EXTRACTION, CHARACTERIZATION AND INDUSTRIAL USES … WORK MICHO... · extraction, characterization and industrial uses of lecithin from three varieties of cucumis melo (melon seed)

EXTRACTION, CHARACTERIZATION AND INDUSTRIAL USES

OF LECITHIN FROM THREE VARIETIES OF

Cucumis melo (MELON SEED) OIL.

BY

NWANKWO MICHAEL OLISA

PG.M.Sc/07/43494

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA, NSUKKA

NOVEMBER, 2009

TITLE PAGE

EXTRACTION, CHARACTERIZATION AND INDUSTRIAL USES OF

LECITHIN FROM THREE VARIETIES OF Cucumis melo (MELON SEED)OIL.

i

ii

CERTIFICATION

Nwankwo Michael. O., post graduate student of the Department of Biochemistry with

Registration number PG/M.Sc/07/43494 has satisfactorily completed the requirements for

the degree of Masters of Science (M.Sc) in Biochemistry. The work embodied in this

dissertation is original and has not been submitted in part or full for any other diploma or

degree of this or any other University.

……………………………… …………………………..

Prof. O. Njoku. Prof. I.N.E Onwurah.

(Supervisor) (Head of Department)

……………………………

External Examiner

iii

DEDICATION

This dissertation is dedicated to God the father, the Son, and the Holy Spirit, and to all

lovers of knowledge.

iv

ABSTRACT

Melon seed, Cucumis melo oil and lecithin were evaluated for their physicochemical and

possible biopharmaceutical uses as an adjunct in self-emulsifying drug delivery systems

(SEDDSs) for future use as safe drug vehicle for poorly aqueous soluble drugs. Melon

seed oil was extracted using standard procedure, while the Lecithin was extracted from

the seed oil. The oil was subjected to some physicochemical characterization and acute

toxicity test. The lecithin also extracted was subjected to physicochemical test as well as

solubility and antioxidant evaluations. From the physicochemical studies, the result of the

physical properties showed that the colour of the oil is yellow, the mean refractive index

of the three oils is 0.0091±0.1specific gravity, 0.9323±0.2 and viscost of 338.89±0.1.The

chemical studies showed an acid value of 0.9327± 0.1 mg KOH/g, saponification value

166.13±0.2 mg KOH/g iodine value, 121.8±0.1Wijs, proxide value, 10.67± 0.1 and Ester

value of 165.13±0.2 mg KOH/g. The lecithin extracted has mean percentage yield of

0.58±0.1%, and has solubility in acetone, chloroform, petroleum ether but slightly soluble

in methanol and water. The acute toxicity test showed that the oil is not toxic, and has no

significant behavioural modification of the animals it was administered up to a dosage of

5000mg/kg body weight. The result in this present study shows that the oil and lecithin

extracted from Cucumis melo have a lot of nutritional and biopharmaceutical

applications. The developed vitamin E SEDDSs formulations containing melon seed oil

showed promise as a possible clinical arsenal for the delivery of poorly water-soluble

drugs. The result obtained demonstrated notable usefulness of both the oil and lecithin in

health, industry and agriculture. The developed vitamin E SEDDSs formulations

containing melon seed oil were found to facilitate maximal delivery, absorption and

bioavailability of lipophilic drugs.

.

v

ACKNOWLEDGEMENT

May all honour, thanksgiving and praises be ascribed to God the father, the Son

and Holy Spirit? To God be the glory. A tree does not make a forest, and so I will everly

submit my loyalty to my Supervisor, Professor Obi Njoku for his zeal and fatherly

training devoid of reservation. I appreciate his youthful pattern of working during his

tenures as the Head of Department, as well as the Dean, Faculty of Biological Sciences.

His invaluable advice helped me immensely, and most especially, his usual constant

prodding made this work a success today.

My inestimable thankfulness must be reserved for my lecturers whose combined

hard work, efforts and ingenuity overwhelmed me during my course work lecture days.

The above are Eze Professor I.C Ononogbu, Professors O. Obidoa, I.N.E Onurah, P.N,

Uzoegwu, L.U.S Ezeanyika, F.C. Chilaka and Doctors. O.F.C Nwodo, E, Alumanah,

V.N, Ogugua, B.C, Nwanguma. Others are Messrs. O.C Enechi, P.A.C Egbuna, V.O.E

Ozougwu , O Ikwuagwu, S.C Ubani, Mrs. C. Anosike, and Mrs. U Njoku.

My Family members and friends whose hardwork and encouragement I cherished

most and whose support spiritual and material spurred me in an inestimable way were

Mr. and Mrs. B.F Nwankwo, Engr. T.N Nwankwo, Professor B.C. Obah Engr. J.A.

Okafor, Dr. Eddy Emegoakor, Messrs. Alloy, Augustine, and Joe-Vin Ngolumuo

Ifediorah, Barrister. D. Ogbueli, Mr Mike Igbozendu, Mr Cyril. E. Nwazuba Miss

Josephine Okaa Omee and late Bro. Cyril Oguejiofor.

I wish to express. My indebtedness to my B.Sc days lecturers, whose input

during my M.Sc preparations marked indelibly in my memory, and who naturally sowed

the academic seed in me. They are Professors (Mrs.) ANC Okaka, F.C. Ezeonu, J.K.

Emeh, E. Ilouno. Others are Doctors (Mrs.) Nebedum, J. Okonkwo, G. Igbokwe, S.

Udedi and a colleague Cally Anagonye.

The people that must be appreciated for their whole support, assistance,

suggestion, and hard work for making this research a success include Messrs E.U.

Nwachi, P.E. Joshua, Dr. A.A, Attama, and Dr. N. Obitte. Mr. Vincent. N. Chigor is the

initiator of the programme, God‟s favour will never leave him.

vi

Other friends and colleagues include Miss Chinelo Edokwe, Mr. Chinedu

Okonkwo, Miss Nneka Onwudiwe, Mr. Raph Ekeanyanwu, Miss Adaorah Umeji., Miss

Ruth Okoro, Mrs Chika Ezugwu,. Mr. Emmanuel Uhuo ,Miss Claribel Igboabuchi and

Miss Adaeze Akuwudike.

Finally, my inexplicable, sincere and invaluable appreciation will undoubtedly go

to Miss U.C.E Chidume of Faculty of Education for her heart-warming company

especially when the project work went lonely. May the Almighty God bless, direct and

protect our collective intentions

Nwankwo Michael .O.

M.Sc. Biochemistry

2009.

