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. Isolation of volatiles from Nigella sativa seeds 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 Biomed. Chromatogr. 2013; 27: 938–945 Figure 1. Schematic diagram of microwave-assisted extraction. Copyright © 2013 John Wiley & Sons, Ltd. 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 wileyonlinelibrary.com/journal/bmc 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. Copyright © 2013 John Wiley & Sons, Ltd. v¼ Vmax S Km þ S (1) Biomed. Chromatogr. 2013; 27: 938–945 Isolation of volatiles from Nigella sativa seeds 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. Copyright © 2013 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc 941 Figure 2. Effects of added water volume on extraction efﬁciency of volatiles from Nigella sativa seeds using microwave-assisted extraction (MAE). Biomed. Chromatogr. 2013; 27: 938–945 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. wileyonlinelibrary.com/journal/bmc Copyright © 2013 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2013; 27: 938–945 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 Ethoxyresorufin concentration (µM) 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 De-ethylation (nmol/min/mg) 0.5 De-ethylation (nmol/min/mg) De-ethylation (nmol/min/mg) 0 De-ethylation (nmol/min/mg) De-ethylation (nmol/min/mg) 0.12 0.5 1 1.5 2 2.5 Ethoxyresorufin concentration (µM) De-ethylation (nmol/min/mg) De-ethylation (nmol/min/mg) Isolation of volatiles from Nigella sativa seeds 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 De-ethylation (nmol/min/mg) De-ethylation (nmol/min/mg) Ethoxyresorufin concentration (µM) 0.06 ME 0.05 0.04 0.03 0.02 0.01 0 0 0.5 1 1.5 2 2.5 Ethoxyresorufin concentration (µM) Biomed. Chromatogr. 2013; 27: 938–945 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. Copyright © 2013 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc 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. wileyonlinelibrary.com/journal/bmc 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 Copyright © 2013 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2013; 27: 938–945 Isolation of volatiles from Nigella sativa seeds 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 Biomed. 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