SYNTHESIS AND CHARACTERIZATION OF FATTY ACID METHYL ESTER FROM LINUM USITATISSIMUM (LINSEED) SEED OIL

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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Borhade

World Journal of Pharmacy and Pharmaceutical

SJIF Impact Factor 2.786

Volume 3, Issue 7, 977-989.

Research Article

ISSN 2278 – 4357

SYNTHESIS AND CHARACTERIZATION OF FATTY ACID METHYL ESTER FROM LINUM USITATISSIMUM (LINSEED) SEED OIL Shobha Borhade* Department of Drug Chemistry, S.M.B.S.T.College, Sangamner, DistAhmednagar,University of Pune, Maharashtra, INDIA-422605

Article Received on 17 April 2014, Revised on 16 May 2014, Accepted on 09 Jun 2014

ABSTRACT Linum usitatissimum (Linseed) seed is popularly used in various food products, because of its nutritional values and potential health benefits. Natural seed of of Linum usitatissimum (Linseed) contains 35 % of oil. Linum usitatissimum (Linseed) seed oil contains 53.21 % alpha-

*Author for Correspondence

linolenic

Shobha Borhade

acid ). Seed oil was extracted by n-hexane & fatty acids present in the

Department of Drug Chemistry, S.M.B.S.T.College,

( omega-3 fatty acid ) & 17.25 % linoleic ( omega-6 fatty

seed oil is necessary to apply for transesterification to convert fatty

Sangamner, DistAhmednagar,

acid methyl ester. Fatty acid methyl ester (FAME) is created through

University of Pune,

an alkali catalyzed reaction between fats or fatty acids and methanol.

Maharashtra, INDIA

In the transesterification of fatty acids, the carboxyl group of an acid and the hydroxyl group of an alcohol are condensed. This makes the

fatty acids methyl ester Now FAME excellent for stability as samples for analysis. Transesterification is a general term used to indicate the direct conversion of triacylglycerols lipids by alcohols to alkyl esters without first isolating the free fatty acids (FFA). The alkaline catalyst, sodium hydroxide (NaOH),was used in the methanolysis of Linum usitatissimum (Linseed) seed oil Physico-chemical properties of FAME of of Linum usitatissimum (Linseed) seed oil was studied .Fatty acid methyl ester was characterized by UV, IR & LCMS. INTRODUCTION Linum usitatissimum (Linseed) seed oil is extracted from the seed by extraction with nhexane. Linum usitatissimum (Linseed) is believed to have originated inEgypt. Now, this plant grows through out Canada and the north western United States.

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Linum usitatissimum (Linseed) Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Malpighiales Family: Linaceae Genus: Linum Species: L. usitatissimum

Linum usitatissimum (Linseed) seed is popularly used in various food products, because of its nutritional values and potential health benefits. Linseed is rich in alpha linolenic acid (ALA), a plant-based omega-3 fatty acid, as well as fiber and lignans (phytoestrogens), making it a possible functional food for reducing cardiovascular risk factors linseed positively affected lipoprotein A (Lp(a)) and insulin sensitivity. They also found a modest but short-lived lowering effect in participants' LDL ("bad") cholesterol levels.Linseed oil is derived from the hard, tiny seeds of the linseed plant. Like fish oil, linseed oil contains an omega-3 fatty acid = alpha-linolenic acid (ALA). In addition to alpha-linolenic acid, linseed oil also contains omega-6 fatty acids. Both acids are essential to health.Linseed have long been used in human and animal diets and in industry as a source of oil and as the basic component or additive of various paints or polymers. Recently there has been a growing interest in the probiotic properties of linseed and in its beneficial effects on coronary heart disease, some kinds of cancer and neurological and hormonal disorders1. The beneficial effects are mostly due to linseed lipids. linseed oil is the richest plant source of linoleic (omega-6) and linolenic (omega-3) polyunsaturated fatty acids (PUFA), which are essential for humans since they cannot be synthesized in the organism and must be ingested in food. Linseed oil is qualitatively different from the more common vegetable oils with high PUFA proportions, such as soya oil, sunflower oil, rape oil, olive oil, etc. Linseed oil is a rich source of the

