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Industrial Crops and Products 57 (2014) 52–58

Contents lists available at ScienceDirect

Industrial Crops and Products

jo u r n al homep age: www.elsev ier .com/ locate / indcrop

hysicochemical properties and stability of black cuminNigella sativa) seed oil as affected by different extraction methods

ustafa Kiralana,∗, Gülcan Özkanb, Ali Bayrakc, Mohamed Fawzy Ramadand,e

Abant Izzet Baysal University, Faculty of Engineering and Architecture, Department of Food Engineering, Bolu, TurkeySuleyman Demirel University, Faculty of Engineering, Department of Food Engineering, Isparta, TurkeyAnkara University, Faculty of Engineering, Department of Food Engineering, Ankara, TurkeyBiochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig 44519, EgyptScientific Research Deanship, Umm Al-Qura University, Makkah, Saudi Arabia

r t i c l e i n f o

rticle history:eceived 1 December 2013eceived in revised form 28 February 2014ccepted 19 March 2014vailable online 12 April 2014

eywords:igella sativa

a b s t r a c t

Black cumin (Nigella sativa) oil (BCO) was recovered using different extraction techniques including sol-vent free system (cold-pressing) and solvent extracted systems (Soxhlet and microwave assisted). Oilswere analyzed for the composition of fatty acids and bioactive compounds (sterols, tocopherols, chloro-phyll, carotenoid and phenolics profile) and for some physicochemical properties [free fatty acid, peroxidevalue (PV), refractive index, and ultraviolet (UV) absorption at K232 and K270]. Antiradical power (AP) ofoils was also evaluated, wherein cold-pressed oil had stronger AP than solvent extracted oils. Phenolicprofiles analyzed by HPLC revealed that thymoquinone was the main phenolic compound wherein high

ilxtractionold-pressingicrowavexidative stability

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 at110 ◦C). The greatest induction period was 19.6 h for Soxhlet-extracted BCO, and the lowest inductionperiod was 3.48 h for cold-pressed BCO. PV of cold-pressed BCO reached 85.3 meq O2/kg oil, while PV ofthe other extracted oils were under 27.0 meq O2/kg oil at the end of storage period.

. Introduction

Black cumin (Nigella sativa) is a spice native to Mediterraneanegion. The used part of plant is the seeds which is utilized world-ide for edible and medicinal applications (Ramadan, 2007; Cemek

t al., 2008). Seeds are added to some food products such asaste, pastry, cheese, pickles and bakery products for flavoringD’Antuono et al., 2002; Cheikh-Rouhou et al., 2007). N. sativa seedomponents have been used to prepare functional cosmetic andietary supplemental products. Studies were conducted on phar-acological properties of N. sativa essential, fixed and cold-pressed

il (Ramadan, 2007; Lutterodt et al., 2010). Black cumin fixed seedil (BCO) is rich in essential fatty acids as well as bioactive sterolsnd tocols (Ramadan and Mörsel, 2002; Ramadan, 2013; Piras et al.,013).

The need for widely usable and easily available bioactive lipids

nd natural antioxidants continues to grow. Methods used foril extraction may alter minor constituents that have functionalroperties and contribute to oxidation stability (OS). N. sativa oil

∗ Corresponding author. Tel.: +90 374 253 4640; fax: +90 374 253 4558.E-mail address: mustafakiralan@yahoo.com (M. Kiralan).

ttp://dx.doi.org/10.1016/j.indcrop.2014.03.026926-6690/© 2014 Elsevier B.V. All rights reserved.

© 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 extrac-tion (Ramadan, 2013). Over the last few years, increased interestin cold-pressed oils has been observed as these oils have highnutritive properties. The cold pressing procedure is becoming aninteresting substitute for conventional practices because of con-sumers’ desire for natural and safe food products (Parry et al., 2006;Lutterodt et al., 2010). Cold pressing is a technology which involvesno heat or chemical treatments during oil extraction. Cold pressingalso involves no refining and may contain a high level of lipophilicphytochemicals including natural antioxidants.

