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Evaluation of the Volatile Oils from Different Local Cultivars of Nigella sativa L.
Grown in Egypt with Emphasis on the Effect of Extraction Method on
Thymoquinone

Article in Journal of Essential Oil Bearing Plants · March 2013

DOI: 10.1080/0972060X.2010.10643805

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 Jeobp 13 (2) 2010 pp 154 - 164 154

ISSN 0972-060X

Evaluation of the Volatile Oils from Different Local Cultivars

of Nigella sativa L. Grown in Egypt with Emphasis on the
Effect of Extraction Method on Thymoquinone

Amr E. Edris
Aroma and Flavor Chemistry Department, National Research Center, Cairo, Egypt
Received 11 July 2009; accepted in revised form 19 October 2009

Abstract: The seeds and the total oil (fixed plus volatile) of Nigella sativa L (Black cumin)
have well known flavoring, neutraceutical and pharmaceutical characteristics. These properties are
attributed mainly to the content of the volatile oil fraction. The aim of this study was to investigate
the content and the chemical composition of N. sativa volatile oil from seeds grown organically in
the major traditional growing locations of that herb in Middle Egypt and a new location in the Upper
Egypt. In addition, the investigation also aimed to verify the effect of the extraction method on the
volatile oil composition with respect to thymoquinone, which is the major constituent and the
reportedly most potent biologically active component of the volatile oil. Results indicated that the
volatile oil content in the seeds varied greatly among the different growing locations from 0.01 % w/
w to 0.4 % w/w of the whole seed. These percentages are equivalent to 0.11 % w/w to 1.8 % w/w of
the corresponding total oil (fixed plus volatile). GC-FID and GC-MS analysis revealed that the
chemical composition of the volatile oils from the different growing locations varied only
quantitatively. The major differences found by GC analysis were for the levels of thymoquinone
(TQ) (30.2 % - 63.3 %) and γ-terpinene (0.4 % - 11.8 %). GC analysis also confirmed that extraction
of the volatile oil directly from the seeds in one step by hydro-distillation led to a dramatic decrease
in the amount of TQ to 2.5 %. This investigation highlights the importance of both the growing
location and the extraction method on the chemical characteristics of N. sativa volatile oil especially
TQ.

Key words: Nigella sativa (Black Cumin), total oil, volatile oil, chemical
composition, extraction method, thymoquinone, γ-terpinene, neutraceutic, food additives,
daily supplements.

Introduction: Nigella sativa L. (Black cumin, Ranunculaceae) is a herbaceous plant

that grows well in Mediterranean countries. The seeds can be added to traditional dishes,
soups and bakery products to impart a characteristic flavor which is described commonly

*Corresponding author (Amr E. Edris)

E- mail: < amr_edris@[Link] >
 Amr E. Edris / Jeobp 13 (2) 2010 pp 154 - 164 155

as peppery, slightly bitter and smoky. The total oil obtained from the seeds, usually by cold
expression, is used as a daily food supplement. This oil is a mixture of the seeds’ fixed oil
and volatile oil. The latter is alleged to be responsible for Nigella’s biological activities
including antitumor 1,2, antioxidant 3,4, anti-inflammatory 5 and immune stimulatory
properties 6. These activities are attributed mainly 7-10 to thymoquinone (TQ) which is usually
the major constituent of the volatile oil. The chemical composition of the volatile oil has
therefore gained much attention by many investigators using different methods of extraction
and analysis 4, 11-14. The composition of the volatile oil varied greatly depending on the
Nigella Species 14-17, fertilization treatment 18,19 and the geographical origin of the herb4.
Egypt is considered to be one of the main producers of N. sativa seeds. The bulk
crop comes from the traditional growing areas located in Middle Egypt (South of Cairo).
Recently, new areas located in Upper Egypt (further south of the traditional locations)
have been evaluated for growing this herb. The chemical composition of the Egyptian
volatile oils of N. sativa has been investigated before 19-21. However, the seeds in these
investigations were collected from the local market or collected from limited controlled
studies on plants grown in experimental stations under specific conditions.
Thus the current work is aimed at characterizing the volatile oils from seeds grown
on a much wider scale from the bulk-producing traditional and new locations. This
investigation also aimed to verify the effect of the extraction method on the levels of TQ in
the volatile oil.

