Microwave pretreatment as a promising strategy for increment of nutraceutical content and extraction yield of oil from milk thistle seed

Industrial Crops & Products 128 (2019) 527–533

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Microwave pretreatment as a promising strategy for increment of nutraceutical content and extraction yield of oil from milk thistle seed

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Bahram Fathi-Achachloueia, , Sodeif Azadmard-Damirchib, Younes Zahedia, Rezvan Shaddela a b

Department of Food Science and Technology, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, P.O. Box 56199-11367, Ardabil, Iran Department of Food Science and Technology, Faculty of Agriculture, University of Tabriz, P.O. Box 51666-16471, Tabriz, Iran

A R T I C LE I N FO

A B S T R A C T

Keywords: Milk thistle oil Microwave pretreatment Physicochemical properties Nutraceuticals content

In this study, seeds of Milk thistle (Silybum marianum L.) as a biomedical plant were pretreated with microwaves (800 W) for 2 and 4 min, to evaluate the process of intensifying oil extraction efficiency, physicochemical properties, nutraceuticals content, and fatty acids profile of milk thistle seeds oil extracted from Iranian ecotype, Khoreslo. Results showed that microwave pretreatment of Milk thistle seed increased the oil extraction yield (by 6%), total phenolic content (by 12.2%), phytosterols (by 25%), and tocopherols (by 37.5%) of the oil obtained by solvent. Some physicochemical properties of seed oil such as chlorophyll content (0.55–1.73 mg pheophytin/ kg oil) and saponification value (179–187 mg KOH/g oil) increased, but acid value (4.24−2.16 mg KOH/g oil), peroxide value (5.11−2.09 meqO2/kg oil), iodine value (107−99 g I2/100 g oil), and the poly unsaturated to saturated fatty acids (PUFA/SFA) ratio of all samples decreased by treatment with microwaves. Moreover, the α, β, γ-, δ δ -tocopherols, and phytosterols content such as cholesterol, campesterol, stigmasterol, cleroesterol, βsitosterol, and Δ7-sterol of milk thistle seed oils increased by microwaves treatment. In conclusion, the results indicated that microwave pretreatment is a promising strategy for amplification of oil extraction yield and the content of nutraceuticals in obtained oil from milk thistle seeds.

1. Introduction Silybum marianum L., commonly known as milk thistle (a member of the Astraceae family), is an annual or biennial flowering plant with acanaceous leaves and a milky sap. Milk thistle is known as a native species in the Mediterranean region of Europe; however, it is naturalized in California and the eastern United States, and also grows in North Africa and the Middle East, especially in Iran (Hadolin et al., 2001; Pepping, 1999). Extract from the mature milk thistle seeds has been shown to have clinical utility in different liver disorders, containing hepatitis, cirrhosis, and alcoholic liver disease (Pepping, 1999). The major active ingredients of milk thistle are flavonolignans; The bioactive flavonolignans are generally called silymarin found in the fruit, seeds, and leaves of the plant (Pepping, 1999; Talbott and Hughes, 2007). The seeds also contain trimethylglycine, essential fatty acids, and betaine which might be helpful for silymarin’s medicinal potentials (Subramaniam et al., 2008). Silymarin has strong antioxidant properties and consists of three isomers namely silybin, silydianin, and silychristin, with silybin being the most bioactive agent. Silymarin has

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shown cholesterol and blood pressure lowering activity, anti-proliferative activity against cancer cells, and chemo-protective activity (Pepping, 1999; Talbott and Hughes, 2007). Milk thistle seeds have a relatively high content of oil (26–31%). Considering this point, it is needed to remove the oil from seed before the extraction of silymarin. Actually, the oil is considered as a by-product of silymarin production (Fathi-Achachlouei and AzadmardDamirchi, 2009). Milk thistle seed oil as a suitable edible oil contains long chain fatty acids (C16-C24), phytosterols such as campesterol, cleroesterol, stigmasterol, Δ-sterol and β-sitosterol, and it is also rich in vitamin E (Hadolin et al., 2001; Pepping, 1999; El-Mallah et al., 2003; Vojtisek et al., 1991). Many reports have represented pretreatment of oilseeds with several methods (microwave, ultrasonic baths, milling, rapid gas decompression, etc.) to amplify the extraction of valuable oilseed components and accessibility of favorable nutraceuticals, like tocopherols and phytosterols in the extracted oil, particularly microwave radiation of seeds which has been introduced as an impressive technique for enhancement the oil extraction efficiency from seeds (AzadmardDamirchi et al., 2010; Đurđević et al., 2017). The advantages of microwave radiation are reducing the processing time and energy

