12.7 mg BHT E/ml) and Wu, Chen, & Huang, 2004). Oils and fats with higher value of P/S
minimum in AN-6 (2.4 mg BHT E/ml) with an average of 7.5 mg BHT index more than 1 are considered as good nutritional value. Several
E/ml. Free radical scavenging capacity was the maximum in geno-
type AN-4 (48%) while the minimum capacity was seen in AN-6
Table 7
(11%). Results indicated that Nigella seed oil have considerable
Principal Components for oil, FAME prole and their characteristics in genotypes of
antioxidant activity. Presence of phenolic and avonoid contents Nigella.
positively contributes to antioxidant activity. Thymoquinone,
Variables PC1 PC2 PC3 PC4 PC5
carvacrol, anethole and 4-terpineol demonstrated adequate radical
scavenging capacity and all are present in Nigella essential and xed Oil (%) 0.424 0.188 0.008 0.579 0.362
oil (Kruk et al., 2000; Badary et al., 2003). (14:0) 0.060 0.505 0.355 0.171 0.166
(16:0) 0.145 0.874 0.007 0.299 0.101
(16:1 n-7) 0.036 0.312 0.486 0.417 0.449
3.3. Fatty acid ratio (17:0) 0.184 0.196 0.234 0.016 0.693
(17:1 n-7) 0.293 0.056 0.206 0.570 0.378
(18:0) 0.282 0.234 0.272 0.719 0.204
To describe the biochemical capacities of metabolic pathway in (18:1 n-9) 0.974 0.110 0.008 0.067 0.103
Nigella oil for fatty acids inter-conversion (Chain lengthing and (18:2 n-6) 0.963 0.070 0.140 0.073 0.125
desaturation), various FA ratios have been calculated. The ER and (18:3 n-3) 0.265 0.038 0.807 0.318 0.190
DR for the different genotypes are shown in Table 5. Genotypes AN- (20:0) 0.208 0.275 0.378 0.347 0.452
(20:2 n-6) 0.042 0.866 0.166 0.121 0.087
4 showed minimum ER (0.037) while maximum (0.094) was SFA 0.019 0.954 0.119 0.002 0.093
observed in AN-23. The lower ER estimates the relative weight of MUFA 0.983 0.049 0.072 0.050 0.044
the elongation pathway from oleic acid to longer chain fatty acids. PUFA 0.929 0.348 0.019 0.052 0.074
Value of DR was, however, maximum in AN-7 (0.87) and minimum EFA 0.943 0.018 0.096 0.074 0.102
ER 0.040 0.836 0.173 0.145 0.088
in AN-15 (0.73) with an average of 0.83. DR is an estimate of relative
DR 0.937 0.310 0.049 0.007 0.120
weight of the desaturation pathway from oleic acid to PUFA i.e. LA ODR 0.982 0.088 0.003 0.048 0.100
and ALA which are more desirable for n-3 and n-6 content. The LDR 0.189 0.028 0.823 0.325 0.204
maximum ODR value (0.91) was observed in AN-7 genotype and M/S 0.913 0.331 0.111 0.072 0.027
the minimum in AN-15 (0.76). Similarly, maximum LDR value was P/S 0.394 0.893 0.155 0.021 0.087
M P/S 0.050 0.963 0.187 0.046 0.072
observed in AN-4 (0.014) and minimum value noted in AN-21 Phenolics 0.332 0.266 0.503 0.314 0.249
(0.004). ODR and LDR is the indicator of the efciency of desatu- Flavonoids 0.304 0.150 0.590 0.043 0.329
ration systems from C18:1 to C18:2, and from C18:2 to C18:3, Antioxidant Content 0.190 0.436 0.658 0.066 0.276
respectively. Scavenging capacity (%) 0.179 0.083 0.189 0.317 0.133
Eigenvalue 8.258 6.054 3.254 2.068 1.688
To describe the potential of Nigella oil for human consumption
Variability (%) 30.584 22.423 12.051 7.661 6.250
various other important fatty acids ratios viz. MUFA/SFA, PUFA/SFA Cumulative % 30.584 53.006 65.057 72.718 78.968
and MUFA PUFA/SFA (M/S, P/S) and (M P)/S have been calcu-
SFA (S): Saturated Fatty Acids; MUFA (M): Mono Unsaturated Fatty Acids; PUFA (P):
lated and presented in Table 5, M/S ratio noted minimum in AN-7 Poly Unsaturated Fatty Acids; EFA: Essentail Fatty Acids; ER: Elongation Ratio; DR:
(0.45) and maximum in AN-15 (1.33). P/S and (M P)/S ratios Desaturation Ratio; ODR: Oleic Desaturation Ratio & LDR: Linoleic Desaturation
were found maximum in AN-23 (6.41 and 7.29, respectively), while Ratio.
204 S.N. Saxena et al. / LWT - Food Science and Technology 78 (2017) 198e207

