FATTY ACID COMPOSITION OF THE LIPID CLASSES OF
SILYBUM
MARIANUM
SEED OIL
Zafar Iqbal*1, Amran Waheed1, Mohammad Akram2, and Khalid Mahmood3 1
Applied Chemistry Research Centre, PCSIR Laboratories Complex, Ferozepur Road, Lahore-54000-Pakistan.
2
Medicinal Botanic Centre, PCSIR Labs Complex, Peshawar, Pakistan. 3
13-Dudley Place, Middlesex Hayes, United Kingdom.
ABSTRACT
Powdered seeds of two varieties of silybum marianum were subjected to soxhlet apparatus using hexane as solvent for oil extraction. Yield of oil from both categories was 33.25 % and 34.50 % respectively. Extracted oils were fractionated into hydrocarbons, wax asters, triglycerides, free fatty acids, 1,3–diglycerides, 1,2–diglycerides and monoglycerides by thin layer chromatography and identified by the comparison with Rf values of respective standards samples. The fatty acids composition of both oils was determined after conversion into their respective methyl esters. These methyl esters were analyzed by gas chromatography. Oil of category 1 silybum marianum contain oleic acid (40.12 %) as a major component followed by linoleic acid (34.70 %), palmitic acid (11.48 %), linolenic acid (4.75 %), stearic acid (4.23 %), arachidic acid (3.24 %) and lauric acid (1.48 %). Similarly in category 2 silybum marianam seed oil oleic acid (39.96 %) was the dominating fatty acid followed by linoleic acid (34.15 %), palmitic acid (12.32 %), linoleinic acid (4.48 %), arachidic acid (3.92 %), lauric acid (1.08 %) and myristic acid (0.24 %) respectively. Furthermore saturated and unsaturated fatty acids in the oil, wax esters, triglycerides, diglycerides, monoglycerides and free fatty acids were also determined.
KEYWORDS: Silybum marianum, Soxhlet, Gas chromatography, Lipid, Oleic acid, Linoleic acid.
Volume 4, Issue 12, 2183-2191. Research Article ISSN 2277– 7105
*Correspondence for
Author
Zafar Iqbal
Applied Chemistry Research Centre, PCSIR Laboratories Complex, Ferozepur Road, Lahore-54000-Pakistan.
Article Received on 09 Nov 2015,
INTRODUCTION
Silybum marianum (L.) Gaertner (milk thistle) is an annual winter or biennial vascular, stout
and erect herb belongs to family Astereaceae.[1] It grows to a height of 2.5 meters and a width of 0.9 meters.[2, 3] It has two varieties clearly distinguished by purplish and white head flowers.[4] After flowering, thick, white, fluffy thistledown develops and scatters the seeds.[2,5]
Silybum marianum grows natively in the Mediterranean area of Europe, North and South America and Australia and is widespread in the other regions of the world.[6, 7] Flowering of silybum marianum begins in the spring and the plant is harvested at the begning of summer.[8]
The seeds are obliquely obovoid and are 6-7 millimetres long and 3 millimeters wide. They are brown in colour, and have a yellowish projecting swollen ring at its tip and a canaliculated hilum at the other end.[9, 10] The seed yields 1.5-3% of flavanolignans including silibinin, silydianin, silychristin and taxifoline, which are collectively called Silymarin.[11] Silymarin has long history for the treatment of hepatobiliary disease. It is purported to have value as a liver protectant to lessen damage from potentially hepatotoxic drugs and for treating liver disorders including toxic liver damage caused by chemicals.[12] It also improve the quality of breast milk.[13,14] Silymarine acts in four different ways as an antioxidant, absorber, regulator and stabilizer of cell membrane permeability that prevents the entering of hepatotoxic substances in to hepatocytes.[15] Silybum marianum seed contains 20-25% fatty acids; of this amount, 50-60% is linoleic acid and 25-30% is oleic acid. Linoleic acid is an essential fatty acid required for the production of prostaglandins, which help to reduce inflammation and aid in normalizing hormonal balance in both women and men.[4,11] Silybum marianum has been widely studied worldwide for their oil contents, amino acids, complex
lipids and food nutrients.[7,15] Hydrocarbons, sterols and fatty acid composition of Egyptian wild silybum marianum were also reported.[6] In Pakistan it has been studied for potential source of diet and climatic variation effects on the growth.[15,16] The effects of heavy metals on the growth of silybum marianum in various polluted areas were also investigated.[17]
MATERIALS AND METHODS Collection of silybum marianum seeds
Wildly growing Silybum marianum purple head flower variety was selected from two different localities of NWFP (Pakistan). Silybum marianum, plants were ripened in the month of April and after careful mowing seeds were collected manually. These seeds were washed with distilled water and dried. Seeds were then grinded to powder and stored for oil extraction.[18]
Extraction of oil
Powder seeds 100 g of category 1 silybum marianum was taken in soxhlet apparatus and n-hexane 400 ml was taken in a one-liter conical flask. After five hours of reflux the solvent was removed under reduced pressure in a rotary evaporator. Yellow colored residual oil thus obtained was dried over anhydrous sodium sulfate and percentage yield was determined on dry basis. Similarly category 2 seed oil was also extracted.
