Study on Silybum marianum Seed through Fatty Acids Comparison, Peroxide Tests, Refractive Index and Oil Percentage

In this study, milk thistle seeds were collected from four regions of Iran (Ahvaz, Lorestan, Kazeroon and Zarghan). The oil extracted from seeds of plant using n-hexane solvent extraction soxhlet. The extracted oil was analyzed by gas chromatography and fatty acids were identified in all four samples. In all four samples, the oil refractive index of Milk thistle oil fatty acids vis. Linoleic acid, Oleic acid, Palmitic acid was evaluated. The Peroxide test and determination of oil percentage was performed in Milk thistle seed oil.


Materials
Milk thistle seed were collected from four regions in Iran, (Ahvaz, Lorestan, Kazeroon and Zarghan (Fars-Iran) in July 2015.After collecting the seeds of milk thistle the seeds were dried and prepared for extraction of oil (Figure 1).Oleic acid composition of oil was identified by gas chromatography, also measure the refractive index, determining the percentage of oil, determining the peroxide value; Milk thistle seed for oils was performed.

Oil extraction
The seeds oil was extracted using Soxhlet. 10,11he seeds were dried from the Milk thistle plant.30 grams of milk thistle seed were powder and then the powder, cartouche putinto Soxhlet, special balloons solvent and 500 ml of n-hexane solvent added into Soxhlet in 70 ° a period of 8 hours.Then solvent flask containing the milk thistle seed oil was separated by Rotary and the weight of oil was determined.Grams of oil obtained from the dried plants are used for oil extraction subtraction.Methyl esters are identified using gas chromatography.

Analysis of fatty acid methyl esters by GC
The FAMES were analyzed by GC according to the method described by Azadmard-Damirchi and Dutta. 12The GC instrument was equipped with a flame ionization detector and a split/split less injector.A film thickness fused-silica capillary column BPX70 (SGE, Austin, TX, USA) was used for analysis.Injector and detector temperatures were 230 and 250 °C, respectively.Oven conditions were 158 °C increased to 220 °C at a rate of 2 °C/min and maintained for 5 minutes.Helium was used as the carrier gas and nitrogen as the make-up gas at a flow rate of 30 ml/min.The FAMES were identified by comparison of their retention times with standard FAMES and the peak areas reported as a percentage of the total fatty acids. 13

Determination of oil peroxide
Peroxide value (PV) was determined according to the AOAC method. 14bout 5 g of oil was weighed into a 250 mL flask.Previously prepared acetic acid/chloroform (CHCl3) solution (30 mL), saturated potassium iodide (0.5 mL) and distilled water (30 mL) were added with occasional shaking.The mixture was titrated with 0.05 mol L−1 Na2S2O3 with vigorous shaking; 0.5 mL of 10 mL L−1 starch solution was added a dtitration was continued with vigorous shaking, to release all iodine from the CHCl3 layer, until the blue color just disappeared.PV was calculated using this equation. 15 (meq O2 kg−1 sample) = (S × 2M × 1000)/m Where S (mL) is the volume of Na2S2O3 consumed (blank corrected), M (mol L−1) is molarity of Na2S2O3 and m (g) is the mass of the test sample. 15

Measure the refractive index
A Metrohm 743 Rancimat (Metrohm AG, Herisau, Switzerland) was used for analysis of the oxidative stability index (OSI).The tests were done with 3 g oil sample at a 120 °C temperature in airflow rate of 15 L h−1. 16

Results and discussions
8][19][20] The climatic conditions and varieties grown greatly influence the oil and fatty acids content. 20Oil obtained from the seeds of milk thistle were analyzed with gas chromatography, and the composition of fatty acids in all four samples of milk thistle oil from the areas was prepared and identified (Table 1).The composition of fatty acids in plant oils contains Milk thistle was included C18:2, C18:1, C16:0.The Linoleic acid achieved from Milk thistle which collected from Lorestan and Ahvaz exhibited more amount compared with Kazeroon and Zarghan.Fatty acid composition of plants collected from Ahvaz, Lorestan, Kazeroon and Zarghan is shown in figure 1 to 4, respectively.

Oxidative stability
Oxidative stability of oil for each four sample was performed.The results are shown in table 2. Results in this experiment showed a significant effect of weather conditions on the oxidation of the oil sample.

Determine percentage of oil
Percentage of oil was determined for each sample and the results are shown in table 3. The highest percent of oil content exhibited from Ahvaz.

Refractive index
Rancimat device used to measure the refractive index of the oil and the results are presented in bellow (Table 4).The less number of double bonds in samples revealed less refractive index and the samples with higher refractive index showed lower melting point and saturation.

Figure 1 :
Figure 1: Milk thistle oil fatty acid composition extracted from Ahvaz

Figure 2 :
Figure 2: Milk thistle oil fatty acid composition extracted from Lorestan