Physicochemical properties and stability of black cumin (Nigella sativa) seed oil as affected by different extraction methods

Industrial Crops and Products 57 (2014) 52–58

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Physicochemical properties and stability of black cumin (Nigella sativa) seed oil as affected by different extraction methods Mustafa Kiralan a,∗ , Gülcan Özkan b , Ali Bayrak c , Mohamed Fawzy Ramadan d,e a

Abant Izzet Baysal University, Faculty of Engineering and Architecture, Department of Food Engineering, Bolu, Turkey Suleyman Demirel University, Faculty of Engineering, Department of Food Engineering, Isparta, Turkey Ankara University, Faculty of Engineering, Department of Food Engineering, Ankara, Turkey d Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig 44519, Egypt e Scientific Research Deanship, Umm Al-Qura University, Makkah, Saudi Arabia b c

a r t i c l e

i n f o

Article history: Received 1 December 2013 Received in revised form 28 February 2014 Accepted 19 March 2014 Available online 12 April 2014 Keywords: Nigella sativa Oil Extraction Cold-pressing Microwave Oxidative stability

a b s t r a c t Black cumin (Nigella sativa) oil (BCO) was recovered using different extraction techniques including solvent free system (cold-pressing) and solvent extracted systems (Soxhlet and microwave assisted). Oils were analyzed for the composition of fatty acids and bioactive compounds (sterols, tocopherols, chlorophyll, carotenoid and phenolics profile) and for some physicochemical properties [free fatty acid, peroxide value (PV), refractive index, and ultraviolet (UV) absorption at K232 and K270 ]. Antiradical power (AP) of oils was also evaluated, wherein cold-pressed oil had stronger AP than solvent extracted oils. Phenolic profiles analyzed by HPLC revealed that thymoquinone was the main phenolic compound wherein high levels of benzoic and p-hydroxy benzoic acids were found in cold pressed-BCO. Oxidative stability (OS) of oils was evaluated during accelerated oxidation conditions (oven test at 60 ◦ C and Rancimat test at 110 ◦ C). The greatest induction period was 19.6 h for Soxhlet-extracted BCO, and the lowest induction period was 3.48 h for cold-pressed BCO. PV of cold-pressed BCO reached 85.3 meq O2 /kg oil, while PV of the other extracted oils were under 27.0 meq O2 /kg oil at the end of storage period. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Black cumin (Nigella sativa) is a spice native to Mediterranean region. The used part of plant is the seeds which is utilized worldwide for edible and medicinal applications (Ramadan, 2007; Cemek et al., 2008). Seeds are added to some food products such as paste, pastry, cheese, pickles and bakery products for flavoring (D’Antuono et al., 2002; Cheikh-Rouhou et al., 2007). N. sativa seed components have been used to prepare functional cosmetic and dietary supplemental products. Studies were conducted on pharmacological properties of N. sativa essential, fixed and cold-pressed oil (Ramadan, 2007; Lutterodt et al., 2010). Black cumin fixed seed oil (BCO) is rich in essential fatty acids as well as bioactive sterols and tocols (Ramadan and Mörsel, 2002; Ramadan, 2013; Piras et al., 2013). The need for widely usable and easily available bioactive lipids and natural antioxidants continues to grow. Methods used for oil extraction may alter minor constituents that have functional properties and contribute to oxidation stability (OS). N. sativa oil

∗ Corresponding author. Tel.: +90 374 253 4640; fax: +90 374 253 4558. E-mail address: [email protected] (M. Kiralan). http://dx.doi.org/10.1016/j.indcrop.2014.03.026 0926-6690/© 2014 Elsevier B.V. All rights reserved.

