Scholarly article on topic 'Synthesis and Characterization of 2-monoacylglycerols from Canarium Oil (Canarium Indicum)'

Synthesis and Characterization of 2-monoacylglycerols from Canarium Oil (Canarium Indicum) Academic research paper on "Chemical sciences"

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Procedia Food Science
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{"Canarium oil" / 2-monoacylglycerols / "structured lipid" / "fatty acids" / "enzymatic reaction."}

Abstract of research paper on Chemical sciences, author of scientific article — Hamidah Rahman, Kusnandar Anggadiredja, Johnner P. Sitompul, Tutus Gusdinar

Abstract This paper studies synthesis and characterization of 2-monoacylglycerols (2-MAGs), generally used for structured lipid production. Synthesis used canarium oil by enzymatic reaction using immobilized lipase from Mucor miehei that has a specific activity on the sn-1 and 3 of the triacylglycerol structure. Characterization of 2-MAGs by thin layer chromatography using a standard of 2-oleoylglycerol followed by isolation and purification to determine the fatty acids composition.The highest composition of fatty acids on 2-MAGs were unsaturated fatty acids, namely oleic acid (939.8ppm) and linoleic acid (445.5ppm), therefore it could be used for the synthesis of structured lipid.

Academic research paper on topic "Synthesis and Characterization of 2-monoacylglycerols from Canarium Oil (Canarium Indicum)"



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Procedia Food Science 3 (2015) 162 - 173

The First International Symposium on Food and Agro-biodiversity (ISFA2014) Synthesis and characterization of 2-monoacylglycerols from Canarium oil (Canarium indicum)

Hamidah Rahman1*-*, Kusnandar Anggadiredja1), Johnner P Sitompul2), Tutus Gusdinar1)

Pharmacy Department, School of Pharmacy, Institute of Technology Bandung 2Chemical Engineering Department, Faculty of Industrial Technology, Institute of Technology Bandung

Jalan Ganesha 10 Bandung, 40132, Indonesia


This paper studies synthesis and characterization of 2-monoacylglycerols (2-MAGs), generally used for structured lipid production. Synthesis used canarium oil by enzymatic reaction using immobilized lipase from Mucor miehei that has a specific activity on the sn-1 and 3 of the triacylglycerol structure. Characterization of 2-MAGs by thin layer chromatography using a standard of 2-oleoylglycerol followed by isolation and purification to determine the fatty acids composition. The highest composition of fatty acids on 2-MAGs were unsaturated fatty acids, namely oleic acid (939.8 ppm) and linoleic acid (445.5 ppm), therefore it could be used for the synthesis of structured lipid.

© 2015 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.Org/licenses/by-nc-nd/4.0/).

Peer-reviewunder responsibility of the organizing committee of Indonesian Food Technologist Community

Keywords: Canarium oil; 2-monoacylglycerols; structured lipid; fatty acids; enzymatic reaction. INTRODUCTION

The important function of fat intake is to provide essential fatty acids for regulating the human body physiology. Essential fatty acids could not be synthesized on humans and animals, therefore humans need to consume from outside, for example from plants source. Biochemical systems in humans could not insert the first double bond at position C3 (n-3) and C6 position (n-6), however was able to extend

Corresponding author. E-mail address:,id

2211-601X © 2015 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (

Peer-review under responsibility of the organizing committee of Indonesian Food Technologist Community doi:10.1016/j.profoo.2015.01.018

carbon chains by elongation reactions and adding the number of double bonds by desaturation reactions. Fatty acids with a carbon number of 18, 20 and 22, with 2 to 6 double bonds in the cis position and the position of the first double bond is located between C3 and C4 and also in position C6 and C7 are classified as essential fatty acids for humans, for instance linoleic acid and a-linolenic acid [1]. Several studies have also reported the function of fatty acids on brain development and to control membrane function in the nervous system. Intake of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) could assist to delay the decline in cognitive function in both animals and humans. a linolenic acid (ALA, 18: 3n-3) and linoleic acid (LA, 18: 2n-6) also showed an increase in performance of the Morris water maze test in rats [2]. Supplements rich unsaturated fatty acids such as oleic acid and linoleic acid derived from olive oil could be used to prevent coronary heart disease [3].

