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Extraction of oil and silybin compounds from milk thistle seeds using supercritical carbon dioxide
J. of Supercritical Fluids 100 (2015) 105–109
Contents lists available at ScienceDirect
The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu
Extraction of oil and silybin compounds from milk thistle seeds using supercritical carbon dioxide Hatice Tu˘gba C¸elik, Metin Gürü ∗ Gazi University, Engineering Faculty, Chemical Engineering Department, Maltepe, 06570 Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 8 January 2015 Received in revised form 19 February 2015 Accepted 20 February 2015 Available online 27 February 2015 Keywords: Milk thistle seeds Silybin A Silybin B Supercritical extraction
a b s t r a c t Milk thistle is a plant that has been used medicinally for over 2000 years. It has been used for the treatment of many diseases such as cancer, liver, kidney, cardiac, brain. Their seeds are rich in silymarin compounds (especially silybin A and silybin B) and fatty acids. The purpose of this study was to extract silybin A and silybin B from milk thistle seeds with the supercritical CO2 . The effect of operating parameters such as temperature (40–80 ◦ C), pressure (160–220 bar), CO2 flow rate (3, 4 and 5 mL/min) and particle size (0.3025, 0.925 and 1.2 mm) on extracted oil, silybin A and silybin B were investigated. Fatty acid composition in milk thistle seed extract was determined at optimum conditions. The results indicated that the optimal conditions were 40 ◦ C temperature, 200 bar pressure, 4 mL/min CO2 flow rate and 0.3025 mm particle size. In these conditions; the amounts of oil, silybin A and silybin B were obtained 327, 2.29 and 1.92 mg/g milk thistle seeds respectively. For the fatty acids obtained by supercritical CO2 extraction, the most abundant compounds were palmitic acid (8.15%); stearic acid (5.51%), oleic acid (24.10%); linoleic acid (54.97%) at optimum conditions. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The milk thistle is an annual or biannual plant of the Asteraceae family. The milk thistle seeds are included in silymarin compounds, especially silybin A and silybin B. Silybin A and silybin B are well known as antioxidant [1,2] and have been used for the treatment of cancer, liver, kidney, cardiac, brain, cirrhosis and poisoning of alcohol, drugs or toxins [3–6]. Also the milk thistle seeds are consisting of fatty acid compounds like oleic acid, linoleic acid, etc. which are good antioxidant substances too. Silymarin compounds [7–11] and fatty acid compounds [12–14] are generally produced by traditional extraction method from plant. Supercritical fluid method is used many industries such as medicine, chemistry, biochemistry and food. Supercritical fluid extraction method has supremacy in comparison to conventional solvent extraction methods. The most widely used supercritical fluid is carbon dioxide which is non-toxic, relatively inert, and non-flammable [15]. The extraction solvents such as petroleum ether and ethyl acetate are widely used in traditional methods but they cannot be removed completely from extract. However, carbon dioxide is gaseous at ambient conditions of temperature and
∗ Corresponding author. Tel.: +90 312 5823555; fax: +90 312 2308434. E-mail addresses: [email protected] (H.T. C¸elik), [email protected] (M. Gürü). http://dx.doi.org/10.1016/j.supflu.2015.02.025 0896-8446/© 2015 Elsevier B.V. All rights reserved.
pressure and completely separated from the extract in these conditions [16,17]. Several investigations were reported for extraction by supercritical CO2 from tea [18,19], jatropha [20,21], Stevia rebaudiana leaves [22], grape [23], mango leaves [24] and watermelon [25]. Hadolin et al. [26] studied the extraction of milk thistle seeds by supercritical CO2 /propan to obtain vitamin E content. Szentmihályi et al. [27] studied the extraction of milk thistle seeds by supercritical CO2 /propan to obtain fatty acid, pheophytin, carotene, tocopherol content and concentrations of some metals. In addition, there are some studies that investigate the effect of supercritical CO2 with together cosolvent such as methanol, ethanol, water on extraction process [28–31]. But there is no study in the literature regarding, the effect of parameters of temperature, pressure, particle size and CO2 flow rate on silybin A and silybin B in oil extracted from the milk thistle seeds by supercritical CO2 without cosolvent. The purpose of this study was to investigate of pharmaceutical importance silybin A and silybin B were extracted from milk thistle seeds by the supercritical CO2 without using cosolvent due to the preventing of cosolvent contamination. The purity of silybin compounds must be affected by cosolvent. The qualitative and quantitative analysis of silybin A and silybin B were performed by HPLC. The fatty acids composition in extract was analyzed by GCFID. The effect of parameters such as temperature, pressure, particle size and CO2 flow rate were investigated on supercritical extraction process.