has been usually produced by conventional solvent extraction (D’Antuono et al., 2002; Ramadan and Mörsel, 2002). However, cold pressing was used to keep away hazardous of solvent extraction (Ramadan, 2013). Over the last few years, increased interest in cold-pressed oils has been observed as these oils have high nutritive properties. The cold pressing procedure is becoming an interesting substitute for conventional practices because of consumers’ desire for natural and safe food products (Parry et al., 2006; Lutterodt et al., 2010). Cold pressing is a technology which involves no heat or chemical treatments during oil extraction. Cold pressing also involves no refining and may contain a high level of lipophilic phytochemicals including natural antioxidants. Microwave-assisted extraction (MAE) is a new extraction technology used for the extraction of nutraceuticals, which is based on combination of microwave and conventional solvent extraction. This technique which is used in extraction of essential oil has many advantages such as short time, less solvent usage and higher extraction yield (Wang and Weller, 2006; Zigoneanu et al., 2008). Liu et al. (2013) isolated volatile compounds from black cumin seeds using MAE. The main compounds emitted were thymoquinone (38.23%), p-cymene (28.61%), 4-isopropyl-9-methoxy1-methyl-1-cyclohexene (5.74%), longifolene (5.33%), ␣-thujene (3.88) and carvacol (2.31%). Atta (2003) used two methods

M. Kiralan et al. / Industrial Crops and Products 57 (2014) 52–58

(Soxhlet and cold pressing) methods to extract oil from N. sativa seeds. Both methods affected the oil quality such as melting point, specific gravity, refractive index, color, free fatty acids, peroxide value (PV), iodine value, saponification number, lipid classes, fatty acid profile and sterol composition. Research on BCO extracted using conventional solvent methods (Cheikh-Rouhou et al., 2007; Ramadan and Mörsel, 2007) and cold pressing (Ramadan et al., 2012; Ramadan, 2013) studied physicochemical properties, antiradical power (AP) and OS of oil. To the best of our knowledge, there is no information about MAE-extracted BCO in the literature. Thus, the aim of this study was to compare the effects of three different extraction methods used to recover BCO on some physicochemical properties, AP and OS of BCO. 2. Material and methods 2.1. Material N. sativa seeds were supplied from a local spice market (Konya, Turkey). Analytical-grade solvents were purchased from Sigma (St. Louis, MO, USA). All reference substances for phytosterols, phenolics (p-hydroxy benzoic acid, benzoic acid, cinnamic acid and thymoquinone), fatty acid methyl ester mix and tocopherols (␣, ␤, ␥ and ␦ isomers) were purchased from Sigma. 2.2. Methods 2.2.1. Oil extraction 2.2.1.1. Cold pressing. Black cumin seeds were pressed at room temperature (25 ◦ C) without any thermal treatment. Mesilla was stored for one night at room temperature to separate oil phase from Mesilla then oil was filtered over anhydrous sodium thiosulphate and cotton filter using glass funnel. 2.2.1.2. Conventional Soxhlet extraction. Seeds were extracted using n-hexane in a Soxhlet apparatus for 4 h. 2.2.1.3. Microwave-assisted extraction (MAE). MAE extraction was carried out with a focused open-vessel microwave system with 500 mL short-necked flask (Milestone, Italy). The maximum output power of the microwave apparatus was 1000 W with 2450 MHz of microwave radiation frequency. The reactor time, temperature and power were controlled using the “easy-WAVE” software package. Temperature was monitored by a shielded thermocouple (ATC300) inserted directly into the sample container and by an external infrared sensor, and controlled by a feedback to the microwave power regulator. According to our pre-experiments we carried out microwave treatment at low temperature. The extraction was continued at 45 ◦ C and atmospheric pressure until no more oil was obtained. Temperature program was as follows: 20 ◦ C to 45 ◦ C in 10 min and hold at 45 ◦ C for 40 min. A cooling system outside the microwave cavity condensed the extraction continuously. For the different extraction techniques, recovered BCO was stored at −18 ◦ C in darkness using amber glass bottles without headspace until analysis. 2.2.2. Determination of physicochemical properties of BCO Free fatty acid (FFA), peroxide value (PV), refractive index (RI) at 20 ◦ C, and UV absorption characteristics (K232 and K270 ) were determined according to AOCS Official Methods (1997) Ca 5a-40, Cd 8-53, Cc 7-25 and Ch 5-91, respectively. Chlorophyll and carotenoid pigments were determined using the method of Mínguez-Mosquera et al. (1991). The chlorophyll and carotenoid fractions in the absorption spectrum were determined at 670 and 470 nm, respectively. Results are given as milligrams per kg of oil. Chlorophyll and carotenoid content were calculated using Eqs. (1) and (2); where