Observations (axes PC1 and PC2: 53.01 %)

10
9 AN-23
8
7
6
5
PC2 (22.42 %)

4
3
2 AN-9 AN-15
AN-19
1 AN-5 AN-6 AN-18 AN-16
AN-8 AN-21 AN-24 AN-14 AN-17
0
AN-11 AN-1AN-13
-1 AN-2
AN-10 AN-22
-2
AN-4
-3 AN-20
AN-7 AN-3
-4
-8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9
PC1 (30.58 %)
Fig. 1. Principal Component Analysis (PCA) plot of Nigella genotypes based on oil, fatty acids and their characteristics.

studies indicated that higher value of P/S index means a smaller
deposition of lipids in the body (Lawton, Delargry, Brockman,
Simith, & Blundell, 2000). The diets high in a polyunsaturated-to-
saturated fatty acid (P/S) ratio ameliorated insulin action in strep-
tozotocin induced diabetic rats fed a high-fat diet, but a high P/S
ratio diet enhances oxidative stress because PUFA are highly sus-
ceptible to lipid per-oxidation (Kang, Shin, Park, & Lee, 2005).
3.4. Correlation analysis
Coefcient of correlation among oil, FAME, SFA, MUFA and PUFA
in all genotypes was nd out to analyze the interrelationship be-
tween different fatty acids and presented in Table 6. Oil content
showed positive association with 10-heptadecenoic acid (17:1 n-7)
and linoleic acid and negative association with oleic acid. Myristic
acid was positively associated with palmitic acid and SFA. Palmitic
acid showed signicant positive correlation with monounsaturated
fatty acid, 9-Hexadecenoic acid (16:1 n-7) and SFA, while signi-
cant negative correlation was observed with 11,14-Eicosadienoic
acid (20:2 n-6) and PUFA. 9-Hexadecenoic acid is negatively
correlated with stearic acid (18:0) and archedic acid (20:0), the
latter was statistically signicant. Similarly, stearic acid was nega-
tively correlated with MUFA. Oleic acid (18:1) showed signicant
negative correlation with linoleic acid (18:2) and PUFA, while sig-
nicant positive correlation was observed with MUFA. Linoleic acid
exhibited positive correlation with PUFA and signicant negative
correlation with MUFA at higher level of signicance. MUFA and
PUFA were negatively correlated to each other.
3.5. Principal component analysis (PCA)
The component traits are expected to be related to the seed
yield, i.e. are expected to be highly correlated either positively or
negatively (Rymuza, Turska, Wielogorska, & Bombik, 2012).
Principal component analysis helps researchers to distinguish sig-
nicant relationship among traits. It helps reduce the number of
variables into related components called principal components.
The cluster analysis is also an appropriate method for determining
family relationships but the main advantage of using PCA over
cluster analysis is that each genotype can be assigned to one group
only. PCA reects the importance of the largest contributor to the
total variation at each axis of differentiation (Sharma, 1998, pp.
1e432). PCA was conducted for oil, FAME prole and their char-
acteristics in Nigella genotypes for selection of contributor to the
total variation. As can be seen from Table 7 and Fig. 1, rst ve
components with Eigenvalues >1 contributed 78.96% of variance
for oil, FAME and their characteristics. A variation of 30.58% was
observed and the traits that contributed positively to PC 1 were,
MUFA, oleic acid (18:1 n-9), M/S ratio, and phenol content, whereas
negative contribution was observed for PUFA, EFA, linoleic acid
(18:2 n-6), DR and ODR. For PC 2, the variability of 22.42% was
observed and (M P)/S, P/S and ER contributed more positively,
while SFA and palmitic acid (16:0) contributes negatively. For PC 3,
12.05% variability was observed and linolenic acid (18:3 n-3), LDR,
avonoids, antioxidant activity (AO) were positive contributions to
PC 3. Stearic acid (18:0) was negatively contributed to this
component. PC 4 and PC 5 showed 7.66% and 6.25% variability,
respectively. According to PC 4, stearic acid, linolenic acid, archidic
acid and SC contributes positively while oil content, 16:1 n-7 con-
tributes negatively. The variable's correlation circle of PCA depicted
the projection of studied variables in factors space (Fig. 2). The
studied twenty seven variables groups over four plains showing
negative and positive factor space in 2D. MUFA, 20:0, 18:1 n-9, M/S
laid on positive plane of the rst and second component axis
(Factor 1 & 2) showing strong positive association. Whereas, rest 22
variables showed negative association with one or the other
parameter by occurring in the negative plane of both the compo-
nent axis.
 S.N. Saxena et al. / LWT - Food Science and Technology 78 (2017) 198e207 205

