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113

Ifeanyi et al. World Journal of Pharmacy and Pharmaceutical Sciences

ISOLATION AND COMPARISM OF FATTY ACID AND

ACYLGLYCEROL CONTENTS IN LOCAL HYBRID

COCONUTS(COCOS NUCIFERA)

Aloh Godwin Sunday1, Obeagu Emmanuel Ifeanyi*2, Eze Obioma Benedict L1

1Lecturer,Department of Biochemistry,Michael Okpara University of

Agriculture,Umudike,Abia State,Nigeria. 2.Diagnostic Laboratory Unit,University Health Services Department, Michael Okpara University of

Agriculture,Umudike,Abia State,Nigeria.

ABSTRACT

INTRODUCTION

The coconut belongs to the palm family, palmae (Gill, 1988). The

coconut is found throughout the west tropical low lands (Anochili and

Tindall. 1986). It probably originated in South-East Asia of the

pacific region from where it was taken to the Atlantic coasts of the

America and tropical areas of Africa where suitable conditions for their

growth existed (Purseglove, 1972). However, the considerable

extension in acreage during the past 100 years is due to the demand for

coconut oil in temperate countries. In West Africa, coconut are grown

along the coastal and inland areas where there is good drainage.

Coconut has different local names. Benin – Ovi oibo, Yoruba - agbon,

Hausa – Kwakara itagara, Igbo – aki oyibo, Efik – isip mbakara and

Fante – kbve (Gill, 1988).

Coconuts are grouped into two main categories: the dwarf and tall varieties, based on

flowering and fruiting habit, and tree height (Tindall and Anochili, 1986).

1. Dwarf varieties (Var. Nana

These are probably mutations from tall types and have occurred in several countries (Brook,

1982). This variety starts bearing fruits earlier than the tall variety. It flowers as early as three

years after four to five years when the trees have reached a height of about 1.5m. They are

normally self-pollinated due to overlapping of the male and female phases, and are therefore

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Article Received on 19 February 2014, Revised on 12 March 2014, Accepted on 31 March 2014

*Correspondence for Author

Obeagu Emmanuel Ifeanyi

Diagnostic Laboratory

Unit,University Health

Services,Michael Okpara

University of

Agriculture,Umudike,Abia

State, Nigeria


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fairly homozygous (Tindall and Anochili, 1986). This quality makes it possible for it to

maintain its dwarf character. The nuts are small but numerous, and 6000 – 8000 nuts are

required to produce 1 ton of copra. They have a short productive life of 30-35 years, although

white head (1966) records plantations, which are still in good bearing after 40 years. They

require better soil and climatic conditions, and in Ceylon have been found to be readily

susceptible to pests and disease. They cross with the tall and the hybtid show considerable

promise (Harries, 1978).

2. Tall Varieties (Var. typica)

This variety is sometimes referred to Var. typical (Purseglove, 1972). They are the most

commonly planted for commercial production, growing to a height of 20 – 30m. They are

slow-maturing, first flowering 6 – 10 years after planting. They are long-lived and may attain

an age of 80 – 100 years (Luntungan, 1978). They are normally cross-pollinated, as there is

usually no overlapping of the male and female phases of the protandrous flowers (Harries,

1978).

The nut is medium to large in size and 4000 -6000 nuts usually yield 1 ton of copra. They are

hardy and will thrive on different soil types and varying environmental conditions (Liyanage,

1967) however, tall palms which have developed in various regions are variable and distinct.

But the common forms may be referred to by the local names of the countries in which they

are grown e.g Jamaica Tall; have an average fruit weight of about 1.7kg and nut weight of

about 0.7kg, of which 50% is endosperm producing 02.kg of copra. They are susceptible to

lethal yellowing.

3. The King Coconut (Var. aurantiaca)

In coconut, the manifestation of hybrid vigor has been observed in growth as well as yield

characteristics (Luntungan, 1982). Inter-varietal hybrid obtained between the tall and dwarf

varieties are more promising than intra-varietal hybrids (Gill, 1989). The inter-varietal

hybrids with the tall variety as the pollen parent are precocious, high yielding and produce

nuts with copra quality, equal to the rill parent. The production of a large number of inter-

varietal hybrids by artificial pollination is discouraged by several factors;

a. The absence of suitable markers to identify the hybrids;

b. The inability of the hybrids to perform well except under favourable management

conditions.


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c. The considerable labour and expenditure involved in the production of hybrids (Robbelen

and Downey, 1989).