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following unsaturated fatty acids: oleic (C18, 16–24 %),linoleic (C18, 18–24 %), and linolenic acid (C18, 36–50 %)2 and it has a relatively low glucosinolate content3.The protein and fiber content in the seed are also important nutritional parameters: the crude protein content in the seed ranges from 25 % to 45 %, while the crude fiber content is about10%4 .The

results

of

the

analyses

of

linseed

oil

have

been

reported5-6

Unsaponifiable lipid constituents of seed oils naturally contain hydrocarbons, terpene alcohols, sterols, tocopherols and other phenolic compounds which may act as oxidation inhibitors under a range of conditions7. The effectiveness of lipid unsaponifiable matters in retarding oil deterioration has been demonstrated by many investigators8-9. In linseed grains, lipids are protected against oxidation by various mechanisms, for example, the presence of antioxidants such as lignans, phenols, tocopherols-(vitamin E) and

flavonoids10- 11

.In addition to

preventing fat rancidity, these antioxidants could increase commercial value of food products and have beneficial effects on human health. When consumed together with essential unsaturated fatty acids, they can reduce the risk of various diseases

12

The antioxidant ability

of phenols, tocopherols (vitamin E) and flavonoids is related to the presence of OH groups which may directly bind to free radicals and chelate metals13 .Linseed improves the quality of hair, nails, and skin, helps to regulate body weight, lowers cholesterol and blood pressure, and prevents arthritis and cancers. The omega 3 fatty acids of linseed oil lower high blood cholesterol and triglycerides. They also decrease the probability of clots in the arteries which may lead to stroke, heart attack, pulmonary embolism, or peripheral vascular disease occurring. They can also lower high blood pressure.It can kill human cancer cells on the same culture.omega 3 fatty acids have been shown to be helpful in the treatment and prevention of arthritis. Linseed oil can relieve asthma noticeably by decreasing inflammation and improving lung function.omega 3 fatty acids can relieve or even eliminate PMS symptoms.omega 3 fatty acids help to decrease allergic response. Linseed oil helps the kidneys to remove sodium and water.Linseed oil is renowned for its ability to improve the texture and quality of the skin, and will also alleviate skin conditions that are caused by the lack of omega 3 fatty acids in the diet. the use of linseed oil can result in increased vitality and more energy. Stamina is improved omega 3 fatty acids prevent excess toxic biochemicals that the body produces under stress.Linseed oil has been shown benefits on lowering heart diseases by reducing cholesterol levels, high blood pressure and slowing down atherosclerosis. Linseed oil may also benefit people suffered from bipolar disorder, cancer, acne and rheumatoid arthritis. In this section, we are going to review how linseed oil may benefit our health, based on recent research findings. Oral linseed oil reduces the www.wjpps.com

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inflammation in rheumatoid arthritis14 .Linseed oil in inflammatory disorders like rheumatoid arthritis15. Dietary level of alpha-linolenic acid (10% linseed oil) results in modest improvements in some bone linseed oil may benefit people on bone health16. ALA significantly decreased hepatic cholesterol but no effect was observed on heart and kidney cholesterol levels17. Further, ALA is capable to alter platelet function and inflammation18 .Linseed oil are probably effective to decrease the risk factors associated with cardiovascular disease19 & did not increase the effect of dietary therapy in correction of glycemia, hypertension and atherogenic lipid levels20.Oil supplements are also believed to benefit patients suffered from different types of cancers21.High doses of linseed oil could also delay in the growth of mammary cancers 22 and is effective in preventing colon tumor development 23

.

Fatty acid methyl ester prepared by transesterification.In the transesterification of oils, a triglyceride reacts with an alcohol in the presence of a strong acid or base, producing a mixture of fatty acids alkyl esters and glycerol 24-25 . Transesterification is the general term used to describe the important class of organic reactions where an ester is transformed into another through interchange of the alkoxy moiety. When the original ester is reacted with an alcohol, The transesterification process is called alcoholysis (Scheme 1) 26 .