Variables (axes F1 and F2: 53.01 %)

1
M+P/S
P/S (20:2 n-6)
0.75 ER

0.5
PUFA M/S
(20:0)
0.25 Oil (%)
F2 (22.42 %)

(18:2 n-6) (18:1 n-9)

(18:3 n-3) LDR
0 MUFA
EFA (17:1 n-7)
ODR SC
Flavonoids
-0.25 (17:0)
(18:0) (16:1 n-7) Phenol
DR
AO
-0.5
(14:0)

-0.75
(16:0)
SFA
-1
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
F1 (30.58 %)
Fig. 2. Principal Component Analysis eVariable's correlation circle. Components 1 and 2 accounted for 30.58% and 22.42% of the total variation, respectively.

3.6. Cluster analysis

Clusters analysis was done on the basis of unweighted pair Dendrogram

group method with arithmetic mean (UPGMA) (Fig. 3). All studied
genotypes were divided on 50% Euclidean distances into 8 clusters 12
for total oil, FAME and their characteristics i.e. SFA, MUFA, PUFA,
EFA, ER, DR, ODR, LDR, M/S, P/S, (M P)/S, PC, FC, SC. Cluster-I
consisted of 9 genotypes (AN-1, AN-2, AN-13, AN-14, AN-16, AN- 10
17, AN-18, AN-20 and AN-22), whereas cluster-II, III, V, VI, VII and
VIII consisted of one genotype each, AN-3, AN-4, AN-6, AN-7, AN-15
8
and AN-23, respectively. Cluster-IV consisted eight genotypes (AN-
Dissimilarity

5, AN-8, AN-9, AN-10, AN-11, AN-19, AN-21 and AN-24). Genotypes

present in cluster-I exhibited low variability for total oil, FAME and 6
their characteristics and posses moderate values for all variables.
Genotypes in cluster-II exhibited highest SFA, 16:0, 16:1, PC and
higher antioxidant activity with lowest P/S ratio. These all charac- 4
ters are interrelated with each other. Palmitic acid is the major
constituent of SFA group, contributed to SFA value. Lowest P/S ratio
2
is mainly due to higher SFA content. Higher antioxidant activity and
free radical scavenging capacity mainly contributed by phenolic
content present in oil. Genotypes present in cluster-III exhibited 0
AN-23
AN-15