In general, fatty acids are found in triglycerides. Chemical, physical and biochemical characteristics of a triglyceride composition is determined by the composition of fatty acids and stereospecific structure in positions sn-1, sn-2 and sn-3 [4], therefore it is important to determine the position or stereospecific distribution and composition of fatty acids in triglyceride. This will be linked to the process of digestion and absorption of fatty acids in the body. For nutritional purposes, there is an interest to produce structured lipid by locating medium chain fatty acids at sn-1 dan sn-3 positions and functional long chain fatty acids at sn-2 position. This structure is claimed have a beneficial value on human health because less accumulation of fats in the body as well as a reduction in serum cholesterol. Structured lipid with eocasapentaenoic acid (EPA, 20:5n:3) and docosahexaenoic acid (DHA, 22:6n:3) in position sn-2 were more readily absorbed than other structured lipid with similar fatty acid composition but with a random fatty acid distribution [5].

Structured lipids (SL) or structured triacylglycerols (STAGs) are triglycerides that have been modified by changing the composition and or distribution of the fatty acid position on the glycerol backbone that could be done by chemical or enzymatic catalysts or genetic engineering. The general objective of structured lipids product is to improve the nutritional value and the physical properties that can be used for food applications as expected. Structured lipids have been also widely used for therapeutic purposes, for instance to increase the function of the immune system, reduces the risk of cancer, thrombosis prevention, lowering cholesterol and others [6].

Synthesis of SL was carried by modification of natural triglycerides or native state by incorporating a new fatty acids into triglycerides in order to obtain a new triglyceride structure or restructuring

triglycerides by changing the position of fatty acids [7]. Interesterification reaction is used to produce SL by chemically and/or enzymatically catalyzed reactions and/or genetic engineering. Interesterification including reactions acidolysis, alcoholysis, glycerolysis and transesterification. Acidolysis reaction occurs between a triglyceride with a fatty acid, alcoholysis reaction between an alcohol with the triglycerides, whereas if it acted as an alcohol called glycerolysis. If there is a reaction between the two triglycerides is called transesterification [8].

Structured lipid could be synthesized by utilize triglycerides backbone of vegetable oils. Previous studies have incorporated of caprylic acid into triglycerides of olive oil to obtain a new triglyceride structure which faster absorbed and metabolized to produce energy quickly [9]. The same study was also performed by using borage oil as a source of triglycerides backbone and uses an enzyme catalyst because by using chemical catalyst the reaction product take place with random [10]. Other researches were the synthesis of structured lipid from sesame oil [11], from rice bran oil [7], and soybean oil [13]. As long as our knowledge, we have not yet found the report of production structured lipid of canarium oil, however the results of our previous study obtained high composition of oleic acid (omega 9), linoleic acid (omega 6) and linolenic acid (omega 3) in canarium oil therefore making it possible to produce structured lipids by utilize canarium oil.

Canarium is a native plant to eastern Indonesia, Papau New Guinea, the Solomon Islands and Vanuatu. Furthermore, these plants spread in several parts of the country such as the Philippines and Malaysia and has approximately 100 species worldwide [14]. Canarium tree from Canarium indicum species produce seeds commonly used as a snack food. The main composition of canarium seed is oil by 6570%, therefore this seed can be used as a source of vegetable oil. Based on that canarium can be comparable to other plants that have a high oil composition in the seeds, for instance almond, cashew, walnut, brazilnut, hazelnuts, pecans and macademia [15] and canarium seed have a potency as a source of essential fatty acids. The benefit of this research is to obtain a structured lipid product for healthy food application by using canarium oil as a raw material that will improve the economic value of canarium directly.



Canarium oil as a substrate was obtained from the extraction of canarium kernels derived from the Moluccas province. Extraction for obtain canarium oil was carried out using a mechanical pressing.