Corresponding author. E-mail addresses: [email protected], [email protected] (B. Fathi-Achachlouei).

https://doi.org/10.1016/j.indcrop.2018.11.034 Received 7 September 2018; Received in revised form 12 November 2018; Accepted 13 November 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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2.3. Oil extraction

consumption, since the energy is transported instantly throughout the volume of materials, resulting to heat generation throughout the material and it is likely to attain quick and uniform heating treatment of relatively thick materials (Azadmard-Damirchi et al., 2010; Đurđević et al., 2017). Microwaves utilize radio waves to transmit energy and change it to heat at a definite frequency which is approximately 300 MHz to 300 GHz (Đurđević et al., 2017). By using this frequency range, waves are mostly absorbed by the moisture content of oilseed when heated up inside the materials because of the microwave effect, evaporates and generates gigantic pressure on the membrane of seed cell. The pressure impels the cell membrane from inside and finally the cell membrane is ruptured, which facilitates draining out of the active components from the permanent pores (Đurđević et al., 2017). Consequently, penetration of the solvent into the seed cell membrane as well as enabling of oil to move across the permeable cell walls would be facilitated (Đurđević et al., 2017; Uquiche et al., 2008). Microwave radiation have already been used in the food industry with enhancing success in oil extraction, pasteurization, blanching, baking, drying, cooking, and thawing of miscellaneous food products (Azadmard-Damirchi et al., 2011). Microwave pretreatment of oilseeds have been previously studied for extraction and characterization oil obtained from pumpkin seeds (Ali et al., 2017), sunflower seed (Anjum et al., 2006), rapeseed (Azadmard-Damirchi et al., 2010), pomegranate seed (Đurđević et al., 2017), chilean hazelnuts (Uquiche et al., 2008), Nigella sativa L. seeds (Mazaheri et al., 2019), and even black cumin seeds (Bakhshabadi et al., 2017). Considering literature review, there is not any information on the application of microwaves pretreatment of oilseed to improve the extraction of nutraceuticals from milk thistle seed oil. Therefore, the objective of this study was to evaluate the effects of microwave pretreatment prior to the oil extraction process by solvent on the process of enhancing oil extraction yield, physicochemical properties, phytosterols, tocopherols, fatty acids profile, and maintenance the most natural antioxidants and phenolic compounds in the milk thistle seeds oil extracted from Iranian ecotype, Khoreslo. The oil extracted from untreated Milk thistle seed by solvent was used for comparison.

Oil samples were extracted from milk thistle following the method mentioned by Azadmard-Damirchi et al. (2005), after some modifications. Briefly, extraction of oil from powdered milk thistle seeds (100 g) was carried out using 300 ml of hexane at 25°C in an Erlenmeyer flask covered by aluminum foil on a platform shaker for 1 h. Then, the obtained mixture was filtrated using defatted filter papers under vacuum in a Buchner funnel. Removal of the solvent was done under declined pressure at 35°C. To minimize the oxidation of compounds, experiments were performed on extracted oil samples as quickly as possible upon oil extraction; if not the extracted oil samples were kept at 4°C until use. Three 100-g sets of seeds were used for extraction. 2.4. Determination of physicochemical properties To determine the peroxide value (PV) (method Cd 8–53), acid value (AV) (method Cd 3d-63), saponification value (SV) (method Cd 3–25), iodine value (IV) (method Cd 1–25), and refractive index (RI) (using Abbé refractometer at 30°C) the methods from American Oil Chemist’s Society (American Oil Chemists’ Society (AOCS), 1997) were used. Chlorophyll content (as mg pheophytin/kg oil) was specified according to Pokoprny et al. (1995). 2.5. Determination of total phenolic content (TPC) One gram of the milk thistle seed oil samples was extracted via 3 ml of MeOH by whirling at 25 °C (Parry et al., 2006). The obtained samples were centrifuged to collect the MeOH extracts. Re-extraction of oil residues was then done twice with 3 mL × 2 of a MeOH solution. The three provided MeOH extracts were blended; The testing sample solutions was prepared by adding MeOH to achieve the ultimate volume of 10 mL. The TPC of milk thistle seed oils was determined using the FolinCiocalteu (FC) reagent, according to Yu et al. (2003). Gallic acid as the standard was used for measurement. 2.6. Fatty acids measurement 2.6.1. Preparation of fatty acid methyl esters Fatty acid methyl esters of the oil samples were prepared using the method described by Savage et al. (1997) and Savage and McNeil (1998).