The coconut can be adapt to a range of climatic conditions and thrives well between latitude

20o North and 20o South of the equator. The highest yields are generally obtained from trees

grown at 600m to 900m above sea level. The coconut frows well in areas, which have an

evenly distributed rainfall of about 100 to 225cm per annum. It is not suited to regions, which

have a long dry season. A mean annual temperature of about 27oC is required, while

temperature below 20oC cause fruit abnormalities (Luntungan, 1982). The coconut requires

warm humid conditions with about 70% relative humidity. Coconut palm grows well on

drained, rich alluvial or loamy soils which permit unrestricted root development and aeration.

Violent storms such as cyclones, hurricanes or typhones are hazards in several coconut

growing countries.

From the base of the stem arise numerous adventitious roots, some of which appears above

the ground level. The first roots tend to penetrate the soil vertically and subsequent roots

usually spread horizontally (Anthony, 1988). The roots are widely and superficially spread to

absorb nutrients from the upper layer of the soil. The trunk carries a crown of leaves at the

top, and each leaf is compound with a mid—rib (rachis) which bears pinnately arranged

leaflets measuring about one metre (Adams and Bamford, 1999). No visible trunk is formed

until the palm is several years old and a trunk can only be formed when the apical meristem

has attained its full diameter since there is no cambium (Tandall and Anochili, 1986). The

palm like other monocotyledons is composed of a series of joints, each having a nod, a leaf

and internodes flowers appear in an inflorescence which is enclosed in a boat shaped sheath,

or spathe. The inflorescence is composed of many male (staminate) flowers at that apex of

the inflorescence stalk and a few female (pistillate), flowers at its base, making the plant

monoecious (Gill 1988). The mature fruit is a fibrous drupe, about 20 – 30cm long weighing

1.2 – 2.0kg and consisting of:

1. Epicarp or outer skin which is tough, smooth, hard and may be green, yellow, orange, red

or brown.

2. Mesocarp or fibrous layer which is pale brown.

3. Endocarp or shell which is ovoid, hard, stony and dark brown and

4. S single seed with a thin brown testa closely oppressed to endocarp and adhering firmly to

endocarp or “meat”, which is firm, white, oily, 1 – 2cm thick and supply copra and oil.


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(Purseglove, 1972). In center of the seed is a large cavity partially iled with coconut water,

which is completely absorbed about 6 months after harvesting. The endocarp contains sugar

or carbohydrate, fats, oil and protein as food reserves (Robbelen, 1989). See table 1.1 and the

figure below.

Table 1.1 : CHEMICAL COMPOSITION OF COCONUT

Source: (Robbelen and Downey, 1989)

Source: Luntungan, H.T. (1982).

Rhinoceros beetle or coconut black beetle, Oryctes rhinoceros found throughout South – east

Asia burrows into the terminal bud, damaging the unopen leaves and may kill young palms

(Bock, et al, 1970). The larvae of palm weevils, Rhynchoporus spp, of which the most

important are R. Ferrugineous and R. Schach in Malaya damage the trunk and crown,

especially those of youmg palms (Purseglove, 1872). Others like termites, red ring nematodes

and rats are destructive bur controls are achieved with insecticides e.g DDT BHG. Coconuts

suffer from a number of diseases such as Bud disease, caused by phytophthora palmivora

which causes wilting and collapse of the youngest leaves, followed by similar symptoms

successive lower leaves recently described lethal bole rot caused Maraemiellus cocophilus

pegler along the East Africa coast and lethal yellowing, which was formerly called shedding

of young and maturing nuts (Bock et al., 1970).

It provides food, drink, oil, medicine, fibre, timber, thatch, mats, fuel, and domestic utensils

(Purseglove, 1972). The main producers are Philippines and Indonesia. Coconut is essentially

an oil crop though every part of the palm is useful to mankind. Coconut oil due to its high

content of lauric and myristic acid, has a high saponification value, a low iodine number, and

is used extensively for edible and industrial purposes (Varner and Bonna, 1976). Coconut oil

ENDO

– SPERM

COPRA COCONUT CAKE

DRINKING COCONUT

WATER

SWEET TODDY

Mill Expeller Chekku Water 36.3 6.8 11.0 11.0 13.3 95.4 84.4 Protein 4.5 7.8 19.1 19.1 14.3 0.1 0.1

Fat 41.6 63.7 6.0 10.0 26.7 0.1 0.1 Carbohydrate 13.0 16.1 45.3 43.8 32.8 4.0 15.1