Enzyme immobilized lipase from Mucor miehei (Lypozyme®) and standard 2-oleoylglycerol (2-monoolein) were suplied from Sigma-Aldrich Singapore. Standard fatty acids (octanoic and decanoic) were suplied from Sigma-Aldrich while fatty acids (lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic, and linolenic) were suplied from Nacalai Japan. 2,4-dibromoacetophenone (PBPB) and 18-crown-6-ether used for derivatization in High Performance Lipid Chromatography (HPLC) analysis were suplied from Sigma-Aldrich Singapore. Acetonitrile was HPLC grade, whereas other solvents (chloroform, n-hexane, dry ethanol, acetone, methanol) were analytical grade. Plate of silica gel GF254 thin layer chromatography for identification and isolation of 2-MAGs, and for visualizing the spots using iodine vapor and 0.2% dichlorofluoresceine in methanol.


Ethanolysis Reaction

Ethanolysis reaction between triglycerides of canarium oil and dry ethanol was catalized by immobilized lipase based on method that has been done by Esteban according to the procedure in figure 1. In this reaction, the oil (TAG) reacts with ethanol was catalyzed by 1,3 specific lipase to yield 2-MAGs, diacylglycerols (DAGs) and ethyl esters (EEs).

Ethanolysis Reaction

Canarium oil + dry ethanol (1:4 w/w)

+ 10% immobilized lipase enzyme (Lypozyme)

Incubation at 35°C, 248 rpm ;

6 hours

^ Filtration

Purification of 2-MAGs

+ (9 V) EtOH/H2O (90:10 v/v)

3 x (9V) washes with n-hexane

Hexanic Phase (TAG+Esters)

Hydroethanolic Phase (2-MAGs)

Fig. 1. Experimental procedure for ethanolysis reaction and purification 2-MAGs

A typical reaction mixture consisted of 750 mg of canarium oil and 3 g dry ethanol (1: 4 w/w) to act as a substrate of 375 mg lipase enzyme (10% of total substrate). This mixture was placed in an orbital shaker water bath for incubation process at 35oC with a speed of 248 rpm for 6 hours. The reaction was stopped by removing the enzyme by filtration. The final volume was measured to determine the volume of ethanol/water mixture (90:10 v/v) as solvent extraction to isolation and purification of 2-MAGs.

Purification of 2-monoacylglycerols

The procedure for isolation and purification of 2-MAGs from ethanolysis reaction was performed by solvent extraction based procedure [16]. Solvent extraction using ethanol/water mixture (90:10 v/v) nine times to the final volume obtained by filtration, and than washing three times with n-hexane. The volume of n-hexane was nine times of the volume of the filtration for each washing. Two phases were formed during extraction which hexanic phase consist of triglycerides that were not involved in the reaction and ethyl esters, and the subsequent phase was hydroethanolic phase with contain 2-MAGs as a target compound. The remaining of solvent was evaporated using vacuum evaporated rotavapour based on the vapour point of each solvents.The volume was then adjustedto 25 mL by addition of n-hexane and stored under inert atmosphere at-24°C until analysis.

The characterization of reaction products

The products of ethanolysis (MAGs, DAGs, TAGs and EEs) were characterizated by thin layer chromatography (TLC) using chloroform/aceton/methanol (95:4,5:0,5 v/v/v) as the mobile phase. To detect the presence of 2-MAGs using 2-oleoylglycerol as a standard. TLC was carried out on silica gel plates that activated in oven at 150°C for 30 min before used. 200 ^L of the sample was developed on TLC plates in mobile phase and then after drying the bands were located by viewing under 254 nm of UV light. The band corresponding to 2-MAGs was scraped to perform preparative TLC and subsequently used for saponification reaction for fatty acids analysis by HPLC.

Saponification was carried out by weighing about 5 mg of 2-MAGs fraction, then mixture with 100 ^L of a 1 N KOH solution in methanol. The resulting mixture was incubated at 80°C for 30 min, and cooled to ambient temperature. Then, 120 ^L of a 1 M HCl was added to convert the fatty acid salts to their acidic form. Next, 1 mL of distilled water was added and the free fatty acids which liberated was extracted once with 2 mL and twice with 1 mL of chloroform/methanol mixture (2:1 v/v). The solvent was then evaporated under nitrogen and subsequently the fatty acids fraction was derivatized to form p-bromophenacyl esters to HPLC analysis.