AN-20

AN-22
AN-13
AN-16
AN-17
AN-14
AN-18

AN-19

AN-21
AN-24
AN-10
AN-11

higher 14:0, 18:3 n-3, and 20:2 n-6, FC, AO, SC and LDR. Higher
AN-7

AN-1
AN-2

AN-6
AN-9

AN-5
AN-8

AN-3
AN-4

amount of 18:3 n-3 is directly linked with highest LDR value, while
higher AC and SC might be due to contribution of FC. Members of
cluster-IV provides higher oil content, EFA and higher PUFA, 18:2 n- Fig. 3. Dendrogram of 23 Nigella genotypes based on oil, fatty acids and their
characteristics.
206 S.N. Saxena et al. / LWT - Food Science and Technology 78 (2017) 198e207
6, DR and ODR with lowest FC and lower AC and SC. Highest EFA is
contributed by higher level of 18:2 n-6 and PUFA while higher
amount of 18:2 n-6 might be due to higher DR and ODR value,
higher value of both ratios is responsible for conversion of oleic to
linoleic acid (18:2 n-6) and desaturation in fatty acids. Lower AC
and SC might be due to lower value of FC. A signicant positive
correlation is also exhibited between oil content and linoleic acid
(Table-6).
Cluster-V consisted with one genotype exhibited by highest 18:0
and lowest AO and SC. Lowest value of AO and SC might be due to
lower side FC value which is responsible for both attributes.
Cluster-VI with one genotype, categorized with Highest 17:0, DR
and ODR value with higher side SFA and 18:2 n-6 with lowest
MUFA, 18:1 n-9, M/S, (M P)/S. Lowest MUFA is due to lower
amount of its main contributor FA 18:1 n-9. Minimum value of M/S,
(M P)/S might be contributed by a higher side SFA and a higher
side value of 18:2 n-6 and lowest 18:1 n-9 might be due to highest
values of DR and ODR. A higher ODR is responsible for conversion of
oleic to linoleic system.
Cluster-VII consists of one genotype have highest 18:1 n-9, 20:0,
MUFA and M/S with lowest values of 18:0, 18:2, 18:3 n-3, PUFA, EFA,
DR, ODR and lower side LDR value. Lowest DR and ODR values
might be directly affect the conversion of 18:1n-9 to 18:2 n-6 and
lower side LDR is responsible for low conversion of 18:2 n-6 to 18:3
n-6. Minimum value of PUFA might be due to minimum contribu-
tion of 18:2 n-6, which is main contributor to PUFA. A low value of
18:2 n-6 is due to lowest EFA. Highest M/S is due to higher content
of 18:1 n-9 which is main contributor of MUFA.
Cluster-VIII consist of only one genotype with Highest PUFA,
20:2 n-6, P/S, (M P)/S, and ER with higher side value of 18:2 n-6
and oil content. SFA and 16:0 exhibited lowest values. A maximum
amount of 20:2 n-6 might be due to a highest ER value which is
responsible for chain elongation of FA. A higher P/S, (M P)/S
mainly contributed by higher value of PUFA and lowest value of
SFA.
4. Conclusion
Genetic variability in Nigella may be utilized in trait specic
breeding programme. Analysis revealed that genotype AN-23
(cluster-VIII) is most suitable to be used in breeding programme
for edible oil due to its unique and desirable fatty acid combinations
i.e. higher oil content, PUFA, linoleic acid, 11, 14-Eicosadienoic acid,
P/S, (M P)/S and ER with lower contents i.e. saturated; palmitic
and stearic acids. Cluster analysis revealed a group of genotypes
with desirable characters viz., higher oil content, PUFA, and EFA,
18:2 n-6, DR and ODR. These genotypes are suitable for food oil and
industrial purpose and advance breeding programme. Genotypes
AN-4 (cluster-III) has a combination of higher PUFA, linolenic,
linoleic acid, avonoid, higher antioxidant contents and LDR as well
as good amount of FC, AC, SC. This combination may be utilized for
enhancing storage period of oil. The results suggested that a diverse
collection of genotypes may be used for novel applications in edible
oil industries. Nigella seed oil should be considered as one among
newer sources of edible oils that can be important in human
nutrition and health. This underutilized crop has strong potential
for edible oil and herbal industry.
Acknowledgment
Authors are highly thankful to ICAR-National Research Centre on