Experimental
Plant material: Local cultivars of Nigella sativa L. were planted in October in two
different traditional locations in Middle Egypt namely Menia and Asiout which abbreviated
in the text as TL1 and TL2, respectively. From these locations, the bulk crop of N. sativa is
traditionally grown. Seeds were also planted in a third new location, in Upper Egypt namely
Kena which abbreviated in the test as NL. The geographical and climatic details of these
locations and also the agricultural practice used for growing N. sativa are summarized in
(Table 1). The seeds were harvested in May and samples from each location were collected,
packed, labeled and sent for analysis. Samples were authenticated by Prof. A. Shalaby at
the Medicinal and Aromatic Plant Department, National Research Center. A voucher
specimen was deposited at the herbarium of the National Research Center, Cairo, Egypt
(Registration No. 13531).

Chemicals: Furfuryl pentanoate (97 %), n-hexane and hydrocarbon kit C6-C20 were
all obtained from Sigma-Aldrich Chemical Company Inc. (WI, USA). Diethyl ether (99
%) was obtained from (Fluka Chemical Inc GmbH-9471 Buchs). All chemicals were used
without further purification.

Extraction of the volatile oil

One-step extraction method: Batches of one hundred grams of seeds were ground
into a fine powder using an electric grinder. The powder was mixed with 500 ml distilled
water and hydro-distilled for 2-hour using a Clevenger-type apparatus to extract the volatile
 Amr E. Edris / Jeobp 13 (2) 2010 pp 154 - 164 156

oil. Before the beginning of distillation, 1 ml of n-hexane was added on the surface of
water in the side arm of the Clevenger apparatus to collect the condensed droplets of the
volatile oil, thus decrease its partition with water to increase the yield. At the end of the
distillation process, the hexane layer (including the volatile oil) was collected from the
side arm, dried and evaporated under a slow stream of nitrogen to remove the hexane and
yield the pure volatile oil. The latter was weighed then analyzed by GC-FID and GC-MS.
Because of our previous knowledge from the literature 4 that the amount of TQ would be
decreased due to the one-step extraction process, seeds from the new location NL were
chosen randomly among the other locations for determining the quantitative effect of that
extraction method on TQ content.

Two-step extraction method: Seeds from all locations, TL1, TL2 and NL were
subjected to the two-step extraction method. First, batches of one hundred grams of the
different seeds were ground into a fine powder using an electric grinder followed by soaking
in n-hexane at a w/v ratio 1:4. The extraction was performed at room temperature with
gentle stirring for 2h and repeated several times until no oil was left in the seeds. The
solvent portions were filtered, dried over anhydrous sodium sulfate and evaporated under
reduced pressure at 40°C to get the total oil which is a mixture of fixed and volatile oil.
Second, the total oil was mixed with 500 ml of distilled water and hydro-distilled for 2-
hour in a second step using Clevenger-type apparatus to extract the volatile oil from the
fixed oil. This procedure was then continued as previously described for the one-step
extraction method and repeated to collect sufficient volatile oil for analysis. All extraction
methods were performed in triplicate.

Analysis of the volatile oil using GC-FID: 20 μl of each pure volatile oil from the
one-step and the two-step extraction methods were diluted separately with 1ml diethyl
ether in 2ml vials. Then 3 μl (equivalent to 2.99 mg) of 97 % furfuryl pentanoate was
added to each vial as internal standard for quantification using a Hamilton micro-syringe.
2 μl of each mixture was injected, (at a split ratio 10:1) into a Perkin Elmer XL gas
chromatograph equipped with a flame ionization detector (FID). A 60 m x 0.32 mm i.d. x
0.25 μm film thickness fused silica capillary column coated with DB-5 (5 % phenyl, 95 %
methyl polysiloxane) was used to separate the different volatile components. The oven
temperature was programmed from 50°C to 230°C at a rate of 3°C/min. The injector and
detector temperatures were 230°C and 250°C respectively. Helium was used as carrier gas
at a flow rate of 1.0 ml/min.
The quantification of the volatile oil constituents was reported in two different ways,
first: as percent of total FID peak area, second: as mg volatile constituent/g equivalent of
the internal standard. The response factors were not calculated thus the data shown are
considered semi-quantitative. All values reported are mean of three replicates ± S.D.