vii

TABLE OF CONTENT

Title page - - - - - - - - - i

Certification - - - - - - - - - ii

Dedication - - - - - - - - - iii

Abstract - - - - - - - - - iv

Acknowledgement - - - - - - - - v

Table of Content - - - - - - - - vii

List of Tables - - - - - - - -

List of Abbreviations - - - - - - - -

CHAPTER ONE - - - - - - - - 1

1.0 Introduction and Literature Review - - - - - 1

1.1 Lipids, Classification and Functions - - - - - 2

1.1.1 Uses of lipids - - - - - - - 5

1.1.1.1 Oil Extraction - - - - - - - - 9

1.1.1.2 Refining of Crude vegetable oil - - - - - 9

1.2 Bleaching - - - - - - - - 9

1.2.1 Hydrogenation- - - - - - - - 10

1.2.1.1 Fractionation - - - - - - - - 10

1.2.1.2 Interesterification - - - - - - - 11

1.2.1.3 Physical refining - - - - - - - 12

1.2.1.4 Physical and Chemical Properties oil - - - - - 14

1.4 Chemical properties of Melon seed oil - - - - 16

1.4.1 History of Lecithin - - - - - - - 22

1.4..1.2 Biochemical value of lecithin - - - - - 22

1.4.1.3 Dietary and supplemental lecithin - - - - - 23

1.5 Lecithin in Health - - - - - - - 24

1.5.1 Lecithin and Neurological Diseases - - - - - 24

1.5.1.1 Lecithin and respiratory function - - - - - 24

1.5.1.2 The Liver and Lecithin - - - - - - 25

1.5.2 Lecithin and Signal Transduction - - - - - 26

viii

1.5.2.1 Lecithin and biomembrane - - - - - - 27

1.5.2.2 Lecithin and the blood-brain barrier - - - - - 27

1.5.2.4 Lecithin and Fat Metabolism - - - - - - 28

1.5.2.5 Lecithin in Reproduction and Fertility - - - - 29

1.5.2.6 Lecithin Role in Short-term memory - - - - - 29

1.5.2.7 Lecithin and Breastfeeding mother and Child - - - 31

1.5.3. Lecithin and Industrial Roles - - - - - - 31

1.5.3.1 Lecithin in manufacturing processes - - - - - 32

1.6 Lecithin and Pharmaceuticals - - - - - - 33

1.6.1 Lecithin and cosmetics industry - - - - - - 33

1.6.2 Self-Emulsifying Drug Delivery System (SEDDSs) - - - 34

1.6.3 Lecithin and the nutrition industry - - - - - 35

1.6.4 Lecithin as Antioxidant - - - - - - 36

1.6.5 Lecithin and the Beverage Industry - - - - - 37

1.6.6 Lecithin as an Emulsifier and Separating Agent - - - 38

1.7 Lecithin in agriculture - - - - - - - 40

1.8 Melon Seed Oil - - - - - - - 41

1.8.1 Characteristic / Morphology of melon plant (Cucumis melo) - 42

1.8.2 Habitat / Ecology of melon (Cucumis melo) plant - - - 42

1.8.3 Distribution and Local names of Melon - - - - 42

1.8.4 General profile of Cucumis melo - - - - - 43

1.9 Rationale of the Study - - - - - - - 44

1.10 Research Objective - - - - - - - 44

1.11 Aims of the Study - - - - - - - 44

CHAPTER TWO

2.0 Materials and Methods - - - - - - 45

2.1 Material - - - - - - - - 45

2.1.1 Chemicals / Reagents - - - - - - - 45

2.1.2 Equipment / instrument - - - - - - 45

2.1.3 Plant Material - - - - - - - - 45

2.1.4 Preparation of reagents for characterization - - - - 45

ix

2.1.5 Extraction of Melon Seed Oil - - - - - - 46

2.1.6 Determination of Oil Percentage Yield - - - - 46

2.1.7 Characterization of the Oil - - - - - - 47

2.1.8 Physiochemical properties of the melon seed oil - - - 47

2.1.9 Acute toxicity / lethality (LD50) Test - - - - - 50

2.1.10 Extraction of Lecithin from Melon Seed Oil - - - - 50

2.2 Determination of Lecithin Percentage Yield - - - - 50

2.2.1 Physicohemical Properties of Lecithin - - - - 50

2.2.1.1 Lecithin Solubility Test - - - - - - 51

2.2.1.2 Simple Test for Lecithin - - - - - - 51

2.2.1.3 Thin layer chromatography (TLC) - - - - - 51

2.2.1.4 Antioxidant Property of Lecithin-Oil Stability Test - - - 52

2.2.1.5 Preparation of Stable Vit. E SEEDSs - - - - - 52

2.2.1.6 Stable vit E SEEDSs composition - - - - - 54

2.2.1.7 Characterization of SEEDSs - - - - - - 55

CHAPTER THREE

3.1 Masses of the seeds and percentage Yield of the oil - - - 57

3.2 The hull percentage yield of the melon seeds - - - - 58

3.3 Physical properties of the three varieties of the Cucumis melo (melon) 59

3.4 Chemical properties of the three varieties of Cucumis melo (melon) 60

3.5 Percentage Yield of Lecithin - - - - - - 61

3.6 Phosphate test of the three varieties of Cucumis melo (melon) - 62

3.7 Solubility of Lecithin in water and organic solvents - - - 63

3.8 Result of the acute toxicity test - - - - - 64

3.9 Isotropicity/ Stability test - - - - - - 65

3.10 Physicochemical properties of vitamin E SEDDSs formulations - 66

x

CHAPTER FOUR

4.0 Discussion - - - - - - - - 67

4.1 Conclusion - - - - - - - - 70

4.2 Suggestion for further research - - - - - 70

References - - - - - - - - - 71

Appendices - - - - - - - - - 80-88

xi

LIST OF TABLES

Table 1.1 : Some oils used in Industry and Automobiles - - - 6

Table 1.2: Some uses of Oils in Health Care delivery - - - 7

Table 1.3: Some uses of Oils in Agriculture - - - - 8

Table 1.4: Classification of melon plant - - - - - 43

Table 2.1: Stable Vit. E SEDDSs composition - - - - 54

Table 3.1 Masses of the Seeds and Percentage Yield of the Oil - - 57

Table 3.2: The hull percentage yield of the melon seeds - - - 58

Table 3.3: Physical properties of the three varieties of the Cucumis melo (melon) 59

Table 3.4: Chemical properties of the three regional varieties of

Cucumis melo (melon) - - - - - - 60

Table 3.5: Percentage yield of Lecithin - - - - - 61

Table 3.6: Phosphate Test of the three Regional Varieties of

Cucumis melo (Melon) - - - - - - 62

Table 3.7: Solubility of Lecithin in water and organic solvents - - 63

Table 3.8: Result of the Acute Toxicity Test - - - - 64

Table 3.9: Isotropicity/Stability Test - - - - - 65

Table 3.10 Physicochemical properties of the stable Vit E SEDDSs formulations 66

xii

LIST OF FIGURES

Figure 1.1: Flow chart of physical refining of oil - - - - 12

Figure 1.2: Flow chart of chemical refining - - - - - 13

Figure 1.3: The Structures of phosphatidyl Serine and Inositol - - - 19

Figure 1.4: Structures of phosphatidylcholine and Ethanolamine - - - 17

Figure 1.5: The Structure of Lecithin - - - - - - 18

Figure A.1: Melon fruit in the farm intercropped with cassava - - 80

Figure A.2: Melon seed during drying in the sun - - - - 82

Figure A.3: The three regional varieties of lecithin extract - - 83

Figure A.4: Flow chart of lecithin extraction from melon seed oil - - 84

Figure A.5: Graph of Standard lecithin and refined soybean oil - - 85

Figure A.6: Graph of lecithin extract and melon seed oil - - - 86

Figure A.7: Potomicrograph of oil/surfactant ratios - - - - 87

Figure A.8: TLC chromatogram of the three lecithin extracts - - 88

xiii

LIST OF ABBREVIATIONS

AD Alzheimer‟s disease

BHA Butylated Hydroxylanisole

BHT Butylated Hydroxyl toluene

Ca2+

Calcium Ion

COA Coenzyme A

CVD Cardiovascular Disease

DNA Deoxyribonucleic acid

EFAS Essential Fatty Acids

FDA Food and Drug Administration

GIT Gastrointestinal Tract.

GTP Guanosine Triphosphate

HDL High Density Lipoproteins

LCAT Lecithin Cholesterol Acyl Transferase

LDL Low Density Lipoproteins

LS Lipid Soluble

MG Myasthenia Gravis

ND Neurological Disorders

PAF Platelet Activating Factor

PC Phosphatidylcholine

PE Phosphatidyl ethanolamine

PKC Protein Kinase

PLO Pluronic Lecithin Organolgel

PM Phospholipid Membrane

PO4 Phosphate

PUFAS Pohyunsaturated Fatty Acids

RBC Red Blood Corpuscles (Erythrocytes)

RNA Ribonucleic acid

ROS Reactive Oxygen Species

SD Senile Dementia

xiv

SOD Superoxide Dismutase

TD Tardive Dyskinesia

VDL Very Low Density Lipoproteins

Vit Vitamins

WS Water Soluble

1

CHAPTER ONE

1.0 Introduction and Literature Review

Lecithin is an important by-product of vegetable oil processing industries that have

important functions in health, agriculture and in the industries (Dreon et al, 1990).

Lecithin is a mixture of glycerol-phospholipids obtained from animal, vegetable and

microbial sources, containing varying amounts of substances such as triacylglycerols,

fatty acids, glycolipids, sterols and sphingolipids (Meek, 1997). The major source of

commercial lecithin is soybean oil, and is called 1,2-diacylglycero-3-phosphorylcholine

(Dashiell, 2003).

The production of lecithin from oil seed is by hydration of the phosphatides using

water or steam (Shanhani, (1980). Lecithin has diverse roles in human metabolism

(Orthoefer, 1998), especially in the control of nerve activities and breathing (Gordon,

2000), production and quality could be affected by crude oil storage, soil type, nutrient

availability, climatic changes, drying process, and handling manner (Renfree, 2005).

Lecithin also has multifunctional uses in agriculture, food confectioneries,

pharmaceuticals, paints, plastics, and in the textile industries (Lucas, 1996).

Lecithin is an emulsifying, wetting, and dispersing agent. It has antioxidant,

surfactant and lipotropic functions, as well as anti-corrosive and anti-spattering roles

(Eyster, 2007. In the pharmaceutical industries, lecithin is also important in lowering

blood cholesterol levels facilitating optimum absorption of fat-soluble vitamins,

maintaining cell membrane integrity, as well as increasing serum choline levels and it

also gives relief and cure in the severity of neurological diseases (Kidd, 1997). The

important uses of lecithin in health, industries, and agriculture is increasing; therefore,

there is need to explore other sources of lecithin in order to reduce over-dependence on

soybean source (Spiller, 2006). Melon seeds are produced in the eastern, middle belt, and

northern states of Nigeria, and Nigeria is one of the largest producers in the world

(Ofune, 1988). Melon seeds are used for edible purposes in food, cake, seasoning agent,

unlike in the western world where its oil is used for soap, cream production, as well as in

other pharmaceuticals (Van der Vossen et al, 1992). The deterioration of melon seed and

fungal infestation during storage has made farmers to abandon melon production in many

2

parts of Nigeria. Curits, 1964 recognized the possible economic value of the seed oil,

their crude protein and by-products of cucurbitaceous plants, the physicochemical

characteristics of their oils and by-products attracted the attention of Wentz et al (1983).

Bolley et al (1983) characterized their oils as soft drying oils with similarities to soybean

oil. Shanhani et al (1980) indicated the possibility of processing the crude oil obtained

from such seeds for yielding edible oil and other products. Vasconcellos et al (1982)

reported that the oil contents ranged between 35-41 percent. It is believed that if the oil is

extracted, and lecithin is produced from it, this will give added value to the melon seeds

produced in Nigeria, hence the objective of this research.

1.1 Lipids, Classification and Uses

Lipids are broadly defined as any fat-soluble (Lipophilic), naturally-occurring

molecule, such as fats, oils, waxes, cholesterol, sterols, fat-soluble vitamins (such as

vitamins A,D, E and K), monoglycerol diglycerides, phospholipids and others. The main

biological functions of lipids include energy storage, acting as structural components of

cell membranes, and participating as important signaling molecules (Berg et al 2006).

Although the term lipid is sometimes used as a synonym for fats, fats are a

subgroup of lipids called triglycerol and should not be confused with the term fatty acid.

Lipids also encompass molecules such as fatty acids and their derivatives (Including tri-,

di-, and monoacylglyerol and phospholipids), as well as other sterol-containing

metabolites such as cholesterol, (Spiller, 2006).

Lipids are classified in to three groups which are simple, compound and complex

lipids. These three groups are further divided in to eight sub-groups which are:-

Fatty acyls (including fatty acids) are a diverse group of molecules synthesized by

chain-elongation of an acetyl-CoA primer with malonyl-CoA or methylmalonyl-CoA

groups. The fatty acyl structure represents the major lipid building block of complex

lipids and therefore is one of the most fundamental categories of biological lipids. The

carbon chain may be saturated or unsaturated, and may be attached to functional groups

containing oxygen, halogens, nitrogen and sulphur. Examples of biologically- interesting

fatty acyls are the eicosanoids which are in turn derived from arachidonic acid which

include prostaglandins, leukotrienes, and thromboxanes. Other major lipid classes in the

3

fatty acyl category are the fatty esters and fatty amides. Fatty esters include important

biochemical intermediates such as wax, esters, fatty , coenzyme A derivatives, fatty acyl

thioester, ACP derivatives and fatty acyl carnitines. The fatty amides include N-acyl

ethanolamines such as anandamide. (Berg, 2006).