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2. Materials and method 2.1. Materials Milk thistle was obtained from Mu˘gla in Turkey. Whole seeds were separated from flowers. They were dried at room conditions and then ground up the specific particle dimension by a coffee grinder. The specific particle dimensions of samples were 0.3025, 0.925 and 12 mm. Grinding was carried out just before extraction in order to avoid oxidation.
2.2. Supercritical CO2 extraction Extractions were performed in laboratory scale supercritical fluid extraction system (Applied Separation Speed, USA). A 24 mL of extraction vessel was packed with 4 g of the milk thistle seeds particles. The vessel was put in a temperature-controlled oven. CO2 was sent continuously through the system. The extract was collected in vials. The detailed description of the system was given in another our study [18]. The collection vials were changed per every 30 min. The experiments were performed among 120 min in the range of 40–80 ◦ C, 160–220 bar, 3–5 mL CO2 /min, 0.3025–1.2 mm particle size. Extraction time was fixed at 120 min because of there were not more significant changed in the amounts of oil, silybin A and silybin B over 120 min in all experiments.
2.3. Analysis of silybin A and silybin B in the milk thistle seeds extract Silybin A and silybin B were determined by high pressure liquid chromatography (HPLC, Dionex 680) equipped with Acclaim 120 C18 column of 150 mm × 4.6 mm and 3 m particle size. The mobile phase consisted of solvent A (methanol:water = 20:80 (v/v)) and solvent B (methanol) and applying the following gradient: 0–40 min %80 B, 40–45 min %10 B. The detection wavelength (UVD170U detector) was 288 nm. Injection volume and mobile phase flow rate were 20 L and 0.8 mL/min, respectively. Stock standard solution of silybin A and silybin B was prepared in methanol. Silybin A and silybin B in milk thistle seeds extract peaks were identified by comparing their retention times with the reference standards. The analysis was carried out in triplicate. Concentrations of silybin A and silybin B in the samples were estimated with leastsquares equation derived from peak area ratios of individual silybin A and silybin B.
2.4. Analysis of fatty acid in the milk thistle seeds extract The fatty acid composition was determined by conversion of fatty acid methyl esters (FAMEs) based on the using IUPAC method 2.301 [32]. The fatty acid composition was analyzed with gas chromatography which equipped with a flame ionization detector and a HP-88 column (100 m × 0.25 mm I.D., 0.2 m film thickness Agilent Technologies, Spain). The carrier and supporting gas were hydrogen (30 mL/min) and air (350 mL/min), respectively. The temperature of detector was set to 270 ◦ C. The temperature program of the column was set 90 ◦ C for 5 min and a subsequent increase to 190 ◦ C with 10 ◦ C/min rate. The injection was performed at 250 ◦ C with split ratio of 1/50 and injection volume 2 L. The individual fatty acid peaks were identified by comparison their retention times with standard fatty acids mixtures peaks (Supelco Fame Mix). The results were expressed as relative percentages of total fatty acids.
Fig. 1. The effect of temperature on amounts of silybin A, silybin B, total silybin and oil (200 bar, 4 mL/min, 0.925 mm) (×: oil, : total silybin, : silybin A, : silybin B).
3. Results and discussion The effect of process parameters (temperature, pressure, CO2 flow rate and particle size) were investigated on supercritical CO2 extraction of milk thistle seeds. The range of the temperature (40–80 ◦ C), the pressure (160–220 bar), the flow rate of supercritical CO2 (3–5 ml/min) and the particle size (0.3025–1.2 mm) were selected according to the literatures research. Firstly, the effect of temperature was researched on the amounts of oil, silybin A and silybin B extracted from milk thistle seeds. Extractions were accomplished for 40, 60 and 80 ◦ C temperature at 200 bar pressure, 4 mL/min CO2 flow rate, 0.925 mm particle size. For analyzing the effect of one parameter, values of other parameters fixed at nearly middle of the range selected. The maximum amounts of oil, silybin A, silybin B and total silybin (silybin A + silybin B) were recorded as, 129.64, 0.93, 1.21 and 2.14 mg/g seeds, respectively, for temperatures of 40 ◦ C (Fig. 1). It can be observed that amounts of oil, silybin A and total silybin were reduced with temperature rising through 80 ◦ C. When extraction temperature upward from 40 ◦ C to 80 ◦ C; the CO2 density was decreasing ∼29-fold at 200 bar pressure [33]. The decreasing of CO2 density was shown dominant effect than vapor pressure of the active components in oil. Parameter of temperature adversely affected to the yield of oil, silybin A and total silybin. Similar results have been reported for extracted oils from different natural products. Yield of nutmeg oils from nutmeg seeds reduced with rising temperature in the range of 313–323 K at pressure of 15 Mpa [34]. The study of hemp seeds extraction with supercritical CO2 was shown; the yield of oils reduced with increasing temperature in the range of 40–60 ◦ C at pressure of 300 bar [35]. As a result, 40 ◦ C was selected as an optimum temperature value in the following experiments. Secondly, the effect of extraction pressure on the amounts of oil, silybin A and silybin B was investigated. The experiments carried out at pressure 160, 180, 200 and 220 bar at 40 ◦ C for the temperature, 4 mL/min for the CO2 flow rate, 0.925 mm for the particle size. The experimental results were given in Fig. 2. It is evident from Fig. 2 that the amounts of oil and total silybin gone up significantly with increasing pressure from 160 to 180 bar at a constant temperature. The solubility of oil in supercritical CO2 increases with rising of pressure. Above 180 bar, there were no significant rise in amounts of oil and total silybin. From 160 to 180 bar, there were meaningful increases in the amounts of oil and total silybin (23% and 25%, respectively) than from 180 to 220 bar (0.73% and 0.67%, respectively). Similar results had been also reported by Mahmudah et al. [34], Uribe et al. [36], Rebolleda et al. [37].