Fibre 3.6 3.8 12.2 11.8 8.9 - - Minerals 1.0 2.0 5.7 5.3 4.1 0.4 0.3


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is used in margarine, biscuit and cooking oil industries, in the production of toilet articles

such as hair oil, shaving creams, shampoos, soap and cosmetic. The oil is also used in

synthetic resins as well as rubber substitutes detergent, lubricant and fuel production of

petrol, kerosene and oil substitutes is possible from coconut oil (Purseglove, 1972). The cake

obtained after oil extraction can be used as fertilizer for field crops, or fed to cattle, poultry or

young growing swine. The meat of the ripe coconut us in cooking and for household oil

production. The milk or cream obtained from gratings of the meat is used as substitute for

cow’s milk. The liquid or coconut water in the tender nut, is a refreshing drink. Is often

recommended as a medicine in cases of gastroenteritis where it is considered for a substitute

for saline glucose (Purseglove, 1972). Coconut water is a major ingredient in coconut

vinegar, sauce and Lemonade, Sweet toddy obtained by tapping the unopened inflorescence

of the palm, is an excellent beverage and palm sugar or jiggery is obtained by boiling and

evaporating fresh toddy (Tindall and Anochili, 1986). The hard shells of coconut are used in

the manufacture of shell charcoal, activated carbon and shell flour. The fibrous husk of

coconut yields a fibre known as coir which is board. The stem of palm, are used for thatching

houses, fencing and for making basket (Purseglove, 1982).

AIMS AND OBJECTIVES

The aims of this project was to compare the fatty acid and acylglycerol contents of loca and

hybrid coconuts since coconut is one of the edible foods consumed in the whole world for its

fatty acid and acylglycerol contents.

{MATERIALS AND METHODS

MATERIALS USED FOR THE EXPERIMENT

1. Coconut (Cocos nucifera) endosperm

2. Reagents

i. Chloroform – M&B, AnalaR

ii. Hexane-BDH, GPR

iii. Diethyl ether -BDH, GPR

IV. Methanol – M&B, GPR

V. Calcium Oxide -BDH, GPR

vi. Sodium sulpjate –BDH, AnalaR

vii. Ethanol -BDH, GPR

viii. Acetic acid – glacial -BDH, GPR


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ix. Isopropyl either -BDH, GPR

x. Isopropyl alcohol -BDH, GPR

xi. Potassium hydroxide pellets – BDH< AnanaR

xii. Concentration Sulphuric acid -BDH, GPR

xiii. Glycerol – BDH, AnalaR

xiv. Silica gel G, type 60 – MERCK, AnalaR

xv. Lipid standards – Oleic acid, Linoleic acid, palmitic acid, myristic acid, lauric acid,

stearic acid, Glycerol tristearate glycerol trioleate, and glycerol tripalmitae -BDH, GPR

xvi. Sodium thiosulphate -BDH, GPR

xvii. Salicyclic acid -BDH, GPR

xviii.Periodic acid -BDH, GPR

xix. Starch indicator _CVI, GPR

xx. Triolein standard -BDH, GPR

xxi. Periodic solution -BDH, GPR

xxii. Cupric nitrate -BDH, GPR

xxiii.Triethanol amine -BDH, AnalaR

xxiv. Diethyldithiocarbamate -BDH, GPR

3. Instruments, Containers, Equipment Used

i. Deep freezer – model MK3 novum freezer, Britain.

ii. Centrifuge – Haraeus Christ GMDH centrifuge, London.

iii. Spectrophometer – Nova spectrophotometer by Biochrome, London.

iv. Mettler H 35 & P 1000 Electrical balances. London.

v. Conical flask, test tubes funnel, burette.

METHODS

EXTRACTION OF COCONUT OIL (TRIACYLGLYCEROL).

The coconut oil was obtained by freeze thawing following the method described by

Robledeno and Luzuriagal.

Procedure: 120g of fresh coconut kernel from the local and hybrid varieties were grated fine

with an improvised fat grater, placed in a white cloth and the “milk” pressed and wrung out

by hand. The milk was washed thrice in a separating funnel with tap water and only the

floating “cream” was recovered during each washing. The washed cream in a conical flask

was allowed to freeze in a deep freezer – thaw operation gad to be repeated four times but


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this time, at intervals of four hours to get the oil liberated. The oil was next decanted into

another flask and centrifuged briefly at low speed to remove further solid impurities. The

weight and volume of the oil were determined and stored for use.

Separation Of Triacyglycerol (Neutral Lipid) From Probable Polar Lipids Present.

Neutral lipids were separated from polar phospho–lipids using an activated alumina

(triacyglycerol purifier) column, a method that is also employed in the purification of

phospholipids (Merinetti, 1962).

Procedure: A clean, drawn out tipped burette was fitted with glass wool at bottom and

mounted vertically with a cramp.