Glycerolipids are composed mainly of mono-, di-and tri-substituted glycerols, the most

well known being the fatty acid esters of glycerol (triacylglycerols), also known as

triacylglycerol. These comprise the bulk of storage fat in animal tissues. Additional

subclasses are represented by glycosylglycerols, which are characterized by the presence

of one or more sugar residues attached to glycerol via a glycosidic linkage. Examples of

structures in this category are the digalactosyldiacylglycerols found in plant membranes

and seminolipid from mammalian spermatozoa (Holzl: and Doramann 2007).

Glycerophospholipids, also referred to as phospholipids, are ubiquitous in nature and are

key components of the lipid bilayer of cells, as well as being involved in metabolism and

signaling. Glycerophospholipids may be subdivided into distinct classes, based on the

nature of the polar head group at the sn-3 position of the glycerol backbone in eukaryotes

and eubacteria or the sn-1 position in the case of archaebacteria. Example of

glycerophospholipids found in biological membranes are phosphatidylcholine (also

known as PC or GPCho,and lecithin), phosphatidylethanolamine PE or gPEtn) and

phosphatidylserine GPSer). In addition to serving as a primary component of cellular

membranes and binding sites for intra-and inter-cellular proteins, some

glycerophospholipids in eukaryotic cells, such as phosphatidylinositol and phosphatidic

acids are either precursors of, or are themselves, membrane-derived second messengers.

Typically, one or both of these hydroxyl group are acylated with long-chain fatty acids,

but there are also alkyl-linked and alkenyl-linked (plasmalogen) glycerolphospholipids,

as well as diakylether variants in prokaryotes. (Spiller 2006).

Sphingolipids are a complex family of compounds that share a common structural

feature, a sphingoid base backbone that is synthesized de novo from serine and a long-

chain fatty acyl CoA, then converted into ceramides, phosphosphingolipids

glycosphingolipids and other species. The major sphingoid base of mammals is

commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) as a major

subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are

4

typically saturated or mono-unsaturated with chain lengths from 14 to 26 carbon atoms.

The major phosphosphingolipids of mammals are sphingomyelins (ceramide

phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and

fungi have phytoceramidephosphoinositols and mannose containing head groups. The

Glycosphingolipids are a diverse family of molecules composed of one or more sugar

residues linked via a glycosidic bond to the sphingoid base. Examples of these are the

simple and complex glycosphingolidpids such as cerebrosides (Bach and Watchtel 2003).

Sterol lipids, such as cholesterol and its derivatives are an important component of

membrane lipids, along with the glycerophospholipids and sphingomyelins. The steroids,

which also contain the same fused four-ring core structure, have different biological roles

as hormones and singaling molecules. The C18 steroids include the eostrogen family

whereas the C19 steroids comprise the androgens such as testosterone and androsterone.

The C21 subclass includes the progestogens as well as the glucocorticoids and

mineralocorticoids. The secosteroids, comprising various forms of Vitamin D, are

characterized by cleavage of the B ring of the core structure. Other examples of sterols

are the bile acids and their conjugates, which in mammals are oxidized derivatives of

cholesterol and are synthesized in the liver (Wang. 2004).

Prenol lipids are synthesized from the 5-carbon precursors isopentenyl diphosphate and

dimethylallyl diphosphate that are produced mainly via the mevalonic acid pathway. The

simple isoprenoids (linear alcohols, diphosphates, etc) are formed by the successive

addition of C5 units, and are classified according to number of these terpene units.

Structures containing greater than 40 carbons are known as polyterpenes. Carotenoids are

important simple isoprenoids that function as antioxidant and as precursors of vitamin A.

Another biologically important class of molecules is exemplified by the quinones and

hydroquinones, which contain an isoprenoid tail attached to a quinonoid core of non-

isoprenoid origin. Vitamin E and Vitamin K, as well as the ubiquinones, are examples of

this class. Bacteria synthesize polyprenols (called bactoprenols) in which the terminal

isoprenoid unit attached to oxygen remains unsaturated, whereas in animal polyprenols

(dolichols) the terminal isoprenoid is reduced. (Kuzuyama and Seto. 2003).

Saccharolipids describe compounds in which fatty acids are linked directly to a sugar

backbone, forming structures that are compatible with membrane bilayers. in the

5

saccharolipids, a sugar substitutes for the glycerol backbone that is present in

glycerolipids and glycerophosphospholipids. The most familiar saccharolipids are the

acylated glucosamine precursors of the lipid A component of the lipopolysaccharides in

gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine,

which are derivatized with as many as seven fatty-acyl chains. The minimal

lipopolysaccharide required for growth in E. coli is KdO2-Lipid A, a hexa-acylated

disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic

acid (KdO2) residues (Heinz, 1996).

Polyketides are synthesized by polymerization of acetyl and propionyl subunits by

classic enzymes as well as iterative and multimodular enzymes that share mechanistic

features with the fatty acid synthases. They comprise a very large number of secondary

metabolites and natural products from animal, plant, bacterial, fungal and marine sources,

and have great structural diversity, many polyketides are cyclic molecules whose

backbones are often further modified by glycosylation, methylation, hydroxylation,

oxidation, and/or other processes. Many commonly used anti-microbial, anti-parasitic,

and anti-cancer agents are polyketides or polyketide derivatives, such as erythromycins,

tetracyclines, ivermectins, and anti-tumor epothilones (Walsh, 2004).

1.1.1 Uses of lipids

Lipids of fats and oils, steroids, waxes and related compounds have their

functions divided into to three areas which include; health, industry and agriculture.

However, other lipids which are present in their sources in trace quantities function as

enzymes, cofactors; electron carriers, light-absorbing pigments, hydrophobic anchors;

emulsifying agents, hormones and intracellular messengers (Nelson and Cox 2001).

6

Table I.I. Some oils used in Industry and Automobiles

Paints and varnishes Vernonia oil, safflower oil walnut oil, Tung

oil, stillingia oil (Chinese vegetable tallow

oil)

Chemicals Castor oil, cuphea oil, snow ball seed oil,

bladder pod oil crambe oil, Vernonia oil

Candle and lighting Neem oil, orange oil, Tonka bean oil,

Amur cork tree fruit oil.

Insectcides Balanos oil

Biofuel and Biodiesel Melon seed oil, Jojoba oil plam kernel oil,

Palm oil and Jatropha oil.

Lubricants Castor oil, olive oil, Ramtil oil, Dammar

oil, Jojoba oil, Tall oil.

(Source: Nelson, 1981)

7

Table 1.2 Some Uses of Oils in Health Care Delivery

Medicinal and Antisepitcs Lemon oil, wheat germ oil cashew oil,

Almond oil, Borneo tallow nut oil, shea

butter, Snowball seed oil, Corriander seed

oil, Perilla seed oil, Amur Cork tree fruit

oil, chaulmoogra oil, Brucia havanica oil

burdock oil

Pharmaceuticals Soybean oil, melon seed oil, cashew nut

oil, cocoa butter; Almond oil.

Cosmetic and Skin Care Hazelnut oil, coconut oil, cotton seed oil

Acai oil, Amaranth oil Borneo tallow oil,

Avocado oil Cohune oil, Rape seed oil,

Perilta seed oil, Olive oil, Carrot seed oil,

lemon oil, Neem oil, Poppy seed oil,

Candle nut oil carrot seed oil shea better.

Soap and Cleaning products Palm kernel oil, Palm oil, Borneo tallow

nut oil, kapok seed oil. Linseed oil, Poppy

seed oil, Daminar oil.

Perfumes and Fragrances Palm oil, Castor oil, Copaiba oil, Honge

oil, Jojoba oil sunflower oil,


8

Table 1.3 Some Uses of oils in Agriculture

Animal feed Soybean oil, melon seed oil, Aglae oil,

Evening prime rose oil

Pesticides Balanos oil

Fertilizers Soybean oil, melon seed oil, palm oil,

safflower oil


9

1.1.1.1 Oil Extraction

The conventional methods for oil extraction involves three basic approaches

namely-Physical, chemical and a combination of both (Owusu-Ansah, 1994). The

physical method employed for oil seeds of high oil content example, groundnut, palm

fruit and kernel etc, while chemical method is primarily used for oil seeds of low oil

content, example soybeans, rice bran, etc (Owusu-Ansah, 1994).

The method used could affect the physical and chemical properties of the oil or fat

to a considerable extent. In selecting solvent for extraction, the solubility of the oil or fat

in the solvent, toxicity and the intended use of the oil are of utmost importance.

Petroleum ether, n-hexane, methanol and chloroform are frequently used (Christie, 1982).

Enzymes have found use in oil extraction. The application of enzymes in oil extraction

can be categorized into: enzyme-assisted processing, enzyme-enhanced solvent

extraction, and enzyme-assisted aqueous extraction (Owusu-Ansah, 1994). In all these

approaches, the enzymes are used to break the cell walls of the oil bearing material to

release the oil.

1.1.1.2 Refining of Crude vegetable Oil

The further processing of edible oils after extraction from the raw materials is

concerned with refining and modification (Young et al., 1994). Refining treatment is

needed to remove or reduce as far as possible, those contaminants of the crude vegetable

oil which will adversely affect the quality of the end-product and the efficient operation

of the modification process. Two methods are in use for the refining of oils and fats.

These are termed physical and chemical from the means by which free fatty acids are

removed from the oil (Young et al., 1994). The fatty acids are distilled off in the physical

process and in the chemical process are neutralized using an alkaline reagent thus

forming soap, which are removed from the oil by phase separation.