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A() is the absorbance and L is the spectrophotometer cell thickness (10 mm), respectively. For chlorophyll (in mg/kg) = For carotenoid (in mg/kg) =

 A670 × 106  613 × 100 × L

;

 A470 × 106  2000 × 100 × L

(1) (2)

All analyses were carried out using three replications and the results were averaged. 2.2.3. Determination of fatty acid composition of BCO The fatty acid composition of the oils was determined by gas chromatography (GC) as fatty acid methyl esters (FAME). FAME were prepared according to the official method of the IUPAC (1987). A chromatographic analysis was performed in a Shimadzu GC-2010 chromatograph using a DB-23 fused-silica capillary column (30 m, 0.25 mm i.d., 0.25 ␮m film thickness, Agilent JandW, USA). Helium was used as a carrier gas at a flow rate of 1.00 mL/min. The column temperature was isothermal at 190 ◦ C wherein the injector and detector temperatures were 230 ◦ C and 240 ◦ C, respectively. FAME were identified by comparison of their retention times with those of the reference standards. 2.2.4. Determination of sterol composition Sterol analysis of oils was carried out according to method of ISO-12228 (1999). An HP 7890A series GC equipped with a flame ionization detector and capillary column, HP-5 (30 m length × 0.32 mm i.d. × 0.25 ␮m film thickness) was used. The carrier gas was He with a flow rate of 2 mL/min and a split ratio of 40:1. The injector and detector temperatures were set to 280 and 290 ◦ C. The GC oven was programmed at 260 ◦ C for 50 min. The result of each sterol compound was expressed as percent concentration. 2.2.5. Determination of tocopherol composition Tocopherols (␣, ␤, ␥ and ␦-tocopherols) were analyzed using modified method following AOCS (1997). Tocopherols were evaluated using high performance liquid chromatography (HPLC) with direct injection of BCO in a mixture of heptane:tetrahydrofuran (95:5, v/v) solution. Detection and quantification was carried out with a SCL-10Avp System controller, SIL–10ADvp Autosampler, LC-10ADvp pump, CTO-10 Avp column heater and fluorescence detector with wavelengths set at 295 nm for excitation and 330 nm for emission. The 15 cm × 4.6 mm i.d. column used was filled with Supelcosil Luna, 5 ␮m (Supelco, Inc. Bellefonte, PA). The mobile phase consisted of heptane/tetrahydrofuran (95:5, v/v) at a flow rate of 1.2 mL/min and the injection volume 10 ␮L. The data were integrated and analyzed using the Shimadzu Class-VP Chromatography Laboratory Automated Software system. Standard of ␣, ␤, ␥ and ␦ isomers of tocopherols were dissolved in hexane and used for identification and quantification of peaks. The amount of tocopherols in the oils was calculated as mg tocopherols per kg oil using external calibration curves (r = 0.999), which were obtained for each tocopherol standard. All chromatographic analysis was carried out for three replications and the results were averaged. 2.2.6. Determination of total phenols (TP) and HPLC characterization of phenolics Phenolics of the BCO samples were isolated from a solution of oil extract in hexane by triple-extraction with water:methanol (80:20, v/v). The solvent was evaporated in a rotary evaporator at 35 ◦ C under vacuum. The residue was dissolved in methanol. The total phenols (TP) content of the extracts was determined according to the Folin–Ciocalteu spectrophotometric method (T70 + UV/VIS spectrophotometer, PG Instruments, England) at 765 nm using a gallic acid calibration curve (r2 = 0.999). The results were expressed as mg of gallic acid per kilogram of oil.

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M. Kiralan et al. / Industrial Crops and Products 57 (2014) 52–58

Table 1 Solvent gradient conditions for HPLC. Final time

A (%)

B (%)

(Initial) 3 18 20 30 40 50 55 65 70 75 80

95 84 84 84 84 84 60 55 50 45 0 0

5 16 16 16 16 16 40 45 50 55 100 100

Solvent A: acetic acid:water (2:98, v/v); solvent B: methanol.

