BANGLADESH RESEARCH PUBLICATIONS JOURNAL ISSN: 1998-2003, Volume: 8, Issue: 2, Page: 180-185, March - April, 2013

THE EFFECT OF DIFFERENT SOLVENT MIXTURES FOR THE EXTRACTION OF SESAME (Sesamum indicum Linn.) SEED LIPIDS MM Uddin1, S Yeasmin1 , M Munsur1, M Mizanur1, F Mazumder1*, M Saifur2, M Raihan2 and M Kamonasish3

MM Uddin, S Yeasmin, M Munsur, M Mizanur, F Mazumder, M Saifur, M Raihan and M Kamonasish (2013). The Effect of Different Solvent Mixtures for the Extraction of Sesame (Sesamum indicum linn) Seed Lipids. Bangladesh Res. Pub. J. 8(2): 180-185. Retrieve from http://www.bdresearchpublications.com/admin/journal/upload/1308126/1308126.pdf

Abstract Studies were carried out on the effect of different solvent mixture for the extraction of sesame seed lipids. The dried kernels of three different varieties of sesame seed lipids were extracted with chloroform-methanol (2:1), methylene chloride-methanol (2:1) and hexane-isopropanol (3:2) mixtures. The total lipid extracts were fractionated into lipid classes by silicic acid column chromatography. The neutral lipids were found to be 97.2, 86.1 and 85.6%, Glycolipids 1.83, 9.7 and 9.64, phospholipids 0.40, 3.5 and 3.6% of the total weight of the lipid extracted by hexane-isopropanol, chloroform-methanol and methylene chloride-methanol mixtures in average in each variety respectively. The fatty acid composition of triglyceride (TG) fraction were analysed by gas liquid chromatography (GLC). It was found that chloroform-methanol and methylene chloride-methanol mixtures extracted comparable amounts of fatty acids in the neutral lipids fraction of the sesame seed lipids whereas hexane-isopropanol mixture extracted less fatty acids irrespective of varieties.

Key words: Sesame seed lipid, Column Chromatography, GLC, Chloroform-Methanol, Methylenechloride-Methanol and Hexane-Isopropanol.

Introduction Sesame (Sesamum indicum Linn.), an oil seed crop belongs to the family Pedaliaceae. It is cultivated widely in China, India, Mexico, Burma, Sudan, Turkey, Bangladesh and in many other countries of the world as an oil seed crop. Sesame prefers to grow on light well drained soil with adequate moisture. In Bangladesh, the soil texture and climatic condition are quite suitable for the cultivation of sesame. As an annual crop, sesame is considered as the second major oil seed crop in Bangladesh both in respect of acreage and production (Annon, 1991). Most of the sesame seeds are used for oil extraction and the rest are used for planting and edible purposes (Rahman et al. 2007). On account of its good agreeable flavour, sesame seeds are also used as nourishing and flavouring agent (Annon, 1988). Sesame seed contains 44-55% edible oil (Mattil et al. 1964) and the oil is used largely as edible oil for cooking and as table oils in salad dressing (Annon, 1950). The oil is also utilized for the manufacture of soap, insecticide and paint (Vaughan 1970). Again the oil is used as substitutes for olive oil and in medicine (Hill 1951). The oil is used for anointing hair and the human body before bathing (Ninan 1989). The results of a recent study indicated that sesame oil may help to reduce high blood pressure and lower the amount of medication needed to control hypertension (Shultz 2003). At present Bangladesh is facing acute shortage of edible and inedible oils and so she is compelled to import the oil seeds, edible oils and inedible industrial oils from abroad. As Corresponding Author: Email: [email protected] 1 BCSIR Laboratories, Binodpur Bazar, Rajshahi-6206, Bangladesh 2 Department of Applied Chemistry & Chemical Engineering University of Rajshahi, Rajshahi – 6205, Bangladesh 3 Bangladesh Sericulture Research and Training Institute, Rajshahi-6207, Bangladesh