1.2 Bleaching

Pigments such as carotenoids, chlorophyll, Gossypol, and related compounds and

the products of degradation and condensation reactions that occur during the handling,

10

storage and treatment of the extracted oils is removed by bleaching. It was later realized

that activated absorbents, in particular are responsible for removing at least partially,

other impurities such as soaps, trace metals, phosphatides and hydroperoxide compounds

containing sulphur. Primary oxidation levels are also reduced by the breakdown of the

oxidation product on the absorbent surface followed by absorption of the carbonyl

compounds that are the secondary oxidation products. The process is usually carried out

by treating the oil with absorbents such as special clays, and charcoals at high

temperature (100 -1100C) and under reduced pressure (Haraldsson, 1983). The operation

however provides lighter coloured oil and prepares it for subsequent processing.

1.2.1 Hydrogenation

The majority of fatty acids which are contained in naturally occurring oils or fats

are unsaturated. Hydrogenation process is used to provide the direct addition of hydrogen

into the unsaturated double bonds of the fatty acids chains within neutral oils. When

hydrogen is added to fatty acid‟s double bond, it becomes saturated with consequent

increases in the oxidative stability and melting point of the oil of which it is a part

(Young et al., 1994). The process involves reacting gaseous hydrogen, liquid oil, and

nickel or copper catalyst by mechanical agitation at a specific temperature (150-1800C)

and pressure (3-5atm) in a closed reaction vessel. The reaction is directed by changes in

conditions affecting mass transfer of hydrogen to catalyst surface and of oil to and from

the surface (Young et al., 1994). The reaction end point is controlled by determining the

refractive index which relates relatively close to the iodine value of zero which is not

desirable because the oil will be brittle and unpalatable (Owusu-Ansal, 1994).

1.2.1.1 Fractionation

Fats and oils are mixtures of triacylglycerols having different fatty acid

compositions. They have melting point spanning from 50-800C, each of the oil having its

own melting range. The melting range limits the use of a particular oil or fat (Young et

al., 1994). Fractionation is a thermo-mechanical process by which the raw materials are

separated into two or more portions which widens the use of the oil. Thermo-mechanical

separation processes include distillation and crystallization. Distillation is commercially

11

unsuitable for the fractionation of triacylglycerol mixtures because of their low vapour

pressures and because of their relative instability at high temperatures. Separation can

however be affected by crystallization. Crystallization of the oil can be done using three

methods namely-the dry process, the Lanza or Lipofrac process and the solvent process

(Young et al., 1994). The difference in efficiency between the three techniques lies in the

degree of contamination of stearin by the Olein after separation in view of the high

viscosity of the crystalline slurry, separation of the Olein from the Stearin is difficult

(Owusu-Ansah, 1982). In solvent process, the oil is crystallized using mainly acetone or

n-hexane as the solvent. In this way, the viscosity is reduced, thereby improving

crystallization and filterability. Fractionation process can be used for extending the

applicability of fats and oils.

1.2.1.2 Interesterification

Natural fats are mixtures of triacylglyerols in which the acyl groups are usually

distributed in a random manner. Under the influence of an appropriate catalyst, the acyl

groups are redistributed first intra-moleculary, and then inter-molecularly until a wholly

random distribution is finally achieved. Interesterification is the name given to this

process in which the arrangement of fatty acid in a triacylglycerol molecule is changed.

Chemical inter-esterification leads to a random distribution of the fatty acids on the

glycerol molecules. This is known as random inter-esterification. Catalysts usually

employed are sodium hydroxide, sodium methoxide or a sodium potassium alloy at 0.2-

0.4% level (Young et al, 1994). The redistribution of acyl groups leads to a change in

physicochemical properties of the triacylglycerol mixtures. An extension of the process

has been introduced by the use of lipases as catalyst for oil and fat inter-esterification

reactions (Xu, 2002). Its advantage over the more conventional procedures lies in the

additional control of product composition (Owusu-Ansah, 1994). Inter-esterification

procedures are used industrially to improve the physical properties of lard, to produce

cocoa butter substitute from cheaper oils (usually combined with hydrogenation and

fractionation), to produce fats containing acetic acid and to produce margarine of

appropriate melting behaviour with a minimum content of polyene acids (Young et al.,

1994).

12

1.2.1.3 Physical Refining

This physical refining involves all the other steps of refining apart from the

neutralization to produce refined,

bleached and deodorized melon seed oil (RBDMSO).

The flow chart is shown below:

Figure 1.1: Flow Chart of Physical refining

Crude oil

0.2% water, heating and stirring

Degummed oil

Bleached earth

Degummed bleached oil

Steam deodourization

Refined, Bleached and deodorized oil

13

Figure 1.2: Flow Chart of Chemical Refining of Oil

Chemical Refining

Crude palm oil + 0.2% water, heating and stirring

Caustic soda,

degummed and Neutralized

Water washed vacuum dried

Neutralized oil (NMSO)

Bleached earth

degummed Neutralized and Bleached

Steam deodourization

Neutralized Bleached, Deodorized oil (NBDMSO)

14

This is represented on the flow chart source (Chow and Ho, 2000). The chemical refining

values these processes.

i) Hydration to remove phosphatides

ii) Neutralization to remove acidity

iii) Bleaching to remove colouring matters

iv) Deodorization to remove smell and taste and finally,

v) Hydrogenation to harden the oil.

These processes are represented below.

1.2.1.4 Physical and Chemical Properties oil

1. Organoleptic properties

Pure oils and their constituent fatty acids are generally colourless; hence do not

possess spectral qualities in the visible range. The presence however, of such substances

as chlorophyll, carotenoids and resins for example always contribute to the colours

noticed in most crude fat samples, but can be easily removed during processing.

Vegetable fats are colourless the natural odours and flavours observed, except

those from very short fatty acids are due mainly to the presence of volatile and non-fatty

breakdown products such as ketones and aldehydes with very low flavour thresholds.

Pure fats are supposed to be tasteless, but the presence of non-fatty products also induces

characteristics taste in them.

2. Solubility/miscibility

Vegetable oils are generally insoluble in water and 90% ethanol, but are freely

miscible and soluble in organic, polar solvents such as diethyl ether, hexane, chloroform

and petroleum ether. This has formed a basis for their extraction from oil seeds as those

solvents usually dissolve the fats. Oil seed such as castor and croton are however, readily

soluble in ethanol but not in petroleum ether. Unfortunately, other unwanted fatty

materials dissolve along with the vegetable fats, but are removed during processing.

Solubility like other properties is determined by the component fatty acids. Long chain

fatty acids are insoluble in water, sparingly soluble in alcohols, acetones and highly

soluble in hydrocarbons and halogenated solvents (with the solubility decreasing with

15

increasing chain length of saturated acids). At very high temperature and pressure

however, fats dissolve in large amounts in water (Njoku, 2001).

3. Viscosity (rheological studies): One of the most distinctive properties of fats is

oilness that is the ability to form lubricant films. Rheological studies refer to the

deformation of fats under the influence of stress which may be applied perpendicular to

the surface (tensile stress) or tangentially (shearing stress).

Viscosity is the ratio of shear stress to shear rate or simply the flow resistance to

fats and it increases by polymerization. The inter-molecular attraction of long chains in

glycerides molecules accounts for the relative high viscosity of oil. The kinematic

viscosity is a function of molecular size and orientation since it increases with increase in

saturation range and chain length of fatty acids. Pure vegetable fats being single phased,

exhibit Newtonian flow behaviour at normal shear rate, but thioxotropic at high shear

rate. Viscosity generally decreases with rise in temperature and knowledge of this helps

in the industrial step up to know what temperature the oil will be pumped to reduce cost

while maintaining quality of the oil.

Specific gravity: This is usually measured at 150C in tropical regions, where this

temperature is usually unobtainable; the specific gravity can be measured at any

temperature t0C up to the temperature of boiling water, and then corrected as shown in the

formula:

S.P. Gravity at (150C) = Sp. Gravity (t

0C -15 x 0.000069)

Specific gravity varies as a rule with narrow limits for various samples of the same fat,

and is used for identification. Oils rich in oleic acid usually have low specific gravity.

Most fats possess specific gravity of the order 0.91-1.00, and thus float in water.

Refractive Index: This is quotient of the sine of the incident angle of light in the air and

the sine of the angle of refraction of light in the substance. Refractive index is an

important characteristic of oils because of ease and speed of determination as well as the

small sample needed for analysis, and the relationship of refractive index values with

temperature. Determination is done at 200C for liquid, at 40

0C for solid fats. It varies

indirectly with the average molecular weight and directly with the degree of unsaturation.

It correlates with iodine number. A correction factor can be included where it is not

possible to work at stipulated temperature. The formula for calculation is thus;

16

Refractive index = R + 0.00380θ

Where R = Refractometer reading

θ = number of degree in centigrade by which the measured temperature is above the

specified temperature, another equation can be used.

θ = Refractive index = 1.4643 – 0.0000665 - IS

A

0001171.0

0096.0

Where

S = Saponification value

A = acid value

I = iodine value

1.4 Chemical properties of Vegetable seed oil

Acid value: This is number of milligrams of KOH required to neutralize 1g of oil or fat.

It indicates the amount of free fatty acid present (Ononogbu, 2002). The presence of free

fatty acids in an oil or fat is an indicator of the previous lipase activity and other

hydrolytic action or oxidation (Gordon, 1993). It can occur in refined oils at about 1.1%

(w/w) up to as much as 15% in crude oil, but typically about 5% in crude oils

(Hammond, 1993).