2. Materials and methods 2.1. Sample Milk thistle seeds gathered (for two years) from Iranian ecotype, Khoreslo, (in Ardabil) in the north-west of Iran. The samples were collected ∼2 kg (1 kg in each year). Each 2-kg seed sample was utterly blended and 100 g of the sample were weighed for oil extraction and subsequently analyzed. Solvents were provided from Merck (Darmstadt, Germany). 5a-cholestane, tocopherols and sterol standards were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA).

2.6.2. Analysis of fatty acid methyl esters by GC Identification of the fatty acid methyl esters were done using a GC instrument, equipped with a flame ionization detector, a split/splitless injector and a film thickness fused-silica capillary column (50 m × 0.22 mm, 0.25 μm), type BPX70 (SGE, Austin, TX, USA). The operation conditions were as follows: injector temperature, 230 °C; detector temperatures, 250 °C; make-up gas, N2 inert gas; carrier gas, helium; flow rate of gases, 30 ml/min. The initial temperature of oven was 158 °C then enhanced to 220 °C at a rate of 2 °C/min and kept for 5 min (Azadmard-Damirchi and Dutta, 2008). The FAMEs were recognized by comparison of their retention times with standard FAMEs and the peak areas reported as a percentage of the total fatty acids.

2.2. Microwave pretreatment Milk thistle seeds were spread on a Pyrex petri dish, in the middle of the microwave oven (Model: MW2300 GF, 800 W). Throughout the microwave radiation, the sample spins inside of the oven. This arrangement allowed the samples to move through the fixed electromagnetic field pattern formed inside the microwave oven, letting uniform energy absorption within the seeds. Samples were microwave pretreated at a constant frequency of 2450 MHz for 2 and 4 min. The selection of these microwave conditions (800 W; 2 and 4 min) was done based on preliminary trials and the previous study published by Azadmard-Damirchi et al. (2010), in which it was examined at the longer time and higher microwave power, the seeds started to make smoke and burning. Milk thistle seed sample with no microwave treatment was utilized as a control.

2.7. Determination of tocopherols by HPLC The amount of tocopherols in the oil samples was analysed by HPLC based on the method described by Fathi-Achachlouei and AzadmardDamirchi (2009) and Azadmard-Damirchi and Dutta (2008).10 mg of the extracted oil were dissolved in 1 ml of n-heptane. 10 μl of former mixture were then injected. The column applied in this method was LiChro CART 250-4. Tocopherols were identified by fluorescence detector at excitation/emission wavelengths of 294 and 320 nm, respectively. Each tocopherol in the analyzed oil samples was detected by 528

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et al., 2008). It has been shown that microwave treatment causes a modification in the cellular wall and possesses greater porosity (Uquiche et al., 2008).

comparing the retention times of reference standards of tocopherols in the chromatograms. Quantification was performed using an external standard method with reference standards of tocopherols. For this purpose, different concentrations of each standard tocopherol were injected and the peak areas were obtained from the HPLC analysis. By the concentration and peak area, the calibration curve obtained. Then, the obtained peak area for tocopherols from oil samples were converted to the concentration by the calibration curve.

