Isolation of volatiles from Nigella sativa seeds using

Transcription

Research article
Received: 22 November 2012,
Revised: 14 January 2013,
Accepted: 28 January 2013
Published online in Wiley Online Library: 30 April 2013
(wileyonlinelibrary.com) DOI 10.1002/bmc.2887
Isolation of volatiles from Nigella sativa seeds
using microwave-assisted extraction: effect of
whole extracts on canine and murine CYP1A
Xue Liua,b, Jong-Hyouk Parkb, A. M. Abd El-Atyc, M. E. Assayedd,
Minoru Shimodae* and Jae-Han Shimb*
ABSTRACT: The volatile components of Nigella sativa seeds were isolated using microwave-assisted extraction (MAE)
and identified using gas chromatography. Further investigations were carried out to demonstrate the effects of whole
extracts on canine (dog) and murine (rat) cytochrome P450 1A (CYP1A). The optimal extraction conditions of MAE were
as follows: 25 mL of water, medium level of microwave oven power and 10 min of extraction time. A total of 32 compounds were identified under the conditions using GC-FID and GC-MS. Thymoquinone (38.23%), p-cymene (28.61%),
4-isopropyl-9-methoxy-1-methyl-1-cyclohexene (5.74%), longifolene (5.33%), a-thujene (3.88) and carvacol (2.31%) were
the main compounds emitted from N. sativa seeds. Various extracts including pure compounds, essential oil, nonpolar
partition, relatively high-polar/nonpolar partition, and polar partition extracts effectively inhibited the reaction of
ethoxyresorufin O-de-ethylation, which is specified for CYP1A activity both in dog and rat. This in vitro data should
be heeded as a signal of possible in vivo interactions. The use of human liver preparations would considerably
strengthen the practical impact of the data generated from this study. Copyright © 2013 John Wiley & Sons, Ltd.
Keywords: Nigella sativa; volatile compounds; microwave-assisted extraction; ethoxyresorufin O-de-ethylation; cytochrome P450 1A;
dog and rat
Introduction
938
Nigella sativa L. is an herbaceous plant belonging to the
botanical family Ranunculaceae. It has long been used as a
natural food additive and remedy in many Middle Eastern and
Far Eastern countries (Liu et al., 2012). N. sativa has been
reported to have many biological activities, including
antioxidation (Burits and Bucar, 2000), anti-inflammatory
(Al-Ghamdi, 2001; Ghannadi et al., 2005), analgesic (Al-Ghamdi,
2001; Ghannadi et al., 2005), gastroprotective (El-Abhar et al.,
2003), antimicrobial (Hanafy and Hatem, 1991), antifungal (Khan
et al., 2003) and anti-tumor (Ait Mbarek et al., 2007). Most of the
activities have been attributed to the volatile oil obtained from
N. sativa preparation (Swamy and Tan, 2000). Therefore, the
chemical composition of N. sativa seeds has been thoroughly
investigated in previous studies, particularly its essential oil or
volatile component. To obtain the essential oil or volatile
component of N. sativa, various kinds of extraction methods
have been used, including solvent extraction, hydrodistillation
extraction (HD), supercritical fluid extraction, steam distillation
extraction, solvent extraction–steam distillation extraction,
supercritical fluid extraction–steam distillation extraction and
headspace solid phase microextraction (Liu, 2011).
The microwave-assisted extraction (MAE) technique has been
developed rapidly over the past 5–10 years as a result of its
inherent advantages, which are reduced extraction time and
solvent volume compared with traditional extraction techniques
(Ballard et al., 2010). The enhancement of product recovery
using microwaves is generally attributed to its heating effect
by the dipole rotation of the solvent in the microwave field. This
Biomed. Chromatogr. 2013; 27: 938–945
effect causes the solvent temperature to rise, which then
increases the solubility of the compound of interest
(Hemwimon et al., 2007). The high temperature reached by
microwave heating can dramatically reduce both the extraction
time and the volume of solvent required (Kaufmann and
* Correspondence to: Jae-Han Shim, Natural Products Chemistry Laboratory,
Biotechnology Research Institute, Chonnam National University, 77 Yongbong-ro,
Buk-gu, Gwangju 500-757, Republic of Korea. E-mail: [email protected]
Minoru Shimoda, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo
University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 1830054, Japan. E-mail: [email protected]
a
The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education,
School of Chemical and Material Engineering, Jiangnan University, 1800 Lihu
Avenue, Wuxi 214122, China
b
Natural Products Chemistry Laboratory, Biotechnology Research Institute, Chonnam
National University, 77 Yongbong-ro, Buk-gu, Gwangju 500-757, Republic of Korea
c
Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University,
12211Giza, Egypt
d
Department of Forensic Medicine and Toxicology, Faculty of Veterinary
Medicine, Menoufiya University, Sadat City Branch, Egypt
e
Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of
Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-0054, Japan
Abbreviations used: CYP1A, cytochrome P450 1A; DE, dichloromethane
extract; DMSO, dimethylsulfoxide; EO, essential oil; HD, hydrodistillation;
HE, n-hexane extract; MAE, microwave-assisted extraction; ME, methanol
extract; TQ, thymoquinone.
