Study of diacyl glycerides profile and content in cooking oils using thin layer chromatography and gel permeation chromatography.
Obesity is widely recognized to contribute to the occurrence of various diseases such as non-insulin dependent diabetes mellitus, coronary heart disease, strokes, respiratory disorders and degenerative joint disease (Hoffmann-La Roche's) [1,2]. Diacyl glyceride (DAG) has been shown to be beneficial in reducing these disorders . Studies on animals and humans indicate that diet containing DAG decreases body weight and body fat accumulation, in particular, visceral fat [2,4-8]. Extensive studies indicate that DAG has the capability of reducing post-meal blood triglyceride levels, which subsequently contributes to a decrease in the overall cholesterol and fat content in the physiological system [9,10,11]. Clinical studies in Japan have shown that DAG can increase the metabolic pathways for energy production, which enables consumption of the fat [6,7].
Structural difference between TAG and DAG can be seen from Scheme 1. TAG contains a three fatty ester and DAG is made up of two fatty acids ester.
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Commercially available DAG oil generally contains ~75 % diacylglycerol, 20% triacylglycerol, 5% monoacylglycerol, <0.2% emulsifiers (polyglycerol esters of fatty acids) and antioxidants (ascorbyl palmitate and tocopherol)1. DAG is made up of oleic (C18:1), linoleic (C18:2), and linolenic (C18:3) acids in a ratio of 7:3:1.
The metabolism of DAG in the intestine differs from that of TAG. The fatty acids of both DAG and TAG are absorbed through the intestinal wall. Then, the triglycerides are converted to mono acylglycerides (MAG) by 1,3 lipases in the small intestine. The free fatty acids and two molecules of MAG pass through the epithelial cells, where a 1,3 lipase reassembles the fatty acids onto the two molecules of MAG to reform TAG. TAG is then packaged into chylomicrons and passed in the lymph and bloodstream for storage as fat, or eventually to the liver for lipid degradation and/or synthesis. The 1,3 diglycerides into two (1 or 3) monoglycerides by the 1,3 lipases in the small intestine, but no resynthesis of TAG from occurs as the glycerol backbone lacks the critical third fatty acid chain, which acts as the substrate for the lipases. These fatty acids are diverted into the portal vein to the liver4,5. In the liver, lipid oxidation of the fatty acids causes breakdown instead of storage of fat.
Today many cooking oils are available in the South Indian market, as shown in Table 1. No study exists that provides an insight into the DAG content in these oils. The present study investigates the lipid profiles of various cooking oil available in South India and identifies the cooking oil that contains significant quantities of DAG content.
Materials and methods
A large number of cooking oils were sourced from retail suppliers in India. 15 [micro]L of cooking oil sample is dissolved 1ml hexane, mixed in a vortex mixer. 15 [micro]L of this mixture is spotted on TLC treated with Petroleum ether: Diethyl ether: Acetic acid (70:30:1) mobile phase. Lipid profiles on the TLC sheet are examined for the presence of neutral lipids comprising TAG, DAG, MAG, FFA and phospholipids and deducted on exposure to iodine fumes.
Result and Discussion
The TLC profiles of various cooking oils are show in Figures 2 to 7. A qualitative representation of various components of both neutral and phospholipids are deducted and shown from the Figures 2 to 5. The profile and retention front separation of various neutral lipids component such as Triacyl glyceride (TAG), Diacyl glyceride), Free fatty acid(FF) and Mono acyl glycerides (MAG) profiles are deduced by comparing with the standard triolene. The lipid components are separated based on relative front, polarity and hydrophobicity. The significance of the presence of DAG is monitored in all TLC sheets and the thickest band represents strong presence of DAG band. The oil samples that show such strong bands have been selected for separation by column chromatography.
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Note--TAG-Triacylglyceride, DAG-Diacylglyceride, FF-Free fatty acids and MAG-Monoacyl glyceride.
We observed high DAG content in Sakthi soya oil, corn premium cooking oil, mahakosh refined sunflower oil, Porna refined sunflower oil and Durga sesame oil based up on the thickest spots on the TLC plates and Table 2. Short listed sample for higher DAG presence.