20gm of activated alumina was activated at 12o for 30minutes and cooled. This was poured

into the burette and dissolved in chloroform to form a 10cm column. On solidification, it was

washed with 50ml of chloroform after which 2.5ml of the lipid extract was applied. The

extract was allowed to enter the column and then eluted with 50ml of chloroform for the

natural lipids. Elution with 50ml of methanol on a similar column would have extracted the

polar lipids. The extract was concentrated to about 5ml and stored for use, this was done on

each of the variety.

Extraction Of Free Fatty Acids.

Free fatty acids were extracted from the triacyglycerol by the Dole method (Dole, 1956). The

Dole’s extraction mixture consist of isopropyl alcohol; Hexane; INH2SO4 (40: 10: 1 v/v)

Procedure: 5ml of Dole’s extraction mixture was pipetted into a clean test tube, 1ml of lipids

extract was added and the mixture vigorously shaken using an incubator shaker. The mixture

was placed in an ice-bath for 10minutes after which 2ml of hexane and 3ml of distilled water

were added. The contents of the tube were again shaken with the shaker for 5minutes. On

standing, the phase separated and 3ml of the upper hexane phade was pipette into another

clean dry test tube. To this was added 1ml of chloroformhexane mixture (5:1 v/v) and

centrifuged at 2,500 rpm for 15 minutes. The upper chloroform-hexane phase was pipette into

another bottle and used for free fatty acid analysis.

Qualitative Analysis Of Triacylglycerol

i. Preparation of plates:

The plates were prepared according to (Boyer and King, 1977) with slight modifications


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Procedure: Silica gel G, type 60 and water used in a 1:2 ratio to prepare the TLC plates. 60gm

of the gel and 120ml of water in a 250ml reagent bottle were shaken vigorously for 10

minutes and used in preparing the plates, were washed, dried and cleaned with acetone

soaked in cotton wool. The gel was spread with Shandon TLC plate spreader for TLC plates.

After a brief air-drying, the plates were activated in the oven ar 121oc for 45 minutes, they

were cooled and used.

ii. One Dimensional TLC of Triacylglycerol Fatty Acids and Fatty Acids Standards

A shandon TLC tank was saturated with a solvent system of Hexane-ether-acetic acid

(60:40:1) for 30 minutes after it had been made airtight with Vaseline or grease along the

edges. 2.0% solutions of these lipids standard in chloroform were prepared, mytistic, lauric,

palmitic, oleic, stearic and lioleic acids. A drop in each of the lipids standards was applied

alongside that form coconut at a distance 2cm from the base of the plate. It took 1 hour 30

minutes for the solvent front to reach 2cm marked point above the plates. The plates was

briefly air-dried and visualized by iodine vapour as a chromogenic reageant, the Rf values

were calculated, the spots encircled and the plates photographed (fig. 4.1)

iii. One Dimensional TLC of Tricylglycerol Fatty Acids and Fatty Acids Standards

A Shandon TLCtank was saturated with a solvent system of Hexane-ether-acetic acid

(60:40:1) for 30 minutes after it had been made airtight with Vaseline or grease along the

edges. 2.0% solutions of these lipids standard in chloroform were prepared, mytistic, lauric,

palmitic, oleic, stearic and lioleic acids. A drop in each of the lipids standards was applied

alongside that form coconut at a distance 2cm from the base of the plate. It took 1 hour 30

minutes for the solvent front to reach 2cm marked point above the plates. The plates was

briefly air-dried and visualized by iodine vapour as a chromogenic reageant, the Rf values

were calculated, the spots encircled and the plates photographed (fig. 4.1)

iv. One-Dimensional TLC of Triacylglycerol and Triacylglycerol Standards.

The plates and the Shandon TLC Tank were prepared as in (i) and (ii) above but with a

solvent system of hexanedithylether-acetic acid (80: 20: 1).

The triacylglycerol standards (glycerol tripalmitate, glycerol trioleate and glycerol

tristearate),all in chloroform were spotted alongside the coconut sample triglyceride also in

chloroform at a distance of 2cm from the base of the plate. It took 1hour 20 minutes for the

solvent front to reach the marked point from the top of the plate. It was removed, briefly air-


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dried and visualized in the iodine vapour. The plate photographed (fig. 4.2) the spots

encircled and the Rf values calculated.

MANUAL QUANTIFICATION OF TRIACYLGLYCEROL CONTENT.