A

In the transesterification of vegetable oils, a triglyceride reacts with an alcohol in the presence of a strong acid or base, producing a mixture of fatty acids alkyl esters and glycerol 27-28

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The transesterification process is catalyzed by BrØnsted acids, preferably by sulfonic

29

and

sulfuric acids These catalysts give very high yields in alkyl esters, but the reactions are slow, requiring, tipically, temperatures above 100 °C and more than 3 h to reach complete conversion. The base-catalyzed transesterification of vegetable oils proceeds faster than the acid-catalyzed reaction30-31.Dueto this reason, together with the fact that the alkaline catalysts are less corrosives than acidic compounds, industrial processes usually favor base catalysts, such as alkaline metal alkoxides

32-33

and hydroxides

34-38

as well as sodium or potassium

carbonates 39-40 The overall process is a sequence of three consecutive and reversible reactions, in which diand monoglycerides are formed as intermediates

41

. The stoichiometric reaction requires 1

mol of a triglyceride and 3 mol of the alcohol. However, an excess of the alcohol is used to increase the yields of the alkyl esters and to allow its phase separation from the glycerol formed. Fatty acid methyl esters can be transformed into a lot of useful chemicals, and raw materials for further synthesis

41

.The alkanolamides, whose production consumes the major

part of the methyl esters produced in the world, have a direct application as non-ionic surfactants, emulsifying, thickening and plastifying agents, etc . The fatty alcohols are applied as pharmaceutical and cosmetics additives (C16-C18) as well as lubricants and plastifying agents (C6-C12), depending on the length of their carbon chain

42

. The isopropyl

esters are also applied as plastifying agents and emollients. However, they can not be produced in a convenient way by esterification of fatty acids, as an azeotrope formed by water and isopropanol avoids the recycling of the alcohol. The fatty acid methyl esters are further used in the manufacture of carbohydrate fatty acid esters (sucrose polyesters), which can be applied as non-ionic surfactants or edible non-calorific oils 43-46 and can be used as an alternative fuel substitute for diesel engines (biodiesel) 47-52 . The glycerol also has important applications, in cosmetics, toothpastes, pharmaceuticals, food, lacquers, plastics, alkyl resins, tobacco, explosives, cellulose processing, etc

53

.

Fatty acid methyl ester obtained from natural seed oil. Fatty acid methyl ester are conventionally produced by the transesterification of Linum usitatissimum (Linseed) seed using methanol in presence of suitable catalyst.

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MATERIAL AND METHODS Collection of materials The dried Linum usitatissimum (Linseed) seeds were obtained from local market in Ahmednagar, Dist Ahmednagar, Maharashtra, India. They are dried in room, clean and stored in a sealed vessel wrapped with polyethylene bag at 40C. Linum usitatissimum (Linseed) seed oil , Methanol, NaOH, High purity water , Sulphuric acid.The instrument used were UV spectrophotometer with wavelength range 200-450 nm. IR spectra of FAME was taken in the range of 4000cm-1 to 600 cm-1 on perkin Elmer 221 IR spectrophotometer using KBR pellet techniques & LCMS chromatogram. Extraction of oil After cleaning and removal of the sand and foreign material , the dried Linum usitatissimum (Linseed) seeds were ground to a fine powder using a grinde. The oil was extracted with nhexane ( 1:4 w/v ) by continous extraction in a soxhlet apparatus for 12 hours. Experimental set up The experimental set up as a 1000 ml three necked round bottom flask was used as a reactor. The flask was placed in heating mantle whose temperature about 55-600C. Two side necked was equipped with a condenser and other was used as a thermometer for temperature measurement. Stirrer placed at the centre for stirring the reaction mixture.( fig.1 ) Pretreatment of oil Linum usitatissimum (Linseed) seed oil is first filtered to remove solid material then it is preheated at 10500C for 25 min to remove moisture ( Presence of moisture responsible for saponification in the reaction ) Transesterification – Based catalysed reaction : Mixing of alcohol and Catalyst Weight 12 kg of Linum usitatissimum (Linseed) seed oil and pour it into the reactor for preliminary heating to temperature of about 60-700C. In beaker dissolve 45.6 grams of NaOH ( 3.8 grams per liter of oil , got by 3.5 grams stoichiometric equivqlent and 0.3 grams for neutralizing FFA in 2.4 L methanol, 200 ml per lit of oil ). Add the NaOH slowly. This combined mixture makes sodium methoxide. Adding sodium methoxide in Linum usitatissimum (Linseed) seed oil provide rigorous mixing with the use of stirrer. The cloudy looking of free fatty acids called glycerine, will sink to the bottom and the methyl ester a translucent liquid will remain on top. When the separation appears not to be advancing any