The DAG was separated from these shortlisted oils using a gel permeation chromatography method reported previously13. The column was prepared with 1.2 cm diameter tube loaded with 12 g of silica gel (100*20 mesh) in hexane. 250 mg of the oils shown in Table 2, were taken in 1ml hexane respectively and passed on silica gel column (60-120 mesh) and eluted with Petroleum ether, diethyl ether and acetic acid (70:30:1) mixture. Fractions were collected and analyzed on TLC and exposed to iodine fumes to determine the lipid profiles. The TAG, MAG DAG and FF fractions were pooled individually and dried under rotating evaporator. Phospholipids was not found in the elution mixture initially, hence, highly polar solvent 100 % methanol was added to separate the phospholipids that had bound to the silica gel. Phospholipids fractions were dried and dissolved in to n-hexane and passed through a silica gel column (60-120 mesh) with packing size of 100*20 mm with chloroform: methanol: water (65:35:4) mobile phase. All the fractions were analyzed for phosphotidylcholine, phosphotidyl ethanolamine, and phosphotidyl serine on TLC with same mobile phase. The contaminant fractions were eluted by reloading the DAG fractions on to silica gel column in order to obtain DAG fractions. Without other contaminants is obtained. The lipid profile and DAG content are shown in Table 3 and Table 4 respectively. Figure 6 depicts the TLC profile of the fractions from Porna rice bran oil.
[FIGURE 8 OMITTED]
The composition of crude rice bran oil was found to be 81.3-84.3% triglycerides, 2-3% diglycerides, 5-6% monoglycerides, 2-3% free fatty acids, 0.3% waxes, 0.8% glycolipids, 1.6% phospholipids, 4% unsaponifiables. The separated DAG fractions were dried and the total weight is 2.46 %. The 20 mg DAG lipid was esterified with 2ml HCl and 10 ml methanol and analyzed for GC-MS and the results suggest that DAG contains Myristic (14:0), Palmitic (16:0), Stearic (18:0), Oleic (18:1), Linoleic (18:2), Linolenic (18:3) and Arachidic.
From this study, it was found that rice bran oil of Poorna brand contains 2.46 % of Diacyl glycerides (DAG) and is the highest compared to the other oils. The fatty acid profile of the DAG obtained from rice bran oil was found to contain a mixture of Myristic (14:0), Palmitic (16:0), Stearic (18:0), Oleic (18:1), Linoleic (18:2), Linolenic (18:3) and Arachidic. The study indicates that most of the cooking oils available in south India contain lesser quantities of DAG.
We thank Krishnamoorthy Muthusamy, Ramyadevi Thangavelu, Kavitha Seetharaman and Selvaraj Palanisamy for their invaluable insights and part financial support to this work.
 Masanobu Hibi, Hideto Takase, Shinichi Meguro, Ichiro Tokimitsu. The effects of diacylglycerol oil on fat oxidation and energy expenditure in humans and animals. BioFactors, Volume 35 Issue 2, Pages, 11 Mar (2009); 175-177.
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Chandramohan Marimuthu *, Sriraj Srinivasan, Karthik Periyasamy, Brindha Ganesan, Jayaramani Manickam, Vinothini Gnanasekaran and Kathiravan Veeramalai
Microcore Research Laboratories India Pvt. Ltd. 9th km, 30 Feet Road, 204--A Poondurai Main Road, Checkmedu, Erode--638115, Tamil Nadu, India
* Corresponding Author E-mail: firstname.lastname@example.org