3.2. Physicochemical properties Physicochemical properties of milk thistle seeds oil with or without pretreatment by microwave are shown in Table 1. Refractive index (RI) is a parameter that shows the purification and quality of oil. Different oils and fats show their particular refractive indices; so, this property is utilized to detect and specify the purity of oils and fats (Bakhshabadi et al., 2017). As shown in Table 1, applying pretreatment by microwaves had no significant effect on the refractive index of milk thistle seed oil (p > 0.05). Bakhshabadi et al. (2017) ascribed no changes in refractive index of oils obtained from microwave treatment to the resemblance of fatty acid combinations in the non-treated and treated samples. Some physicochemical properties of seed oil such as chlorophyll content (0.55–1.73 mg pheophytin/kg oil), saponification value (179–187 mg KOH/g oil), and total phenolic content (381–422 mg GAE/100 g oil) increased, but acid value (4.24−2.16 mg KOH/g oil), peroxide value (5.11−2.09 meqO2/kgoil), and iodine value (107−99 g I2/100 g oil) declined by pretreatment with microwaves. It has also been shown that the exposure time of the milk thistle oilseeds to microwave radiation had a significant effect (p < 0.05) on the aforementioned physicochemical characteristics of milk thistle seed oil (Table 1). Anjum et al. (2006) studied the effects of microwave roasting on the physicochemical properties and oxidative resistance of sunflower seed oil. They reported that the unsaponifiable matter, refractive index, and iodine value of the oils remarkably decreased with increasing microwave roasting time. They also observed that high roasting time increments free fatty acid content and saponification value of sunflower oil. Peroxide value (PV) is the most widely used values for evaluation of edible oil quality and indicates the stability of oil against the oxidation. A peroxide value of more than 10 meqO2/kg oil is known as unacceptable (Shahidi, 2005). In this study, the PV of milk thistle oils varied from 5.11 to 2.09 meqO2/kg oil which the lowest value was associated to milk thistle pretreated by microwave for 4 min. The main reason of reduction in PV may be due to increasing of total phenolic content in treated milk thistle seeds oil by microwave pretreatment (Azadmard-Damirchi et al., 2010). Iodine value (IV) indicates the degree of unsaturation of oil. The IV of the milk thistle oil ranged from 107 to 99 g I2/100 g oil, which decreased with pretreatment by microwaves.

2.8. Phytosterol analysis 2.8.1. Saponification of oil samples To the analysis of phytosterols, oil samples were saponified based on the method described by Azadmard-Damirchi et al. (2005), with minor modifications. Approximately, 30 mg of oil sample was weighed and blended with 3 ml of 2 M KOH in 95% ethanol in a small glass tube. The prepared sample was then shaken for 15 min in a 90°C water bath. Upon cooling the tubes, preparation of the samples was continued by adding 2 ml of water and 1.5 ml of hexane and then vortex mixing vigorously. Subsequently, the hexane layer including unsaponifiables was separated for further analysis after centrifugation of the mixture at 3000 rpm for 5 min. 2.8.2. Trimethylsilyl ether derivatives of the phytosterols Preparation of trimethylsilyl (TMS) ether derivatives of phytosterol classes were done according to the proposed method of AzadmardDamirchi et al. (2005). 2.8.3. GC analysis of the phytosterols Quantification of phytosterols were accomplished by GC as TMSether derivatives using a fused-silica capillary column (DB-5MS, 30 m0.25 mm, 0.50 lm; J&W Scientific, Folsom, CA, USA) according to the method of Azadmard-Damirchi and Dutta (2006). The column was connected to a Chrompack CP 9002 gas chromatograph (Middleburg, The Netherlands) which was equipped with a flame-ionization detector. The conditions were as follows: (a) injector zone, 260°C; (b) oven initial temperature 60°C for 1 min, increased to a final temperature of 310°C at 40°C/min held for 27 min; (c) nitrogen as the make-up gas and helium as the carrier gas with flow rate of 30 ml/min and (d) detector batch temperature of 310°C. Quantification was conducted using the 5acholestane as the internal standard. 2.9. Statistical analysis Data was analyzed via the general linear model (GLM) using SAS 9.1 software based on the completely randomized design (CRD). The differences among of means were declared by the Duncan’s test and p < 0.05 was considered as significance level.