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such sesame seed oil can play a vital role in bridging the vegetable oil gap in the country which necessitates thorough studies on the extraction, characterization, processing and fractionation of the lipids. Methods of extraction and use of solvent mixtures for extracting sesame seed lipid have also assumed importance in view of its increasing demand for using it as edible oil. A number of organic solvents can be used for extracting plant and animal lipids but all of them are not recommended as suitable solvent for the extraction of lipids. Chloroform-methanol (2:1) mixture has been extensively used for the extraction of plant and animal lipids. But recently through out the world the use of chloroform as extracting solvent has been restricted due to its hepatoxic action. Several solvent mixtures, such as hexane-isopropanol (3:2) and methylene chloridemethanol (2:1) have been suggested to avoid the use of chloroform. So, we have attempted to find out the effect of different solvent mixtures for the extraction of sesame seed lipids through which a suitable solvent mixture can be identified for better an effective extraction of lipids.

Materials and Methods Three different (black, white and brown) varieties of ripe matured sesame seed were collected from the local market. The seeds were then cleaned and deshelled manually. The kernels thus obtained were crushed into smaller particles in an iron mortar and dried in an oven at a temperature of 1050C for about half an hour to about 4-6% moisture. The moisture content in the fresh kernel was determined by IUPAC methods (Annon, 1979). The lipids were extracted from the seed kernels by using three different solvent mixtures namely chloroform-methanol (2:1), methylene chloride-methanol (2:1) and hexaneisopropanol (3:2) (Folch et al. 1957). The solvent containing the lipids were filtered. The lipids were then recovered from the filtrate by evaporating the solvent using a rotary vacuum evaporator under reduced pressure and the percentage of lipid content was calculated. The physical and chemical characteristics of the lipids were determined by the standard AOCS methods (Annon, 1980).

Separation of lipid classes by column and thin layer chromatography The total lipids extracted with three solvent mixtures were fractionated into three major lipid classes on a silicic acid column (Rouser et al. 1966). The silicic acid (E. Merck Darmstadt, W. Germany, 70-230 mesh) was washed with water and methanol to remove fine particles and impurities. It was activated at 1200C overnight and again for 1 hour immediately before the column was prepared. For each column 25 gm silicic acid was washed with 250 ml of chloroform-methanol (7:1 v/v), 120 ml chloroform-methanol (15:1 v/v) and 160 ml chloroform. A slurry of 25 gm of silicic acid in chloroform was poured into the column (2.2 cm i.d). After the column was washed with 100 ml diethyl ether and 325 ml 4% diethyl ether in petroleum ether (b.p. 60-700C), 150 mg of the total lipids were dissolved in 5ml chloroform and quantitatively transferred to the column. The neutral lipid was eluted by 80 ml of chloroform, glycolipids by 200 ml of acetone and phospholipids with 175 ml of methanol (Robertson et al. 1978). The elution was controlled with a flow rate of 1.5 – 2 ml/min. The elution of each fraction was monitored by microslide thin layer chromatography (TLC) to ensure uniformity of separation of each lipid classes and the eluted solvents were collected in weighed flasks. The fractions thus obtained were evaporated in a rotary vacuum evaporator and were dried under reduced pressure before being weighed. The purity of the lipid classes was further checked by TLC on 20 cm × 20 cm plates coated with a layer (0.5 mm) of silica gel G. Neutral lipids were separated further by high performance preparative TLC using hexane-diethylether-acetic acid (80:20:2) as the developing solvent system and individual lipid class and their fatty acids were quantitively determined by GLC with added internal standard (Rouser et al. 1966, Robertson et al. 1978).