Copyright © 2013 John Wiley & Sons, Ltd.
Christen, 2002). Benkaci-Ali et al. (2007) applied MAE in the
study of N. sativa seeds, and many compounds were identified
by GC-MS. However, they only carried out the kinetic study of
the extraction procedure, and there was no method optimization.
The moistened volume was neglected, which is an important
factor for MAE (Kaufmann and Christen, 2002). In contrast to
other samples that contained enough water for the extraction,
N. sativa contains moisture levels ranging from 5.52 to 7.43%
(Abdel-Aal and Attia, 1993; Salem, 2001; Takruri and Dameh,
1998). Therefore, solvent volume must be considered for N.
sativa when it is extracted using MAE.
The cytochrome P450 (CYP) enzymes are a superfamily of
hemoproteins that are the terminal oxidases of the mixed function
oxidase system found on the membrane of the smooth
endoplasmic reticulum preferentially expressed in the centrilobular
area of the liver (Oinonen and Lindros, 1998). Among the CYP
families 1–3, CYP1A participates in the metabolism of many
environmental carcinogens and mutagens, such as polycyclic
aromatic hydrocarbons (Lee et al., 1998). Many examples of
herbs or food products that interact with CYP enzymes have
been evaluated in the relevant literature (Koul et al., 2000;
Subehan et al., 2006; Abd El-Aty et al., 2008). Herbs contain a
diverse array of active constituents, each with the potential to
modulate the activity of specific cytochrome P450 enzymes
(Abd El-Aty et al., 2008). In general, studies in animals (rodents
and canine) are often undertaken to provide possible clinical
insight without complete validation of the human relevance
(Abd El-Aty et al., 2008). It was reported that N. sativa oil had a
protective effect against the carbon tetrachloride-mediated
suppression of hepatic CYPs in rats and this protective effect
was partly related to the reduction of nitric oxide via the
down-regulation of inducible nitric oxide synthase, in addition to
the reduction of tumor necrosis factor-a and the up-regulation of
the anti-inflmmatory IL-10 (Ibrahim et al., 2008).
The present study was aimed at applying the modern MAE
method for the investigation of secondary volatile components in
N. sativa seeds. Moreover, the in vitro activity of 7-ethoxyresorufin
O-de-ethylation for CYP1A, in the absence and presence of
whole N. sativa extracts using canine and murine liver
microsomes was determined.
center of the cavity. A sample flask was connected to a Clevenger
apparatus, which was located outside the microwave oven to condense
and collect the volatile components. The MAE apparatus is illustrated in
Fig. 1. The experimental MAE variables were optimized by the univariate
method in order to maximize the yield and quality of essential oil.
In a typical MAE procedure, a 25 g aliquot of ground seeds was placed
in a 250 mL round-bottomed flask; 25 mL of distilled water was added to
moisturize the seeds for 30 min. Then, the flask was connected to a
Clevenger apparatus and heated using medium-level power for 10 min.
The volatile distillate was eluted out by n-hexane and dried through
anhydrous sodium sulfate. The n-hexane was removed under vacuum
conditions. The essential oil obtained was refrigerated prior to analysis.
Sample preparation for CYP
For examination of biological activity, essential oil was obtained by MAE
with optimized extraction conditions. The ground seeds were extracted
successively with n-hexane, dichloromethane and methanol. The
solvent of each extracted solution was separately removed from
extracts with an evaporator under vacuum conditions (Scheme 1). The
essential oil and extracts were dissolved in dimethylsulfoxide (DMSO)
with appropriate concentration and stored in a refrigerator at 20 C.
Gas chromatography conditions
An HP 4890 gas chromatograph (Hewlett-Packard, Pale Alto, CA, USA)
equipped with a flame ionization detector (FID) was used for the
determination of the volatile compounds in N. sativa seeds. Separations
were carried out on an HP-5 column (30 m 0.25 mm id 0.25 mm film
thickness, J&W Scientific Products, Santa Clara, CA, USA). The injector
and detector temperatures were set to 250 and 300 C, respectively.
The oven temperature was held at 60 C for 10 min and increased to
180 C at a rate of 4 C/min, then increased to 250 C at a rate of 25 C/min,
Condenser
Experimental
Chemicals and reagents
Nigella sativa seeds were acquired from the Popy Trading Co. (Dhaka,
Bangladesh). Thymoquinone, p-cymene, 7-ethoxyresorufin, glucose-6phosephate, glucose-6-phosphate dehydrogenase, and nicotinamide
adenine dinucleotide phosphate were purchased from Sigma Chemical
Co. (St Louis, MO, USA). Water was purified using an Ultima Duo 200
water purification system (Balmann, Ulsan, Republic of Korea). The other
chemicals and reagents were of analytical, biochemical or HPLC grade.