Seed Spices, Ajmer for providing support to carry out this study.
References
Akram Khan, M. (1999). Chemical composition and medicinal properties of Nigella
sativa L. Inammopharmacology, 7, 15e35.
Al-Kayssi, A. W., Shihab, R. M., & Mustafa, S. H. (2011). Impact of soil water stress on
nigellone oil content of black cumin seeds grown in calcareous-gypsiferous
soils. Agriculture Water Management, 100, 46e57.
Amin, I., Norazaidah, Y., & Hainida, K. I. E. (2006). Antioxidant activity and phenolic
content of raw and blanched Amaranthus species. Food Chemistry, 94, 47e52.
Atta, M. B. (2003). Some characteristics of nigella (Nigella sativa L.) seed cultivated
in Egypt and its lipid prole. Food Chemistry, 83, 63e68.
Badary, A., Taha, R. A., Ayman, M., El-Din, G., & Abdel-Wahab, M. H. (2003). Thy-
moquinone is a potent superoxide anion scavenger. Drug Chemical Toxicology,
26(2), 87e98.
Badr, N., Arzoo, S., & Bakeet, Z. A. N. (2014). Characteristics and fatty acid compo-
sition of commonly consumed cooking oil marketed locally in Riyadh City. In-
ternational Journal of Biosciences, 4(9), 227e238.
Bourgou, S., Bettaieb, I., Saidani, M., & Marzouk, B. (2010). Fatty acids, essential oil
and phenolics modication of black cumin fruit under NaCl stress conditions.
Journal of Agricultural and Food Chemistry, 58, 12399e12406.
Chang, N. W., & Huang, P. C. (1998). Effects of the ratio of polyunsaturated and
monounsaturated fatty acid to saturated fatty acid on rat plasma and liver lipid
concentrations. Lipids, 33, 481e487.
Chang, N. W., Wu, C. T., Chen, F. N., & Huang, P. (2004). High polyunsaturated and
monounsaturated fatty acid to saturated fatty acid ratio increases plasma very
low density lipoprotein lipids and reduces the hepatic hypertriglyceridemic
effect of dietary cholesterol in rats. Nutrition Research, 24, 73e83.
Chang, C., Yang, M., Wen, H., & Chern, J. (2002). Estimation of total avonoid content
in propolis by two complementary colorimetric methods. Journal of Food and
Drug Analysis, 10, 178e182.
Cheikh-Rouhou, S., Besbes, S., Hentati, B., Blecker, C., Deroanne, C., Hamadi, Attia,
et al. (2007). Nigella sativa L. Chemical composition and phytochemical char-
acteristics of lipid fraction. Food Chemistry, 101, 673e681.
Denny, E. C. C., Andre, G. V. C., Maria Do, C. G. P., Sergio Matta, D. S., Marco, T. C. S.,
Neuza, M. B. C., et al. (2006). Lipid prole of rats fed high-fat diets based on
axseed, peanut, trout or chicken skin. Nutrition, 22, 197e205.
Dubey, P. N., Singh, B., Mishra, B. K., Kant, K., & Solanki, R. K. (2016). Nigella (Nigella
sativa L.): A high value seed spice with immense medicinal potential. Indian
Journal of Agricultural Sciences, 86(8), 967e979.
Edris, A. E. (2007). Pharmaceutical and therapeutic potentials of essential oils and
their individual volatile constituents: A review. Phytotherapy Research, 21(4),
308e323.
Fararh, K. M., Atoji, Y., Shimizu, Y., & Takewaki, T. (2008). Insulinotropic properties of
Nigella sativa oil in streptozotocin plus nicotinamide diabetic hamster. Research
in Veterinary Science, 73, 279e282.
Fritsche, K. L. (2008). Too much linoleic acid promotes inammationddoesnt it?
Prostaglandins, Leukotrienes and Essential Fatty Acids, 79, 173e175.
Gharby, S., Harhar, H., Guillaume, D., Roudani, A., Boulbaroud, S., Ibrahimi, M., et al.
(2015). Chemical investigation of Nigella sativa L. seed oil produced in Morocco.
Journal of the Saudi Society of Agricultural Sciences, 14, 172e177.
Gomez, C. C., Bermejo, L. M., & Kohen, V. (2011). Importance of a balanced omega 6/
omega 3 ratio for the maintenance of health: Nutritional recommendations.
Nutricion Hospitalaria, 26, 323e329.
Hamrouni-Sellami, I., Kchouk, M. E., & Marzouk, B. (2003). Lipid and aroma
composition of black cumin (Nigella sativa L.) seeds from Tunisia. Journal of Food
Biochemistry, 32, 335e352.
Iqbal, M. S., Ghafoor, A., Ullah, I., & Ahmad, H. (2014). Quantication and compo-
sitional diversity of fatty acid methyl esters prole in Nigella sativa L. germ-