Analysis of fatty acids The fatty acid composition of the triglyceride (TG) fraction of each variety was analysed as their methyl esters, which was prepared by the borontriflouride methanol method (Gofur et al. 1993). The analysis was carried out by gas liquid chromatography (GLC) instrument equipped with a flame ionization detector. Nitrogen carrier gas was used at a flow rate of 25 ml/min. Fatty acid esters were separated on a 1.8 m × 2 mm i.d. glass http://www.bdresearchpublications.com/journal/

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column packed with 6% BDS (Butanediol succinate polyesters) on solid support Anakoram ABS 100/120 mesh. Analysis was carried out at isothermal column temperature of 1900C, injector and detector temperature for all GLC analysis was 2000C. The peaks were identified by comparison with standard methyl esters for retention times, by plotting the log of retention times against equivalent carbon length (ECL). The peak areas were determined by multiplying peak height by peak width at half height. The percentage of each peak was calculated as the percentage of the total area of all the peaks. For the pancreatic lipase hydrolysis of the triglyceride about 9 mg pancreatin was added to 50 mg of weighed triglycerides in a screw cap vial (Luddy et. el. 1964). Then 1 ml of IM trisbuffer (trishydroxymethyl amino methane, pH adjusted to 8.0) 0.1 ml 22% calcium chloride solution and 0.25 ml of 0.1% bile salt solution were added. The preheating and shaking were carried out at 400C. At the end of reaction the content of the vial was acidified with 0.5 ml of 6N HCl and immediately transferred to a small separating funnel and extracted with diethyl ether. The extract was washed with water, dried over anhydrous sodium sulphate and evaporated. The extract of the hydrolysate was separated on a 20 cm X 20 cm plate coated with a layer of 0.5 mm thick of silica gel G with a solvent system petroleum ether – diethyl ether – acetic acid (70:30:1). The developed plate was sprayed with 2,7-dichlorofluorescein to locate the 2-monoglyceride by comparison with standard reference samples. The band was scrapped off and extracted with chloroform. The eluant was evaporated to dryness under a stream of nitrogen converted to methyl esters by the method of described earlier (William and Lloyd 1964). The fatty acids of 2-monoglyceride were analysed by gas liquid chromatography.

Results and Discussion Sesame seed kernels of three different varieties have been studied to evaluate the lipid compositions extracted by three solvent mixtures. The physico-chemical properties of the extracted lipids were determined and the results were presented in Table1. From the results it was found that the physico-chemical properties were not appreciably influenced by solvent mixtures except some varietal variations. Table 1. Physical and Chemical characteristics of sesame seed lipids extracted by three solvent mixture Variety

Black

White

Brown

Extracting solvent mixtures ChloroformMethanol Methylene chlorideMethanol Hexaneisopropanol ChloroformMethanol Methylene chlorideMethanol Hexaneisopropanol ChloroformMethanol Methylene chlorideMethanol Hexaneisopropanol

Specific Refractive gravity index at at 280C 280C

Saponification value

Unsaponifiable matter (%)

Iodine value

Peroxide value m.eq./kg.

0.917

1.4562

192

1.6

112

1.23

0.916

1.4560

190

1.7

110

1.21

0.917

1.4561

191

1.6

111

1.22

0.915

1.4565

190

1.7

110

1.25

0.913

1.4563

192

1.6

112

1.23

0.914

1.4564

190

1.6

110

1.24

0.914

1.4563

192

1.8

113

1.22

0.915

1.4562

190

1.6

112

1.24

0.914

1.4563

192

1.7

113

1.23

Total seed lipids were fractionated into three major lipid classes. Neutral lipids, glycolipids, and phospholipids by silicic acid column chromatography and the results were shown in http://www.bdresearchpublications.com/journal/

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Table 2. From the results it was observed that the quantity of the lipids extracted by chloroform methanol (2:1) and methylene chloride-methanol (2:1) mixtures did not differ significantly but the hexane-isopropanol (3:2) mixture was lower than those of other two solvent mixtures irrespective of varieties. The fractionation of total sesame seed lipids revealed that neutral lipids accounted for 97.2, 86.2 and 85.6% of the total weight of the lipids extracted by hexane-isopropanol, methylenechloride-methanol and chloroformmethanol mixtures respectably. But the lower amounts of glycolipids and phospholipids were extracted by hexane-isopropanol mixture compared to those of other two solvent mixtures. Table 2. Effect of different solvent mixtures on the lipid composition of sesame seed kernel Variety