H.T. C¸elik, M. Gürü / J. of Supercritical Fluids 100 (2015) 105–109
Fig. 2. The effect of pressure on amounts of silybin A, silybin B, total silybin and oil (40 ◦ C, 4 mL/min, 0.925 mm) (×: oil, : total silybin, : silybin A, : silybin B).
It was seen obviously that the amount of silybin B increased from the pressure of 160–180 bar. However, the amount of silybin B decreased at 180–220 bar pressure range. In spite of the fact that the amount of silybin A diminished between the pressure range of 160 and 180 bar, its concentration increased at 180–220 bar pressure range. It can be seen from Wallace et al. study that obtained the silybin A/silybin B ratio in milk thistle seeds oil was changed with extraction conditions. In this study, silybin A/silybin B ratio was recorded as 0.6 and 1 for extraction solvent of ethanol and methanol respectively by using traditional extraction methods [7]. As can be seen in our study, the silybin A/silybin B ratio was changed with temperature and pressure (Figs. 1 and 2). The yield of oil and total silybin were not significantly increased above 180 bar. The rising of pressure requires more energy in these pressure ranges for supercritical extraction system and this is not economic. For the reason optimum pressure value was determined as a 180 bar in the following experiments. Thirdly, the influence of the CO2 flow rate was investigated at a temperature of 40 ◦ C, pressure of 180 bar and particle size of 0.925 mm. The amounts of oil and total silybin were 148, 166, 170 mg oil/g seeds and 1.29, 2.25, 2.29 mg total silybin/g seeds for 3, 4 and 5 mL/min, respectively (Fig. 3). The amounts of oil and total silybin increased with rising CO2 flow rate until 4 mL/min. Rising of CO2 flow rate was caused turbulence in extractor and increased inter-molecular interaction between CO2 and the solute. External mass transfer resistance was
Fig. 3. The effect of CO2 flow rate on amounts of silybin A, silybin B, total silybin and oil (40 ◦ C, 180 bar, 0.925 mm) (×: oil, : total silybin, : silybin A, : silybin B).
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Fig. 4. The effect of particle size on amounts of silybin A, silybin B, total silybin and oil (40 ◦ C, 180 bar, 4 mL/min) (×: oil, : total silybin, : silybin A, : silybin B).
reduced by raising the CO2 flow rate and the intraparticle diffusion resistance was dominant. There was no significant rise in the amounts of the oil and total silybin above 4 mL/min (Fig. 3). It was explained that contact time was become shorter between CO2 and the seeds. Similar results had been also reported by I˙ c¸en and Gürü [18,19], Mahmudah et al. [34]. As shown in Fig. 3, amount of silybin A was increased with rising of CO2 flow rate. Amount of silybin B was raised until 4 mL/min. However the amount of silybin B was not affected significantly above 4 mL/min. The silybin A/silybin B ratio was also affected from parameter of CO2 flow rate. The optimum CO2 flow rate value was obtained as a 4 mL/min in the following experiments. In order to investigate the effect of particle size on the amounts of oil, total silybin, silybin A and silybin B, temperature, pressure, CO2 flow rate and extraction time were fixed as 40 ◦ C, 180 bar and 4 mL/min, respectively. Experiments were done according to the particle size of 1.2, 0.925 and 0.305 mm and results were given in Fig. 4. Particle size was diminished from 1.2 to 0.3025 mm, the amounts of oil and total silybin increased 74% and 68%, respectively (Fig. 4). The amounts of silybin A and silybin B were obtained 0.16 (for 1.2 mm) mg silybin A/g seeds and 2.29 (for 0.3025 mm) mg silybin A/g seeds; 1.18 (for 1.2 mm) mg silybin B/g seeds and 1.92 (for 0.3025 mm) mg silybin B/g seeds. The amounts of oil, total silybin, silybin A and silybin B increased with decreasing particle sizes because of rising contract area between CO2 and milk thistle seeds. The particle size is significant in extraction controlled by internal mass transfer resistances. Results about the effect of the particle size on the extraction process were similar with literature [35,38]. As a result of the experiments, the amounts of oil, total silybin, silybin A and silybin B extracted from milk thistle seeds were optimal with values of 327, 4.21, 2.29 and 1.92 mg/g milk thistle seeds, respectively, at 40 ◦ C, 180 bar, 4 mL/min and 0.3025 mm. The fatty acid compositions were determined using gas chromatography equipped with a flame ionization detector at optimum conditions. The individual fatty acid peaks were identified by comparison their retention times with standard fatty acids mixtures peaks. The relative percentages of total fatty acids compositions of milk thistle seeds oil in supercritical CO2 extracted were given Table 1. As shown in Table 1, the most abundant compounds were found as palmitic acid (8.15%), stearic acid (5.51%), oleic acid (24.10%), linoleic acid (54.97%) at optimum conditions.