This was carried out according to (Kessler Lederer, 1965). As well as Fletchers (1968)

method for the measurement of serum triacylglycerol. The method involves the oxidation of

glycerol to formaldehyde which is measured calorimetrically with colour reagent. The lipid

extract must therefore be free from other sources of glycerol, particularly phosphoslipid,

Activated alumina was used to absorb these interfering substances.

Reagents

1. Triacylglyceride purifier (Activated alumina)

2. Isopropanol

3. Triolein standard, contains 300mg triolein (glycerol trioleate) dissolve in 100ml

anhydrous isopropanol.

4. Potassium hydroxide 1 N

5. Periodic solution

6. Colour Reagent

METHOD

Test: 0.4g of activated alumina was placed in a test tube and 2.5ml of isopropanol added,

followed by 0-1ml of the triglyceride sample. The mixture is shaken manually for at least 5

minutes.

Standard: 0.4g of activated alumina, 2.4ml of isopropanol, 0.1ml of water and 0.1ml of

triolein standard were mixed and shaken manually for at least 5 minutes.

Blank: 0.4g of activated alumina 2.5ml of isopropanol and 0.1ml of water were mixed and

shaken manually for at least 5 minutes.

Centrifuge the tubes at about 3000 rpm for 5 minutes to obtain a clear supernatant. Carefully

transfer 0.1ml of clear supernatant into a similar test tube, taking care not to vary over

absorbent 0.25ml of Potassium hydroxide at 60oC for 5 minutes in a water bath to soporific

the triglyceride. After cooling the tubes, 0.25ml of periodic solution was added and mixed

immediately after each addition. After 20 minutes, 0.1ml of colour reagent was added and

each in a 60oc water bath for 30 minutes.


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After cooling, the absorbance of the standard and test verse the blank were read in a

spectrophometer of 410nm.

The triglyceride content in mg/ 100ml was calculated from the relation.

Coconut triglyceride in mg/ 100ml was calculated from the relation.

Coconut triglyceride mg/ 100m= x 300

PREPARATION OF MONOACYLGLYCEROL FROM TRIACYLGLYCEROL

Monoacylglycerols were prepared from the triacylglycerols by a transesterification reaction

between coconut oil and glycerol according to the optimum condition found by Arida,

(1973).

Procedure: Two grams of coconut oil was heated to approximately 180oC. 0.4g of glycerol,

preheated to 120oC was added at once followed by 0.001g of Calcium oxide (CaO). The

mixture was stirred for two hours at 200oC. The reaction mixture was cooked and allowed to

stand for twenty four hours.

Removal of Free Glycerol

One gram of the reaction mixture was melted at about 60oC and washed three times with 5ml

portion of aqueous 1% Sodium sulphate previously heated to 60oC.

Extraction of Monoacylglycerol

The glycerol free product was dissolved in hexane in the ratio of 6g hexane to 1g of

monoglyceride reaction mixture. The monoglyceride fraction was extracted from the solution

several times (6 times) with portion of aqueous ethanol (65%)

Ethyl alcohol from the combined monoglyceride extracts was removed using a water bath at

60oC instead of Rotary evaporationwhich was not available.the residue was allowed to dry in

a vacuum oven at 800c to constant weight.

EXTRACTION OF FREE FATTY ACID FROM MONOACYLGLYCEROLS.

Free fatty acids extracted by the Dole’s method (1956) as described in 3.4 before.

QUALITATIVE ANALYSIS OF MONOGLYCERIDES FOR FATTY ACIDS BY

ONE-DIMENSIONAL TLC.

The free fatty acids extracted above was spotted on the TLC plate alongside the fatty acid

standards – oleic acid, linoleic acid, stearic acid, palmitric acid, myristic acid lauric acid. The


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run lasted 2 hours in a solvent system consisting of hexane-diethyl-acetic acid (80: 1 v/v).

The plate was briefly air-dried and visualized in iodine vapour. The plate photographed (fig.

4.3), the spots encircled and the Rf- values were calculated.

3.10 Quantitative Estimation of Total Monoacylglycerol contents prepared from coconut

oil.

The total monoacylglyceride contents were estimated by the method described Bartman,

(1956).

Method

Reagents

1. Periodic acid -1.35g dissolved in 25ml of H2O, 190ml acetic acid added mixed store in

dark.

2. Sodium thiosulphate (Na2S2O2) 0.1N – 2g dissolved in 25ml of H2O.

3. Salicyclic acid- 0.25g dissolved in 200 of H2O.

4. 10 NH2SO4 – 27.8ml of conc. H2SO4 were made up to 10ml of H2O.