The phenolic profiles in BCO samples were determined using HPLC. Phenolics of the BCO samples were isolated from a solution of oil extract in hexane by triple-extraction with water:methanol (80:20, v/v). The solvent was evaporated in a rotary evaporator at 35 ◦ C under vacuum. The residue was dissolved in methanol, and then filtered by a 0.45-␮m pore size membrane filter (Vivascience AG, Hannover, Germany). Detection and quantification was carried out using HPLC with a SCL-10Avp System controller, a SIL–10AD vp Autosampler, a LC-10AD vp pump, a DGU-14a degasser, a CTO-10 A vp column heater and a diode array detector (DAD) with wavelengths set at 278 nm. The 250 × 4.6 mm i.d., 5 ␮m column used was filled with Luna Prodigy, 5 ␮m. The flow rate was 1 mL/min, injection volume was 10 ␮L and the column temperature was set at 30 ◦ C. Gradient elution of two solvents was used: solvent A consisted of acetic acid:water (2:98, v/v), solvent B: methanol and the gradient program used is given in Table 1. The data were integrated and analyzed using the Shimadzu Class-VP Chromatography Laboratory Automated Software system. The amount of phenolic compounds in the extract was calculated as mg/100 g using external calibration curves, constructed for each pure phenolic standard. All extractions and chromatographic analysis was carried out twice and the results were averaged. 2.2.7. Antiradical power (AP) of BCO The AP was measured using 2,2-diphenyl-1-picrylhydrazyl(DPPH• ) radical scavenging method and the results were given as % of inhibition (Gülcin, 2012). A 50 ␮L aliquot of BCO phenolic extract, in Tris–HCl buffer (50 mM, pH 7.4) was mixed with 450 ␮L of Tris–HCl buffer (50 mM) and 1.0 mL of DPPH• (0.1 mM, in methanol). After 30 min incubation in darkness and at ambient temperature, the resultant absorbance was recorded at 517 nm. The percentage inhibition was calculated using the following equation: Inhibition (%) =





(Absorbance of control − Absorbance of sample) × 100 Absorbance of control

Estimated DPPH• inhibition (%) values are presented as the average of quadruplicate analyses. All spectrophotometric analyses were repeated three times for each type of extract and the results were averaged. 2.2.8. Determination of OS of BCO (accelerated oxidation experiments) Oil samples (60 g) were placed in a series of glass bottles stored for 27 days. The oxidation reaction was accelerated in a forced-draft air oven set at 60 ± 2 ◦ C for up to 27 days. Oxidation was monitored in three day intervals over 27 days storage and analyzed for PV, conjugated dienes and trienes value to follow the oxidative changes. In addition, OS was measured with a Rancimat 743 apparatus (Metrohm Co., Basilea, Switzerland) according to AOCS Official Method (1997) Cd 12b-92 to determine the induction time for BCO samples. The temperature was set at 110 ◦ C and 20 L/h air flow, the oil sample was 3 g and the stability was expressed as oxidation induction time (h). 3. Results and discussion 3.1. Impact of extraction method on physicochemical properties of BCO Physicochemical properties of BCO are presented in Table 2. Level of FFA for cold-pressed BCO (7.49%) was lower than oils recovered from solvent and MAE extraction (9.51% and 9.28%, respectively). FFA level of BCO from solvent extraction were lower than results of Cheikh-Rouhou et al. (2007) but higher than results of Atta (2003). PV of oils obtained using cold pressing, MAE and Soxhlet extraction were 31.32, 21.45 and 25.23 meq O2 /kg oil, respectively. The PV for solvent-extracted oil was higher than those of solvent-extracted BCO reported by Atta (2003) (10.7 meq O2 /kg oil), Cheikh-Rouhou et al. (2007) (4.35–5.65 meq O2 /kg oil) and Khoddami et al. (2011) (6.72–9.78 meq O2 /kg oil). Furthermore, PV of cold-pressed BCO had higher value (31.3 meq O2 /kg oil) than for cold-pressed BCO reported by Atta (2003). RI value was the highest for cold-pressed BCO (1.47326), followed by microwave and Soxhlet-extracted BCO having values of 1.47176 and 1.47142, respectively. These values coincide with those obtained by Atta (2003). Specific extinction values (K232 and K270 ) of BCO samples were similar. K232 value of oils changed between 3.11 and 3.71 and K270 value of oils was recorded between 0.58 and 0.66. These specific extinction values were found to be higher than those reported by Khoddami et al. (2011) and were similar with the findings of Ramadan and Mörsel (2004). 3.2. Impact of extraction method on composition of fatty acids and bioactive lipids Table 3 reports the results of BCO fatty acid composition. The major fatty acids in BCO were linoleic (C18:2) and oleic (C18:1)