Iodine value: Iodine value is the number of grammes of iodine that combines with 100g

of oil or fat. It gives the degree of unsaturation of the fat or oil (Ononogbu, 2002). This is

based on the fact that halogen addition occurs at unsaturated bonds until these are

completely saturated. Not all unsaturated bonds are alike in reactivity, and those near a

carboxyl group hardly absorb iodine. These acids are however rare. When the double

bonds are conjugated, they react more slowly than non-conjugated double bonds

(Gordon, 1993). Several methods for determining iodine value are available; those in

common use being the test of Wijs, Hanus and Rosenmundkuhnhem (Gordon, 1993). The

differences between the methods stated are in the halogenating agents. Fats and oils can

be classified by their iodine values. The iodine values of edible oil range from about 7 to

over 200. Oils with values below 70 are usually referred to as fats because they are solid

at room temperature. Another group which reflect their iodine value is into drying (higher

than 150), semi-drying (between 100-150), non-drying (between 70-100) and fat (70),

17

(Simpson and Corner –Orgarzally 1986). Phosphoglyceride that has molecule of chlorine

attached to its phosphate group, is a major constituent of cell membranes, lecithin and

other phosphoglycerides form a lipid bilayer, in which the water-soluble phosphate

groups orient toward the aqueous (water) environment both inside and outside the cell,

while the water insoluble fatty acids stay in the lipid environment sandwiched between

them. This forms a barrier that helps regulate which substances can pass into and out of

the cell. In food, lecithin helps to keep the oil from separating from the water –soluble

ingredients. It is used by the food industry as an additive to margarine, salad dressing,

chocolate, frozen dressers and baked foods.

Saponfication value: This is the number of miligramme of potassium hydroxide

required to neutralize the fatty acids resulting from complex hydrolysis of 1g of oil or fat.

It is a measure of both free and combined acids. The esters of low molecular weight fatty

acids require most alkali for saponification, so that the saponification value is inversely

proportional to the mean molecular weight of the fatty acids in the triacylglycerols

present (Gordon 1993). Because many oils have similar saponification values, the test is

not universally useful in establishing identity or indicating adulteration and should

always be considered along with the iodine value for these purposes.

Peroxide value: This is the milliequivalent of peroxide oxygen per 100g of fat. It is used

to indicate the degree to which a fat has been oxidized. Oxidation of unsaturated oil or fat

takes place via the formation of hydroperoxides. The hydroperoxides subsequently

decompose in to secondary oxidation products, the majority of which have unpleasant

odour and flavour. Although hydro-peroxides themselves have no off-flavours, they are

an important aspect of rancidity development and is determined as the peroxide value. It

is usually less than 10 per gramme of a fat sample when the sample is fresh.

Unsaponifiable matter: Unsaponifiable matter is the whole quantity of substances

present in the oil or fat which after saponfication by potassium hydroxide and extraction

by a specified solvent, are not soluble in aqueous alkali and non-volatiles under the

condition of test. The unsaponfiable matter of a fat includes, sterols, higher aliphatic

alcohols, pigments, hydrocarbons as well as any foreign organic matter non-volatile at

1000C (eg mineral oils). Refined oils contain lower amounts of unsaponifiable matter. Its

18

determination can be useful in indicating contamination and adulteration of the oil with a

mineral oil or other non-triglyceride contaminants.

Phospholipids: These are lipids attached to a chemical group containing phosphorus

called phosphate group. The phosphoglycerides are the major class of phosphatides like

triglycerides; they have a backbone of glycerol. However, they have only two fatty acids

attached to them. In place of the third fatty acid is a phosphate group, which is then

attached to a variety of the molecules. The specific function of a phosphoglycerides

depends on the molecule that is attached to the phosphate group.

The fatty acid end of phosphoglycerides is soluble in fat, whereas the phosphate

end is water-soluble. This allows phosphoglycerides to mix in both water and fat-a

property that makes them important for many functions in the body and in food. For

example, lecithin, a phosphoglyceride that has a molecule of choline attached to its

phosphate group, is a major constituent of cell membranes, lecithin and other

phosphoglcerides form a lipid bilayer, in which the water-soluble phosphate groups orient

towards the aqueous (water) environment both inside and outside the cell, while the water

insoluble fatty acids stay in the lipid environment sandwhiched between them. This

forms a barrier that helps regulate which substances can pass into and out of the cell. In

food, lecithin helps to keep the oil from separating from the water-soluble ingredients. It

is used by the food industry as an additive to margarine, salad dressing, chocolate, frozen

dressers and baked foods. Hence, lecithin (Phosphotidyl choline) is a component of the

phospholipids with the highest degree of abundance.

19

Phosphatidyl Serine

Phosphatidyl Inositol

Figure 1.3The Structures of Phosphatidyl Serine and Inositol

20

Figure 1.4: Structures of Phosphatidyl choline and ethanolamine

21

Figure 1.5 The Structure of Lecithin

22

1.4.1 History of Lecithin

Lecithin was first discovered and developed in Europe, however; it attracted

interest in East Asia at a rather early date. The earliest reference on lecithin date back to

1897 when Hanai, a Japanese agricultural chemist wrote a four-paged article in English

titled physiological observation on lecithin. Soybean was reported to be a good

concentrated source.

The earliest known production of commercial lecithin in East Asia was about

1923-1926 (Hanai, 1928). When commercial soybean processing plant was in operation

at Imienpo, North Manchuria, extracted with ethyl alcohol, then the phosphatides

(lecithin) was by using Tcherdynzev process. Oil was extracted with ethyl alcohol, and

then the phosphatides were removed using calcium chloride. In 1935, two research

scientists Sorenson and Baal patented an extraction process using hexane. The new

hexane yielded less lecithin product, though hexane has a much lower carbohydrate

content and a much better colour, odour and flavour (less bitter). Hence it found more

wide-spread acceptance (Sorenson and Baal, 1935).

1.4.1.2 Biochemical value of lecithin

The nutritional value of lecithin rests on a plethora of factors, for instance,

choline, a member of vitamin B-complex group is used by the body in forming the

acetylcholine (Meek, 1997), which is required for nerve and brain functions. In the liver,

the lipotropic (fat-loving) nature of lecithin supports efficient uses of fat by the liver,

prevents the formation of atheroma (Sebaceous cyst) in the blood vessels as well as the

ductus lactiferi in the milk ducts of lactating mothers. Lecithin, especially from soybean

source contains Omega-3 and 6-polyunsaturated fatty acids (PUFAs) which together help

in relieving/curring neurological diseases especially at the senile age, performs serious

roles in the capacitation/ripening of male (human) spermatozoa, and providing energy-

rich nourishment to the female ova, thereby boosting fertility in humans.

In respiration, modified lecithin, dilinoleyl facilitates breathing by reducing

friction within the lungs. It maintains sound communication within the brain cells in

signal transduction by being able to permeate the blood-brain barrier, and sharpens our

reasoning and learning faculties. Lecithin also stabilizes the membrane function by

23

performing its lipotropic function on the phospholipids components of the membrane.

Other uses of lecithin have prompted the literature review of lecithin to be broadly looked

at under three sub-headings as lecithin in health, industries and agriculture. The diverse

properties of lecithin were the basis of its involvement in health, industries and

agriculture.

1.4.1.3 Dietary and supplemental lecithin

A renowned nutritional maxim once says “you are what you eat”. People are

therefore advised to consume brewer‟s yeast, whole grains, legumes, eggs, vegetables,

fish, wheat germ, nuts, meat especially organ meat so as to improve on their daily

requirement of lecithin (Lucas, 1996). Latest advances in nutrition has implicated red

meat, organ meat and saturated fats as initiating the pathogenesis of most killer diseases

like atherosclerosis, cardiovascular diseases (CVD) and cancer, but lecithin when

adequately consumed has palliative/curative measures against these deadly diseases

(Berg et al., 2006). Lecithin is low in fat and cholesterol and therefore is helpful to breast

feeding mothers, pregnant women and the elderly, who benefit most from the emulsifying

property of lecithin (Lawrence and Ruth, 1996).

The lecithin available in commercial stores for use as supplement is extracted

from soybean. Lecithin is packaged in two forms: liquid (gel capsules) and granulated

lecithin, which is the best form of supplemental lecithin. The granulated lecithin is 97%

phosphatidyl choline unlike the gel or liquid type which is usually 60% phosphatides and

37% oil (Renfree, 2005). However, other natural sources of lecithin are oil palm fruit and

oil, most vegetable oils, sea foods and soybean products. Supplemental lecithin can be

obtained from yoghurt and other synthetic lecithin.

Supplemental lecithin which contains essential fatty acids (EFAs) helps in

activating the part of the brain that controls thought and reasoning ability. Moreover,

when dietary supplement from Omega-3 fatty acids are taken, it will also help to reduce

the accumulation of tau proteins which causes neurofibrilatory tingle. This intake will

therefore activates the way the brain cells function. (Selkoe, 2004).

24

1.5 Lecithin in Health

1.5.1 Lecithin and Neurological Diseases

Lecithin is a good source of choline, a B-vitamin with a powerful lipotropic

activity. Choline is utilized by virtually every cell for synthesis of various phospholipids,

proteins and the neurotransmitter substance acetylcholine (Green, 2003). Acetylcholine

deficiencies are linked with neurological disorders of tardive dysknesia (involuntary

facial grimaces and body jerking), Huntington‟s chorea (the disease that killed wood

cynthric Davis 1996), Friedrick‟s ataxia (speech impairment, irregular movements, and

paralysis), Olivapontocerebellasatrophy (wasting away of the brain) Alzheimer‟s disease

(a mind destroying disease that starts with memory difficulties), and Myasthenia gravis

(progressive paralysis) (Citron, 2004). Omega-3 fatty acids are components of the long

chain polyunsaturated fatty acids (PUFAs), which reduce the level of accumulation of β-

amyloid and Tau proteins that cause Alzheimer‟s disease symptoms.

However, these omega-3 supplements lower the level of preseneline, an enzyme

responsible for cutting beta-amyloid from its parent that is the amyloid precursor proteins

(Green et al., 2006).