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Table 1 Fatty acid composition of milk thistle seeds oil in supercritical CO2 extracted samples at optimum conditions. Fatty acid composition
%
C14:0 Myristic acid C16:0 Palmitic acid C16:0 Palmitoleic acid C17:0 Margaric acid C17:1 Heptadesenoic acid C18:0 Stearic acid C18:1 Oleic acid C18:2 Linoleic acid C20:0 Arachidic acid C18:3 Linolenic acid C20:1 Gadoleic acid C22:0 Behenic acid C24:0 Lignoceric acid
0.09 8.15 0.10 0.07 0.03 5.51 24.10 54.97 3.03 0.17 0.95 2.27 0.59
4. Conclusion Milk thistle grows on rocky places, uncultivated ground and tracksides. This plant is cheap material. Silybin compounds in milk thistle seeds were used for the treatment of cancer, liver, kidney, cardiac, and brain. In addition to this, milk thistle seeds involve fatty acids compounds. Linoleic acid and oleic acid are fatty acids compounds and they are used to show the antioxidant effect of natural phenols. In this study, supercritical CO2 extraction was performed to extract oil, silybin A and silybin B from milk thistle seeds. The parameters such as temperature, pressure, CO2 flow rate, particle size were shown to be effective on the amounts of oil, silybin A and silybin B. The maximum amount of oil, Silybin A and B were found as 327, 2.29 and 1.92 mg/g seeds respectively at 40 ◦ C, 180 bar, 4 mL/min, and 0.3025 mm. Percent of the most abundant compounds of fatty acid were found as palmitic acid (8.15%), stearic acid (5.51%), oleic acid (24.10%), linoleic acid (54.97%) at optimum conditions. Consequently, this study is thought to be promising for obtaining valuable compounds from milk thistle seeds to use in treatment of several diseases. It can be concluded that the Supercritical CO2 extraction can be considered as an applicable, feasible and environment friendly method to obtain silybin A, silybin B from milk thistle seeds due to cheap raw material, low operating cost and the non-toxic, inert and environment friendly effects of CO2 . Acknowledgement This work has been supported by Gazi University Project (06/2013-04). References [1] P. Corchete, Silybum marianum (L.) Gaertn: the source of silymarin, in: K.G. Ramawat, J.M. Merillon (Eds.), Bioactive Molecules and Medicinal Plants, Springer Ed, 2008, pp. 124–148. [2] A. Karkanis, D. Bilalis, A. Efthimiadou, Cultivation of milk thistle (Silybum marianum L. Gaertn.), a medicinal weed, Industrial Crops and Products 34 (2011) 825–830. [3] R. Agarwal, C. Agarwal, H. Ichikawa, R.P. Singh, B.B. Aggarwal, Anticancer potential of silymarin: from bench to bed side, Anticancer Research 26 (2006) 4457–4498. [4] R. Gazák, D. Walterová, V. Kren, Silybin and silymarin – new and emerging applications in medicine, Current Medicinal Chemistry 14 (2007) 315–338. [5] Y.P. Pei, J. Chen, W. Li, Progress in research and application in silymarin, Medicinal and Aromatic Plant Science and Biotechnology 3 (2009) 1–8. [6] A. Meghreji. Moin, C.N. Patel, J.B.R. Dave Badmanaban, J.A. Patel, Validated method for silymarin by spectrophotometry in bulk drug and pharmaceutical formulations, J. Chemical and Pharmaceutical Research 2 (2010) 396–400. [7] D.J. Wallace, E. Carrier, Clausen extraction of nutraceuticals from milk thistle: part II. Extraction with organic solvents, Applied Biochemistry and Biotechnology 105–108 (2003) 891–903. [8] J.F. Barreto, D.J. Carrier, N. Wallace, E. Sunny, C. Clausen, Extraction of nutraceuticals from milk thistle: I. Hot water extraction, Applied Biochemistry and Biotechnology (2003) 105–108.