Microwave-assisted extraction (MAE) is a new extraction tech-nology used for the extraction of nutraceuticals, which is basedon combination of microwave and conventional solvent extrac-tion. This technique which is used in extraction of essential oil hasmany advantages such as short time, less solvent usage and higherextraction 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 thymo-quinone (38.23%), p-cymene (28.61%), 4-isopropyl-9-methoxy-1-methyl-1-cyclohexene (5.74%), longifolene (5.33%), �-thujene(3.88) and carvacol (2.31%). Atta (2003) used two methods

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M. Kiralan et al. / Industrial C

Soxhlet and cold pressing) methods to extract oil from N. sativaeeds. Both methods affected the oil quality such as melting point,pecific gravity, refractive index, color, free fatty acids, peroxidealue (PV), iodine value, saponification number, lipid classes, fattycid profile and sterol composition. Research on BCO extractedsing conventional solvent methods (Cheikh-Rouhou et al., 2007;amadan and Mörsel, 2007) and cold pressing (Ramadan et al.,012; Ramadan, 2013) studied physicochemical properties, anti-adical power (AP) and OS of oil. To the best of our knowledge, theres no information about MAE-extracted BCO in the literature. Thus,he aim of this study was to compare the effects of three differentxtraction methods used to recover BCO on some physicochemicalroperties, AP and OS of BCO.

. Material and methods

.1. Material

N. sativa seeds were supplied from a local spice market (Konya,urkey). Analytical-grade solvents were purchased from Sigma (St.ouis, MO, USA). All reference substances for phytosterols, phen-lics (p-hydroxy benzoic acid, benzoic acid, cinnamic acid andhymoquinone), fatty acid methyl ester mix and tocopherols (�,, � and � isomers) were purchased from Sigma.

.2. Methods

.2.1. Oil extraction

.2.1.1. Cold pressing. Black cumin seeds were pressed at roomemperature (25 ◦C) without any thermal treatment. Mesilla wastored for one night at room temperature to separate oil phase fromesilla then oil was filtered over anhydrous sodium thiosulphate

nd cotton filter using glass funnel.

.2.1.2. Conventional Soxhlet extraction. Seeds were extractedsing n-hexane in a Soxhlet apparatus for 4 h.

.2.1.3. Microwave-assisted extraction (MAE). MAE extraction wasarried out with a focused open-vessel microwave system with00 mL short-necked flask (Milestone, Italy). The maximum outputower of the microwave apparatus was 1000 W with 2450 MHz oficrowave radiation frequency. The reactor time, temperature and

ower were controlled using the “easy-WAVE” software package.emperature was monitored by a shielded thermocouple (ATC-00) inserted directly into the sample container and by an external

nfrared sensor, and controlled by a feedback to the microwaveower regulator. According to our pre-experiments we carried outicrowave treatment at low temperature. The extraction was con-

inued at 45 ◦C and atmospheric pressure until no more oil wasbtained. Temperature program was as follows: 20 ◦C to 45 ◦C in0 min and hold at 45 ◦C for 40 min. A cooling system outside theicrowave cavity condensed the extraction continuously.For the different extraction techniques, recovered BCO was

tored at −18 ◦C in darkness using amber glass bottles withouteadspace until analysis.

.2.2. Determination of physicochemical properties of BCOFree fatty acid (FFA), peroxide value (PV), refractive index (RI) at

0 ◦C, and UV absorption characteristics (K232 and K270) were deter-ined according to AOCS Official Methods (1997) Ca 5a-40, Cd 8-53,

c 7-25 and Ch 5-91, respectively. Chlorophyll and carotenoid pig-ents were determined using the method of Mínguez-Mosquera

t al. (1991). The chlorophyll and carotenoid fractions in the absorp-ion spectrum were determined at 670 and 470 nm, respectively.esults are given as milligrams per kg of oil. Chlorophyll andarotenoid content were calculated using Eqs. (1) and (2); where

nd Products 57 (2014) 52–58 53

A(�) is the absorbance and L is the spectrophotometer cell thickness(10 mm), respectively.