Table 2 Physicochemical properties of BCO extracted using different methods. Physicochemical property Free fatty acid (as oleic acid %) PV (meq O2 /kg oil) Refractive index (at 20 ◦ C) K232 K270 Chlorophyll (mg/kg) Carotenoid (mg/kg)

Extraction method Cold-pressing 7.49 31.32 1.47326 3.71 0.66 0.30 0.18

± ± ± ± ± ± ±

0.96 0.74 0.00 0.12 0.05 0.00 0.00

MAE 9.51 21.45 1.47176 3.17 0.58 0.43 0.24

Soxhlet ± ± ± ± ± ± ±

0.36 0.79 0.00 0.07 0.05 0.00 0.00

9.28 25.23 1.47142 3.11 0.66 0.81 0.40

± ± ± ± ± ± ±

0.63 1.56 0.00 0.06 0.06 0.01 0.00

M. Kiralan et al. / Industrial Crops and Products 57 (2014) 52–58 Table 3 Fatty acid (%), tocopherols (mg/kg) and sterols (%) profile of BCO as affected by extraction method. Extraction methods Cold-pressing C14:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:2 C24:0 ␣-Tocopherol ␤-Tocopherol ␥-Tocopherol ␦-Tocopherol Campestrol Stigmasterol ␤-Sitosterol 5 -Avenasterol 7 -Stigmasterol 7 -Avenasterol

0.13 12.01 0.25 0.06 0.03 2.77 23.95 57.49 0.25 0.15 0.27 2.33 0.31 7.30 15.47 34.23 8.37 14.88 17.48 58.05 7.27 1.24 1.62

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.00 0.09 0.01 0.00 0.00 0.09 0.12 0.08 0.00 0.00 0.01 0.04 0.02 0.46 0.29 0.21 0.12 0.19 0.54 1.01 0.74 0.10 0.31

MAE 0.14 11.85 0.23 0.07 0.04 2.95 24.13 57.18 0.23 0.16 0.29 2.45 0.28 4.80 8.00 9.57 1.80 13.47 17.49 57.49 8.80 1.14 1.28

Soxhlet ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.00 0.07 0.01 0.00 0.00 0.04 0.11 0.07 0.00 0.01 0.00 0.01 0.02 0.36 0.36 0.51 0.10 0.43 0.43 0.82 0.23 0.26 0.25

0.14 11.84 0.24 0.07 0.04 2.81 23.85 57.52 0.27 0.16 0.31 2.49 0.26 5.33 7.80 8.57 1.63 14.71 18.70 57.41 7.34 0.89 1.58

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.06 0.00 0.00 0.00 0.02 0.07 0.07 0.01 0.00 0.01 0.01 0.02 0.12 0.17 0.21 0.06 0.34 0.59 0.17 0.58 0.09 0.34