The seeds were quickly ground with a food mixer (Hanil Co., Seoul,
Republic of Korea), and sieved through a standard sieve of 0.6 mm pore size.
The ground seeds were placed in a brown glass bottle and stored at 4 C.
Essential oil
Water
Reflux tube
Microwave oven
Sample
Extraction procedure of MAE
Figure 1. Schematic diagram of microwave-assisted extraction.
wileyonlinelibrary.com/journal/bmc
939
The extraction was performed in an adapted commercial kitchen microwave
oven. The maximum output power of this microwave oven was 700 W
with 2450 MHz of microwave irradiation frequency, and a power divider
of three levels (low, medium, high). The dimensions of the cavity were
28 25 18 cm. A 4 cm diameter hole was carefully drilled in the top
X. Liu et al.
Scheme 1. Sample preparation of CYP inhibition study.
and kept constant at 250 C for 15 min. The N2 carrier gas flow rate was
1 mL/min.
An Agilent Technology 6890 N gas chromatograph (USA) equipped
with an Agilent 5973 MSD was used for the identification of each
component. Separations were carried out on an HP-5MS column
(30 m 0.25 mm id 0.25 mm film thickness, J&W Scientific Products)
with the same oven temperature program as GC-FID. High-purity
helium (99.999%) at a constant flow rate of 1 mL/min was used as the
carrier gas. Electron impact mass spectral (EI-MS) analysis was carried
out at an ionization energy of 70 eV at 250 C. All data were obtained
by collecting the full-scan mass spectra within the scan range
40–550 amu. Compounds were identified using the Wiley 6th edition
(Wiley, New York, NY, USA) mass spectral library and retention indices.
Determination of CYP1A activity
Microsomal fractions. Canine and murine microsomal fractions were
prepared as described by van der Hoeven and Coon (Van Der Hoeven
and Coon, 1974). Samples were stored at 80 C until used. The protein
concentration and CYP content were determined as described by
Bradford (1976), and Omura and Sato (Omura and Sato, 1964),
respectively. This experiment was conducted in accordance with the
guidelines for the care and use of laboratory animals of the Faculty of
Agriculture at the Tokyo University of Agriculture and Technology.
940
Enzyme assay. The enzyme kinetics of CYP1A were examined using
ethoxyresorufin O-de-ethylation. The reaction proceeded at 37 C in
50 mM sodium/potassium phosphate buffer (pH 7.4), containing an
NADPH-generating system (0.5 mM b-NADP+, 5.0 mM glucose-6-phosphate,
1.5 U/mL glucose-6-phosphate dehydrogenase, 5 mM MgCl2) and about
0.02 mg of microsomal protein in a total volume of 1 mL. Preincubation
for 5 min at 37 C was carried out before the reaction was started by the
addition of the substrate with 0.1 M DMSO (vehicle solution) or the
extract. The concentrations of ethoxyresorufin in the assay system
ranged from 0.065 to 2.07 mM. After the incubation at 37 C for 15 min,
the reaction was quenched by adding 3 mL of methanol and placed in
an ice-bath for 5 min. After centrifugation at 2000g for 5 min, 1 mL of the
resulting supernatant was transferred to a clear test tube, and 4 mL of
95% methanol–tris-buffer (pH 8.0) was added. Resorufin concentrations
in the mixture were determined by a fluorometric method using a
spectrofluorometer (RF 1500; Shimadzu Corporation, Kyoto, Japan; Burke
et al., 1977). The excitation wavelength was set at 550 and 586 nm.
Reversible inhibition test. Thymoquinone (TQ), essential oil (EO),
n-hexane extract (HE), dichloromethane extract (DE) and methanol
extract (ME) were all dissolved in DMSO and then added to the assay
system just before the addition of substrate. The concentrations in the
assay system of TQ, EO, HE, DE and ME were 1.5, 4.0, 20, 50 and
100 mg/mL, respectively.
Enzyme kinetic analysis
As the double reciprocal plot analysis indicated noncompetitive
inhibition, the following equations were used to analyze the enzyme
kinetics of ethoxyresorufin O-de-ethylation in the absence or existence
of extract, where Vmax and Km are the maximal velocity and Michaelis
constant, and S and I are concentrations of the substrate and inhibitors,
respectively. Ki is an inhibitory constant (dissociation constant of inhibitors).