1.5.1.1 Lecithin and respiratory function

Proteins and mucopolysaccharides constitute the surfactant system of the lungs

which reduces the cohesive force between water molecules at the alveolar surface to

prevent the air spaces from collapsing at low lung volumes (Ito et al., 2005). Between

50% and 60% of lungs surfactant is a specialized form of phosphatidylcholine,

dipalmitoylphosphatidylcholine, in which both fatty acids are palmitic acid (Harry

Lawson, 1995). Other phospholipids include phosphatidyl glycerol (10% of total lipid)

and small amount of phosphatidyl inositol, phosphatidyl serine, phosphatidyl

ethanolamine and sphingomyelin. Specialized type II alveolar cells of the lungs

synthesize the phospholipids. Once secreted, the surfactant lipids coat the air-water

interface as a phospholipids monolayer to lower the surface tension within the alveoli.

(Yeagle., 1992). The full complement of enzymes needed to synthesizes surfactant

phosphatidyl choline is not expressed until near term in humans. For this reason, pre-

25

mature infants often do not synthesize sufficient surfactant and develop collapsed lung

alveoli -respiratory distress syndrome (RDS). This syndrome can be treated by infusion

of lecithin into the lungs (Ito, 2005).

1.5.1.2 The Liver and Lecithin

Liver exports lecithin in two secretions; bile and plasma lipoproteins, the amount

of lecithin exported per day in humans is approximately 10-20% of the liver lecithin pool,

divided equally between the two secretions (Russel, 2003). Lecithin secreted in bile (12g

per day) plays a role in the micellar solublization of cholesterol, free fatty acids, 2-

monoglycerides, steroids and fat-soluble vitamins. The liver packages triglycerides for

export as low-density lipoproteins (LDLs) which are surrounded by a lipoprotein envelop

rich in phosphatidyl choline and phosphatidyl ethanolamine (Salonem, 2003). Circulating

high-density lipoproteins (HDLs) transfer apoprotein C to VLDL, this apoprotein

activates lipoprotein lipases in capillary endothelia resulting in the hydrolysis of

triglycerides. As triglycerides are removed from VLDL, the ratio of envelop (and thus

phosphatidylcholine) to triglycerides increases and a transport lipoprotein of intermediate

size is formed (Berg et al., 2006). The excess surface components (phospholipids,

proteins and cholesterol) are released and enter HDL. The intermediate size lipoproteins

are converted to low density lipoproteins (LDL) by the liver. Both LDL and HDL are rich

in lecithin contents. Lecithin is a lipotropic and antioxidant compound that helps in the

normal functioning of the liver cells. The liver synthesizes lipoproteins which it secrets

into the plasma. Chronic alcoholic intoxication produces frequently cirrhosis and

concomitantly alterations in the liver metabolism. However, prolonged ingestion of

alcoholic beverages, carbon tetrachloride intoxication, hepatotoxic drugs such as cis-

platinum, bupremorphine, among others as well as metabolic insulin resistance syndrome

can produce alterations in lipid metabolism inducing liver steatosis and/or necrosis

(Dreon et al., 2004).

In liver, fatty infiltration virus C aggression can be present, as well as in Reye‟s

syndrome. Under certain pathological conditions, as occur in chronic ethanol exposure,

reactive oxygen species (ROS) production is increased and the level of antioxidant

substances and enzymes are reduced. This imbalance between ROS production and its

26

removal constitutes the process called Oxidative Stress (OS). Almost all these noxious

agents previously referred, produce alterations on liver lipid metabolism, but basically on

liver proteins, glycophospholipids, ceramides, including an important number or enzymes

that participate in its metabolism producing finally liver injury (Janakay, 1999). It is

known that chronic alcoholic liver disease develops because the presence of alcohol and

its metabolities damage its parenchymal and non-parenchymal hepatic cells.

Furthermore, alcohol produces its toxic effect on the intestinal wall, allowing among

other changes, the passage of bacterial toxins to the Splanchnic blood flow, reaching the

liver through the portal circulation (Duel, 1951).

The continued production of ROS by the liver due to alcohol ingestion destroys

the liver and its enzymes (Halliwell and Gutteridge, 1999). The liver mitochondrial

transport of GSH in the presence of alcohol weakens the antioxidant property of the liver

(Kelly, 1998). The alcoholic cirrhotic liver is only healed by taking balanced diet and

supplemental lecithin.

1.5.2 Lecithin and Signal Transduction

Lecithin is the major phospholipids of all mammalian cell membrane. Lecithin

therefore has an important role to play in cellular signal transduction following hormone-

receptor interaction (Wang, 2004). Phosphatidylinositol, located in the plasma membrane

is sequentially phosphorylated by phosphatidyl inositol kinase to yield phosphatidyl

inositol-4- phosphate and phosphatidyl inositol-4, 5-bisphosphate. Upon receptor

activation, phosphorylase C is activated through a linking guanosine triphosphate (GTP)

–binding protein, phosphatidyl inositol 4,5-bisphosphate is hydroylsed to yield inositol-

1,4,5 triphosphate and diacylglycerol, Inositol-1,4,5 triphosphate acts as a second

messenger to stimulate release of calcium ions (Ca2+

) from intracellular Ca2+

stores,

which in combination with diacylglcerol, activates protein kinase C (PKC). Protein

kinase C is able to phosphorylate a wide variety of cellular proteins, resulting in

increased or decreased enzymatic activity of specific protein inositol-1,4,5-triphosphate is

further phosphorylated by a 3-kinase to yield inositol-1, 3, 4, 5-tetraphosphate (Merill,

2002). This compound may also act as a second messenger to make free Ca2+

available to

the inositol-1, 4, 5-triphosphate-sensitive endoplasmic reticulum store. Upon receptor

27

activation, a phospholipase C is activated, this breaks down phosphatidylcholine

(lecithin), generating additional diacylglycerol and enhancing the phosphatidyl inositide-

based signaling system. The signaling is ended when sphingomyelin breakdown

generates sphingosine, an inhibitor of protein Kinase C (Offermanns, 2003).

1.5.2.1 Lecithin and biomembrane

Lecithin is the major structural component of all biological membranes and is

present in the membrane bilayer as phospholipids (Bach and Wachtel, 2003). In an

aqueous environment, the polar head groups (phosphate and esterified alcohol) and the

non-polar tails of the fatty acyl chains cause the phospholipids to spontaneously arrange

in a bilayer or micelle formation which serves to limit the contact of the hydrocarbon

chains with water. The polar phosphate groups arrange themselves on the outer surface of

the vesicle with the acyl chains on the inside. The hydrophobic chain does not allow

hydrophobic molecules to pass through membranes without a transport system such as a

carrier or channel (Hoch, 1992).

Apart from a structural role, the phospholipids also serve as a source of energy for

the cell and in intracellular signaling (Wang, 2004). Phosphatidylcholine, phosphatidyl

ethanolamine, sphingomyelin, phosphatidyl serine and phosphatidyl inositol are the

major membrane phospholipids and are found in varying concentrations in cell and

organelle membrane (Carty et al., 1996). In addition, the phospholipids composition of

each side of the membrane bilayer is different. In erythrocytes, the outside layer contains

more phosphatidyl ethanolamine and phosphatidylserine. The asymmetric arrangement

may be attributable to the unidirectional nature of their synthesis, preferential association

with specific membrane proteins, and/or the differences between the intra and extra-

cellular environment (Alexander et al., 2004). The phospholipids membrane is a dynamic

structure in which the lipids and proteins are able to undergo rapid lateral motion,

however, transverse motion across the bilayer occurs very slowly (Yeagle, 1992).

1.5.2.2 Lecithin and the blood-brain barrier

This is a lipid barrier that protects the brain by restricting the passage of

electrolytes and other water-soluble substances. It is considered to be selectively

28

permeable to most molecules and ions that are lipid soluble (Zeisel, 2004). The blood-

brain barrier is made of brain microvessels, endothelial cells, which are far less

permeable than capillaries in other organs, such as the kidney, liver and muscle. The

above components restrict the passage of most small polar molecules e.g. histamine,

catecholamine, small peptides and macromolecules (e.g. proteins) from the

cerebrovascular circulation to the brain (Cooper et al., 2004). Lecithin is able to pass the

blood-brain barrier, diffusing into the cholinergic nerve endings where it adds its choline

component for the formation of the neurotransmitter substance acetylcholine. As a

general rule, then, a drug must have a certain degree of lipid solubility if it is to penetrate

this barrier and gain access to the brain (Vance, 2002).

1.5.2.4 Lecithin and Fat Metabolism

The emulsifying action of lecithin is essential for the body‟s control of cholesterol

and triglyceride level. Lecithin reduces large, dangerous cholesterol globules and

increases smaller, healthier high density lipoproteins (HDL) particles. In animal trials,

lecithin administration for atherosclerotic arteries resulted in an increases of

phospholipids synthesis. The phospholipids detach the deposited cholesterol and help to

remove the obstruction (Polichetti, 1996). On the other hand, the enzyme Lecithin

Cholesterol acyl transferase (LCAT), maintains cholesterol and phospholipids in balance.

The LCAT enzyme is found in the low-density lipoproteins (LDL), the so-called “bad”

lipoproteins, is rich in cholesterol, whereas HDL is rich in phospholipids, (Wilson et al.,

1998). Research, however, has shown that lecithin can be partially absorbed intact by the

intestine, and incorporated preferentially into HDL. Also, soy lecithin is known to act as

good substrate for lecithin cholesterol acyl transferase (LCAT) activity.

This enzyme is associated with the irreversible formation of HDL from LDL; It

has the capacity to carry cholesterol from peripheral tissue such as the aorta back to the

liver where cholesterol can be converted to bile acids. Laboratory animals study have

shown also that the feeding of lecithin to rats increased (LCAT) activity which increased

cholesterol removal from the blood via faecal bile acid excretion.(Polichetti, 1996).