5. Potassium iodide -7.5g were dissolved in 50ml of water.

6. Starch indicator solution -1.0g were dissolved and made up to 100ml of water

7. Chlorform.

1.0g of the sample were dissolved in 190ml of chloroformand transferred to the separating

flask, 19ml of water was added stopper and shaken vigorously for 1 minute. The mixture is

allowed stand for 30 minutes for clear separation and the bottom chloroform sample layer is

collected. This was done for each of the sample 5ml of periodic acids (HLO4) solution were

pipetted into six beakers. 5ml of chloroform were added to the second beaker and beaker and

5ml of water to the third as blanks. 5ml of different chloroform samples used for the test were

added to the fourth, fifth and sixth beakers. Shake gently and stand the mixture for 30

minutes.

Add 2ml of Potassium iodide shake gently and stand for less than 2 minutes away from

strong sunlight. After which 10ml of H2O were added and filtrated with Sodium thiosulphate,

using 0.2ml of starch solution as indicator.

Coconut monoglyceride in mg/ 100ml

=

Where, B = titer of CHCL3 blank.


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S = titer of sample.

N = normally of Na2S2O3.

W = g sample in CHCL3 aliquot.

M = molecular weight of monoglyceride

QUALITATIVE ESTIMATION OF TOTAL NON-ESTERIFIED FATTY CONTENT

IN COCONUT OIL

Standard curve

The estimation of the total fatty acid content in coconut oil was done by meand of standard

curve for the standard free fatty acid-palmitric. 100mg of palmitric acid were dissolved in

100ml of chloroform. The fatty acid standard solutions were diluted serially to form

concentration range of 20- 100Nm as in table 3.1

Table 3.1: Serial Dilution For Free Fatty Acid Standard Curve

The fatty acid standards treated according to Nucombe’s modification of the method for the

determination of non-esterified fatty acids developed for the analysis of free fatty acid in the

body fluid or plasma. The method is based on the selective transfer of Copper salt of fatty

acid into chloroform. The free fatty acids transferred from the aqueous phase into chloroform

in which the copper complex fatty acid formed is monitored colorimetrically.

Reagents

Copper Reagent: This is prepared by mixing the following solution A, B, C (10: 1: 9 v/v).

Solution A: 6.45g of cupric nitrate (Cu(NO3)2H2O) was made up to 100ml in water.

Solution B: 20ml acetic acids was made up to 100ml in water.

Tubes Standard solution (ml) Chloroform (ml) Final volume

(ml) Final conc. (g/ml)

1. 0.50 4.50 5.00 10.00 2. 1.00 4.00 5.00 20.00 3. 1.50 3.50 5.50 30.00 4. 2.00 3.00 5.00 40.00 5. 2.50 2.50 5.00 50.00 6. 3.00 2.00 5.00 60.00 7. 3.50 1.50 5.00 70.00 8. 4.00 1.00 5.00 80.00 9. 4.50 0.50 5.00 90.00 10. 50.00 0.00 5.00 100.00


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Solution C: 14.92g of triethanolamine was made up to 100ml in water.

(2) Diethyldithiocarbamate (the colour reagent) 0.05 was made to 50ml in butanol or butan-

01.

Procedure: 5ml of chloroform were pipetted into each of the test tubes as in table 3.1, to this

were added 0.5ml of fatty acid, a 2.5ml of fatty acid standard and 2.5ml of copper reagent. In

the blank are placed 5ml of chloroform, 0.5ml of water and 2.5ml of copper reagent solution.

The tubes were stopped, shaken vigorously for 2 minutes and centrifuged for 5 minutes. After

centrifuge, the aqueous and chloroform layers are separated. 3ml of chloroform layer solution

were pippetted into test tubes and 0.5ml of diethiocarbamate reagent added. After mixing, the

coloured sample solution is measured at 440nm against the blank in a spectrophometer.

Analysis Of Fatty Acid Content In The Samples Analysis of the fatty acid content in the samples was treated according to Ducombe’s

modification of the method for the determination of non-esterified fatty acid content in

plasma.

Reagent

1.Copper Reagent Solution: This was made by missing solution A, B and C (10: 1: 9, v/v).

Solution A: 6.45g of Cu(NO3)23H2O was made up to 100 in water.

Solution B: 20ml of acetic acid in 100ml of H2O.

Solution C: 14.92g of Triethanolamine in 100ml of H2O.

Diethyldithiocabamate: 0.05g of Sodium diethyldithiocabamate in 50ml of butanol 2-01.