Table 1: List of cooking oils available in South India. S.No Details Brand 1 Refined Groundnut oil Gold winner Maharaja Usha Pilot Mahaa Ganesh SVS Refined groundnut oil Idhayam Mantra groundnut oil 2 Filtered Maharaja Groundnut oil Sun GN oil Sunland Agmark Gold winner Nutrella Dhara health Usha Nature Fresh actilite Sunrich Sundropsuperlite Gemini Marshal Mahakosh Aachi 3 Rice bran oil Porna Sundrop heart 4 Safflower oil Saffola 5 Sesame oil/Gingelly oil Anandham Maharaja Eniya Vanaraja Idhayam 6 Palmolein oil Ruchi Gold 7 Coconut oil Parachute VVD Gold PKG Agmark Shanthi 8 Soya oil Sundrop nutrilite 9 Mustard oil Dhara Fortune Supreme 10 Corn oil Corn premiumcooking oil S.No Manufacturer 1 Kaleesuwari refineries, Chennai Naga oil mills, Erode B.S.P Refineries, Salem Kingrefineries, Gobichettipalayam P.V.P Refineries, Erode SVS Oil mills, Chennai VVV & Sons, Virudhunagar 2 Naga oil mills, Erode. SGN Oils, Thally,Hosur K.T.V oil mills, Chennai Kaleesuwari refineries, Chennai Ruchi Soya industries, Mumbai Dhara vegetable oils P.Ltd, Karnataka B.S.P Refineries, Salem Tina Agro industries, New Delhi Ruchi Soya industries, Mumbai Sundrop, Chennai Sunraja oil industries, Erode King refineries, Gobichettipalayama Ruchi Soya industries Ltd, Mumbai Sunraja oil industries Pvt Ltd, Erode 3 S.K.M Ltd , Erode Priyanka refineries (p) Ltd, Andhra 4 Marico Ltd, Jalgaon 5 V.V.V Anand & Sons, Viruthunagar Maharaja oil mills, Karaikudi Evergreen Enterprises, Erode Maharaja oil mills, Karaikudi V.V.V & sons, Virudhunagar 6 Ruchi Soya industries Ltd, Thiruvallur 7 Parachute(P)Ltd, Pudhucherry VVD & Sons Pvt Ltd, Tuticorin PKG coconut product, Kangayam Madhan Agro industries, Kangayam 8 Sakthi Pollachi 9 Dhara vegetable oils, Chennai Adani mills Raja chemicals, Chennai 10 Avitaa Food products, Chatrapatti Table 2: Short listed cooking oils with higher DAG. (+ + +) Significant, (+ +) moderate. S.No Cooking Oil Brand DAG band 1 Soya Sakthi +++ 2 Corn Corn premium cooking oil +++ 3 Refined Sunflower Mahakosh +++ 4 Gingelly Commercial ++ 5 Refined rice bran oil Porna +++ 6 Refined sunflower Gemini ++ 7 Groundnut Idhayam Mantra ++ 8 Sesame oil Durga +++ Note : (+ + +) Significant, (+ +) moderate Table 3: Lipid fractions (mg) Lipid fractions in mg of various cooking oils. TL- Total lipid, NL- Neutral lipid, PL- Phospholipid, TAG- Triacylglyceride, DAG- Diacylglyceride, FF-Free fatty acids, MAG Monoacyl glyceride. Cooking Oil & Brand TL NL PL T.L T.L (mg) (mg) (mg) recovery recovery (mg) (%) Soya (Sakthi) 250 234 12 246 98.4 Corn (premium cooking oil) 250 238 13 251 100 R. Sunflower (Mahakosh) 250 235 8 243 97.2 Gingelly (Commercial) 250 233 12 245 98 R.Rice bran oil (Porna) 250 239 8 247 98.8 Refined sunflower (Gemini) 250 237 8.2 245 98.1 Groundnut (Idhayam 250 235 9 244 97.6 Mantra) Sesame oil (Durga) 250 242 0 242 96.8 Cooking Oil & Brand TAG DAG FF MAG (mg) (mg) (mg) (mg) Soya (Sakthi) 233 5.01 0.23 1.30 Corn (premium cooking oil) 231 4.15 0.23 1.10 R. Sunflower (Mahakosh) 233 3.50 0.12 1.20 Gingelly (Commercial) 233 5.30 0.23 1.45 R.Rice bran oil (Porna) 235 6.15 0.24 1.23 Refined sunflower (Gemini) 237 4.31 0.26 1.24 Groundnut (Idhayam 239 4.10 0.00 0.00 Mantra) Sesame oil (Durga) 233 5.00 0.23 0.00 Table 4: Lipid fractions (%). S.No Cooking Oil & Brand TAG % DAG % MAG (%) FF (%) PL (%) 1 Soya (Sakthi) 93 2 0.5 0.1 4.8 2 Corn (premium cooking oil) 92.4 1.66 0.4 0.1 5.2 3 R. Sunflower (Mahakosh) 93 1.4 0.5 0 3.2 4 Gingelly (Commercial) 93 2.12 0.6 0.1 4.8 5 R.Rice bran oil (Porna) 94 2.46 0.5 0.1 3.2 6 Refined sunflower (Gemini) 94.8 1.72 0.5 0.1 3.29 7 Groundnut (Idhayam Mantra) 95.6 1.64 0 0 3.6 8 Sesame oil (Durga) 93 2 0 0.1 0
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|Author:||Marimuthu, Chandramohan; Srinivasan, Sriraj; Periyasamy, Karthik; Ganesan, Brindha; Manickam, Jayara|
|Publication:||International Journal of Biotechnology & Biochemistry|
|Date:||Jan 1, 2010|
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