29

1.5.2.5 Lecithin in Reproduction and Fertility

Lecithin also plays a role in male fertility. Test tube studies have shown that

lecithin has the ability to restore normal structure and movement to abnormal sperm cells

and nearly double the acrosomal response. (Orthoefer, 1998). Lecithin made from

soybean contains Omega-3- and -6- essential fatty acids (EFAs), which generally help in

the maturation/capacitation of sperm cells of male humans (Go and Wolf, 1984). Lecithin

as a phospholipids, contains choline and is involved in the availability of platelet

activating factor (PAF), which is a choline phospholipids. In other words, (PAF),is a

constituent of the class of compound that we call „‟lecithin‟‟. Platelet activating factor

(PAF) is involved in reproduction in three ways-(1) in implanting of the egg in the

uterine wall. (2) In foetal maturation, and (3) in inducing of labour (Lalkhan, 2008). The

acrosome is the cap-like membrane-bound structure covering the anterior portion of the

head of a spermatozoa. It contains enzymes involved in the penetration of the ovum.

1.5.2.6 Lecithin Role in Short-term memory

Lecithin is the primary source of phosophatidyl choline which is essential for

mental and nerve functions. Choline is a precursor for the biosynthesis of acetylcholine,

the neurotransmitter essential for memory. The complex functions of the brain depend

upon the presence of neurotransmitter. Acetylcholine as a neurotransmitter is the

chemical messenger between the brain, nerves and organs. Acetylcholine may also be

involved in a wide range of other normal brain activities that include learning, memory,

sensation, motor Co-ordination, and sleep, as well as the bodily functions of respiration,

circulation and digestion (Carty, and Jolitz, 1996). The most important sources in the

body for the formation of acetylcholine are lecithin and choline. Ingestion of free choline,

however, is less effective than ingestion of lecithin. That is why lecithin is the preferred

dietary component. Over 99% of the choline present in the diet is in the form of lecithin

(Phosphatidyl choline). Choline present in supplemental lecithin brings about a higher

blood level of this substance than that produced by other choline supplements (Spiller,

2006). Lecithin is involved in the myelination (formation of sheath round the axon or

nerve bodies) throughout intra-uterine life, infancy and early childhood, especially at the

6th

month of intra-uterine life. Myelin is a fat-rich substance that coats and insulates

30

neurons to enable them transmit messages faster and more efficiently. Hence, myelinated

neurons conduct messages faster than unmyelinated neurons (Cooper et al., 2004).

It was thought that there may be an inter-relationship between the cholingergic

nerve processes and memory. Short-term memory, in particular, is dependent upon

neurotransmitters. While lecithin is not a learning drug, choline administration does result

in improved memory as evidenced in learning exercises. Selected positive effect have

been observed with memory, cognition and mobility test with Parkinson‟s disease

patients. Animal studies also show that lecithin and choline improve memory and

learning ability. Rats born to mothers consuming supplemental lecithin possess improved

learning ability (Orthoefer, 1998).

Acetylcholine deficiency is commonly associated with Alzheimer‟s disease and

other degenerative senile conditions that involve memory and neurological abnormalities.

Lecithin is unique in being readily available as a nutrient, simple to administer through

the diet and an effective but harmless food supplement with no known undesirable side

effect. (Berg, 2006). Lecithin has been reputed to be a brain food. Students who use

lecithin before examinations, to improve their memory and enhance their ability to study

effectively have been fully vindicated by recent research in the mid 2000 years, brain

researchers found that lecithin was more intimately involved in mental and nervous

functions than previously thought. (Nahorski, 2006). Choline is an important nutrient

that is actively transported from mother to foetus across the placenta, and from mother to

infant through the breast milk. With rats, lecithin added to the diet resulted in improved

learning of the infant. Surprisingly, the improved memory capability of man has been

shown to extend even to old age (Orthoefer, 1998).

This therefore implies that the potential balanced diet of pregnant mothers, more

especially those watching their cholesterol level should contain ample servings of

lecithin. This will not only replace the synthetic statin drugs, but will also go a long way

to improving the formation of foetus which will sustain it for memory improvement

during infancy (Zeisel, 1997).

31

1.5.2.7 Lecithin and Breastfeeding mother and Child

Lecithin has been in use in beauty products for many years as a natural emulsifier

in creams and cosmetics. As a supplement, the natural fatty acids present in lecithin may

make it useful for maintaining youthful skin and for assisting the treatment of psoriasis.

Among mothers, fatty accumulation around the eyes has been an unattractive skin

problem for many people. This might also be effectively dealt with by increasing the

body‟s intake of lecithin (Zeisel, 2004). In breastfeeding mothers, lecithin helps to reduce

the plugging of the nipples duct. In positive case, the mother should limit her

polyunsaturated fats intake, and be taking one (1) table spoonful of lecithin per day.

Lecithin helps to emulsify fats within the lactiferous ducts and sinuses in breast and at the

same time solublizing the fats that may plug the nipple duct thereby preventing breast

engorgement in breastfeeding mothers (Lawrence and Ruth, 1999). On the part of the

breastfed child, there is high chance of his suckling to his fill within each feeding bout.

The presence of Lecithin promotes the absorption of Vitamins A and D, and influence the

utilization of other fat-soluble nutrients such as Vitamins E and K in the intestinal tract

(Kidd, 1997). Lecithin (Phosphatidyl choline) found in the cell membranes helps to

maintain the surface tension of cells and wastes both in and out of cells. This will ensure

thorough homeostasis for the child and therefore sound health (Gordon, 2000).

1.5.3 Industrial Roles of Lecithin

Industrially, lecithin have diverse roles which was as a result of their properties,

in animal feeds, bakery and candy manufacturing industries, the emulsifying dispersing,

wetting, conditioning, surfactant and antioxidant properties of lecithin made them

essential ingredients in production. In chewing gum, chocolate food, edible oil, ice

cream, instant food, insecticides; inks, leather macaroni and noodles manufacturing,

lecithin must be used for their anti-spattering, wetting, emulsifying and other protective

roles on the end-products such as, margarine, paints, petroleum products, plastics, rubber

and textiles. The above require the anticorrosive, lubricating, anti-wearing, dispersing,

plasticity, softening and conditioning properties of lecithin therefore are of immense

applications in industrial manufacture of consumable and useable products (Szuhaj 1989)

32

1.5.3.1 Lecithin and manufacturing processes

In paint making, lecithin is a wetting agent, dispersing agent, suspending agent,

emulsifier and stabilizer, in both oil base and water base (latex and resin emulsion)

paints. It facilitates rapid pigment wetting and dispersion, saves time in grinding and

mixing, permits increased pigmentation, stabilizes viscosity, aids in brushing and

improves remixing after storage. Margarine contains lecithin as an emulsifier anti-

spattering and browning agent; it improves frying properties and spreadability and

shortening action in table margarine.

In petroleum products, it is used as an antioxidant, detergent, emulsifier and anti

corrosive. It is also used for lubricity and anti-wear. It is added to gasoline to stabilize

tetra ethyl lead IV and for inhibition of corrosion. After reaction with aliphatic amines, it

is used as a detergent in motor oils because of its lubricity. Also, it is used in

miscellaneous oils including house hold lubricants and cutting oil. In fuel oils for

surfactant and inhibitory effect and in drilling muds as an emulsifier.

However, in plastics, rubber and textiles industries, lecithin is used for pigment

dispersion and as a slip or release agent. It may also be sprayed on molds. It has

surfactant effects on organosols and plasticity in rubber. It is a wetting and dispersing

agent as well as mold release agent. It increases plasticity and facilitates working. It

emulsifies latex, mixes and aids in preparing solvent dispersion and in vulcanizing. In

textiles, it is used for emulsifying, wetting, softening and conditioning especially in

sizing and finishing. It impacts soft smooth handle and is also used as a spray to reduce

lotion dust.

In natural and mutation cheeses, lecithin is an effective emulsifier and slice

parting agent. Lecithin is a good browning agent, emulsifier, phosphate dispersant and

dietary supplement. In spreads and salad products, lecithin acts as an emulsifier and

controls crystallization. The packaging of materials welcomes lecithin as sealant and

release agent. In processing, such as as frying surfaces, extruders, conveyors, boilers,

dryers and blenders, lecithin is an effective lubricant and internal or external release

agent. The uses of lecithin must not be complete without mentioning dietary and health

implications. Lecithin is consumed as a whole food in liquid, capsule and/or granular

33

forms. The early 1950s interest in lecithin was as a cholesterol lowering agent. However,

today‟s interest in lecithin is in the area of aging and memory.

1.6 Lecithin and Pharmaceuticals

1.6.1 Lecithin and Cosmetics

Lecithin is used in the cosmetics industry for the making of eyeshadows,

moisturizing preparations, make-up bases, lipsticks, hair-conditioners, shampoos (non-

colouring), skin care preparations (excluding shaving preparations, night skin care

preparations, mascara, blushers (all types), make-up preparation (not eyes), face powder,

tonics, dressings and neck preparations), paste masks (mild packs). Other hair grooming

aids, bath capsules, hair sprays (aerosol fixatives). Bath oils, tablets and salts; cleansing

products (cold creams, cleansing lotions, liquids and pads) eyeliners, hair preparations,

skin freshner. Eye brows:- pencils, fragrance preparation, make-up fixatives. Lecithin

are applied in the above products because of their emulsifying, release, instantizing, anti-

spattering, separating, wetting, stabilizing, conditioning and dispersing properties.

(Gottschalck et al, 2004).

Lecithin generally acts as vehicle for delivering topical analgestics for it allows

the drug to permeate the skin. A typical example is the use of lecithin in the transdermal

cream called pluronic lecithin organogel (PLO) (Mark, 1994). PLOs have a unique ability

to pass through the epidermal barrier and deliver drugs (Hanin, 1990). The drug as gel

uses the mechanism of gel permeation to entering the skin as well as slight

disorganization of the skin layers. Generally, Lecithin are components of most nasal,

buccal, oral, ocular, trans-dermal, rectal and pulmonary drug preparations for they act not

only as excipient, but also as permeation enhancers, vehicle for dispersion, low density

lipoprotein carriers and emollient in suppositories etc (Bundgaart, 1992). Lecithin

reduces the brittleness of suppositories, protects against alcohol cirrhosis of the liver,

lowers serum cholesterol level and improves mental and physical performance (Novak,

1991).