3.3. Fatty acid composition The composition of fatty acids of milk thistle seeds specified by GC has been presented in Fig. 1. Nine fatty acids at different levels were found in milk thistle seeds extracted oils (Table 2). Linoleic acid (18:2n6) was the prominent fatty acid followed by oleic acid (18:1n-9), palmitic acid (C16:0), and stearic acid (18:0). The oil samples possessed high poly unsaturated fatty acids (PUFA) content (49.95%) and low saturated fatty acids (19.41%). The results obtained are in line with the previously published findings (Fathi-Achachlouei and AzadmardDamirchi, 2009; Parry et al., 2006). The prominent fatty acid extracted by n-hexane extraction was Linoleic acid (approximately 50.0%). Fatty acid composition of milk thistle seeds extracted oil was influenced by applied microwave pretreatment as linoleic acid slightly decreased from 49.74% (non-treated seeds) to 48.66% (treated seeds by microwave for4 min) and oleic acid negligibly declined from 28.90% (nontreated seeds) to 28.22% (treated seeds by microwave for 4 min); Conversely, palmitic and stearic acids increased from 7.45% (nontreated seeds) to 8.65% (treated seeds by microwave for 4 min) and from 5.60% (non-treated seeds) to 6.10% (treated seeds by microwave

3. Results and discussion 3.1. Oil extraction yield Results exhibited that pretreatment by microwave can enhance oil extraction yield. Furthermore, data showed that the oil extraction yield increased with increasing the treatment time of extraction process; 2 and 4 min pretreated milk thistle samples gave 32.33% and 35.41% oil by solvent in comparison to non-treated seeds oil (29.43%), respectively (Table 1). Obtained results were in line with previously published data regarding the intensification of oil extraction by pretreatment of rapeseeds with microwaves (Azadmard-Damirchi et al., 2010). Accordingly, increment of the treatment time had a positive and a significant effect (p < 0.05) on the extraction yield (Azadmard-Damirchi et al., 2010). The oil extraction yield in untreated milk thistle seeds is low since the undamaged cell wall brings about a major resistance to the oil extraction by solvent (Azadmard-Damirchi et al., 2010; Uquiche 529

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Table 1 Physicochemical properties of Milk thistle seed oil with or without pretreatment by microwave. Oil Properties

Milk thistle

Milk thistle after pretreatment by microwave for 2 min

Extraction yield (%) Refractive index Chlorophyll content (mg pheophytin/kg oil) Acid value (mg KOH/g oil) Peroxide value (meq O2/kg oil) Iodine value (g I2/100 g oil) Saponification value (mg KOH/g oil) Total Phenolic content (mg GAE/100 g oil)

c

4 min b

35.41 ± 0.37a 1.345 + 0.02a 1.73 ± 0.20a 2.16 ± 0.30c 2.09 ± 0.25c 99.33 ± 1.22c 187.47 + 1.22a 434.47 ± 2.54a

32.33 ± 0.35 1.351 + 0.03a 1.34 ± 0.10b 3.21 ± 0.18b 3.28 ± 0.30b 103.14 ± 1.23b 183.72 + 1.33b 412.26 ± 2.34b

29.43 ± 0.32 1.350 + 0.01a 0.55 ± 0.01c 4.24 ± 0.21a 5.11 ± 0.20a 107.31 ± 1.35a 179.68 + 1.42c 381.44 ± 2.25c

Mean ± standard deviation. Different letters within the same row represent significant differences (p < 0.05) according to the Duncan test.

for 4 min) for n-hexane extraction, respectively. This trend was likely due to PUFA degradation which was in good agreement with published investigation by Ali et al. (2017). Kanitkar (2010) found that the fatty acid compositions of oils extracted from microwaves pretreated rice bran varied slightly from formerly published values, but microwave extracted rice bran oil also had a remarkable amount of arachidic acid (C21:0) as compared with former extracted rice bran oil. Linoleic and palmitic acids are usually utilized as indicators of the extent of oil deterioration. As observed by Anjum et al. (2006), the microwave pretreatment can incredibly influence the levels of oleic and linoleic acids compared with palmitic and stearic acids. Accordingly, upon passing the pretreatment time, the percentage of the linoleic acid was reduced; while, the amount of oleic acid was increased. Tan et al. (2001) reported the ratio of C18:2/C16:0 for microwave-radiated oil declined with increased heating power settings. Yoshida et al. (1995) observed a declining trend in the amount of PUFA in soybean oil within roasting time. The contents of total SFA and UFA (MUFA + PUFA) in the studied oils were increased and decreased, respectively after pretreatment seeds by microwaves as compared with non-treated seeds (control). Furthermore, a reduction trend in the percentage of the UFA and an elevation trend in amount of SFA was observed at longer pretreatment time (Table 2). These results were in line with former reports regarding quickly decrease in contents of PUFA and increase in contents of SFA during lipid peroxidation (Vaidya and Choe, 2011a, 2011b). However, non-treated seeds (control) had the highest content of unsaturated fatty acids compared with the other treated seeds by microwave during 2 and 4 min (p < 0.05) (Fig. 2), as well as the higher ratio of UFA/SFA (4.11) was belonged to non-treated milk thistle seeds