Analysis of of fatty acids composition in canarium oil and 2-MAGs product with HPLC based on the procedure [17] with some modifications. Analysis systems were achieved with a Hewlett-Packard 1100 and coupled to an ultraviolet detector. The detector was set to ambient temperature and at wavelength 254 nm. The fatty acids were separated with a Zorbax-C8 column (0.15 m x 4.6 mm) and the column oven temperature was ambient temperature. The mobile phase was acetonitril/water (87:13 v/v) with a constant mobile phase flowrate of 1.5 mL/min and the injection volume was 20 ^L. The derivatization was conducted before analysis by changing the fatty acids to p-bromophenacyl esters. Approximately 100 ^L of a 0.5 mg/mL solution of 18-crown-6-ether in acetonitrile was added to 3 mL of a solution containing 0.15 mg/mL of PBPB in acetonitrile. Next, 300 ^L of solution containing the fatty acids was combined with the mixture of PBPB and 18-crown-6-ether together with approximately 200 mg of K2CO3. The mixture was held at 80°C in a sealed vial for 30 min and then cooled in an ice bath for 15 min. The mixture was filtered through a 0.45 ^m membrane filter and then analyzed by HPLC.


The general procedure to produce structured lipids is by using vegetable oils as a source of triglycerides backbone. Structured lipids have been applied to production of infant formula, enteral and parenteral nutrition, and other food products application. The results of our previous study obtained significant oil composition was about 70% in canarium seed by using a mechanical pressing extraction. Analysis of fatty acids of canarium oil was conducted by gas chromatography and obtained the largest concentration respectively were oleic acid (50.65%), palmitic acid (23.58%), linolenic acid (12.45%) and stearic acid (11.40%) [18]. Based on these data, we used the canarium oil for provide fatty acids for the production of structured lipids.

Production of structured lipids could be done with acidolysis reaction between long chain and medium chain fatty acids of triacylglycerols and catalyzed by a 1.3 specific lipase. The procedure was very simple because it used a direct route synthesis of structured lipids. The main problem of this procedure was the phenomenon of acyl migration which will reduce the yield of SL. Acyl migration is a phenomenon which the acyl groups will migration in triglycerides that will affect the regiospecific of triacylglycerols that expected. In order to avoid this problem, an alternative procedure to minimized the acyl migration by conducting the synthesis in two steps [5]. The first step was alcoholysis or ethanolysis reaction between triacylglycerols (oil) and ethanol using 1.3 lipase as a catalyst. One of the

reaction product was 2-MAGs as a target compound in this work. The second step was esteriflcation reaction between 2-MAGs and fatty acids that will be incorporated at sn-1 and sn-3 of triacylglycerols backbone catalyzed by the same enzyme. In this study, we only perform the first step of the reaction was the synthesis of 2-MAGs with ethanolysis enzymatic reaction using immobilized 1,3-specific lipases. Product 2-MAG was an intermediate product or as a starting material to produce a structured lipid that will be carried out at the next work.

Isolation and purification of 2-MAGs was conducted by solvent extraction using ethanol/ water mixture (90:10 v/v) with consideration of the solvent has a low toxicity and food grade. Separation of 2-MAGs from other reaction products by washing using n-hexane, so the unreacted triacylglycerols (TAG), diacylglycerols (DAG) and ethyl esters will be in hexanic phase while 2-MAGs will be in hydroethanolic phase based on the following equation :

Canarium oil (TAG) + Ethanol sn'13 specific lipasS MAG + DAG + Ethyl Esters

Results of thin layer chromatography using silica gel plates showed that the hydroethanolic phase containing 2-MAGs with a yield of 74%, however in hexanic phase still contain 2-MAGs. Our next work will optimize the extraction process (Figure 2).


Fig. 2. Separation of reaction products (MAGs, DAGs, TAGs and EE) by thin layer chromatography using chloroform/aceton/methanol (95/4.5/0.5) as mobile phase. (1,2,3) and by preparative TLC (4). Spots were visualized by iodine vapor (1) and by 0.2% dichlorofluoresceine in methanol (2,3). Hydroethanolic phase is coded by A and hexanic phase is coded by B. Comparison with spots using 2-oleoyl glycerol (C, 3) and visualization of image (adapted from [19] (5).