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Contents lists available at ScienceDirect
The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu
Extraction of oil and silybin compounds from milk thistle seeds using supercritical carbon dioxide Hatice Tu˘gba C¸elik, Metin Gürü ∗ Gazi University, Engineering Faculty, Chemical Engineering Department, Maltepe, 06570 Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 8 January 2015 Received in revised form 19 February 2015 Accepted 20 February 2015 Available online 27 February 2015 Keywords: Milk thistle seeds Silybin A Silybin B Supercritical extraction
a b s t r a c t Milk thistle is a plant that has been used medicinally for over 2000 years. It has been used for the treatment of many diseases such as cancer, liver, kidney, cardiac, brain. Their seeds are rich in silymarin compounds (especially silybin A and silybin B) and fatty acids. The purpose of this study was to extract silybin A and silybin B from milk thistle seeds with the supercritical CO2 . The effect of operating parameters such as temperature (40–80 ◦ C), pressure (160–220 bar), CO2 flow rate (3, 4 and 5 mL/min) and particle size (0.3025, 0.925 and 1.2 mm) on extracted oil, silybin A and silybin B were investigated. Fatty acid composition in milk thistle seed extract was determined at optimum conditions. The results indicated that the optimal conditions were 40 ◦ C temperature, 200 bar pressure, 4 mL/min CO2 flow rate and 0.3025 mm particle size. In these conditions; the amounts of oil, silybin A and silybin B were obtained 327, 2.29 and 1.92 mg/g milk thistle seeds respectively. For the fatty acids obtained by supercritical CO2 extraction, the most abundant compounds were palmitic acid (8.15%); stearic acid (5.51%), oleic acid (24.10%); linoleic acid (54.97%) at optimum conditions. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The milk thistle is an annual or biannual plant of the Asteraceae family. The milk thistle seeds are included in silymarin compounds, especially silybin A and silybin B. Silybin A and silybin B are well known as antioxidant [1,2] and have been used for the treatment of cancer, liver, kidney, cardiac, brain, cirrhosis and poisoning of alcohol, drugs or toxins [3–6]. Also the milk thistle seeds are consisting of fatty acid compounds like oleic acid, linoleic acid, etc. which are good antioxidant substances too. Silymarin compounds [7–11] and fatty acid compounds [12–14] are generally produced by traditional extraction method from plant. Supercritical fluid method is used many industries such as medicine, chemistry, biochemistry and food. Supercritical fluid extraction method has supremacy in comparison to conventional solvent extraction methods. The most widely used supercritical fluid is carbon dioxide which is non-toxic, relatively inert, and non-flammable [15]. The extraction solvents such as petroleum ether and ethyl acetate are widely used in traditional methods but they cannot be removed completely from extract. However, carbon dioxide is gaseous at ambient conditions of temperature and
∗ Corresponding author. Tel.: +90 312 5823555; fax: +90 312 2308434. E-mail addresses: [email protected] (H.T. C¸elik), [email protected] (M. Gürü). http://dx.doi.org/10.1016/j.supflu.2015.02.025 0896-8446/© 2015 Elsevier B.V. All rights reserved.
pressure and completely separated from the extract in these conditions [16,17]. Several investigations were reported for extraction by supercritical CO2 from tea [18,19], jatropha [20,21], Stevia rebaudiana leaves [22], grape [23], mango leaves [24] and watermelon [25]. Hadolin et al. [26] studied the extraction of milk thistle seeds by supercritical CO2 /propan to obtain vitamin E content. Szentmihályi et al. [27] studied the extraction of milk thistle seeds by supercritical CO2 /propan to obtain fatty acid, pheophytin, carotene, tocopherol content and concentrations of some metals. In addition, there are some studies that investigate the effect of supercritical CO2 with together cosolvent such as methanol, ethanol, water on extraction process [28–31]. But there is no study in the literature regarding, the effect of parameters of temperature, pressure, particle size and CO2 flow rate on silybin A and silybin B in oil extracted from the milk thistle seeds by supercritical CO2 without cosolvent. The purpose of this study was to investigate of pharmaceutical importance silybin A and silybin B were extracted from milk thistle seeds by the supercritical CO2 without using cosolvent due to the preventing of cosolvent contamination. The purity of silybin compounds must be affected by cosolvent. The qualitative and quantitative analysis of silybin A and silybin B were performed by HPLC. The fatty acids composition in extract was analyzed by GCFID. The effect of parameters such as temperature, pressure, particle size and CO2 flow rate were investigated on supercritical extraction process.