For chlorophyll (in mg/kg) =(

A670 × 106613 × 100 × L

); (1)

For carotenoid (in mg/kg) =(

A470 × 1062000 × 100 × L

)(2)

All analyses were carried out using three replications and theresults were averaged.

2.2.3. Determination of fatty acid composition of BCOThe fatty acid composition of the oils was determined by gas

chromatography (GC) as fatty acid methyl esters (FAME). FAMEwere prepared according to the official method of the IUPAC (1987).A chromatographic analysis was performed in a Shimadzu GC-2010chromatograph using a DB-23 fused-silica capillary column (30 m,0.25 mm i.d., 0.25 �m film thickness, Agilent JandW, USA). Heliumwas used as a carrier gas at a flow rate of 1.00 mL/min. The col-umn temperature was isothermal at 190 ◦C wherein the injectorand detector temperatures were 230 ◦C and 240 ◦C, respectively.FAME were identified by comparison of their retention times withthose of the reference standards.

2.2.4. Determination of sterol compositionSterol analysis of oils was carried out according to method

of ISO-12228 (1999). An HP 7890A series GC equipped witha flame ionization detector and capillary column, HP-5 (30 mlength × 0.32 mm i.d. × 0.25 �m film thickness) was used. The car-rier 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 ofeach sterol compound was expressed as percent concentration.

2.2.5. Determination of tocopherol compositionTocopherols (�, �, � and �-tocopherols) were analyzed using

modified method following AOCS (1997). Tocopherols were eval-uated using high performance liquid chromatography (HPLC) withdirect injection of BCO in a mixture of heptane:tetrahydrofuran(95:5, v/v) solution. Detection and quantification was carried outwith a SCL-10Avp System controller, SIL–10ADvp Autosampler,LC-10ADvp pump, CTO-10 Avp column heater and fluorescencedetector with wavelengths set at 295 nm for excitation and 330 nmfor emission. The 15 cm × 4.6 mm i.d. column used was filled withSupelcosil Luna, 5 �m (Supelco, Inc. Bellefonte, PA). The mobilephase consisted of heptane/tetrahydrofuran (95:5, v/v) at a flowrate of 1.2 mL/min and the injection volume 10 �L. The data wereintegrated and analyzed using the Shimadzu Class-VP Chromatog-raphy Laboratory Automated Software system. Standard of �, �, �and � isomers of tocopherols were dissolved in hexane and usedfor identification and quantification of peaks. The amount of toco-pherols in the oils was calculated as mg tocopherols per kg oilusing external calibration curves (r = 0.999), which were obtainedfor each tocopherol standard. All chromatographic analysis wascarried out for three replications and the results were averaged.

2.2.6. Determination of total phenols (TP) and HPLCcharacterization of phenolics

Phenolics of the BCO samples were isolated from a solution of oilextract in hexane by triple-extraction with water:methanol (80:20,v/v). The solvent was evaporated in a rotary evaporator at 35 ◦Cunder vacuum. The residue was dissolved in methanol. The totalphenols (TP) content of the extracts was determined according

to the Folin–Ciocalteu spectrophotometric method (T70 + UV/VISspectrophotometer, PG Instruments, England) at 765 nm using agallic acid calibration curve (r2 = 0.999). The results were expressedas mg of gallic acid per kilogram of oil.

54 M. Kiralan et al. / Industrial Crops a

Table 1Solvent gradient conditions for HPLC.