Procedure: 5ml of the samples were pipetted into different test tubes, followed by 5ml of

chloroform and 2.5ml of copper reagent. In a tube serves as the blank were placed 5ml of

chloroform, 0.5ml of water, and 2.5ml of copper reagent. The tubes were closed, shaken for 2

to 3 minutes and then centrifuged for 5 minutes. After centrifuging, for a few minutes, the

aqueous and chloroform layers were separated. Three (3ml) of the chloro form solution were

pipetted into different test tubes. Then 0.5ml of diethyldithiocarbamate reagent was added.

After mixing, the OD of the coloured sample solution was read off at 440nm against the

blank.

By extrapolation, the OD obtained was read off (in terms of concentration) on the fatty acid

standard curve.


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RESULTS

THIN LAYER CHROMATOGRAPHY (TLC) OF STANDARD FATTY ACIDS AND

SAMPLES (KING AND DWARF HYBRID)

(FIG. 4.1).

PLATE I

Rf values Spot 1 (Linoleic acid) = 0.61

Spot 2 (Stearic acid) = 0.53

Spot 3 (Oleic acid) = 0.60

Spot 4 (Palmitic acid) = 0.59

Spot 5 (Myristic acid) = 0.60

Spot 6 (Lauric acid) = 0.60

King hybrid

Spot 1 = 0.59

Spot 2 = 0.60

Spot 3 = 0.61

King hybrid coconuts contain palmitic acid, linoleic acid oleic and myristic acid or lauric

acid.

Dwarf hybrid

Spot 1 = 0.60

Spot 2 = 0.61

Spot 3 = 0.67

Dwarf hybrid coconuts contain linoleic acid or lauric acid.

Thin Layer Chromatography (Tlc) Of Standard Fatty Acids And Samples (King And

Dwarf Hybrid)

(FIG 4.1)

PLATE 1

FIG. 4.1

Keys:

Lin = Linoleic acid

St = Stearic acid

Ole = Oleic acid

Pal = Palmitic acid


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Myr = Myristic acid

Lau = Lauric acid

K.h = King Hybrid coconut

D.h = Dwarf hybrid coconut

PLATE II

Thin layer chromatography (tlc) of standards fatty acids and samples.

(FIG. 4.2)

Rf values Spot 1 (Linoleic acid) = 0.61

Spot 2 (Stearic acid) = 0.56

Spot 3 (Oleic acid) = 0.60

Spot 4 (Palmitic acid) = 0.58

Spot 5 (Myristic acid) = 0.60

Spot 6 (Lauric acid) = 0.62

Local Tall

Spot 1 = 0.60

Spot 2 = 0.61

Spot 3 = 0.68

Local coconuts contain oleic acid or myristic acid and linoleic acid.

PLATE III

The one dimensional tlc of triacylglycerol and triacylglycerol standard (fig. 4.3)

Table 4.0: one dimensional tlc of triacylglycerol

Ratio front values of ratio of front values of

Triaculglycerol standard samples (coconut)

Tripalmitate local tall

0.22 0.20

0.35 0.30

0.51 0.34

0.87 0.87

Trioleate king Hybrid

0.25 0.22

0.35 0.30


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0.51 0.35

0.87 0.85

Tristearate Dwarf Hybrid

0.22 0.22

0.34 0.30

0.51 0.35

0.87 0.85

The table above shows that the different breeds of coconut contain the standard

Triacylglycerol (tripalmitate, trioleate and tristearate) and some other impurities.

The one dimensional of triacylglycerol, triacylglycerol, standards and samples (local

tall, king hybrid and dwarf hybrid coconut).

PLATE 3

FIG. 4.3

Keys:

L.T = Local Tall coconut

Tp = Tripalmitate

To = Triolein

Ts = Tristearate

K.h = King Hybrid coconut

D.h = Dwarf Hybrid coconut

4.3 ONE DIMENSIONAL TLC MONOACYLGLYCEROL FOR FREE FATTY ACID.

First spot (Local Tall) = 0.80

Second spot (King hybrid) = 0.80

Third spot (Dwarf hybrid) = 0.80

Fourth spot (Linoleic acid) = 0.79

Fifth spot (Oleic acid) = 0.80

Sixth spot (Palmitic acid) = 0.82

Seventh spot (Stearic acid) = 0.81

Eighth spot (Myristic acid) = 0.80

Ninth spot (Lauric acid) = 0.80


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Quantification Of Triacylglycerol Content

Table 4.1: Triacylglycerol Content In Local Tall And Hybrid Coconuts

Breeds of Coconuts Triacylglycerol Content (mg/ 100ml)

Local Tall 733.30

Dwarf Hybrid 333.30

King Hybrid 600.00

Table 4.1 shows the triacylglycerol content of different breeds of coconut (Local Tall, Dwarf

and King Hybrid) in mg/ 100ml.