plasms. Journal of the American Oil Chemists Society, 91, 1975e1986.
Iqbal, M. S., Nadeem, S., Mehmboob, S., Ghafoor, A., Rajoka, M. I., Qureshi, A. S., et al.
(2011). Exploration of genotype specic ngerprinting of Nigella sativa L. using
RAPD markers. Turkish Journal of Agriculture and Forestry, 35(6), 569e578.
Jerry, H. L. (2001). Number cruncher statistical systems (ncss). Kaysville, Utah: Sta-
tistical Software. www.ncss.com.
Kang, M. J., Shin, M. S., Park, J. N., & Lee, S. S. (2005). The effects of polyunsaturated:
Saturated fatty acids ratios and peroxidisability index values of dietary fats on
serum lipid proles and hepatic enzyme activities in rats. British Journal of
Nutrition, 94, 526e532.
Kanter, M., Meral, I., Dede, S., Gunduz, H., Cemek, M., Ozbek, H., et al. (2003). Effect
of Nigella sativa L. and Urtica dioica L., on lipid peroxidation, antioxidant
enzyme systems and some liver enzymes in CC14-treated rats. Journal of Vet-
erinary Medicine, 50, 264e268.
Khan, N., Sharma, S., & Sultana, S. (2009). Nigella sativa (black cumin) ameliorates
potassium bromate-induced early events of carcinogenesis diminution of
oxidative stress. Human and Experimental Toxicology, 22, 193e203.
Kruk, I., Michalska, T., & Klanda, A. (2000). The effect of thymol and its derivatives
on reaction generating reactive oxygen species. Chemosphere, 41, 1059e1064.
Lawton, C. L., Delargry, H. J., Brockman, J., Simith, R. C., & Blundell, J. E. (2000). The
degree of saturation of fatty acids of fatty acids inuences in post ingestive
satiety. British Journal of Nutrition, 83(5), 473e482.
Mashhadian, N. V., & Rakhshandeh, H. (2005). Antibacterial and antifungal effects of
Nigella sativa extracts against S. aureus, P. aeroginosa and C. albicans. Pakistan
Journal of Medical Sciences, 21(1), 47e52.
Matthaus, B., & Ozcan, M. M. (2011). Fatty acids, tocopherol, and sterol content of
 S.N. Saxena et al. / LWT - Food Science and Technology 78 (2017) 198e207
some Nigella species seed oil. Czechoslovakia Journal of Food Sciences, 29,
145e150.
Padhye, S., Banerjee, S., Ahmad, A., Mohammad, R., & Sarkar, F. H. (2008). From here
to eternity-the secret of pharaohs: Therapeutic potential of black cumin seeds
and beyond. Cancer Therapy, 6, 495e510.
Panse, V. C., & Sukhatme, P. V. (1978). In ICAR (Ed.), Statistical methods for agricul-
tural workers.III rev. New Delhi krishikosh.egranth.ac.in/bitstream/1/2049376/1/
44688.pdf.
Pleines, S., & Friedt, W. (1988). Breeding for improved C18 fatty acid composition in
rapeseed (Brassica napus L.). European Journal of Lipid Science and Technology,
90, 167e171.
Ramadan, M. F., & Morsel, J. T. (2003). Analysis of glycolipids from black cumin
(Nigella sativa L.), coriander (Coriandrum sativum L.) and Niger (Guizotia abys-
sinica Cass.) oil seeds. Food Chemistry, 80, 197e204.
Rymuza, K., Turska, E., Wielogorska, G., & Bombik, A. (2012). Use of principal
component analysis for the assessment of spring wheat characteristics. Acta
207
Scientiarum Polonorum-Agricultura, 11, 79e90.
Sharma, J. R. (1998). Statistical and biometrical techniques in plant breeding. Dar-
yaganj, New Delhi: New Age International (P) Ltd, Publishers, 1-432.
Shimada, K., Fujikawa, K., Yahara, K., & Nakamura, T. (1992). Antioxidative proper-
ties of xanthin on autoxidation of soybean oil in cyclodextrin emulsion. Journal
of Agricultural and Food Chemistry, 40, 945e948.
Silvers, K. M., & Scott, K. M. (2002). Fish consumption and self reported physical and
mental health status. Public Health Nutrition, 5, 427e443.
Velasco, L., & Goffman, D. (2000). Tochopherol, plastochromanol and fatty acid
patterns in genus Linum. Plant Systematics and Evolution, 221, 77e88.
Velasco, L., Goffman, F. D., & Becker, H. C. (1998). Variability for the fatty acid
composition of the seed oil in germplasm collection of the genus Brassica.
Genetic Resources and Crop Evolution, 45, 371e382.
Wardlaw, G. M. (2003). Contemporary nutrition (5th ed.). New York: Mc Graw Hill.
Higher Education.