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2. Materials and method 2.1. Materials Milk thistle was obtained from Mu˘gla in Turkey. Whole seeds were separated from flowers. They were dried at room conditions and then ground up the specific particle dimension by a coffee grinder. The specific particle dimensions of samples were 0.3025, 0.925 and 12 mm. Grinding was carried out just before extraction in order to avoid oxidation.
2.2. Supercritical CO2 extraction Extractions were performed in laboratory scale supercritical fluid extraction system (Applied Separation Speed, USA). A 24 mL of extraction vessel was packed with 4 g of the milk thistle seeds particles. The vessel was put in a temperature-controlled oven. CO2 was sent continuously through the system. The extract was collected in vials. The detailed description of the system was given in another our study [18]. The collection vials were changed per every 30 min. The experiments were performed among 120 min in the range of 40–80 ◦ C, 160–220 bar, 3–5 mL CO2 /min, 0.3025–1.2 mm particle size. Extraction time was fixed at 120 min because of there were not more significant changed in the amounts of oil, silybin A and silybin B over 120 min in all experiments.
2.3. Analysis of silybin A and silybin B in the milk thistle seeds extract Silybin A and silybin B were determined by high pressure liquid chromatography (HPLC, Dionex 680) equipped with Acclaim 120 C18 column of 150 mm × 4.6 mm and 3 m particle size. The mobile phase consisted of solvent A (methanol:water = 20:80 (v/v)) and solvent B (methanol) and applying the following gradient: 0–40 min %80 B, 40–45 min %10 B. The detection wavelength (UVD170U detector) was 288 nm. Injection volume and mobile phase flow rate were 20 L and 0.8 mL/min, respectively. Stock standard solution of silybin A and silybin B was prepared in methanol. Silybin A and silybin B in milk thistle seeds extract peaks were identified by comparing their retention times with the reference standards. The analysis was carried out in triplicate. Concentrations of silybin A and silybin B in the samples were estimated with leastsquares equation derived from peak area ratios of individual silybin A and silybin B.
2.4. Analysis of fatty acid in the milk thistle seeds extract The fatty acid composition was determined by conversion of fatty acid methyl esters (FAMEs) based on the using IUPAC method 2.301 [32]. The fatty acid composition was analyzed with gas chromatography which equipped with a flame ionization detector and a HP-88 column (100 m × 0.25 mm I.D., 0.2 m film thickness Agilent Technologies, Spain). The carrier and supporting gas were hydrogen (30 mL/min) and air (350 mL/min), respectively. The temperature of detector was set to 270 ◦ C. The temperature program of the column was set 90 ◦ C for 5 min and a subsequent increase to 190 ◦ C with 10 ◦ C/min rate. The injection was performed at 250 ◦ C with split ratio of 1/50 and injection volume 2 L. The individual fatty acid peaks were identified by comparison their retention times with standard fatty acids mixtures peaks (Supelco Fame Mix). The results were expressed as relative percentages of total fatty acids.
Fig. 1. The effect of temperature on amounts of silybin A, silybin B, total silybin and oil (200 bar, 4 mL/min, 0.925 mm) (×: oil, : total silybin, : silybin A, : silybin B).
3. Results and discussion The effect of process parameters (temperature, pressure, CO2 flow rate and particle size) were investigated on supercritical CO2 extraction of milk thistle seeds. The range of the temperature (40–80 ◦ C), the pressure (160–220 bar), the flow rate of supercritical CO2 (3–5 ml/min) and the particle size (0.3025–1.2 mm) were selected according to the literatures research. Firstly, the effect of temperature was researched on the amounts of oil, silybin A and silybin B extracted from milk thistle seeds. Extractions were accomplished for 40, 60 and 80 ◦ C temperature at 200 bar pressure, 4 mL/min CO2 flow rate, 0.925 mm particle size. For analyzing the effect of one parameter, values of other parameters fixed at nearly middle of the range selected. The maximum amounts of oil, silybin A, silybin B and total silybin (silybin A + silybin B) were recorded as, 129.64, 0.93, 1.21 and 2.14 mg/g seeds, respectively, for temperatures of 40 ◦ C (Fig. 1). It can be observed that amounts of oil, silybin A and total silybin were reduced with temperature rising through 80 ◦ C. When extraction temperature upward from 40 ◦ C to 80 ◦ C; the CO2 density was decreasing ∼29-fold at 200 bar pressure [33]. The decreasing of CO2 density was shown dominant effect than vapor pressure of the active components in oil. Parameter of temperature adversely affected to the yield of oil, silybin A and total silybin. Similar results have been reported for extracted oils from different natural products. Yield of nutmeg oils from nutmeg seeds reduced with rising temperature in the range of 313–323 K at pressure of 15 Mpa [34]. The study of hemp seeds extraction with supercritical CO2 was shown; the yield of oils reduced with increasing temperature in the range of 40–60 ◦ C at pressure of 300 bar [35]. As a result, 40 ◦ C was selected as an optimum temperature value in the following experiments. Secondly, the effect of extraction pressure on the amounts of oil, silybin A and silybin B was investigated. The experiments carried out at pressure 160, 180, 200 and 220 bar at 40 ◦ C for the temperature, 4 mL/min for the CO2 flow rate, 0.925 mm for the particle size. The experimental results were given in Fig. 2. It is evident from Fig. 2 that the amounts of oil and total silybin gone up significantly with increasing pressure from 160 to 180 bar at a constant temperature. The solubility of oil in supercritical CO2 increases with rising of pressure. Above 180 bar, there were no significant rise in amounts of oil and total silybin. From 160 to 180 bar, there were meaningful increases in the amounts of oil and total silybin (23% and 25%, respectively) than from 180 to 220 bar (0.73% and 0.67%, respectively). Similar results had been also reported by Mahmudah et al. [34], Uribe et al. [36], Rebolleda et al. [37].
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Fig. 2. The effect of pressure on amounts of silybin A, silybin B, total silybin and oil (40 ◦ C, 4 mL/min, 0.925 mm) (×: oil, : total silybin, : silybin A, : silybin B).
It was seen obviously that the amount of silybin B increased from the pressure of 160–180 bar. However, the amount of silybin B decreased at 180–220 bar pressure range. In spite of the fact that the amount of silybin A diminished between the pressure range of 160 and 180 bar, its concentration increased at 180–220 bar pressure range. It can be seen from Wallace et al. study that obtained the silybin A/silybin B ratio in milk thistle seeds oil was changed with extraction conditions. In this study, silybin A/silybin B ratio was recorded as 0.6 and 1 for extraction solvent of ethanol and methanol respectively by using traditional extraction methods [7]. As can be seen in our study, the silybin A/silybin B ratio was changed with temperature and pressure (Figs. 1 and 2). The yield of oil and total silybin were not significantly increased above 180 bar. The rising of pressure requires more energy in these pressure ranges for supercritical extraction system and this is not economic. For the reason optimum pressure value was determined as a 180 bar in the following experiments. Thirdly, the influence of the CO2 flow rate was investigated at a temperature of 40 ◦ C, pressure of 180 bar and particle size of 0.925 mm. The amounts of oil and total silybin were 148, 166, 170 mg oil/g seeds and 1.29, 2.25, 2.29 mg total silybin/g seeds for 3, 4 and 5 mL/min, respectively (Fig. 3). The amounts of oil and total silybin increased with rising CO2 flow rate until 4 mL/min. Rising of CO2 flow rate was caused turbulence in extractor and increased inter-molecular interaction between CO2 and the solute. External mass transfer resistance was
Fig. 3. The effect of CO2 flow rate on amounts of silybin A, silybin B, total silybin and oil (40 ◦ C, 180 bar, 0.925 mm) (×: oil, : total silybin, : silybin A, : silybin B).
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Fig. 4. The effect of particle size on amounts of silybin A, silybin B, total silybin and oil (40 ◦ C, 180 bar, 4 mL/min) (×: oil, : total silybin, : silybin A, : silybin B).
reduced by raising the CO2 flow rate and the intraparticle diffusion resistance was dominant. There was no significant rise in the amounts of the oil and total silybin above 4 mL/min (Fig. 3). It was explained that contact time was become shorter between CO2 and the seeds. Similar results had been also reported by I˙ c¸en and Gürü [18,19], Mahmudah et al. [34]. As shown in Fig. 3, amount of silybin A was increased with rising of CO2 flow rate. Amount of silybin B was raised until 4 mL/min. However the amount of silybin B was not affected significantly above 4 mL/min. The silybin A/silybin B ratio was also affected from parameter of CO2 flow rate. The optimum CO2 flow rate value was obtained as a 4 mL/min in the following experiments. In order to investigate the effect of particle size on the amounts of oil, total silybin, silybin A and silybin B, temperature, pressure, CO2 flow rate and extraction time were fixed as 40 ◦ C, 180 bar and 4 mL/min, respectively. Experiments were done according to the particle size of 1.2, 0.925 and 0.305 mm and results were given in Fig. 4. Particle size was diminished from 1.2 to 0.3025 mm, the amounts of oil and total silybin increased 74% and 68%, respectively (Fig. 4). The amounts of silybin A and silybin B were obtained 0.16 (for 1.2 mm) mg silybin A/g seeds and 2.29 (for 0.3025 mm) mg silybin A/g seeds; 1.18 (for 1.2 mm) mg silybin B/g seeds and 1.92 (for 0.3025 mm) mg silybin B/g seeds. The amounts of oil, total silybin, silybin A and silybin B increased with decreasing particle sizes because of rising contract area between CO2 and milk thistle seeds. The particle size is significant in extraction controlled by internal mass transfer resistances. Results about the effect of the particle size on the extraction process were similar with literature [35,38]. As a result of the experiments, the amounts of oil, total silybin, silybin A and silybin B extracted from milk thistle seeds were optimal with values of 327, 4.21, 2.29 and 1.92 mg/g milk thistle seeds, respectively, at 40 ◦ C, 180 bar, 4 mL/min and 0.3025 mm. The fatty acid compositions were determined using gas chromatography equipped with a flame ionization detector at optimum conditions. The individual fatty acid peaks were identified by comparison their retention times with standard fatty acids mixtures peaks. The relative percentages of total fatty acids compositions of milk thistle seeds oil in supercritical CO2 extracted were given Table 1. As shown in Table 1, the most abundant compounds were found as palmitic acid (8.15%), stearic acid (5.51%), oleic acid (24.10%), linoleic acid (54.97%) at optimum conditions.