Black

White

Brown

Extracting solvent mixtures ChloroformMethanol Methylene chloride-Methanol Hexaneisopropanol ChloroformMethanol Methylene chloride-Methanol Hexaneisopropanol ChloroformMethanol Methylene chloride-Methanol Hexaneisopropanol

Total lipids (%)

Neutral lipids (%)

Glycolipids (%)

Phospholipids (%)

42.5

85.6

9.7

3.5

42.6

86.0

9.5

3.4

40.0

97.0

1.83

0.40

42.6

86.5

9.7

3.20

43.0

85.2

9.71

3.65

40.0

97.2

1.93

0.42

42.4

86.4

9.64

3.80

42.6

85.6

9.65

3.70

40.1

97.4

1.90

0.41

Mean value of three experimental results The weight percentage of neutral lipid classes in sesame seed kernel were given in Table 3. Table 3. Weight percentage of neutral lipid classes in sesame seed kernel Variety

Black

White

Brown

Extracting solvent mixtures

Hydrocarbon & sterol esters

ChloroformMethanol Methylene chlorideMethanol Hexaneisopropanol ChloroformMethanol Methylene chlorideMethanol Hexaneisopropanol ChloroformMethanol Methylene chlorideMethanol Hexaneisopropanol

Neutral lipids (wt. %) Fatty Triglycerides acids

Free sterols

Partial glycerides

0.8

91.5

3.2

0.7

1.5

0.7

91.3

2.1

0.8

1.8

0.5

95.5

1.0

0.5

2.4

0.6

91.7

4.2

0.7

1.3

0.5

91.4

4.1

0.6

1.5

0.5

90.5

1.2

0.7

2.2

0.6

90.2

6.4

0.6

1.2

0.4

90.5

6.2

0.7

1.5

0.5

95.8

3.5

0.8

2.3

From the results it was observed that the level of triglyceride fraction was slightly higher and the fatty acid fraction was lower in the neutral lipid extracted by hexane-isopropanol than those of other two solvent mixtures. The amount of triglyceride and fatty acid http://www.bdresearchpublications.com/journal/

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fractions extracted by chloroform-methanol and methylenechloride-methanol mixtures were almost similar (Table-3). The highest fatty acid level attained in the neutral lipid fraction of the chloroform-methanol mixture in comparison with hexane-isopropanol mixture. Table 4. Fatty acid compositions of sesame seed lipids obtained by different solvent mixtures Variety

Black

White

Brown

Extracting solvent mixtures ChloroformMethanol Methylene chlorideMethanol Hexaneisopropanol ChloroformMethanol Methylene chlorideMethanol Hexaneisopropanol ChloroformMethanol Methylene chlorideMethanol Hexaneisopropanol

C16:1

Fatty acids (wt. %) C18:0 C18:1

C18:2

C20:0

9.0

4.3

44.2

40.3

0.7

9.2

4.2

44.1

40.2

0.8

9.4

4.1

43.9

40.6

0.6

9.1

4.5

43.8

40.1

0.6

9.2

4.3

43.5

40.4

0.7

9.4

4.1

43.4

40.3

0.8

9.2

4.2

44.3

40.4

0.7

9.0

4.0

44.5

40.1

0.6

9.1

3.9

44.2

40.3

0.7

Conclusion Sesame is one of the most important oil seed crops of Bangladesh. At present Bangladesh is facing acute shortage of edible oil. So, sesame oil can play a vital role in bridging the oil gap in the country. But a suitable solvent mixture for better and effective extraction of lipids from sesame seeds was urgently felt in view of its increasing demand for using it as edible oil. Accordingly was taken to extract the lipids by using three solvent mixtures to find out the most suitable one.

Acknowledgement The authors express their gratitude to the Director, BCSIR Laboratories, Rajshahi for providing all research facilities.

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