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more, stop mixing. Let the mixture settle overnight. Meanwhile another batch can be started as the reactor is not being used. The liquid on the top is fatty acid methyl ester, but before using it any remaining soaps or salt which have to be removed. The glycerin which has sunk to the bottom can be used in the production of cosmetics. B

KOH

Methanol & Ethanol

Fatty acid methyl ester Reactor Flash

Extract of seed powder

Glycerol & Seed powder

Block Diagram for Production of Fatty Acid Methyl Ester ( FAME ) Rinsing Fatty Acid Methyl Ester Fatty acid methyl ester was poured off into a separate clean container, where it was washed free of any remaining soaps and salts. Warm water was added to the methyl ester. It was stirred lightly and then allowed to settle. The warm water was heated in the main reactor itself. The water was drained out from the bottom. The process was repeated until the discarded rinse water reached pH level of 6-7 and no soap bubbles appeared in it. If the liquid remaining is cloudy, there is water being retained in the methyl ester and it will need to be reheated slowly to evaporate out the water. Any white substances forming at the bottom or any bubbles forming at the surface is a sign of soaps and should be removed or the liquid should be re-washed. The cleaned methyl ester is characterizwd by UV, IR and LCMS. Physico-chemical Properties of FAME Physico-chemical Properties of FAMS of Linum usitatissimum (Linseed) seed

oil was

studied. Density was determined picnometrically, where as refractive index was determined at 400C with Abbey Refractometer equipped with a thermostated circulator. Viscosity was determined by Ostwald method .( Table 1.)

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Absorption spectra of FAME The absorption spectra of FAME of Linum usitatissimum (Linseed) seed oil was recorded against a blank solution ans shown in Fig. 2 The absorption spectra was recorded in the wavelength range 220-520 nm. FAME of Linum usitatissimum (Linseed) seed oil shows the absorption maximum

at 208.20 nm shows absorption 2.130.

Infrared Spectra of FAME The infrared spectra of fatty acid methyl ester of Linum usitatissimum (Linseed) seed oil was taken in the range of 4000 cm-1 to 600 cm-1 on perkin Elmer 221 IR Spectrophotometer using KBR pellet techniques. The characteristic bands observed are in Table 2. Fig 3. Shows IR spectra of FAME of Linum usitatissimum (Linseed) seed oil LCMS of FAME HPLC was applied for testing the presence of number of organic components available of methanolic extract of FAME of Linum usitatissimum (Linseed) seed oil. Method Column : YMC ,ODS, 50x4.6 mm, 3µ, Column ID : E-AC-1 / 12 / COL-03 Mobile phase : A : 0.05 % TFA in water & B : 0.05 % TFA in Acetonitrile Inj , Volume : 5.0 µL, Flow Rate : 1.2 mL / minute, Gradient program : 20 % B to 100 % B in 3.0 minute, At 4.50 min B conc is 20 % hold till 5.0 min Mass parameters : Scan mode +ve, CDL temperature 250 0C , Heat block temperature 200 0C Interface temperature 250 0C ( Table 3. Peak Table , Fig 4) RESULT AND DISCUSSION The extraction of oil from Linum usitatissimum (Linseed) seeds were done by Soxlet method using n-hexane as a solvent at 400C. yield obtained 46 % of oil.Extracted Linum usitatissimum (Linseed) seed oil consisted of 94 % pure triglyceride esters and the rest were free fatty acids. The physico-chemical properties were determined. Fatty acid methyl ester of Linum usitatissimum (Linseed) seed oil shows moisture 0.001 % and ash content 0.02 Wt %.FAME has density 0.7989 gm/cc and Refractive index at 400C 1.2894. Viscosity at 400C is 1.6231. Acid value, Iodine value and Saponification value are 0.34 mg KOH / gm, 91 g / 100 g of oil and 142 mg KOH / g of oil. UV spectra of FAME of Linum usitatissimum (Linseed) seed oil was performed at 208.20 nm wavelength. At 208 nm absorbance was 2.130.. IR spectra was done in the range of 4000cm-1 to 600cm-1 shows P-O Stretch at 1016.42, C=C