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Table 1 Fatty acid composition of milk thistle seeds oil in supercritical CO2 extracted samples at optimum conditions. Fatty acid composition
%
C14:0 Myristic acid C16:0 Palmitic acid C16:0 Palmitoleic acid C17:0 Margaric acid C17:1 Heptadesenoic acid C18:0 Stearic acid C18:1 Oleic acid C18:2 Linoleic acid C20:0 Arachidic acid C18:3 Linolenic acid C20:1 Gadoleic acid C22:0 Behenic acid C24:0 Lignoceric acid
0.09 8.15 0.10 0.07 0.03 5.51 24.10 54.97 3.03 0.17 0.95 2.27 0.59
4. Conclusion Milk thistle grows on rocky places, uncultivated ground and tracksides. This plant is cheap material. Silybin compounds in milk thistle seeds were used for the treatment of cancer, liver, kidney, cardiac, and brain. In addition to this, milk thistle seeds involve fatty acids compounds. Linoleic acid and oleic acid are fatty acids compounds and they are used to show the antioxidant effect of natural phenols. In this study, supercritical CO2 extraction was performed to extract oil, silybin A and silybin B from milk thistle seeds. The parameters such as temperature, pressure, CO2 flow rate, particle size were shown to be effective on the amounts of oil, silybin A and silybin B. The maximum amount of oil, Silybin A and B were found as 327, 2.29 and 1.92 mg/g seeds respectively at 40 ◦ C, 180 bar, 4 mL/min, and 0.3025 mm. Percent of the most abundant compounds of fatty acid were found as palmitic acid (8.15%), stearic acid (5.51%), oleic acid (24.10%), linoleic acid (54.97%) at optimum conditions. Consequently, this study is thought to be promising for obtaining valuable compounds from milk thistle seeds to use in treatment of several diseases. It can be concluded that the Supercritical CO2 extraction can be considered as an applicable, feasible and environment friendly method to obtain silybin A, silybin B from milk thistle seeds due to cheap raw material, low operating cost and the non-toxic, inert and environment friendly effects of CO2 . Acknowledgement This work has been supported by Gazi University Project (06/2013-04). References [1] P. Corchete, Silybum marianum (L.) Gaertn: the source of silymarin, in: K.G. Ramawat, J.M. Merillon (Eds.), Bioactive Molecules and Medicinal Plants, Springer Ed, 2008, pp. 124–148. [2] A. Karkanis, D. Bilalis, A. Efthimiadou, Cultivation of milk thistle (Silybum marianum L. Gaertn.), a medicinal weed, Industrial Crops and Products 34 (2011) 825–830. [3] R. Agarwal, C. Agarwal, H. Ichikawa, R.P. Singh, B.B. Aggarwal, Anticancer potential of silymarin: from bench to bed side, Anticancer Research 26 (2006) 4457–4498. [4] R. Gazák, D. Walterová, V. Kren, Silybin and silymarin – new and emerging applications in medicine, Current Medicinal Chemistry 14 (2007) 315–338. [5] Y.P. Pei, J. Chen, W. Li, Progress in research and application in silymarin, Medicinal and Aromatic Plant Science and Biotechnology 3 (2009) 1–8. [6] A. Meghreji. Moin, C.N. Patel, J.B.R. Dave Badmanaban, J.A. Patel, Validated method for silymarin by spectrophotometry in bulk drug and pharmaceutical formulations, J. Chemical and Pharmaceutical Research 2 (2010) 396–400. [7] D.J. Wallace, E. Carrier, Clausen extraction of nutraceuticals from milk thistle: part II. Extraction with organic solvents, Applied Biochemistry and Biotechnology 105–108 (2003) 891–903. [8] J.F. Barreto, D.J. Carrier, N. Wallace, E. Sunny, C. Clausen, Extraction of nutraceuticals from milk thistle: I. Hot water extraction, Applied Biochemistry and Biotechnology (2003) 105–108.
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