Table 2 Fatty acids composition (g/100 g) and distribution (%) in oil extracted from milk thistle seed samples. Fatty acids

Milk thistle

Milk thistle after pretreatment by microwave for 2 min 4 min

C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 C24:0 SFA† MUFA† PUFA†

7.45 ± 0.072c 5.60 ± 0.04c 28.90 ± 0.03a 49.74 ± 0.13a 0.21 ± 0.005a 3.33 ± 0.03a 0.83 ± 0.04a 2.37 ± 0.03a 0.66 ± 0.02a 19.41 ± 0.16c 29.73 ± 0.04a 49.95 ± 0.19a

8.03 ± 0.074b 5.74 ± 0.03b 28.58 ± 0.05b 49.24 ± 0.12b 0.21 ± 0.004a 3.32 ± 0.05a 0.82 ± 0.03a 2.33 ± 0.02a 0.67 ± 0.04a 20.09 ± 0.25b 29.40 ± 0.46b 49.45 ± 0.26b

8.65 ± 0.075a 6.10 ± 0.05a 28.22 ± 0.04c 48.66 ± 0.14c 0.20 ± 0.006a 3.31 ± 0.04a 0.80 ± 0.02a 2.30 ± 0.03a 0.64 ± 0.03a 21.00 ± 0.26a 29.02 ± 0.27c 48.76 ± 0.47c

Mean ± standard deviation. Different letters within the same row represent significant differences (p < 0.05) according to the Duncan test. † SFA: Saturated fatty acids, MUFA: Monounsaturated fatty acids, PUFA: Polyunsaturated fatty acids.

(control) (Fig. 3). Non-treated milk thistle seeds (control) and treated seeds by microwave during 4 min (p < 0.05) had less and more saturated fatty acids content, respectively. Control had significantly (p < 0.05) the highest amount of PUFA and treated seeds by microwave for 4 min had the lowest amount of PUFA content among different treatments (p < 0.05) (Table 2). The longer the pretreatment time, the lower was the percentage of the UFA and or UFA/SFA and the higher

Fig. 1. Gas chromatogram of fatty acid methyl esters in oil extracted from milk thistle seed samples (C16:0, palmitic; C18:0, stearic; C18:1 oleic; C18:2, linoleic; C18:3, linolenic; C20:0, eicosanoic; C20:1, eicosenoic; C22:0, behenic; C24:0, lignoceric fatty acid methyl esters). 530

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Fig. 2. Unsaturated fatty acids (%) of the three different Milk thistle seeds oil treated by microwaves (2 min and 4 min) and non-treated (Control).

Fig. 4. High performance liquid chromatogram of tocopherols of milk thistle oil. 1: α -tocopherol; 2: β -tocopherol; 3: γ -tocopherol; 4: δ –tocopherol. Table 3 Tocopherol content (ppm) in the oil extracted from milk thistle seed samples.

Fig. 3. UFA/SFA of the three different Milk thistle seeds oil treated by microwaves (2 min and 4 min) and non-treated (Control).