34

1.6.3 Self-Emulsifying Drug Delivery System (SEDDSs)

In pharmaceutical products formulations, drug solubility is very essential for the

bioavailability of poorly soluble drugs. This age-long problem of poor solubility and

bioavailability especially of drugs of oral delivery has been solved by the latest

pharmaceutical technology called Self-Emulsifying Drug Delivery Systems (SEDDSs).

SEDDSs are isotropic mixtures of oils and surfactants, sometimes containing co-

surfactants and can be used for the design of formulations in other to improve the oral

absorption of highly lipophilic compounds. SEDDSs emulsify spontaneously to produce

fine oil-in-water emulsions when introduced into an aqueous phase under gentle

agitation. SEDDSs can be orally administered in soft or hard gelatin capsules and form

fine, relatively stable oil-in-water emulsions upon aqueous dilution (Holm et al.,. 2006).

In recent years, the formulation of poorly soluble compounds presented

interesting challenges for formulation scientists in the pharmaceutical industry. Up to

40% of new chemical compounds discovered by the pharmaceutical industry are poorly

soluble lipophilic compounds, which leads to poor oral bioavailability, high intra-and

inter-subject variability and lack of dose proportionality. The oral formulation of such

compounds involve a number of attempts as decreasing particle size, use of wetting

agents, co-precipitation and preparation of solid dispersion have been made to modify

the dissolution profile and thereby improve the absorption rate. Recently, much attention

has focused on lipid-based formulations to improve the bioavailability of poorly water-

soluble drugs. Among many such delivery options, like incorporation of drugs in oils,

surfactant dispersion, emulsions or liposome. Of all, the most popular approaches are the

Self-Emulsifying Drug Delivery Systems (SEDDSs) (Attama et al 2003).

Self-Emulsifying Drug Delivery Systems (SEDDSs) are mixtures of oil and

surfactants ideally isotropic and sometimes containing co-surfactants, which emulsify

spontaneously to produce fine oil-in-water emulsion when introduced into aqueous phase.

Self-Emulsifying formulations spread readily in the gastrointestinal (GI) tract, and the

digestive motility of the stomach and the intestine provide the agitation necessary for self

emulsification. These systems advantageously presents the drug in dissolved form and the

small droplet size provide a large interfacial areas for the drug absorption. Self-

Emulsifying Drug Delivery Systems typically produce emulsions with a droplet size

35

between 100-300nm, while self-micro emulsifying drug delivery systems (SMEDDSs)

form transparent emulsions with a droplet size of less than 50nm. When compared with

emulsions, which are sensitive and meta-stable dispersed forms, self-emulsifying drug

delivery systems are physically stable formulations that are easy to manufacture. Thus,

for lipophilic drug compounds that exhibit dissolution rate-limited absorption, these

systems may offer an improvement in the rate and extent of absorption and result in more

reproducible blood-time profile (Shen and Zhong, 2006).

1.6.3 Lecithin and the nutrition industry

Most food processing industries especially, the cocoa and beverage industries

employ the versatile characteristics of lecithin in their daily production processes. In

bakery, lecithin is an emulsifier, stabilizer, conditioning and release agent as well as

antioxidant. In yeast-raised dough, for example, it improves moisture absorption, ease of

handling, fermentation tolerance, shortening value of fat, volume and uniformity, and

shelf life. In biscuits and crunchers, pies and loaves, it promotes fat distribution and

shortening action, facilitate mixing and acts as a release agent.

In candies, confections made with oil or fat use lecithin as emulsifier and

distributes fats in caramels, nut brittles and nougats etc. it prevents fats separation and

greasiness. It has fixative action for flavours. Chocolate manufacturers utilize lecithin as

wetting agent, viscosity modulator, increases shelf life, counteracts moisture thickening

and aids release of moulded foods.

Lecithin acts as emulsifier, wetting agent and antioxidants in edible fats and oils.

It extends the shelflife especially in animal fat, increases lubricity (shortening value),

improves stability of compound shortenings, and lowers melting point of vegetable oils.

In foods, lecithin is a dietary addition rich in polyunsaturated phosphatidyl choline,

phosphatidyl ethanolamine, phosphatidyl inositol, and organically combined phosphorus

with emulsifying and antioxidant properties. It enhances fats and vitamin A absorption.

Instant foods contain lecithin for wetting, dispersing, emulsifying and stabilization in

beverage powder and mixes including milk powder, dessert powder, and powdered soup

etc. in ice creams and yoghurts, lecithin emulsifies, stabilizes, improves smoothness and

melting properties as well as counteracts sandiness in storage.

36

In the manufacturing of insecticides, lecithin improves emulsification, spreading,

and penetration ability of the insecticides as well as adhesion. The dye industries apply

lecithin as a coupling agent, especially for water-soluble colours in fatty media. Also, in

milk industries, lecithin is a wetting, dispersing and suspending agent promoting

uniformity, colour intensity and ease of remixing (especially printing inks). Macaroni and

indomie noodles contain lecithin as a conditioning agent and antioxidant, it improves

machining, counteracts disintegration and syneresis. It also improves colour retention.

1.6.4 Lecithin as Antioxidant

Antioxidants are compounds that protect other compounds from oxygen attacks

by themselves reacting with oxygen. They are preservatives that specifically retard

deterioration, rancidity, and/or discolouration of foods due to oxidation. They are also a

group of food additives added to the food to increase its shelf-life, retention of nutrients

and prevention of bacterial spoilage. Oxidation of foods occurs when oxygen is added to

unsaturated sites of molecules. Oxygen, light, heat, heavy metals, pigments, alkaline

conditions, and degree of unsaturation are catalysts in this process (Konat and Wiggins,

1985).

Among the many substances that have more recently been suggested for the

progress of rancidity in fats, vegetable lecithin is probably one of the first. It is somewhat

surprising that in the past years, so little information of an experimental nature has been

published to support the claims of this substance as an inhibitor of oxidation and to

explain the manner of its action. (Chaudiere and Ferrari-Iliou 1999).

Molecules that are easily attacked by antioxidants are – DNA, RNA, lipids (fats)

and proteins. Generally, antioxidants react with the oxidants and protect the above

susceptible molecules from being damaged. Examples of antioxidants are – Vitamins A

BI B5, B6, C, E, amino acid cysteine, food antioxidants – BHT and BHA minerals like

selenium and Zinc (Chaudiere and Ferrari- Iliou. 1999).

Sollmann, using the Warburg apparatus for the oxygen absorption method

reported without supporting data that lecithin inhibited the oxidation of cotton seed oil

catalyzed by cobaltic oleate, and that when exposed to oxygen at temperatures above

650C for one-half hour, it no longer exhibited anti-oxygenic properties.

37

More recently, Evans has found lecithin an excellent antioxidant for cotton seed

oil whose oxidation was accelerated by the presence of cobaltic oleate peroxide. He

proposed explanation of the effectiveness of lecithin as due to the formation of a

compound with the cobaltic oleate peroxide raises question as to the validity of using

such accelerators in the assay of inhibitors for edible oils.

In our antioxidant test, oil stability test was determined using oxygen absorption

method (AOM), which showed the antioxidant property of lecithin. Pure lecithin and

refined soybean oil were used as control and the melon seed oil lecithin-extract and

melon seed oil were also used as the test sample 0.01g-0.05g both the control and test

were each put in to 20ml of oil in a test tube. The ten (10) test tubes were aerated using

vacuum pump. At the end of every 1 hour, each test tube‟s lecithin-oil mixture was

tested for peroxide value. A plot of the lecithin concentrations against the peroxide

values for the control and the test are respectively shown in the result section. Lecithin

has a powerful antioxidant property in foods (Njoku, 1996).

1.6.5 Lecithin and the Beverage industry

Beverage can be defined as any type of drink except water, example tea, milk,

wine and beer. Scientifically, beverage can be looked upon as any non-toxic liquid

except drugs which when taken orally stimulates the biological systems. Beverages are

generally appreciated for their flavour and for the pharmacological action of their active

ingredients and their biochemical consequences. Beverages may be classified into

alcoholic and non-alcoholic on the basis of the presence of ethyl alcohol. The malt

alcoholic beverages which include beer, ale and stout have an alcohol content of between

2.75-4.75% whereas the non-alcoholic beverage which include tea, cocoa and coffee, the

carbonated „soft drinks‟ contain no alcohol. The spirits, whisky, brandy, rum and gin

contain higher alcohol percentages than the malt alcoholic beverages (Ononogbu, 2002).

Beverages use the mediatory action of cyclic AMP to generate heat energy in the system

and encourage lipolysis in the body. Beverages are consumed to replace body water and

for enjoyment. The flavour of each is the most important. Lecithin (Phosphatidyl

choline) is incorporated into most food drinks for it is required by infants, teenagers and

most especially at old age when it performs supplemental roles. The fascinating aspect of

38

most beverages is that they all contain phospholipids as phosphatidyl choline which

enriches the body and maintains homeostasis of mammals. Beverages in addition to

phospholipids contain appreciable amount of sterols and fatty acids. Precisely,

phosphatidylcholine (lecithin) as a phospholipids is used for the fortification of beverages

to increase their food value as well as nutritional quality (Ononogbu, 2002).

1.6.6 Lecithin as an Emulsifier and Separating Agent

Lecithin acts as an emulsifier for the management of reducing fat deposits in the

lactiferous sinus and ductal system located behind the nipple and areola in the lactating

breast. As the body‟s natural emulsifier, lecithin helps to dissolve fats and cholesterol.

Just as a detergent breaks down fat globules to be washed away in water, lecithin breaks

down fat particles. Fats and oils are essential part of the diet, yet they must function

within a watery environment of the body. