Buletin USAMV-CN, 62/2006 (225-230) ISSN 1454-2382
COMPARATIVE STUDIES ON FATTY ACID FINGERPRINT FROM TOTAL LIPIDS AND PHYTOSTEROL ESTERS OF SOME EDIBLE PLANT OILS Dulf F. V., C. Bele, Sonia Spinean, Veronica S. Chedea, G. Zegrean, Carmen Socaciu University of Agricultural Sciences and Veterinary Medicine, 3-5 Mãnãstur Str., 400372 Cluj-Napoca, Romania, e-mail:
[email protected] Key words: fatty acids, phytosterol esters, GC-analysis, edible plant oils Abstract: The aim of our study was the identification of two categories of bioactive molecules, characteristic to functional oils: sunflower, soybean, rapeseed, linseed, and hempseed oil, processed from seeds in Romania. Two separation and purification techniques, column chromatography and thin layer chromatography, were applied and the profiles of fatty acids from total lipids and phytosterol esters fraction was determined by GC-FID. Transesterification procedures were used for derivatization of fatty acids for GC analysis. The main fatty acids from the studied oil samples were: oleic, linoleic and palmitic fatty acids. Hemp oil is one of the few seed oils that contains both of omega-6 and omega-3, gamma-linolenic acid being one of relatively rare fatty acids. A 3.07% content of γ-linolenic acid was found. Hemp oil proved to be the most rich in phytosterols, been recommended as a functional food.
INTRODUCTION
Vegetable oils are mainly constituted by triacylglycerols (95-98%) and complex mixtures of minor compounds (2-5%) of a wide range of chemical nature. These minor constituents show a broad qualitative and quantitative composition, depending on the vegetal species from they were obtained. The main groups of minor constituents present in vegetable oil are: sterols, fatty alcohols, wax esters, hydrocarbons, tocopherols and tocotrienols, phenolic compounds, volatiles, pigments, minor glyceridic compounds, phospholipids and triterpenic acids. (Moreda, 2000) Fatty acids occur predominantly as esters of glycerol, i.e., triacylglycerol, in natural fats of animal and plant origin. There are many different types of fatty acids, but the most common include myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1) and linoleic acid (18:2) ( Christie W. W. ,2003) The human body can manufacture most of the fats it needs, including cholesterol, saturated fatty acids and unsaturated fatty acids. But there are two fatty acids which cannot be manufactured in the body, and which must be obtained from dietary sources: linoleic acid and alpha-linolenic acid. These are the essential fatty acids. Two distinct families of essential fatty acids exist in the human body: the omega-3 family and omega-6 family. The omega-3 family comes from alpha-linolenic acid, and the omega-6 family comes from linoleic acid. Leading experts such as the Scientific Review Committee of Health Canada and the World Health Organization have recommended that the
ratio of intake of ω-6 PUFAs to ω-3 PUFAs shoud range from 4:1 to 10:1. (Simopoulos AP., 2003). Sterols are a group of naturally occurring substances. Phytosterols (PS) are compounds present in all plants and in food products with plant origin. In all plant tissues, phytosterols occur in five common forms as the free alcohol (FS), as fatty-acid esters (SE), as steryl glycosides (SG) and as acylated steryl glycosides (ASG). The last three forms (SE, SG and ASG) are generically called “phytosterol congujates”. (Moreau et al., 2002; Abidi, S.L., 2001). Our studies are focused on identification of, two categories of bioactive molecules, characteristic to functional oils: sunflower, soybean, rapeseed, linseed, and hempseed oil, processed from seeds in Romania. The authors determined the fingerprint of fatty acids from total lipids and phytosterol esters fraction of these five functional oils, by gas chromatographic techniques, using flame ionization detection (FID) to detect the fatty acid methyl esters (FAMEs). MATERIALS AND METHODS
Sampling and Reagents Four edible oils (linseed, hempseed, rapeseed and soybean oil), processed from seeds in Romania, were selected for analysis. They were obtained by traditional methods avoiding organic solvents and they were not rafined. Methanol, petroleum ether, hexane, chloroform and other reagents were analytically grade and purchased from Merck- Germany. Sample preparation for fatty acids and sterol analysis
To analyze the fatty acids from total lipids fraction by gas chromatographic technique (GS), the oils was subjected to transesterification procedures with sodium methoxide (CH3ONa) in anhydrous methanol (CH3OH). By this derivatization procedure (transmethilation) were obtained the fatty acid methyl esters (FAMEs), the more volatile forms of fatty acids. The FAMEs from sterol esters fraction were obtained by the same procedures after the esterified sterols were separated by column chromatography from the oil samples (this fraction was eluted whit petroleum ether:diethyl ether= 95:5 solvent mixture). The dried organic phase (whit FAMEs) was purified then by column chromatography. The column was packed with Silica gel 40 ( 0.063-0.200 mm) ( Merck- Germany). The FAMEs was eluted with a petroleum ether:diethyl ether = 90:10(v/v) solvent mixtures and the free sterols were eluted with petroleum ether:diethyl ether = 50:50(v/v). After the fractions were collected, the solvent mixtures were evaporated and the separated fractions were re-dissolved in hexane and submitted to GC analysis.
GC-FID analysis FAMEs obtained by transesterifaction of oils samples and esterified sterols fraction were analyzed by GC-FID. A SHIMADZU GC-17-A gas-chromatograph with FID detector was used.
FAMEs were separated on a CHROMPACK WCOT 25M×0.25mm ID, 0.2µm film thickness capillary column, using a temperature program from 150oC / 5 min, 4oC/min until 235oC and 5 min at 235oC. Were respected the following conditions: injector temperature 260oC; FID temperature 260oC and the carrier gas – Helium.
RESULTS AND DISCUSSIONS
The main result of the present work is the determination of fatty acids profiles from total lipids and phytosterol esters fractions by GC, to five edible oils from Romania. Determination and knowledge of the fingerprint of these two categories of bioactive molecules with health benefit is very important for the industry and for the consumer.
Results of GC-FID analysis The fingerprints of the fatty acids (from total lipid and esterified sterols fractions) in studied oils are presented in chromatograms from Fig. 1, 2, 3, 4, 5.
2.1. 2.2. Fig. 1. GC-FID chromatograms of FAMEs in sunflower oil (1.1.-total lipids fraction; 1.2.-esterified sterols fraction)
3.1. 3.2. Fig. 2. GC-FID chromatogram of total FAMEs in soybean oil (2.1.-total lipids fraction; 2.2.-esterified sterols fraction)
4.1. 4.2. Fig. 3. GC-FID chromatogram of total FAMEs in rapeseed oil (3.1.-total lipids fraction; 3.2.-esterified sterols fraction)
5.1. 5.2. Fig. 4. GC-FID chromatogram of total FAMEs in linseed oil (4.1.-total lipids fraction; 4.2.-esterified sterols fraction)
6.1. 6.2. Fig. 5. GC-FID chromatogram of total FAMEs in hemp oil (5.1.-total lipids fraction; 5.2.-esterified sterols fraction) The fatty acid composition (from total lipids fraction) of studied oils are presented in Table 1 and Fig. 6 (% of total fatty acids).
In Table 2 are presented the ratio between ω-6/ ω-3 fatty acids from total lipids fractions of studied edible oils. In Table 3 and Fig. 7 are presented the fatty acids composition(from esterified sterols fraction) of studied edible oils from Romania (% of total fatty acids). In Table 4 are presented the ratio between ω-6/ ω-3 fatty acids from esterified sterols fraction of the studied edible oils. We identified, in both of the fractions (total lipids and esterified sterols), different concentrations of MUFAs and PUFAs (Table 1), depending on the biological characteristic of each oil. The main fatty acids from the studied samples were: oleic, linoleic and palmitic fatty acids. Table 1. The fatty acid composition ( from total lipids fraction) of five edible oils from Romania Fatty acid composition of oils ( % of total fatty acids ) Retention Time Fatty Acid Abreviation (Rt)(min.) Sunflower Soybean Rapeseed Linseed Hemp
Miristic Palmitic Palmitoleic Stearic Oleic Vaccenic Linoleic γ-linolenic α-linolenic Stearidonic Arachic Gadoleic Behenic Erucic SATURATED MUFAs1 PUFAs2
100% 80%
14:0 16:0 16:1 18:0 18:1 18:1is 18:2 ω-6 18:3 ω-6 18:3 ω-3 18:4 ω-3 20:0 20:1 22:0 22:1
5.65 0.05 2.75 26.89 0.35 64.17 0.09 8.40 27.29 64.26
9.76 4.01 27.90 traces 45.20 13.11 13.77 27.9 58.31
Gamma-Linolenic Acid (18:3, omega-6) Saturated Fatty Acids
60% 40% 20% 0%
0.07 4.42 0.25 2.4 62.25 traces 18.9 9.36 0.64 1.06 0.3 0.2 7.83 63.76 28.26
100% 80%
6.37 4.37 20.23 traces 18.69 50.31 10.74 20.23 69
6.69 2.96 14.33 traces 55.62 3.07 14.84 0.86 0.88 0.36 0.33 10.86 14.69 74.39
Gamma-Linolenic Acid (18:3, omega-6) Saturated Fatty Acids
60%
Linoleic Acid (18:2, omega-6) Stearidonic Acid (18:4, omega-3) Oleic Acid (18:1, omega-9) Alpha-Linolenic Acid (18:3, omega-3)
40% 20% 0%
Linoleic Acid (18:2, omega-6) Stearidonic Acid (18:4, omega-3) Oleic Acid (18:1, omega-9) Alpha-Linolenic Acid (18:3, omega-3)
Fig. 6. The fatty acid composition (from total lipids fraction) of five edible oils from Romania Fig. 7. Fatty acid composition of esterified sterols fraction
1 2
5.72 9.949 10.292 14.497 14.904 15.148 16.000 16.519 17.323 17.919 18.891 19.229 23.042 23.359
Monounsaturated Fatty Acids Polyunsaturated Fatty Acids
Table 2. The ratio between ω-6/ ω-3 fatty acids from total lipids fractions of studied edible oils. OILS Sunflower Soybean Rapeseed Linseed Hemp 3.4 / 1 2/1 1 / 2.7 3.7 / 1 Ratio between ω-6/ ω-3 Table 3. The fatty acids composition( from esterified sterols fraction) of five edible oils from Romania Fatty Acid Abreviat Fatty acid composition of esterified sterols fraction ( % of total fatty ion acids ) Sunflower Soybean Rapeseed Linseed Hemp Miristic 14:0 Palmitic 16:0 5.01 11.09 5.43 4.37 4.43 Palmitoleic 16:1 0.36 Stearic 18:0 2.65 8.75 4.81 5.97 3.36 Oleic 18:1 25.01 21.26 30.24 21.95 13.84 Vaccenic 18:1is 3.13 1.82 0.30 0.51 Linoleic 18:2 ω-6 65.91 45.14 43.98 32.61 53.49 γ-linolenic 18:3 ω-6 2.77 α-linolenic 18:3 ω-3 1.03 9.71 8.22 30.54 16.71 Stearidonic 18:4 ω-3 1.23 Arachic 20:0 1.17 3.45 2.70 Gadoleic 20:1 0.74 0.78 0.89 Behenic 22:0 0.12 Erucic 22:1 3.42 SATURATED 7.66 19.84 11.53 13.79 10.49 MUFAs3 25.37 24.39 36.22 23.03 15.24 PUFAs4 66.97 55.77 52.25 63.18 74.27
Table 4. The ratio between ω-6/ ω-3 fatty acids from the esterified sterols fraction of the studied edible oils. OILS Sunflower Soybean Rapeseed Linseed Hemp 4.6/ 1 5.3 / 1 1.07 / 1 3.1 / 1 Ratio between ω-6/ ω-3
Sunflower oil is characterized by its high linoleic acid, moderate oleic acid, and low linolenic acid concentration. Linseed oil can be considered to be a good source of the omega3 fatty acids and the sunflower can be a good sources of the omega-6 fatty acids. The sterol esters fraction of the rapeseed oil contains a higher percent of the linoleic acid (18:2 omega-6) than the total lipids fraction from the same oil sample. Hemp oil is one of the few seed oils that contains both of omega-6 and omega-3, gamma-linolenic acid being one of relatively rare fatty acids. A 3.07% content of γ-linolenic acid in total lipids fraction and 2.77% of γlinolenic acid in esterified sterols fraction respectively, were found. From the four edible oils which we studied, in both of the fractions which were analyzed, the hemp oil contains the highest percent of PUFAs (74.39% in total lipids fraction, and 74.27% in esterified sterols fraction, respectively).
3 4
Monounsaturated fatty acids Polyunsaturated fatty acids
CONCLUSIONS
The pattern of (18:1)/(18:2 ω-6)/ 18:3 ω-6/18:3 ω-3, can be useful as authenticity markers and GC-fingerprint, an appropriated method of oil authenticity. The hempseed, soybean and rapeseed oils can be considered good sources of ω-6/ ω-3 fatty acids because the ratio between these acids is closed to the ideal one, mentionated in literature. This ratio of fatty acids has been shown to be beneficial for the prevention of heart disease and cancer, especially omega 3 fatty acids. Hemp oil is one of the few seed oils that contains both of omega-6 and omega-3, gamma-linolenic acid being one of relatively rare fatty acids, and it was proved to be the most rich in phytosterols, been recommended as a functional food. BIBLIOGRAPHY 1. 2. 3.
4. 5.
Abidi, S.L., 2001, Chromatographic analysis of plant sterols in foods and vegetable oils,Review, J. Chromatogr. A, 935 , 173-201 Christie, W. W., 2003, Lipid Analysis: Isolation, Separation, Identification and Structural Analysis of Lipids. The Oily Press, Bridgewater, pp: 1-416. Moreau, R. A., B. D. Whitaker, K. B. Hicks, 2002, Phytosterols, phytostanols, and their conjugates in foods: structural diversity, quantitative analysis, and health-promoting uses. Progress in Lipid Research, 41, 457-500. Moreda W., 2000, Chromatographic analysis of minor constituents in vegetable oils,( Review), Journal of Chromatography A, 881 (2000) 131-148 Simopoulos A. P., L. G. Cleland (eds), 2003, "omega-6/omega-3 Essential Fatty Acid Ratio: The Scientific Evidence." World Rev Nutr Diet. Basel, Karger, 2003, Vol 92.