Final time A (%) B (%)

(Initial) 95 53 84 1618 84 1620 84 1630 84 1640 84 1650 60 4055 55 4565 50 5070 45 5575 0 100

S

Ho(3tAoAAlwiactgLcnAa

2

(al4itp

I

TP

80 0 100

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

The phenolic profiles in BCO samples were determined usingPLC. Phenolics of the BCO samples were isolated from a solutionf oil extract in hexane by triple-extraction with water:methanol80:20, v/v). The solvent was evaporated in a rotary evaporator at5 ◦C under vacuum. The residue was dissolved in methanol, andhen filtered by a 0.45-�m pore size membrane filter (VivascienceG, Hannover, Germany). Detection and quantification was carriedut using HPLC with a SCL-10Avp System controller, a SIL–10AD vputosampler, a LC-10AD vp pump, a DGU-14a degasser, a CTO-10

vp column heater and a diode array detector (DAD) with wave-engths set at 278 nm. The 250 × 4.6 mm i.d., 5 �m column used

as filled with Luna Prodigy, 5 �m. The flow rate was 1 mL/min,njection volume was 10 �L and the column temperature was sett 30 ◦C. Gradient elution of two solvents was used: solvent Aonsisted of acetic acid:water (2:98, v/v), solvent B: methanol andhe gradient program used is given in Table 1. The data were inte-rated and analyzed using the Shimadzu Class-VP Chromatographyaboratory Automated Software system. The amount of phenolicompounds in the extract was calculated as mg/100 g using exter-al calibration curves, constructed for each pure phenolic standard.ll extractions and chromatographic analysis was carried out twicend the results were averaged.

.2.7. Antiradical power (AP) of BCOThe AP was measured using 2,2-diphenyl-1-picrylhydrazyl-

DPPH•) radical scavenging method and the results were givens % of inhibition (Gülcin, 2012). A 50 �L aliquot of BCO pheno-ic extract, in Tris–HCl buffer (50 mM, pH 7.4) was mixed with50 �L of Tris–HCl buffer (50 mM) and 1.0 mL of DPPH• (0.1 mM,

n methanol). After 30 min incubation in darkness and at ambientemperature, the resultant absorbance was recorded at 517 nm. Theercentage inhibition was calculated using the following equation:

nhibition (%)

=[

(Absorbance of control − Absorbance of sample)Absorbance of control

]× 100

able 2hysicochemical properties of BCO extracted using different methods.

Physicochemicalproperty

Cold-pressing

Free fatty acid (as oleic acid %) 7.49 ± 0.96

PV (meq O2/kg oil) 31.32 ± 0.74

Refractive index (at 20 ◦C) 1.47326 ± 0.00

K232 3.71 ± 0.12

K270 0.66 ± 0.05

Chlorophyll (mg/kg) 0.30 ± 0.00

Carotenoid (mg/kg) 0.18 ± 0.00

nd Products 57 (2014) 52–58

Estimated DPPH• inhibition (%) values are presented as theaverage of quadruplicate analyses. All spectrophotometric analyseswere repeated three times for each type of extract and the resultswere averaged.

2.2.8. Determination of OS of BCO (accelerated oxidationexperiments)

Oil samples (60 g) were placed in a series of glass bottles storedfor 27 days. The oxidation reaction was accelerated in a forced-draftair oven set at 60 ± 2 ◦C for up to 27 days. Oxidation was mon-itored in three day intervals over 27 days storage and analyzedfor PV, conjugated dienes and trienes value to follow the oxidativechanges.

In addition, OS was measured with a Rancimat 743 appara-tus (Metrohm Co., Basilea, Switzerland) according to AOCS OfficialMethod (1997) Cd 12b-92 to determine the induction time for BCOsamples. 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 oxidationinduction time (h).

3. Results and discussion

3.1. Impact of extraction method on physicochemical propertiesof BCO

Physicochemical properties of BCO are presented in Table 2.Level of FFA for cold-pressed BCO (7.49%) was lower than oilsrecovered from solvent and MAE extraction (9.51% and 9.28%,respectively). FFA level of BCO from solvent extraction were lowerthan results of Cheikh-Rouhou et al. (2007) but higher than resultsof Atta (2003). PV of oils obtained using cold pressing, MAE andSoxhlet extraction were 31.32, 21.45 and 25.23 meq O2/kg oil,respectively. The PV for solvent-extracted oil was higher than thoseof solvent-extracted BCO reported by Atta (2003) (10.7 meq O2/kgoil), Cheikh-Rouhou et al. (2007) (4.35–5.65 meq O2/kg oil) andKhoddami et al. (2011) (6.72–9.78 meq O2/kg oil). Furthermore, PVof cold-pressed BCO had higher value (31.3 meq O2/kg oil) thanfor cold-pressed BCO reported by Atta (2003). RI value was thehighest for cold-pressed BCO (1.47326), followed by microwaveand 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 sampleswere similar. K232 value of oils changed between 3.11 and 3.71 andK270 value of oils was recorded between 0.58 and 0.66. These spe-cific extinction values were found to be higher than those reportedby Khoddami et al. (2011) and were similar with the findings ofRamadan 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. Themajor fatty acids in BCO were linoleic (C18:2) and oleic (C18:1)

Extraction method

MAE Soxhlet

9.51 ± 0.36 9.28 ± 0.6321.45 ± 0.79 25.23 ± 1.56

1.47176 ± 0.00 1.47142 ± 0.003.17 ± 0.07 3.11 ± 0.060.58 ± 0.05 0.66 ± 0.060.43 ± 0.00 0.81 ± 0.010.24 ± 0.00 0.40 ± 0.00

M. Kiralan et al. / Industrial Crops a

Table 3Fatty acid (%), tocopherols (mg/kg) and sterols (%) profile of BCO as affected byextraction method.

Extraction methods

Cold-pressing MAE Soxhlet

C14:0 0.13 ± 0.00 0.14 ± 0.00 0.14 ± 0.01C16:0 12.01 ± 0.09 11.85 ± 0.07 11.84 ± 0.06C16:1 0.25 ± 0.01 0.23 ± 0.01 0.24 ± 0.00C17:0 0.06 ± 0.00 0.07 ± 0.00 0.07 ± 0.00C17:1 0.03 ± 0.00 0.04 ± 0.00 0.04 ± 0.00C18:0 2.77 ± 0.09 2.95 ± 0.04 2.81 ± 0.02C18:1 23.95 ± 0.12 24.13 ± 0.11 23.85 ± 0.07C18:2 57.49 ± 0.08 57.18 ± 0.07 57.52 ± 0.07C18:3 0.25 ± 0.00 0.23 ± 0.00 0.27 ± 0.01C20:0 0.15 ± 0.00 0.16 ± 0.01 0.16 ± 0.00C20:1 0.27 ± 0.01 0.29 ± 0.00 0.31 ± 0.01C20:2 2.33 ± 0.04 2.45 ± 0.01 2.49 ± 0.01C24:0 0.31 ± 0.02 0.28 ± 0.02 0.26 ± 0.02�-Tocopherol 7.30 ± 0.46 4.80 ± 0.36 5.33 ± 0.12�-Tocopherol 15.47 ± 0.29 8.00 ± 0.36 7.80 ± 0.17�-Tocopherol 34.23 ± 0.21 9.57 ± 0.51 8.57 ± 0.21�-Tocopherol 8.37 ± 0.12 1.80 ± 0.10 1.63 ± 0.06Campestrol 14.88 ± 0.19 13.47 ± 0.43 14.71 ± 0.34Stigmasterol 17.48 ± 0.54 17.49 ± 0.43 18.70 ± 0.59�-Sitosterol 58.05 ± 1.01 57.49 ± 0.82 57.41 ± 0.17�5-Avenasterol 7.27 ± 0.74 8.80 ± 0.23 7.34 ± 0.58�7-Stigmasterol 1.24 ± 0.10 1.14 ± 0.26 0.89 ± 0.09

fmpCoS(tRwlC((aoo(to(tml“mocac

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The lowest level of cinnamic acid was found in cold pressed-

�7-Avenasterol 1.62 ± 0.31 1.28 ± 0.25 1.58 ± 0.34

or unsaturated fatty acids, while palmitic acid (C16:0) was theajor saturated fatty acid. These results are in agreement with

reviously reported data (Atta, 2003; Ramadan and Mörsel, 2004;heikh-Rouhou et al., 2007; Ramadan et al., 2012). The percentagef linoleic acid ranged between 57.1% for MAE-oil and 57.5% foroxhlet-extracted oil. Similar values were reported by Atta (2003)47.5–49.0%), Ramadan and Mörsel (2004) (57.3%) but higher thanhose reported by Cheikh-Rouhou et al. (2007) (49.1–50.3%) andamadan et al. (2012) (55.3%). Percentages for oleic acid in BCOere 23.8% for Soxhlet-extracted oil and 24.1% for MAE-oil. Simi-

ar results were reported by Ramadan and Mörsel (2004) (24.1%),heikh-Rouhou et al. (2007) (23.7–25.0%), and Ramadan et al.2012) (24.1%). The content of palmitic acid ranged from 11.84Soxhlet-extracted BCO) to 12.0% (cold-pressed BCO). These valuesre similar to those reported by Atta (2003) (12.1%) for cold-pressedil and is relatively lower than that reported for soxhlet-extractedil (13.0%) (Ramadan and Mörsel, 2004), for cold-pressed BCO12.5%) (Ramadan et al., 2012). The values were also lower thanhose reported by Cheikh-Rouhou et al. (2007) (17.2% for Tunisianil and 18.4% for Iranian oil), but higher than that reported by Atta2003) for Soxhlet-extracted oil (9.9%). BCO was characterized byhe relative high levels of polyunsaturated fatty acids (PUFA) and

onounsaturated fatty acids (MUFA). MUFA have been shown toower “bad” LDL cholesterol (low density lipoproteins) and retaingood” HDL cholesterol (high density lipoproteins). This is theajor benefit of olive oil over the highly polyunsaturated edible

ils, wherein PUFA reduce both the “bad” as well as the “good”holesterol levels in the blood (Ramadan et al., 2010). The fattycid profile and high amounts of PUFA makes the BCO a specialomponent for nutritional applications.

Tocochromanols (vitamin E) are the most important lipid solu-le antioxidants and they represent an essential nutrient for humanealth. These molecules are important for scavenging free radi-als and inhibiting lipid peroxidation in biological membranes. Theocochromanols comprise eight chemically distinct compoundshat are separated into tocopherols and tocotrienols, according

o saturation of the hydrophobic tails. Tocopherols contain aully saturated tail, whereas tocotrienols have three unsaturatedouble bonds. The different tocopherol and tocotrienol isomers,

nd Products 57 (2014) 52–58 55

that is, alpha (�), beta (�), gamma (�), and delta (�), are dis-tinguished by the locations of methyl groups on the chromanolring. Tocopherol content of BCO is given in Table 3. The con-tent of �-tocopherol was the highest, with levels ranging from8.57 to 34.23 ppm. However, �-tocopherol was the lowest toco-pherol isomer found in solvent-extracted oils (1.80 ppm in MAE-oiland 1.63 ppm in Soxhlet-extracted oil). This result was lowerthan those reported by Ramadan and Mörsel (2004) for solvent-extracted oil and by Ramadan et al. (2012) for cold-pressedoils.

Levels of sterols in vegetable oils are used for the iden-tification of oils, oil derivatives and for the determination ofoil quality. Furthermore, the concentration of sterols has beenreported to be little affected by environmental factors and/orby cultivation of new breeding lines (Ramadan and Mörsel,2007). The values obtained for sterol composition are listed inTable 3. �-sitosterol was the major sterol found in all oil samples(57.4–58.1%), followed by stigmasterol and campesterol rangedbetween 17.5–18.7% and 13.5–14.9%, respectively. Besides thesecompounds, �5-avenasterol, �7-stigmasterol and �7-avenasterolwere also found in all samples. �-sitosterol content of oils wasfound higher than those reported in Tunisian and Iranian BCO (44.5and 53.9%, respectively) (Cheikh-Rouhou et al., 2007). Stigmas-terol and campesterol were most abundant sterol compounds after�-sitosterol in Tunisian and Iranian BCO. Stigmasterol and campes-terol content of Tunisian and Iranian oil were found to be 20.9 and13.7%, and 16.5 and 12.1%, respectively. Stigmasterol content of BCOwas similar with Iranian oil and lower than Tunisian oil. Campes-terol level of BCO was within the values reported by Cheikh-Rouhouet al. (2007).

Chlorophyll and carotenoid were found in the highest amounts(0.81 and 0.40 mg/kg, respectively) in Soxhlet-extracted BCO, butmeasured in lower values (0.30 and 0.18 mg/kg, respectively)in cold-pressed BCO. Chlorophyll amount of BCO samples werelower 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. TPcontents were in the range of 15.1 (MAE-oil) and 36.0 (cold-pressedoil) mg gallic acid/kg oil. Total phenol content of oils was lowerthan the results for Tunisian and Iranian BCO (245 and 309 mggallic acid/kg oil, respectively). Inhibition rate results were in linewith the results of total phenol content. The highest inhibition rateobserved 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 4and Fig. 1. Thymoquinone was main identified phenolic com-pound. Cold pressed-oil contained the highest level of thymo-quinone (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 thy-moquinone in the present study were found to be lower thanthose reported by Lutterodt et al. (2010) who reported that thehighest amount of thymoquinone was found to be 8.73 mg/gof oil, and the lowest thymoquinone concentration was 3.48.Similarly to thymoquinone, the highest level of benzoic acidsand p-hydroxy benzoic acid, were found in cold pressed-oil(4.15 and 1.50 �g/g, respectively). The level of phenolic acids forSoxhlet-extracted oil (2.65 and 0.20 �g/g, respectively) and MAE-oil (2.15 and 0.20 �g/g, respectively) were relatively the same.

oil (0.03 �g/g), while the level of cinnamic acid for MAE-oil andSoxhlet-extracted oil (0.05 and 0.06 �g/g, respectively) were rela-tively the same.

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

Table 4Total phenolics content, inhibition rate, phenolic composition and induction time of BCO as affected by extraction method.

Extraction methods

Cold-pressing MAE Soxhlet

Total phenol (mg gallic acid/kg oil) 36.05 ± 0.50 15.19 ± 0.38 21.44 ± 0.80Inhibition rate (%) 78.45 ± 0.79 61.69 ± 0.88 65.58 ± 0.40p-Hydroxy benzoic acid (�g/g) 1.50 ± 0.00 0.20 ± 0.00 0.20 ± 0.00Benzoic acid (�g/g) 4.15 ± 0.07 2.15 ± 0.07 2.65 ± 0.07

3

tMLBfw

coiTosa

F(

Cinnamic acid (�g/g) 0.03 ± 0.00

Thymoquinone (�g/g) 14.40 ± 0.57

Induction time (h, at 110 ◦C) 3.48 ± 0.21

.4. Impact of extraction method and storage on OS of BCO

The results of induction period are given in Table 4. Inductionime was the highest (19.6 h) for Soxhlet-extracted oil, followed by

AE-oil (18.4 h) and was the lowest for cold-pressed BCO (3.48 h).utterodt et al. (2010) showed that induction periods of differentCO were in the range between 76 and 157 h at 80 ◦C. Because dif-

erent temperatures were used; we cannot compare our resultsith the results of Lutterodt et al. (2010).

PV is widely used assay for the measurement of oxidative ran-idity in oils and fats. Hydroperoxide is the primary product of lipidxidation; therefore, determination of PV can be used as oxidativendex during the early stage of lipid oxidation (Mohdaly et al., 2010).

◦

he results for PV for oven test at 60 C are presented in Fig. 2A. PVf cold-pressed BCO had higher PV (85.3 meq O2/kg oil) than PV ofolvent-extracted oils (25.8 meq O2/kg oil for Soxhlet-extracted oilnd 26.8 meq O2/kg oil for MAE-oil) at the end of storage period.

ig. 1. HPLC Chromatograms of (A) standard phenolic compounds (1) p-hydroxy benzoicC) MAE-BCO; and (D) Soxhlet-extracted BCO.

0.05 ± 0.00 0.06 ± 0.005.65 ± 0.07 6.20 ± 0.28

18.46 ± 0.22 19.62 ± 0.11

Organic solvents extract actually more polar lipids than cold press-ing. Thus, synergism of polar lipids with other antioxidant mayincrease OS of solvent-extracted oils. Ramadan and Mörsel (2004)reported that crude solvent extracted BCO reached up 51 meq O2/kgoil during storage at 60 ◦C.

Conjugated dienes (CD) and trienes (CT) are good parameter forthe determination of OS of oils. Formation of hydroperoxides iscoincidental with conjugation of double bonds in PUFA, measuredby absorptivity at the UV spectrum (Ramadan and Mörsel, 2004).Lipids containing methylene-interrupted dienes or polyenes showa shift in their double bond position during oxidation. The result-ing CD exhibit intense absorption at 232 nm, similarly CT absorbat 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 thelower will be the OS (Mohdaly et al., 2010).

Fig. 2B and C show the formation of CD and CT during Shaal oventest. Cold-pressed BCO contained the highest value for K232 and

acid, (2) benzoic acid, (3) cinnamic acid, (4) thymoquinone; (B) cold-pressed BCO;

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

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 standarddeviation.

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270 (12.8 and 1.4, respectively) and these results are in line withhe results of PV after 27 days of oven storage (60 ◦C). Soxhlet and

AE-oils remained stable during storage time and this tendencybserved by Ramadan and Mörsel (2004). Maximum values of K232nd K270 were 3.94, 0.78 for Soxhlet-extracted oil and 3.94, 0.73 forAE-oil.According to the results from oven test and Rancimat test, BCO

s highly stable to oxidation. Lutterodt et al. (2010) emphasizedhat crude BCO may be served as a natural antioxidant. Oxidationtability of sunflower (Ramadan, 2013) and corn oil (Ramadan and

ahdan, 2012) were increased by blending with BCO. Oxidationtability of cold-pressed BCO was lower than solvent-extractedils. The results of AP (Table 4) showed that cold-pressed BCOad stronger AP than solvent-extracted oils. Beside strong AP, TPnd tocopherol content of cold-pressed BCO were higher thanolvent-extracted oils. OS of oils depends on some factors such asocopherols and phenolics content and profile. As known, phenolicompounds contribute to the overall antioxidant capacity of oils. Inhis research, no correlation between total phenolics, tocopherolsnd OS during accelerated oxidation test. However, good correla-ion observed between total phenols and AP. Individual phenolicompounds contribute to OS of oils. No relationship was observedetween TP, thymoquinone content and OS for BCO extracted usingifferent methods (Lutterodt et al., 2010).

. Conclusion

Composition of BCO extracted using different techniques hadeen investigated. The results of our study showed that the extrac-ion technique affect the composition and the quality of BCO.old-pressed BCO was more susceptible to accelerated oxidationhan solvent-extracted oils. MAE-oil is rich in thymoquinone and

ay be used a natural thymoquinone source.

cknowledgment

We thank Scientific Research Projects Fund of Abant Izzet Baysalniversity in Turkey for providing fund support of the project underontract grant number 2012.09.01.491.

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