Tocopherols

Milk thistle

Milk thistle after pretreatment by microwave for 2 min 4 min

α-Tocopherol β-Tocopherol γ-Tocopherol δ-Tocopherol Total

465.78 ± 0.95c 51.74 ± 0.69c 35.71 ± 0.56c 80.75 ± 0.50c 633.98c

724.76 ± 1.35a 100.81 ± 1.59a 57.47 ± 0.86a 132.18 ± 1.20a 1015.22a

678.62 ± 1.68b 80.75 ± 1.47b 46.34 ± 0.76b 114.64 ± 0.80b 920.35b

Mean ± standard deviation. Different letters within the same row represent significant differences (p < 0.05) according to the Duncan test.

was that of SFA (Figs. 2 and 3 and Table 2). This trend was probably a negative point of pretreatment by microwaves due to UFA degradation and was in agreement with many authors that have announced the effects of microwave pretreatment on the combination of fatty acid in vegetable oils (Ali et al., 2017; Kanitkar, 2010; Anjum et al., 2006; Yoshida et al., 1995). In addition, the ratio of polyunsaturated to saturated fatty acids (PUFA/SFA) of all samples decreased with pretreatment time by microwaves, which enabled to figure out the oil oxidation and degradation (Ali et al., 2017). Given the above, one of the reasons for using low intensity and short duration of pretreatment with microwave was to prevent the severe negative effects on the nutrient compositions of extracted oil from milk thistle seeds, especially on fatty acids profile, which can be recommended for the oil industry.

100, 35 to 57, and 80 to 132 μg/g oil, respectively. Tested milk thistle seed oils possessed a higher content of α-tocopherol compared with the other type of tocopherols (Table3). Total content of tocopherol in the oil extracted from treated seeds by microwave during 2 min was much more than that of control and treated seeds by microwave during 4 min (p < 0.05). The obtained results concur with previously published data (Azadmard-Damirchi et al., 2010). In fact, due to more duration of radiation with microwave, higher temperatures were induced which may cause breaking the structure of double bonds in tocopherols and a reduction in the tocopherol content. According to Lee et al. (2004), by increasing the roasting time up to 160 °C, the levels of α-tocopherol content in safflower oil gradually elevated but further heating up to 180 °C had a reversed result. Yen (1990) found that the tocopherols in sesame oils pretreated by microwave incremented by the roasting temperature up to 200 °C, but decreased up to 260 °C. The levels of α, β, and γ -tocopherol in rice bran oil were enhanced significantly (p < 0.05) by pretreatment of the rice bran in microwave for up to 30 s (Ko et al., 2003). However, it has been reported that the level of α-, β-, γ- and δ -tocopherols declined within microwave heating and α-tocopherol displayed the highest rate of loss because of unsaturation of the TAG (triacylglycerols) system under their process situations (Yen, 1990; Yoshida et al., 1995; Anjum et al., 2006; Azadmard-Damirchi et al., 2011). Considering the results of PV measurement, the authors claim that the main reason for low PV of treated milk thistle seeds oil by microwaves during 2 and 4 min were because of a high level of tocopherols and total phenolic content in the treated seeds oil (Table 1). AzadmardDamirchi et al. (2010) reported that the microwave pretreatment of rapeseeds before the oil extraction elevated significantly (p < 0.05) the

3.4. Tocopherols analysis Tocopherols are one of the main functional ingredients as radicalchain breaking antioxidants in vegetable oils and foods as well as in the body, e.g. lipoproteins and membranes (Kamal-Eldin and Appelqvist, 1996). Tocopherols may decline the risk of cardiovascular diseases and definite types of cancer because of its antioxidant features and different functions at the molecular level (Burton, 1994; Burton and Traber, 1990). Milk thistle seed oil is a very good source of tocopherols and the tocopherol amount of the milk thistle seeds oil is comparable with other vegetable oil resources like sunflower oil (Fathi-Achachlouei and Azadmard-Damirchi, 2009; Gunstone, 2000). In this study, tocopherols composition and their content were determined in all the treated milk thistle seeds oil samples (Fig. 4). The results of tocopherols measurement in different treated milk thistle seeds oil are presented in Table 3. The level of α, β, γ, and δ-tocopherols ranged from 465 to 724, 51 to 531

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(> 160 °C) (Spielmeyer et al., 2009). Phytosterols were characterized as their TMS-ether derivatives by GC (Fig. 5). Six phytosterols – cholesterol, campesterol, stigmasterol, cleroesterol, β-sitosterol, and Δ7-sterol were detected in the analyzed milk thistle oil samples (Table 4). β-sitosterol was predominant (32–34%), followed by Δ7-sterol (20–21%) (Table 4). These results were in line with previously published results (Fathi-Achachlouei and Azadmard-Damirchi, 2009). Phytosterol content in oil samples extracted increased over microwave treatment time (Table 4). Total phytosterol contents of oil extracted by solvent from non-treated oil and microwave pretreated-oil for 2 and 4 min were 1816, 2116, and 2422 ppm, respectively. These results revealed that microwaves pretreatment of milk thistle would be as a promising tool to enrich the phytosterol content of extracted oil. It is worth mentioning that some of the phytosterols, can act as antioxidants (Azadmard-Damirchi et al., 2010; Fathi-Achachlouei and Azadmard-Damirchi, 2009) which might be another explanation for the elevated stability of the extracted oil from microwave pretreated milk thistle seed (Table 4).

Fig. 5. Gas chromatogram of TMS ether derivatives of phytosterols in oil extracted from milk thistle seed samples. (Peak identification: IS: 5a-cholestane (internal standard); 1: Cholesterol; 2: Campesterol; 3: Stigmasterol; 4: Clerosterol; 5: β -Sitosterol; 6: Δ7-Sterol). Table 4 Phytosterol distribution and content in oil extracted from milk thistle seed samples. Phytosterols

Milk thistle

Milk thistle after pretreatment by microwave for 2 min 4 min

Cholesterol Campesterol Stigmasterol Cleroesterol β-Sitosterol Δ7-Sterol Unknown Total

161Ac(8.8)B 81c(4.5) 115c(6.3) 63c(3.5) 630c(34.7) 390c(21.5) 376c(20.7) 1816c

175b(8.3) 126b(5.9) 148b(7) 86b(4.1) 716b(33.8) 438b(20.7) 427b(20.2) 2116b

4. Conclusions In current study, the effect of microwave pretreatment of milk thistle on oil extraction yield and its physicochemical properties, nutraceuticals content, and fatty acids profile were investigated. Obtained results suggest that microwave pretreatment may increase oil extraction yield. Among physicochemical properties of milk thistle seed oil, chlorophyll content, saponification value, and total phenolic content increased, whereas acid value, peroxide value, and iodine value declined and the refractive index had no change by pretreatment of the seeds with microwaves compared with non-treated samples. Also, the polyunsaturated to saturated fatty acids (PUFA/SFA) ratio of all samples decreased by microwaves pretreatment. Moreover, the amount of nutraceuticals including tocopherols and phytosterols increased remarkably in treated seeds oil. However, despite the fact that more duration of radiation with microwave (4 min) increases some of the physicochemical parameters and antioxidant compounds in oil, but because of prevention the severe negative effects on the nutrient compositions of extracted oil, especially on unsaturated fatty acids, and a reduction in the tocopherol content, short duration (2 min) of pretreatment with microwave is recommended to apply in the oil industry. Overall, it can be concluded that microwave pretreatment is a promising tactic for amplification of oil extraction and content of nutraceuticals in oil obtained from milk thistle seeds.

190a(7.8) 165a(6.8) 184a(7.6) 119a(4.9) 788a(32.5) 492a(20.3) 484a(19.9) 2422a

Means of triplicate analyses (CV is generally less than 2%). (a–c) Denotes statistically significant differences (p < 0.05). A Values are in μg/goil. B Values are percentages.

tocopherols contents in oils. As a point of view, the oilseed cell membrane damaging by microwave pretreatment qualify incremented the release of tocopherols and intensify their amount in extracted oil. Furthermore, amount of individual tocopherols in treated seeds oil by microwaves during 2 min was also remarkably higher than two others. The results obtained on α- and γ-tocopherols amount are in agreement with formerly published result, which has been reported the content of α- and γ-tocopherol in milk thistle seeds oil as 156 and 35 ppm, respectively (Parry et al., 2006). However, in the above report β-tocopherol was not evaluated and the δ-tocopherol content was about 7 ppm (Parry et al., 2006).

Acknowledgment This study was financed by the University of Mohaghegh Ardabili.

3.5. Phytosterols analysis

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