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Stretch at 1643.24, Alkynes ( RHC =CH2) at 2104.19, O-CH3 at 2842.88, Alkanes ( -CH3 ) at 2954.74, N-H Stretch at 3398.34, Primary free N-H at 3411.84 & 3436.91, N-H Stretch at 3454.27. LCMS is suitable analytical method for determining FAME of Linum usitatissimum (Linseed) seed oil. It shows 31.96 % at 0.525 retention time, 03.72 % at 0.596, 34.31 % at 0.668 , 03.38 % at 0.798 , 06.48 % at 0.967, 06.48 % at 0.967, 02.39 % at 1.063, 06.49 % at 2.464 and 00.94 % at 2.648. Table 1. Physico-chemical properties of FAMS of Linum usitatissimum (Linseed) seed oil Sr.No. 1) 2) 3) 4) 5) 6) 7) 8) Table 2.

Properties Moisture Ash content Density Refractive index at 400C Viscosity at 400C Acid value Iodine value Saponification value

Cst mg KOH / gm g / 100 g of oil mg KOH / g of oil

Values 0.001 0.02 0.7989 1.2894 1.6231 0.34 91 142

IR of FAMS of Linum usitatissimum (Linseed) seed oil

Sr.No.

Frequency Wavenumber 1016.42 1643.24 2104.19 2842.88 2954.74 3398.34 3411.84 3436.91 3454.27

1) 2) 3) 4) 5) 6) 7) 8) 9) Table 3.

Unit % Wt % gm / cc

Expected Elements P-0 Stretch C=C Stretch Alkynes ( RHC =CH2) O-CH3 Alkanes ( -CH3 ) N-H Stretch Primary free N-H Primary free N-H N-H Stretch

LCMS of FAMS of Linum usitatissimum (Linseed) seed oil

Sr.No. 1) 2) 3) 4) 5) 6) 7) 8)

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Ret. Time 0.525 0.596 0.668 0.798 0.967 1.063 2.464 2.648

Area 60856 7087 65321 6428 12339 4567 12363 1784

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Area % 31.96 03.72 34.31 03.38 06.48 02.39 06.49 00.94

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Figure 1. Experimental set for preparation of Linum usitatissimum (Linseed) methyl ester A B C S T TC

-------------------------

Speed controller Stirrer Condensed ( Water condenser ) Sample inlet Temp. indicator Temp. controlled

Figure 2 . UV Spectra of FAMS of Linum usitatissimum (Linseed) seed oil

Figure 3 . IR of FAMS of Linum usitatissimum (Linseed) seed oil

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Figure 4 . LCMS of FAMS of Linum usitatissimum (Linseed) seed oil REFERENCES 1. Huang, S., D. Milles,Gamma-linolenic acid: Metabolism and its roles in nutrition and medicine. Champaign, IL, AOCS Press.1996. 2. Huang, S., A. Ziboh, Gamma-linolenic acid: Recent advances in biotechnology and clinical applications. Champaign, IL, AOCS Press, 2001. 3. Schuster, A., W. Friedt,

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