for unsaturated fatty acids, while palmitic acid (C16:0) was the major saturated fatty acid. These results are in agreement with previously reported data (Atta, 2003; Ramadan and Mörsel, 2004; Cheikh-Rouhou et al., 2007; Ramadan et al., 2012). The percentage of linoleic acid ranged between 57.1% for MAE-oil and 57.5% for Soxhlet-extracted oil. Similar values were reported by Atta (2003) (47.5–49.0%), Ramadan and Mörsel (2004) (57.3%) but higher than those reported by Cheikh-Rouhou et al. (2007) (49.1–50.3%) and Ramadan et al. (2012) (55.3%). Percentages for oleic acid in BCO were 23.8% for Soxhlet-extracted oil and 24.1% for MAE-oil. Similar results were reported by Ramadan and Mörsel (2004) (24.1%), Cheikh-Rouhou et al. (2007) (23.7–25.0%), and Ramadan et al. (2012) (24.1%). The content of palmitic acid ranged from 11.84 (Soxhlet-extracted BCO) to 12.0% (cold-pressed BCO). These values are similar to those reported by Atta (2003) (12.1%) for cold-pressed oil and is relatively lower than that reported for soxhlet-extracted oil (13.0%) (Ramadan and Mörsel, 2004), for cold-pressed BCO (12.5%) (Ramadan et al., 2012). The values were also lower than those reported by Cheikh-Rouhou et al. (2007) (17.2% for Tunisian oil and 18.4% for Iranian oil), but higher than that reported by Atta (2003) for Soxhlet-extracted oil (9.9%). BCO was characterized by the relative high levels of polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids (MUFA). MUFA have been shown to lower “bad” LDL cholesterol (low density lipoproteins) and retain “good” HDL cholesterol (high density lipoproteins). This is the major benefit of olive oil over the highly polyunsaturated edible oils, wherein PUFA reduce both the “bad” as well as the “good” cholesterol levels in the blood (Ramadan et al., 2010). The fatty acid profile and high amounts of PUFA makes the BCO a special component for nutritional applications. Tocochromanols (vitamin E) are the most important lipid soluble antioxidants and they represent an essential nutrient for human health. These molecules are important for scavenging free radicals and inhibiting lipid peroxidation in biological membranes. The tocochromanols comprise eight chemically distinct compounds that are separated into tocopherols and tocotrienols, according to saturation of the hydrophobic tails. Tocopherols contain a fully saturated tail, whereas tocotrienols have three unsaturated double bonds. The different tocopherol and tocotrienol isomers,

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that is, alpha (␣), beta (␤), gamma (␥), and delta (␦), are distinguished by the locations of methyl groups on the chromanol ring. Tocopherol content of BCO is given in Table 3. The content of ␥-tocopherol was the highest, with levels ranging from 8.57 to 34.23 ppm. However, ␦-tocopherol was the lowest tocopherol isomer found in solvent-extracted oils (1.80 ppm in MAE-oil and 1.63 ppm in Soxhlet-extracted oil). This result was lower than those reported by Ramadan and Mörsel (2004) for solventextracted oil and by Ramadan et al. (2012) for cold-pressed oils. Levels of sterols in vegetable oils are used for the identification of oils, oil derivatives and for the determination of oil quality. Furthermore, the concentration of sterols has been reported to be little affected by environmental factors and/or by cultivation of new breeding lines (Ramadan and Mörsel, 2007). The values obtained for sterol composition are listed in Table 3. ␤-sitosterol was the major sterol found in all oil samples (57.4–58.1%), followed by stigmasterol and campesterol ranged between 17.5–18.7% and 13.5–14.9%, respectively. Besides these compounds, 5 -avenasterol, 7 -stigmasterol and 7 -avenasterol were also found in all samples. ␤-sitosterol content of oils was found higher than those reported in Tunisian and Iranian BCO (44.5 and 53.9%, respectively) (Cheikh-Rouhou et al., 2007). Stigmasterol and campesterol were most abundant sterol compounds after ␤-sitosterol in Tunisian and Iranian BCO. Stigmasterol and campesterol content of Tunisian and Iranian oil were found to be 20.9 and 13.7%, and 16.5 and 12.1%, respectively. Stigmasterol content of BCO was similar with Iranian oil and lower than Tunisian oil. Campesterol level of BCO was within the values reported by Cheikh-Rouhou et al. (2007). Chlorophyll and carotenoid were found in the highest amounts (0.81 and 0.40 mg/kg, respectively) in Soxhlet-extracted BCO, but measured in lower values (0.30 and 0.18 mg/kg, respectively) in cold-pressed BCO. Chlorophyll amount of BCO samples were lower than oils obtained from Tunisian and Iranian N, sativa seeds (Cheikh-Rouhou et al., 2007).