Quantification Estimation Of Total Monoacylglycerol Content

Table 4.2: Total Monoacylglycerol Content

No. of beaker Monoacylglyceride (mg/ 100ml)

King Hybrid 0.0115

Dwarf Hybrid 0.0125

Local Tall 0.0120

Table 4.2 shows the total monoacylglycerol content in different breeds of coconuts. The

monoacylglycerol was noticed to be very low in the coconut species, with the dwarf hybrid

having the highest.

TABLE 4.3: The Final Concentration And The Absorbance Of The Standard Free

Fatty Acids.

Tubes Final Conc. Nm/L Absorbance

10.00 0.18

20.00 0.30

30.00 0.48

40.00 0.60

50.00 0.80

60.00 0.84

70.00 0.96

80.00 1.10

90.00 1.18

100.00 1.29


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The above table shows the final concentration of the standard free fatty acids and the

absorbance.

Table 4.4: Absorbance For Analysis Of Fatty Acid Content In The Sample.

Tubes Samples Absorbance Conc. (mg/ 100ml)

1. Local Tall 0.60 43.5

2. Dwarf hybrid 0.58 42.5

3. King hybrid 0.59 43.0

Table 4.4 shows the absorbance for the analysis of fatty acid content in the coconut sample

with the concentration read from the fatty acid standard curve.

DISCUSSION

This project was designed to compare the fatty acid and acylglycerol contents of local and

hybrid coconuts. Thin layer chromatography (TLC) and other quantitative methods were

combined to assess the presence and quantities of fatty acids and acylglycerols in both

species of coconuts.

Within the limits of available fatty acid standards, TLC analysis revealed the presence of

myristic, lauric, oleic and linoleic acids in the oils of both local and hybrid coconuts. The

presence of pallmitic and stearic acids was inferred from the TLC analysis of the

acylglycerols which in addition indicated glycerol trioleate, revealed glycerol tristearate and

glycerol tripalmitate respectively. This inference is considered reasonable because the TLC of

the fatty acids from both local and hybrid coconuts revealed spots whose ratio front (RF)

values did not match those of the fatty acid standards used. These were believed to be other

fatty acids present in the oils. The solvent fronts also spots common to both the standards and

the samples. They were believed to be impurities soluble in the solvent used for the TLC.

These “extra” spots were also present in the chromatograms developed after the TLC analysis

of the acylglycerols. They are likely to be other acylglycerols which could not be identified

because there were no corresponding acylglycerol standards. Owing to the non-availability of

monocylglycerol standards, a TLC of monoacylglycerols was not done. Instead, they were

hydrolysed and analysed by TLC for the presence of fatty acids. Myristic, Lauric, Oleic and

Linoleic acids were identified, suggesting the presence in the oils of glycerol monomyristate,

monooleate nad monolinoleate in both the local anad hybrid coconut oils.


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pQuantitatively, the local coconut was found to have much higher triacylglycerol content-

733.30mg/ 100ml in comparison with 466.70mg/ 100ml in the hybrid coconut. This is

surprising since the hybrid species was thought to be endowed with higher biosynthetic

capacity. However, this can be rationalized on the basis of the generally greater access to

more sunlight for enhanced photosynthesis. Since the hybrid species are generally shorter,

they may have been shielded from sunlight by taller plants hence lower photosynthetic

activity resulting in lower acylglycerol content. It is also possible that the local breed is

genetically endowed to synthesize more tracylglycerol in their endosperms than the hybrid

ones. Generally, monoacylglycerol levels were found to be low in both local and hybrid

coconuts constituting 0.011mg/ 100ml and 0.012mg/ 100ml of the oils from both species.

This finding is consistent with what is known of plant oils, i.e, they are mainly triacylglycerol

with only trace amountsof fatty acids, mono-and diacylglycerols.

Although the triacylglycerol contents of the local and hybrid coconuts differed markedly

(733.30mg/ 100ml and 466.67mg/ 100ml respectively), their total free fatty acids were almost

equal 43.50mg/L and 43.00mg/L respectively. This would suggest that the fatty acids in the

local coconuts might be in a form that it is easily hydrolysable.

CONCLUSION

The local and hybrid coconuts contain the same types of fatty acids but not in equal amounts.

Since the local species contain more triacylglycerol per mg endosperm, it is more desirable to

use it as a source of coconut oils. The only disadvantage may be difficulty of harvesting its

fruits since it is usually taller and requires climbing the tree with its associated dangers. If

they could be modified (genetically) to bear fruits without growing very tall, they will be

economically and nutritionally better than the hybrid ones we have today.

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