For each extract, three reaction velocity–substrate concentration curves
were simultaneously analyzed using MULTI software (Yamaoka et al.,
1981) to estimate the kinetic parameter values, including Vmax, Km and Ki.
v¼
Vmax S
Km þ S
(1)
v¼
Effect of microwave oven power
V S
max Km 1 þ KIi þ S
(2)
In the case of noncompetitive inhibition, the metabolic rate can be
expressed by eqns (1) and (3) in the absence or presence of the inhibitors.
v¼
Vmax S
ðKm þ SÞ 1 þ KIi
(3)
The power level of the microwave oven was changed by a
simple power divider. It was used to change the working
frequency of the microwave source and achieve three different
control levels. The extraction efficiency of all targets increased
when the microwave oven power increased to a medium level
and then decreased thereafter (Fig. 3). This might be caused
by the volatile loss by fast heating during the extraction procedure. The volatile components were expelled by strong boiling
water vapor even when the condenser was extra long. Therefore, medium level power was selected for further study.
Effect of extraction time
Optimization of MAE
First, the effects of moistened water volume (0, 25 and 50 mL) were
studied. Then, the extraction power of the microwave oven and
extraction time were successively optimized. All the optimization
procedures were performed in triplicate. All of the extracts were
dissolved and diluted to the concentration of 0.25 g seeds/mL with
n-hexane. The tested solution was analyzed by GC-FID.
Effect of water volume
Volatile components of N. sativa seeds extracted by MAE
The ground seeds were extracted under the optimized conditions
of MAE for three replicates. The extracts were dissolved in an
appropriate volume of n-hexane and analyzed by GC-FID and
12000
10000
8000
6000
0
0
10000
Thymoquinone
4000
Longifolene
2000
3
4
14000
10000
p-Cymene
2
Figure 3. Effects of microwave oven power level on the extraction
efficiency of volatiles from N. sativa seeds.
12000
6000
1
Power level (1.Weak, 2.Medium, 3.Strong)
12000
8000
p-Cymene
Thymoquinone
Longifolene
4000
2000
Peak area
Peak area
When the extraction of MAE was performed without adding
water, a high yield of dark brown essential oil was obtained;
however, a very small amount of thymoquinone was extracted.
Most of the selected compounds had the best extraction
efficiency when the added volume of water was equal to the
sample weight (25 mL; Fig. 2). Solvent-free microwave extraction
was applied for extracting volatiles from plant materials, and
good results were obtained (Lucchesi et al., 2004; Phutdhawong
et al., 2007; Farhat et al., 2011). In previous reports, MAE was
applied to fresh aromatic herbs (initial moisture >80%), which
can supply enough water for the extraction (Lucchesi et al.,
2004). The microwave oven used in the study by Farhat et al.
(2011) was specially designed, and the extracts were moved
out of the microwave oven by gravity to obtain essential oil
from orange peel (initial moisture >90%). However, proximate
analysis of whole, mature N. sativa seeds showed that the
moisture content ranged from 5.52 to 7.43% (Abdel-Aal and Attia,
1993; Salem, 2001; Takruri and Dameh, 1998). Therefore, 25 mL of
water was selected to moisten the seeds before extraction.
Figure 4 shows that longer extraction time was better for the
extraction of most compounds, except for thymoquinone. The
extraction efficiency of thymoquinone decreased considerably
after 10 min. This might have been due to degradation at high
temperature. Since thymoquinone is one of the most important
volatile components in N. sativa seeds, and the efficiency of
other components did not increase too much, 10 min was
decided to be the optimum extraction time.
Peak area
Results and discussion
8000
α-Thujene
6000
p-Cymene
Thymoquinone
4000
Longifolene
2000
0
0
0
20
40
60
0
Water addition volume (mL)
10
15
20
Figure 4. Dynamic extraction time study for the extraction of volatiles
from N. sativa seeds.
941
Figure 2. Effects of added water volume on extraction efficiency of volatiles
from Nigella sativa seeds using microwave-assisted extraction (MAE).
5
Extraction time (min)
X. Liu et al.
Table 1. Volatile compounds in N. sativa seeds using MAE followed by GC–MS
No.
Compound name
Retention time (min)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
a-Thujene
a-Pinene
Sabinene
b-Pinene
a-terpinene
p-Cymene
Limonene
g-terpinene
a-p-Dimethylstyrene
Unidentified 1
4-Isopropyl-6-methoxy-1-methyl-1-cyclohexene
1,4-Dimethyl-3-cyclohexenyl methyl ketone
L-4-Terpineol
b-Cyclocitral
Carvone
Thymoquinone
Unidentified 2
Borneol acetate
Carvacrol
a-Longipinene
a-Copaene
Eremophilene
Longifolene
2-Tridecanone
Bisabolene
Epizonarene
4-Methoxy-2,3,6-trimethylphenol
Biformene
Dibutyl phthalate
Diphenyl-2-pyridylmethane
Triphenylamine
Diisooctyl phthalate
7.0
7.2
9.4
9.6
12.4
13.1
13.3
15.5
17.9
18.3
19.9
23.6
24.4
26.3
29.2
30.0
30.8
32.0
34.3
36.1
37.9
39.3
39.6
45.8
46.4
47.3
50.7
68.9
70.4
77.1
86.2
94.9
Retention index
921
926
966
968
1014
1024
1026
1056
1088
1093
1115
1164
1175
1200
1241
1252
1264
1281
1314
1342
1369
1390
1395
1496
1505
1521
1581
1930
1962
2107
>2200
>2200
Percentage (%)
3.88
0.84
0.71
1.36
0.20
28.61
1.58
0.35
T
0.93
5.74
0.58
0.78
1.31
0.10
38.23
0.14
0.21
2.31
1.32
0.04
0.18
5.33
0.12
T
0.12
0.17
T
0.27
0.17
T
0.40
Retention index relative to C8–C22 n-alkanes on HP-5MS capillary column; T, trace (<0.02%).