3.3. Impact of extraction method on phenolics profile and AP The results for TP and inhibition rate are given in Table 4. TP contents were in the range of 15.1 (MAE-oil) and 36.0 (cold-pressed oil) mg gallic acid/kg oil. Total phenol content of oils was lower than the results for Tunisian and Iranian BCO (245 and 309 mg gallic acid/kg oil, respectively). Inhibition rate results were in line with the results of total phenol content. The highest inhibition rate observed for cold-pressed BCO (78.4%), while the lowest rate was (61.7%) for MAE-oil. The profile of phenolics in BCO is shown in Table 4 and Fig. 1. Thymoquinone was main identified phenolic compound. Cold pressed-oil contained the highest level of thymoquinone (14.4 ␮g/g). Soxhlet-extracted oil (6.20 ␮g/g) and MAE-oil (5.65 ␮g/g) have lower levels of thymoquinone. Levels of thymoquinone in the present study were found to be lower than those reported by Lutterodt et al. (2010) who reported that the highest amount of thymoquinone was found to be 8.73 mg/g of oil, and the lowest thymoquinone concentration was 3.48. Similarly to thymoquinone, the highest level of benzoic acids and p-hydroxy benzoic acid, were found in cold pressed-oil (4.15 and 1.50 ␮g/g, respectively). The level of phenolic acids for Soxhlet-extracted oil (2.65 and 0.20 ␮g/g, respectively) and MAEoil (2.15 and 0.20 ␮g/g, respectively) were relatively the same. The lowest level of cinnamic acid was found in cold pressedoil (0.03 ␮g/g), while the level of cinnamic acid for MAE-oil and Soxhlet-extracted oil (0.05 and 0.06 ␮g/g, respectively) were relatively the same.

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Table 4 Total phenolics content, inhibition rate, phenolic composition and induction time of BCO as affected by extraction method. Extraction methods Cold-pressing Total phenol (mg gallic acid/kg oil) Inhibition rate (%) p-Hydroxy benzoic acid (␮g/g) Benzoic acid (␮g/g) Cinnamic acid (␮g/g) Thymoquinone (␮g/g) Induction time (h, at 110 ◦ C)

36.05 78.45 1.50 4.15 0.03 14.40 3.48

± ± ± ± ± ± ±

0.50 0.79 0.00 0.07 0.00 0.57 0.21

3.4. Impact of extraction method and storage on OS of BCO The results of induction period are given in Table 4. Induction time was the highest (19.6 h) for Soxhlet-extracted oil, followed by MAE-oil (18.4 h) and was the lowest for cold-pressed BCO (3.48 h). Lutterodt et al. (2010) showed that induction periods of different BCO were in the range between 76 and 157 h at 80 ◦ C. Because different temperatures were used; we cannot compare our results with the results of Lutterodt et al. (2010). PV is widely used assay for the measurement of oxidative rancidity in oils and fats. Hydroperoxide is the primary product of lipid oxidation; therefore, determination of PV can be used as oxidative index during the early stage of lipid oxidation (Mohdaly et al., 2010). The results for PV for oven test at 60 ◦ C are presented in Fig. 2A. PV of cold-pressed BCO had higher PV (85.3 meq O2 /kg oil) than PV of solvent-extracted oils (25.8 meq O2 /kg oil for Soxhlet-extracted oil and 26.8 meq O2 /kg oil for MAE-oil) at the end of storage period.

MAE 15.19 61.69 0.20 2.15 0.05 5.65 18.46

Soxhlet ± ± ± ± ± ± ±

0.38 0.88 0.00 0.07 0.00 0.07 0.22

21.44 65.58 0.20 2.65 0.06 6.20 19.62

± ± ± ± ± ± ±

0.80 0.40 0.00 0.07 0.00 0.28 0.11

Organic solvents extract actually more polar lipids than cold pressing. Thus, synergism of polar lipids with other antioxidant may increase OS of solvent-extracted oils. Ramadan and Mörsel (2004) reported that crude solvent extracted BCO reached up 51 meq O2 /kg oil during storage at 60 ◦ C. Conjugated dienes (CD) and trienes (CT) are good parameter for the determination of OS of oils. Formation of hydroperoxides is coincidental with conjugation of double bonds in PUFA, measured by absorptivity at the UV spectrum (Ramadan and Mörsel, 2004). Lipids containing methylene-interrupted dienes or polyenes show a shift in their double bond position during oxidation. The resulting CD exhibit intense absorption at 232 nm, similarly CT absorb at 270 nm. The increase in CD and CT contents is proportional to the uptake of oxygen. The greater the levels of CD and CT in oil the lower will be the OS (Mohdaly et al., 2010). Fig. 2B and C show the formation of CD and CT during Shaal oven test. Cold-pressed BCO contained the highest value for K232 and