Analysis of the volatile oil using GC-MS: GC-MS analysis was conducted on a
Hewlett-Packard 5985 GC instrument coupled to an HP model 5972 MS detector. The
ionization voltage was 70 eV and the ion source temperature was 200°C. Data were acquired
 Amr E. Edris / Jeobp 13 (2) 2010 pp 154 - 164 157

in scan mode, scanning from m/z 41 to 350. Other parameters were the same as for the GC
analysis.

Identification of the volatile components: Components were identified using the

National Bureau of Standards (NBS) registry of mass spectral library or other published
mass spectra 22. For further confirmation of the identity of the components, the available
standard samples were also injected and their retention times were matched with those of
the unknowns. These standard components are marked with an asterisk in table 4.

Results and discussion: It was previously indicated, without justification, that direct
extraction of the volatile oil of N. sativa from the ground seeds in one step using hydro-
distillation led to a decrease in the amount of TQ 4,14. The current study looked at seeds
from location NL to verify this important observation and to determine the extent of this
detrimental effect on TQ content semi-quantitatively. NL seeds were extracted using both
the one-step and two-step extraction methods. GC analysis revealed a major difference in
the amount of TQ from the two different extractions (table 2 and figure 1). From the data
in table 2, the one-step extraction method decreased the amount of TQ to 197.0 mg/g
equivalent internal standard compared to 2253.7mg/ g equivalent internal standard from
the two-step method (about 91.0 % reduction). This result came in accordance with and
confirms the previous investigations 4,14, with the privilege of quantification which was
not conducted in these investigations. The mechanism by which the different extraction
methods led to such differences in the amounts of TQ has yet to be justified. However, it
could be correlated with the volatility and/or the binding of TQ to the cells of the oil
glands in the seeds of N. sativa. Further work is required before this issue can be resolved.
Based on the above result, characterization of the volatile oils of N. sativa from all locations
in this study was conducted after using the two-step extraction method which proved to be
reliable in conserving the content of TQ of the volatile oil.
The content of volatile oils of all samples is illustrated in (table 3). It is evident that
the content from the traditional locations TL1 (0.02 %) and TL2 (0.01 %) is very low
compared with other reported values (0.1 - 1.5 %) 12,23. This could be due to the absence of
chemical fertilization in our study, which utilized organic fertilizers. The volatile oil content
of seeds from the new growing location NL (0.4 %) was much higher than that from the
traditional locations TL1 and TL2, despite receiving the same organic fertilization treatment.
The change in environmental conditions at the new geographical location NL would appear
to be one of the main reasons for the difference in volatile oil contents (table 1).
Table 4 shows the chemical composition of N. sativa volatile oils from the different
growing locations. p-Cymene and TQ are the major constituents in oils from all locations.
This is a characteristic that distinguishes the volatile oil of N. sativa from the other Nigella
species such as N. damascene, N. arvensis or N. orientalis in which p-cymene and TQ
ranges are 0.0 - 0.5 % and 0.0 - 0.1 %, respectively 15-17. However, a rare case was reported
for one cultivar of N. sativa which had only 0.6 % TQ, and 38.3 % trans-anethole 11. This
is likely to be a difference in the chemotype of the seeds rather than the impact of
environmental conditions or agriculture practice.
 Amr E. Edris / Jeobp 13 (2) 2010 pp 154 - 164 158