Unidentified 1: m/z 125 (999), 93 (777), 85 (645), 153 (512), 72 (430), 121 (376), 100 (371), 81 (365), 55 (303). Unidentified 2: m/z 93
(999), 43 (554), 121 (494), 92 (312), 41 (214), 94 (200).
942
Figure 5. Typical GC-MS total ion chromatogram with marked peaks of identified volatile compounds from N. sativa seed extracted by MAE.
See Table 1 for peak identification.
TQ
0.1
0.08
0.06
0.04
0.02
0
1
1.5
2
EO
0.1
0.08
0.06
0.04
0.02
0
0.5
0.12
1
1.5
2
0.1
0.08
0.06
0.04
0.02
0
0.5
0.12
1
1.5
2
0.1
0.08
0.06
0.04
0.02
0
0.5
1
1.5
2
0.04
0.03
0.02
0.01
0
0
0.5
1
1.5
2
2.5
Ethoxyresorufin concentration (µM)
0.06
EO
0.05
0.04
0.03
0.02
0.01
0
0
0.5
1
1.5
2
2.5
0.06
HE
0.05
0.04
0.03
0.02
0.01
0
0
2.5
DE
0
TQ
0.05
2.5
HE
0
0.06
2.5
De-ethylation (nmol/min/mg)
0.12
0
0.5
0
0.12
0.5
1
1.5
2
2.5
0.06
DE
0.05
0.04
0.03
0.02
0.01
0
0
2.5
0.5
1
1.5
2
2.5
0.12
ME
0.1
0.08
0.06
0.04
0.02
0
0
0.5
1
1.5
2
2.5
0.06
ME
0.05
0.04
0.03
0.02
0.01
0
0
0.5
1
1.5
2
2.5
Figure 7. Michaelis–Menten kinetics of ethoxyresorufin O-de-ethylation
with or without analytes in murine hepatic microsomes. Each point and
vertical bar represents mean SD from five microsomes. Open circles
were obtained from reactions without analytes (vehicle addition). Solid
circles were obtained from reactions with analytes at the concentration
of 1.5, 4.0, 20, 50 and 100 mg/mL for TQ, EO, HE, DE and ME, respectively.
The solid curves in the figure were calculated by eqns (1)–(3) in
Experimental section using kinetic parameters shown in Table 2.
943
Figure 6. Michaelis–Menten kinetics of ethoxyresorufin O-de-ethylation
with or without analytes in canine hepatic microsomes. Each point and
vertical bar represents the mean SD from five microsomes. Open circles
were obtained from reactions without analytes (vehicle addition). Solid circles
were obtained from reactions with analytes at the concentrations of 1.5, 4.0,
20, 50 and 100 mg/mL for TQ, EO, HE, DE and ME, respectively. The solid
curves in the figure were calculated by eqns (1)–(3) in the Experimental
section using the kinetic parameters shown in Table 2.
X. Liu et al.
Table 2. Michaelis–Menten kinetic parameters for ethoxyresorufin O-de-ethylation and inhibitory constants of thymoquinone
(TQ), essential oil (EO), n-hexane extract (HE), dichloromethane extract (DE) and methanol extract (ME) using hepatic microsomes
from dogs and rats
Analytes
Dog
TQ
EO
HE
DE
ME
Rat
TQ
EO
HE
DE
ME
Vmax (nmol/min/mg protein)
Km (mM)
Vmax/Km (mL/min/mg protein)
Ki (mM)
0.092 0.028
0.095 0.027
0.098 0.029
0.096 0.025
0.104 0.034
0.099 0.019
0.094 0.025
0.093 0.027
0.102 0.028
0.099 0.026
0.92 0.38
1.05 0.34
1.16 0.56
1.05 0.52
1.11 0.46
7.13 3.74
3.76 0.74
5.16 1.39
3.62 0.77
6.38 2.52
0.078 0.046
0.075 0.042
0.071 0.037
0.072 0.039
0.072 0.042
1.779 1.013
1.653 0.712
1.519 0.719
1.562 0.635
1.551 0.709
0.046 0.012
0.046 0.012
0.048 0.013
0.047 0.012
0.047 0.012
6.33 1.26
57.47 9.50
52.64 6.96
92.02 18.66
79.78 14.55
Vmax, maximal velocity; Km, Michaelis constant; Ki, inhibitory constant (dissociation constant of inhibitors).