Fatty acids profile of 2-MAGs were characterizated using preparative TLC by scraping the band with visualized under ultraviolet light at 254 nm. The band corresponding to 2-MAGs were extracted with chloroform, followed saponification reaction to liberated free fatty acids at sn-2 position. This portion was subsequently analized by HPLC. In general, to perform qualitative and quantitative analysis of fatty acids used gas chromatography prior derivatization to form fatty acids into esters (fatty acid methyl esters, FAME). However, the use of a high temperature of gas chromatography, especially for the analysis of unsaturated fatty acids which could be decompose and the formation of isomers [20] therefore in this study also attempted analysis fatty acid by HPLC.

Table 1. Fatty acids profile of canarium oil and 2-MAGs determined by HPLC

Concentration (ppm)

Fatty acid _

Canarium oil 2-MAGs

8:0 0.5 16.1

10:0 22.8 107.2

12:0 0.8 81.9

18:3 20.0 31.8

14:0 0.4 12.5

16:1 16.4 36.2

18:2 482.4 445.5

16:0 942.4 193.8

18:1 1295.8 939.8

18:0 478.9 72.8

Fatty acids profile of canarium oil and 2-MAGs (Figures 3 and 4) showed a similar fatty acids composition, however the fatty acid composition of the 2-MAGs consist of three other distinct peaks. This was probably because of the influence of enzyme performance, therefore further verification will be required by comparing the fatty acids of product reaction to fatty acid standards and also repeat the same process using standardized triacylglycerols, for instance tripalmitine as a substrate.

The result of fatty acids analysis in the 2-MAGs by HPLC showed the highest composition were the unsaturated fatty acids , namely oleic acid ( 18 : 1n - 9 ) 939.8 ppm , and linoleic acid (18:2n-6) such as in Table 1. The others unsaturated fatty acid in 2-MAGs were linolenic acid (18:2n-3) 31.8 ppm and palmitoleic acid (16:1n-9) 36.2 ppm. This composition was suitable for utilizing on structured lipids. Structured lipid product that was expected to incorporated a short chain and a medium chain fatty acids in position sn-1 and sn-3 on triacylglycerols backbond, while unsaturated fatty acids (PUFA and MUFA) were incorparated at sn-2 position. This structures will be metabolized easily thus providing energy rapidly and does not accumulate in adipose tissue. SL containing medium chain fatty acids and n-3 PUFA could be a therapeutic or medical lipd source, and may be useful in enteral and parenteral nutrition. This SL could decrease serum cholesterol and triglycerides in blood [6].Our next studies will utilize response surface methodology as a tools that enables to evaluation of effects of multiple parameters, alone or in combination thefore, it was applied for the optimization [21].

mAU -]

0 5 10 15 20 25

Fig. 3. Fatty acids composition of canarium oil was analyzed by HPLC

0 5 10 15 20 25 min

Fig. 4. Fatty acids composition of 2-MAGs was analyzed by HPLC CONCLUSION

Synthesis of 2-MAGs could be perform by ethanolysis reaction between canarium oil and dry ethanol in an orbital shaker water bath incubator. Characterization of 2-MAG carried out by preparative thin layer chromatography using the standar 2-oleoylglycerol and obtained 2-MAG yield of 74% after purification by solvent extraction. Analysis of the fatty acids composition between canarium oil and 2-MAG using high performance liquid chromatography showed a similar composition. The highest composition of fatty acids of 2-MAGs were unsaturated fatty acids, namely oleic acid (18:1n-9) and linoleic acid (18:2n-6) that could be used for production of structured lipids .


Thanks to the Food, Health and Medicine Research Center, Institute of Technology Bandung, that has funded this research. The authors also gratefully acknowledge the funding from the Government of Korea through KRIBB (Korea Research Institute of Bioscience and Biotechnology). The author (HR) is a lecturer of Public Health Department of North Maluku Muhammadiyah University, and thank you for further study permit at ITB and for BPPS scholarship from the Ministry of Education and Culture of Indonesia.


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Presented at ISFA (September 16-17, 2014-Semarang, Indonesia) as paper #48, "Managing Biosafety and Biodiversity of Food from Local to Global Industries"