The volatile oils from the traditional locations TL1 and TL2 were characterized by
the highest content of TQ (63.3 % and 56.4 % respectively) while oil from the new location
NL was about two-fold lower in TQ content (30.2 %). Oil from NL was characterized by
the highest content of p-cymene (30.5 %) and γ-terpinene (11.8 %) compared with TL1
and TL2.
To our knowledge, this is the first report of such a high content of γ-terpinene in the
volatile oil of N. sativa. The level of γ-terpinene from the traditional locations TL1 and
TL2 did not exceed 0.50 % (table 4) and its content in other investigations ranged from 0.2
% to 2.4 % 11,15,24. In term of bioavailability and neutraceutical benefit of the oil, the high
content of γ-terpinene could potentially compensate for the low level of TQ in oil from the
new location NL. That is because γ-terpinene has a high antioxidant activity 25-26 due to the
two active methylene groups that are capable of quenching free radicals (figure 2).
The high levels of p-cymene and γ-terpinene in sample NL (see table 4) is consistent
with the relatively low level of TQ (30.2 %) in that sample. This can be rationalized, in
part, by the biosynthetic route of TQ 15,27 which proceeds via dehydrogenation
(aromatization) of γ-terpinene to p-cymene followed by hydroxylation to thymol and finally
oxidation to TQ. The last two steps in that bioconversion route seem to proceed much
more slowly under the environmental conditions of location NL, leading to the accumulation
of large amounts of γ-terpinene and p-cymene at the expense of TQ. The type of sandy soil
that produced seeds of sample NL may play a role on the chemical composition of its oil.
Sand is well known to reflect sun heat back to the environment which consequently affects
Nigella plants which is exposed under this condition to direct and indirect sun heat. This
factor was moderately affect sample TL1 (sandy-loamy soil) and nearly absent in sample
TL2 (clay soil) which absorb heat with small re-emissions. In addition to soil type, genetic
variation in the seeds is considered to be another possible factor that could contribute to
variation in essential oil compositions.
A follow up evaluation for the volatile oil from the new location NL is planned over
different seasons to asses the reproducibility of its chemical composition.
Currently, we are correlating the data obtained in this study with the oxidative stability
of the parent total oils that bears these investigated volatile oils. That is expected to determine
some potential new factors (e.g. TQ, γ-terpinene and the content of the volatile oil) that
can contribute to the stability of Nigella’s total oil especially during storage. Results will
be published soon in a separate article.

Conclusions: The investigation characterized the content and volatile oil composition
of N. sativa seeds grown organically in two traditional locations and one new location in
Egypt. The volatile oil content from the traditional locations is low while the content of
TQ is high. Conversely, the volatile oil content from the new growing location is much
higher but its content of TQ is lower. The latter oil is characterized by an unusually high
content of γ-terpinene, which has not been previously reported. These results also highlight
the possibilities of maximizing the neutraceutical and pharmaceutical value of N. sativa
seeds and total oil via selection of growing locations that can produce seeds possessing the
highest content of volatile oil, TQ, and/or γ-terpinene. The study also highlighted the
 Amr E. Edris / Jeobp 13 (2) 2010 pp 154 - 164 159

fundamental importance of the two-step extraction method in maximizing the amount of

TQ in the volatile oil. The relevance of this investigation is not restricted to just local or
regional levels, as Egypt in one of the main exporters of organically grown seeds of N.
sativa to different parts of the world.

Acknowledgments: The author is grateful for Associate-Professor Noel Davies,

Organic Mass Spectrometry Facility, Central Science Laboratory, University of Tasmania,
Australia, for his kind help in editing the English language of this manuscript and his
constructive opinions.

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 Table 1. Locations, climatic conditions and agricultural practices used for growing Nigella sativa L. in Egypt

Growing Distance (km) Latitude(°N) Fertilization Soil type Irrigation Temperature Humidity
Location South of Cairo treatment average average
value°C* relative%

TL1 225 28.07 Organic compost sandy-loamy drip 17.2 54.25

TL2 375 27.1 Organic compost clay drip 18.4 40.62
NL 640 26.1 Organic compost sandy drip 20.7 44.00

TL1 and TL2: traditional locations where the bulk crop is cultivated (in Middle Egypt)
NL: new location (in Upper Egypt).
*Temperature average value: is the average of the high temperature daily value and the low temperature daily value during
crop cultivation period (8 months from October to May).
Amr E. Edris / Jeobp 13 (2) 2010 pp 154 - 164
161
 Amr E. Edris / Jeobp 13 (2) 2010 pp 154 - 164 162

Table 2. Effect of extraction methods on the content of

thymoquinone in the volatile oil of Nigella sativa*

Component Extraction methods

One-step method Two-step method

hydro-distillation of the seeds hydro-distillation of the total oil**

area % mg/g equivalent area % mg/g equivalent

internal standard internal standard

Thymoquinone 2.5±0.1 197.0±1.8 30.2±1.2 2253.7±9.9

* seeds were collected from the growing locations NL

** the total oil is the product of extracting the seeds using n-hexane, it comprises the
fixed and the volatile oils

Table 3. Content of the volatile oils of Nigella sativa seeds from different location

Growing location Content of volatile oils

Relative to seed weight Relative to the total oil weight
(g/100 g seeds) (g/100 g seeds)

TL1 0.02±0.08 0.15±0.1

TL2 0.01±0.04 0.11±0.08
NL 0.40±0.1 1.8±0.2

TL1, TL2: traditional locations (in Middle Egypt)

NL: new location (in Upper Egypt)
 Table 4. Chemical composition of the volatile oils of Nigella sativa from different locations

Component RI Volatile oil composition from the different locations

NL TL1 TL2
area % mg/g area % mg/g area % mg/g
equivalent equivalent equivalent
of int. std of int. std of int. std

α-Thujene 933 9.61±0.4 668.3±5.6 2.1±0.3 77.6±2.3 3.6±0.2 194.1±4.2

α-Pinene* 941 2.216±0.1 153.3±1.3 0.5±0.05 19.6±0.9 0.9±0.06 49.4±2.1
Sabinene 980 1.03±0.01 72.4±0.4 0.4±0.03 18.1±0.4 0.6±0.04 36.6±0.7
β-Pinene* 986 2.57±0.08 179.1±1.4 1.0±0.02 60.1±0.8 1.4±0.08 80.5±1.3
Myrcene* 995 0.10±0.01 7.4±0.5 0.05±0.01 4.1±0.01 nd nd
α-Terpinene 1024 0.57±0.02 41.9±0.7 0.2±0.03 20.5±0.4 0.08±0.05 9.4±0.5
p-Cymene* 1037 30.5±0.7 2261.5±8.1 18.8±1.4 707.3±3.8 24.1±0.7 1322.7±7.8
Limonene* 1039 3.23±0.2 220.3±1.2 2.40±0.1 107.2±1.7 1.7±0.09 78.7±1.4
γ-Terpinene 1069 11.8±0.07 836.0±2.6 0.4±0.05 16.0±0.6 0.50±0.05 28.7±0.9
Linalool* 1105 0.59±0.01 42.5±0.8 0.4±0.2 34.3±0.4 0.6±0.02 42.9±0.9
iso-3-Thujanol 1130 3.65±0.1 265.9±0.3 5.7±0.3 281.1±0.8 4.5±0.2 285.0±0.5
Terpinene-4-ol* 1181 0.20±0.02 10.4±1.3 0.4±0.02 18.5±0.2 0.4±0.02 20.0±0.4
α-Terpineol* 1197 0.34±0.02 25.1±0.2 0.76±0.04 37.6±0.5 0.60±0.05 34.3±0.8
Thymoquinone* 1274 30.2±1.2 2253.7±9.9 63.3±2.5 4205.0±8.9 56.4±0.9 3271.5±8.5
trans-Anethole* 1300 0.08±0.05 7.5±0.2 0.2±0.01 15.8±0.1 0.1±0.01 8.1±0.1
Amr E. Edris / Jeobp 13 (2) 2010 pp 154 - 164

Thymol* 1335 nd nd 0.6±0.08 29.0±0.2 0.4±0.04 23.5±0.1

Carvacrol* 1343 1.52±0.21 116.9±0.6 0.5±0.15 22.3±0.1 1.3±0.3 62.8±0.4
α-Longipinene 1361 0.32±0.01 23.3±0.2 0.4±0.01 16.9±0.2 0.2±0.05 12.1±0.1
Longifolene 1427 1.07±0.03 77.9±0.8 2.1±0.02 87.2±0.5 1.3±0.07 52.4±0.9

*The identification is confirmed by authentic sample injection in addition to GC-MS

NL: new location (in Upper Egypt)
163

TL1, TL2: traditional locations (in Middle Egypt)

 Amr E. Edris / Jeobp 13 (2) 2010 pp 154 - 164 164

Fig. 1. GC-FID chromatograms showing the effect of extraction

method on the TQ content of the volatile oil of N. sativa

Upper chromatogram: two-step extraction method for sample NL; volatile oil
extracted by hydro-distillation of the total oil.
Lower chromatogram: one-step extraction method for sample NL; volatile oil
extracted by hydro-distillation of the seeds.
Peak numbers correspond to components listed in Table 4.
*: mg/g equivalent of internal standard

Fig. 2. Chemical structure of γ-terpinene showing the

position of the two active methylenen groups

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