GC-MS. Table 1 lists the identified compounds extracted from N.
sativa seeds by MAE followed by GC-MS. Figure 5 shows a typical
GC-MS chromatogram of essential oil by MAE. A total of 32
compounds were identified. The amount of identified compounds
from N. sativa seeds was lower than that reported by Benkaci-Ali
et al. (2007). This might have been due to the different origin of
the seeds and the lower concentration of sample solution injected
into GC-MS. Benkaci-Ali et al. (2007) also studied the performances
of HD and MAE on the extraction of volatile components from N.
sativa seeds. The MAE was carried out without optimization work
in their study. The yield of essential oil was 0.2%, similar to that
obtained by HD. MAE mainly improved the extraction efficiency
of p-cymene in their study, but the amount of volatile compounds
was less than that obtained by HD. This contrasts with our results.
In our study, the amount of volatiles was similar between the two
methods after optimization, and the content of thymoquinone
was substantially increased. This finding matches the results of
Benkaci-Ali’s study.
Thymoquinone (38.23%) and p-cymene (28.61%) were the
most abundant components, and 4-isopropyl-9-methoxy-1methyl-1-cyclohexene (5.74%), longifolene (5.33%), a-thujene
(3.88) and carvacol (2.31%) were the other main compounds
emitted from N. sativa seeds. The biological activity of N. sativa
seeds was studied with emphasis on its main essential
oil component, thymoquinone (Hajhashemi et al., 2004;
Burits and Bucar, 2000), which has been investigated for its
anti-oxidant, anti-inflammatory and anti-cancer activities
since its first extraction in 1960s from N. sativa (Woo et al.,
2012). p-Cymene possesses low antifungal activity and has
no phytotoxic effect (Kordali et al., 2008), and has also been
revealed to have strong antagonistic effects in antioxidant
capacity when it is paired with thymoquinone (Milos and
Makota, 2012).
Ethoxyresorufin O-de-ethylation assay involves the oxidative
de-ethylation of 7-ethoxyresorufin to resorufin, catalyzed
by CYP1A, which is the principal P-450 isozyme involved in
the O-de-ethylation of the substrate (Petrulis et al., 2011). The
CYP1A isoform is primarily involved in the metabolism of
various foods such as caffeine-containing foods and drugs such
as paracetamol, theophylline, mexiletine and quinolones (Yang
et al., 2002). Figures 6 and 7 showed the profiles of
ethoxyresorufin O-de-ethylation and the inhibition of CYP1A
biological activities by TQ, EO, HE, DE and ME in hepatic
microsomes from dogs and rats. Table 2 shows the kinetic
parameters for the inhibition by TQ, EO, HE, DE and ME, and
each value represents the mean SD (n = 5). For each analyte,
three reaction velocity–substrate concentration curves were
simultaneously analyzed using a nonlinear least squares fitting
program to estimate the kinetic parameter values Vmax, Km
and Ki. All of the tested analytes, which were represented as
pure compound (TQ), commonly used essential oil (EO), nonpolar partition (HE), relatively higher polar nonpolar partition (DE)
and polar partition (ME) in N. sativa seeds, effectively inhibited
the reaction of ethoxyresorufin O-de-ethylation in a
noncompetitive manner in murine and competitive manner in
canine hepatic microsomes. Although we did not characterize
each extract for secondary metabolites, it might be possible
that the inhibitory effect comes from thymoquinone as it did
show a similar pattern to various extracts. As the present study
was carried out in vitro on two animal models, the extrapolation
to the expected pharmacological effects in humans might be
considered more reliable (Levy et al., 2007). Consequently,
attention should be paid to the possible drug interaction in
patients who concurrently use N. sativa as a whole herb and/or
its major components. Clearly, the clinical significance of in vitro
interactions needs to be determined by appropriate measures
(Nair et al., 2007).
Biological activity; reversible inhibition
944
Nigella sativa has been a widely used herb since ancient times as
a food additive or natural remedy for a wide range of diseases,
but the mechanism of its action is still being studied. In this
study, we examined the inhibitory effect on CYP1A activities,
which could indicate the effects of whole N. sativa seeds.
Conclusions
Nigella sativa is a very attractive plant that has been employed
for thousands of years, and is attracting more and more
attention from scientists. MAE extraction technique for the
extraction of volatile components from N. sativa seeds was
carried out. The optimal extraction conditions of MAE were
25 mL of water, a medium level of microwave oven power,
and 10 min of extraction time. The established MAE method
successfully shortened the extraction time, and it is also suitable
for industrial purposes if an industrial microwave oven is
available. A total of 32 compounds were identified using MAE
coupled with GC-FID and GC-MS from N. sativa. Thymoquinone
and p-cymene were the highest-yielding compounds.
