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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 [3]. 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.


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.







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.


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.



[2] 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.

[3] Seong-Koon Lo, Chin-Ping Tan, Kamariah Long, Mohd. Suria Affandi Yusoff and Oi-Ming Lai Diacylglycerol Oil-Properties, Processes and Products.Journal of Food and Bioprocess Technology. Issue Volume 1, Number 3/ September, (2008); 223-233.

[4] MATSUO Noboru Diacylglycerol as a tool for health-nutritional functions of diacylglycerol oil. Recent research developments in lipids Vol. 7 (2004) 87101.

[5] Draft assessment report Application 505-diacylglycerol approved oil as per Food standards Australia New Zealand (FSANZ). FSANZ, 27 August (2003).

[6] KC Maki et al. Consumption of diacylglycerol oil as part of a mildly reducedenergy diet enhances loss of body weight and fat compared with a triacylglycerol control oil. Am Journal of Clinical Nutrition (2002); 76:12301236.

[7] Murase et al. Anti-obesity effect of dietary diacylglycerol in C57BL/6J mice: dietary diacylglycerol stimulates intestinal lipid metabolism. J Lipid Res. (2002); 43:1312-1319

[8] Kevin C Maki, Michael H Davidson, Rikio Tsushima, Noboru Matsuo, Ichiro Tokimitsu, Denise M Umporowicz, Mary R Dicklin, Gregory S Foster, Kate A Ingram, Barbara D Anderson, Scott D Frost and Marjorie Bell.Consumption of diacylglycerol oil as part of a reduced-energy diet enhances loss of body weight and fat in comparison with consumption of a triacylglycerol control oil1, 2, 3 Journal Clinical Nutrition (2002);76:1230-6.

[9] N. Tada et al. Dynamics of post-prandial remnant-like lipoprotein particles in serum after loading of diacylglycerols. Clin Chim Acta. (2001); 311: 109-117

[10] K. Yamamoto et al. Long-term ingestion of dietary diacylglycerol lowers serum triacylglycerol in type II diabetic patients with hypertriglyceridemia. Journal Clinical Nutrition (2001); 131: 3204-3207.

[11] H. Taguchi et al. Double-blind controlled study on the effects of dietary diacylglycerol on post-prandial serum and chylomicron triacylglycerol responses in healthy humans. J Am Col Nutr. (2000); 19: 789-796

[12] T. Nagao et al. Dietary diacylglycerol suppresses accumulation of body fat compared to triacylglycerol in men in a double-blind controlled trial. Journal of Nutritional (2000); 130:792-797.

[13] Morris Kates. Techniques of lipidology isolation, analysis, and identification of lipids.(1986)

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:
Table 1: List of cooking oils available in South India.

S.No   Details                   Brand

1      Refined Groundnut oil     Gold winner
                                 Mahaa Ganesh
                                 SVS Refined groundnut
                                 Idhayam Mantra
                                 groundnut oil

2      Filtered                  Maharaja
       Groundnut oil             Sun GN oil
                                 Sunland Agmark
                                 Gold winner
                                 Dhara health

                                 Nature Fresh actilite

3      Rice bran oil             Porna
                                 Sundrop heart

4      Safflower oil             Saffola

5      Sesame oil/Gingelly oil   Anandham

6      Palmolein oil             Ruchi Gold

7      Coconut oil               Parachute
                                 VVD Gold
                                 PKG Agmark

8      Soya oil                  Sundrop nutrilite

9      Mustard oil               Dhara

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,

       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,

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

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

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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Article Details
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Author:Marimuthu, Chandramohan; Srinivasan, Sriraj; Periyasamy, Karthik; Ganesan, Brindha; Manickam, Jayara
Publication:International Journal of Biotechnology & Biochemistry
Article Type:Report
Geographic Code:9INDI
Date:Jan 1, 2010
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