Removal of non-lipid impurity from oil
The oil thus obtained was washed3 with chloroform, methanol, and 0.9 % aqueous sodium chloride (3:48:47) to remove the non-lipid impurities. The non-lipid impurities were dissolved in the mixture of chloroform and methanol. This washing was repeated twice and the oil so obtained was kept in an inert atmosphere for further analysis.
Fractionation of lipids
Esterification of fatty acid of lipids
Fatty acids of each fraction were converted into Methyl esters in the presence of boron-triflouride. The esters were extracted with n- hexane and washed with saturated solution of sodium chloride. After the washing the extracts were dried by adding anhydrous sodium sulphate and then filtered.[20]
Identification of fatty acids by GLC
The methyl esters of the oil and lipid fractions were analyzed for the respective fatty acid composition by gas chromatography. The apparatus used for this purpose was Shimadzu GC- 14 A, equipped with flame ionization detector (FID) and a capillary column PEG (25mx 0.2mm i.d). The temperatures programming of the column oven was set as 180 oC – 220 oC with rise of 4 oC/ min Nitrogen was used as the carrier gas and its flow was adjusted to 20 ml/min at split ratio of 1:50. The temperatures of detector and injector were set at 250 oC and 230 oC respectively. The peaks were recorded on Shimadzu C-R4A Chromatopac and identified by comparison of their retention time with those of the standard methyl esters analyzed under the same conditions.
RESULT AND DISCUSSION
presence of fatty acids from C12 to C20 (Table 3, 4). These results reflected that oleic acid was predominant in whole oil of both categories. It was 40.12% and 39.96% of the oil in category 1 and 2 respectively. Similar trend was observed in their fractions. Category 1 contain oleic acid in wax ester (47.95 %), triglycerides (47.75 %), 1,3 diglycerides (47.26 %), 1,2 diglycerides (47.66 %), monoglycerides (47.80 %) and free fatty acids (44.80 %) (Table-3). While category 2 contain oleic acid in wax ester (50.85%), triglycerides (46.54%), 1,3 diglycerides (44.79 %), 1,2 diglycerides (56.28 %), monoglycerides (53.87 %) and free fatty acids (66.26 %) (Table-4). Mallah et al., 2003[9] and Hussain et al., 2003[19], observed this trend of oleic acid dominance.
Other major fatty acids found in category 1 seed oils were linoleic acids (34.70 %), palmitic acid (11.48 %), linoleinic acid (4.75 %), stearic acid (4.23 %), arachidic acid (3.24 %) and lauric acid (1.48 %). While category 2 silybum marianum oil have lenolenic acid (34.15 %), palmitic acid (12.32 %), linoleic acid (4.48 %), arachidic acid (3.92 %), stearic acid (3.85 %), Myristic (1.08 %) and lauric acid (0.24 %). There was a little difference in the yield and composition of the oils. The only major difference was that category 2 oil contains lauric acid while it was absent in the category 1. Saturated and unsaturated fatty acid percentage was 20.43 and 79.59 in category 1 while 21.41 and 78.59 was in category 2 silybum marianum oil. Hammouda et al. 1994[8], reported oleic acid was found major constituent while Funes et al. 1979[10] and Hassan et al.2003[19] reported linoleic acid as major constituents of silybum marianum seed oil. The quantitative analysis of fatty acids showed that the percentages of
category 1 silybum marianum seed oil. While category 2 silybum marianum seed oil contains palmitic acid (12.32 %), arachidic acid (3.92 %), stearic acid (3.85 %), myristic acid (1.08 %) and lauric acid (0.24 %). Lauric acid was present in category 2 silybum marianm oil while it was absent in the category 1 seed oil. palmitic acid (9.4 %) and stearic acid (6.6 %) were present in silybum marianum seed oil.[8]
The difference in fatty acid composition may be due to geographical origin of varieties.[22] We conclude from our whole research work that the oil of silybum marianum can be produced on a large scale and can be used for industries purposes. Our finding on fatty acid composition of lipids support that the oil can be used for edible purpose after other investigation like, microbiology anti fungal activities etc.
Table (1): Physical characteristics of silybum marianum seed oil of both categories. Category 1 (%) Category 2 (%)
Moisture Content 4.42 4.50
Oil Content 33.25 34.50
Table (2): Rf value of the different lipids classes of seed oil of silybum marianum
(Category I and 2).
Lipid Classes Rf value of Category 1 Rf value of Category 2
Wax Esters (WE) 0.93 0.92
Triglyceries (TG) 0.61 0.60
Free fatty acids (FFA) 0.41 0.40
1-3 Diglycerides (1,3-DG) 0.33 0.32
1-2 Diglycerides (1,2-DG) 0.29 0.28
Mono-glycerides (MG) 0.18 0.17
Table (3): Fatty acid compositions of whole oil and its lipid class of silybum marianum seed oil (Category – 1)
Lipid fractions
Fatty acids Whole oil W. E T.G. F.F.A. 1,3–D.G. 1,2-D.G. M.G.
C14:0 1.48 0.52 0.60 1.70 1.12 0.89 1.24
C16:0 11.48 13.91 13.95 6.67 11.27 10.56 12.80
C18.0 4.23 5.87 6.97 2.89 4.08 5.03 4.31
C18:1 40.12 47.95 47.75 44.80 47.26 47.66 47.80 C18:2 34.70 24.80 23.72 34.22 29.12 28.64 27.06
C18:3 4.75 4.82 4.92 7.25 4.39 5.06 4.78
Table (4): Fatty acid compositions of lipid class of silybum marianum seed oil (Category 2) Lipid fractions
Fatty acid Whole oil W.E. T.G. F.F.A. 1,3 –D.G. 1,2 –D.G. M.G.
C12 0.24 1.89 0.14 0.26 1.44 0.05 0.47
C14 1.08 1.12 0.40 --- 3.04 0.23 0.16
C16 12.32 28.01 16.98 19.97 19.12 21.65 25.45
C18:0 3.85 12.56 3.24 1.70 0.11 4.28 9.00
C18:1 39.96 50.85 46.54 66.26 44.79 56.28 53.87 C18:2 34.15 3.11 10.05 6.64 27.42 8.67 7.90
C18:3 4.48 2.42 5.20 5.13 2.58 5.03 2.80
C20.0 3.92 --- 1.18 --- 1.46 3.77 ---
Table (5): Percentage of saturated and unsaturated fatty acid of silybum marianum (Category–1).
Fractions Percentage of
saturated fatty acid
Percentage of unsaturated fatty acid
Whole Oil 20.43 79.57
Wax Ester 22.37 77.63
Triglyceries 23.61 76.39
Free fatty acids 13.73 86.27
1,3- Diglyceride 19.23 80.77
1,2- Diglyceride 18.64 81.36
Monoglyceride 20.36 79.64
Table (6): Percentage of saturated and unsaturated fatty acid of silybum marianum (Category–2).
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Wax Ester 43.58 56.42
Triglyceride 15.94 84.06
Free fatty acids 21.93 78.07
1,3- Diglyceride 25.17 74.83
1,2- Diglyceride 29.98 70.02
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