Fig. 1. HPLC Chromatograms of (A) standard phenolic compounds (1) p-hydroxy benzoic acid, (2) benzoic acid, (3) cinnamic acid, (4) thymoquinone; (B) cold-pressed BCO; (C) MAE-BCO; and (D) Soxhlet-extracted BCO.

M. Kiralan et al. / Industrial Crops and Products 57 (2014) 52–58

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Fig. 2. Changes in PV (A), K232 (B), and K270 (C) for different extracted BCO during storage at 60 ◦ C. Error bars show the variations of three determinations in terms of standard deviation.

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K270 (12.8 and 1.4, respectively) and these results are in line with the results of PV after 27 days of oven storage (60 ◦ C). Soxhlet and MAE-oils remained stable during storage time and this tendency observed by Ramadan and Mörsel (2004). Maximum values of K232 and K270 were 3.94, 0.78 for Soxhlet-extracted oil and 3.94, 0.73 for MAE-oil. According to the results from oven test and Rancimat test, BCO is highly stable to oxidation. Lutterodt et al. (2010) emphasized that crude BCO may be served as a natural antioxidant. Oxidation stability of sunflower (Ramadan, 2013) and corn oil (Ramadan and Wahdan, 2012) were increased by blending with BCO. Oxidation stability of cold-pressed BCO was lower than solvent-extracted oils. The results of AP (Table 4) showed that cold-pressed BCO had stronger AP than solvent-extracted oils. Beside strong AP, TP and tocopherol content of cold-pressed BCO were higher than solvent-extracted oils. OS of oils depends on some factors such as tocopherols and phenolics content and profile. As known, phenolic compounds contribute to the overall antioxidant capacity of oils. In this research, no correlation between total phenolics, tocopherols and OS during accelerated oxidation test. However, good correlation observed between total phenols and AP. Individual phenolic compounds contribute to OS of oils. No relationship was observed between TP, thymoquinone content and OS for BCO extracted using different methods (Lutterodt et al., 2010). 4. Conclusion Composition of BCO extracted using different techniques had been investigated. The results of our study showed that the extraction technique affect the composition and the quality of BCO. Cold-pressed BCO was more susceptible to accelerated oxidation than solvent-extracted oils. MAE-oil is rich in thymoquinone and may be used a natural thymoquinone source. Acknowledgment We thank Scientific Research Projects Fund of Abant Izzet Baysal University in Turkey for providing fund support of the project under contract grant number 2012.09.01.491. References AOCS, 1997. Determination of tocopherols and tocotrienols in vegetable oils and fats by HPLC. In: Official Methods and Recommended Practices of the American Oil Chemists’ Society, fifth ed. American Oil Chemists’ Society (AOCS), Champaign, IL, pp. Ce 8–89. Atta, M.B., 2003. Some characteristics of nigella (Nigella sativa L.) seed cultivated in Egypt and its lipid profile. Food Chem. 83, 63–68. Cemek, M., Büyükokuro˘glu, M.E., Bayıro˘glu, F., Koc¸, M., Arora, R., 2008. Herbal Radiomodulators: Applications in Medicine, Homeland Defence and Space. CABI, Wallingford, UK, pp. 56. Cheikh-Rouhou, S., Besbes, S., Hentati, B., Blecker, C., Deroanne, C., Attia, H., 2007. Nigella sativa L.: chemical composition and physicochemical characteristics of lipid fraction. Food Chem. 101, 673–681. D’Antuono, L.F., Moretti, A., Lovato, A.F.S., 2002. Seed yield, yield components, oil content and essential oil content and composition of

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