Moreover, the essential oil, n-hexane, dichloromethane and
methanol extracts from N. sativa showed great inhibitory effects
on the catalytic activity of cytochrome P450 1A in canine and
murine hepatic microsomes. It is prudent to take note of such
in vitro interactions as a cautionary signal in the best interests
of public health when N. sativa extracts, essential oil and
thymoquinone are handled with CYP1A.
References
945
Abdel-Aal ESM and Attia RS. Characterization of balck cumin (Nigella
sativa) seeds. Alexandria Science Exchange 1993; 14: 483–496.
Abd El-Aty AM, Shah SS, Kim BM, Choi JH, Cho HJ, Yi H, Chan BJ, Shin HC,
Lee KB, Shimoda M and Shim JH. In vitro inhibitory potential of
decursin and decursinol angelate on the catalytic activity of
cytochrome P-450 1A1/2, 2D15, and 3A12 isoforms in canine hepatic
microsomes. Archives of Pharmacal Research 2008; 11: 1425–1435.
Ait Mbarek L, Ait Mouse H, Elabbadi N, Bensalah M, Gamouch A,
Aboufatima R, Benharrel A, Chait A, Kamal M, Dalal A and Zyad A.
Anti-tumor properties of blackseed (Nigella sativa L.) extracts.
Brazilian Journal of Medical and Biological Research 2007; 40: 839–847.
Al-Ghamdi MS. The anti-inflammatory, analgesic and antipyretic activity
of Nigella sativa. Journal of Ethnopharmacology 2001; 76: 45–48.
Ballard TS, Mallikarjunan P, Zhou K and O’Keefe S. Microwave-assisted
extraction of phenolic antioxidant compounds from peanut skins.
Food Chemistry 2010; 120: 1185–1192.
Benkaci-Ali F, Baaliouamer A, Meklati BY and Chemat F. Chemical composition
of seed essential oils from Algerian Nigella sativa extracted by microwave
and hydrodistillation. Flavour and Fragrance Journal 2007; 22: 148–153.
Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein–dye
binding. Analytical Biochemistry 1976; 72: 248–254.
Burits M and Bucar F. Antioxidant activity of Nigella sativa essential oil.
Phytotherapy Research 2000; 14: 323–328.
Burke MD, Prough RA and Mayer RT. Characteristics of a microsonal
cytochrome P-448-mediated reaction. Ethoyresorufin O-de-ethylation.
Drug Metabolism and Disposition 1977; 5: 1–8.
El-Abhar HS, Abdallah DM and Saleh S. Gastroprotective activity of Nigella
sativa oil and its constituent, thymoquinone, against gastric mucosal
injury induced by ischaemia/reperfusion in rats. Journal of
Ethnopharmacology 2003; 84: 251–258.
Farhat A, Fabiano-Tixier AS, El Maataoui M, Maingonnat JF, Romdhane M
and Chemat F. Microwave steam diffusion for extraction of essential
oil from orange peel: kinetic data, extract’s global yield and
mechanism. Food Chemistry 2011; 125: 255–261.
Ghannadi A, Hajhashemi V and Jafarabadi H. An investigation of the
analgesic and anti-inflammatory effects of Nigella sativa seed
polyphenols. Journal of Medical Food 2005; 8: 488–493.
Hajhashemi V, Ghannadi A and Jafarabadi H. Black cumin seed essential
oil, as a potent analgesic and antiinflammatory drug. Phytotherapy
Research 2004; 18: 195–199.
Hanafy MSM and Hatem ME. Studies on the antimicrobial activity of Nigella
sativa seed (black cumin). Journal of Ethnopharmacology 1991; 34: 275–278.
Hemwimon S, Pavasant P and Shtipruk A. Microwave-assisted extraction
of antioxidative anthraquinones from roots of Morinda citrifolia.
Separation and Purification Technology 2007; 54: 44–50.
Ibrahim ZS, Ishizuka M, Soliman M, Elbohi K, Sobhy W, Muzandu K,
Elkattawy AM, Sakanoto KQ and Fujita S. Protection by Nigella sativa
against carbon tetrachloride-induced downregulation of hepatic
cytochrome P450 isozymes in rats. The Japanese Journal of Veterinary
Research 2008; 56: 119–128.
Kaufmann B and Christen P. Recent extraction techniques for natural
products: microwave-assisted extraction and pressurised solvent
extraction. Phytochemical Analysis 2002; 13: 105–113.
Khan MAU, Ashfaq MK, Zuberi HS, Mahmood MS and Gilani AH. The
in vivo antifungal activity of the aqueous extract from Nigella sativa
seeds. Phytotherapy Research 2003; 17: 183–186.
Kordali S, Cakir A, Ozer H, Cakmakci R, Kesdek M and Mete E. Antifungal,
phytotoxic and insecticidal properties of essential oil isolated from
Turkish Origanum acutidens and its three components, carvacrol,
thymol and p-cymene, Bioresource Technology 2008; 99: 8788–8795.
Koul S, Koul JL, Taneja SC, Dhar KL, Jamwal DS, Singh K, Reen RK and
Singh J. Structure–activity relationship of piperine and its synthetic
analogues for their inhibitory potentials of rat hepatic microsomal
constitutive and inducible cytochrome P450 activities. Bioorganic &
Medicinal Chemistry 2000; 8: 251–268.
Lee CA, Lawrence P, Kerkvliet NI and Rifkind AB 2,3,7,8Tetrachlorodibenzo-p-dioxin induction of cytochrome P450-dependent
arachidonic acid metabolism in mouse liver microsomes: evidence
for species-specific differences in responses. Toxicology and Applied
Pharmacology 1998; 153: 1–11.
Levy A, Cohen G, Gilat E, Kapon J, Dachir S, Abraham S, Herskovitz M,
Teitelbaum Z and Raveh, L. Extrapolating from animal studies to
the efficacy in humans of a pretreatment combination against
organophosphate poisoning. Archives of Toxicology 2007; 81: 353–359.
Liu X. Improved Extraction Method for Volatiles of Nigella sativa Seeds and
their Biological Activities. Chonnam National University: Gwangju, 2011.
Available from: http://www.dcollection.net/handler/jnu/000000031143.
Liu X, Abd El-Aty AM, Cho SK, Yang A, Park JH and Shim JH. Characterization
of secondary volatile profiles in Nigella sativa seeds from two different
origins using accelerated solvent extraction and gas chromatography–
mass spectrometry. Biomedical Chromatography 2012; 26: 1157–1162.
Lucchesi ME, Chemat F and Smadja J. Solvent-free microwave extraction
of essential oil from aromatic herbs: comparison with conventional
hydro-distillation. Journal of Chromatography 2004; 1043: 323–327.
Milos M and Makota D. Investigation of antioxidant synergisms and
antagonisms among thymol, carvacrol, thymoquinone and p-cymene
in a model system using the Briggs–Rauscher oscillating reaction.
Food Chemistry 2012: 131; 296–299.
Nair VD, Foster BC, Thor Arnason J, Mills EJ and Kanfer I. In vitro evaluation of
human cytochrome P450 and P-glycoprotein-mediated metabolism of
some phytochemicals in extracts and formulations of African potato.
Phytomedicine 2007; 14, 498–507.
Oinonen T and Lindros KO. Zonation of hepatic cytochrome P-450
expression and regulation. Biochemical Journal 1998; 329: 17–35.
Omura T and Sato R. The carbon monoxide-binding pigment of liver
microsomes. The Journal of Biological Chemistry 1964; 239: 2370–2378.
Petrulis JR, Chen G, Benn S, LaMarre J and Bunce NJ. Application of the
ethoxyresorufin-O-de-ethylase (EROD) assay to mixtures of halogenated
aromatic compounds. Environmental Toxicology 2011; 16: 177–184.
Phutdhawong W, Kawaree R, Sanjaiya S, Sengpracha W and Buddhasukh
D. Microwave-assisted isolation of essential oil of Cinnamomum iners
Reinw. ex Bl.: comparison with conventional hydrodistillation.
Molecules 2007; 12: 868–877.
Salem MA. Effect of some heat treatment on nigella seeds characteristics.
1 – Some physical and chemical properties of nigella seed oil. Journal
of Agricultural Research Tanta University 2001; 27: 471–486.
Subehan, Tepy U, Shigetoshi K and Yasuhiro T. Mechanism-based inhibition
of human liver microsomal cytochrome P450 2D6 (CYP2D6) by
alkamides of Piper nigrum. Planta Medica 2006; 72: 527–532.
Swamy SMK and Tan BKH. Cytotoxic and immunopotentiating effects
of ethanolic extract of Nigella sativa L. seeds. Journal of
Ethnopharmacology 2000; 70: 1–7.
Takruri HRH and Dameh MAF. Study of the nutritional value of black
cumin seeds (Nigella sativa L). Journal of the Science of Food and
Agriculture 1998; 76: 404–410.
Van Der Hoeven TA and Coon MJ. Preparation and properties of partially
purified cytochrome P-450 and reduced nicotinamide adenine
dinucleotide phosphate–cytochrome P-450 reductase from rabbit
liver microsomes. The Journal of Biological Chemistry 1974; 249:
6302–6310.
Woo CC, Kumar AP, Sethi G and Tan KHB. Thymoquinone: potential cure
for inflammatory disorders and cancer. Biochemical Pharmacology
2012; 83: 443–451.
Yamaoka K, Tanigawara Y, Nakagawa T and Uno T. Pharmacokinetic
analysis program (MULTI) for microcomputer. Journal of
Pharmacobio-Dynamics 1981; 4: 879–885.
Yang XF, Wang NP and Zeng FD. Effects of the active components of
some Chinese herbs on drug-metabolizing enzymes. Zhongguo
Zhong Yao Za Zhi, 2002; 27, 325–328.

Isolation of volatiles from Nigella sativa seeds using

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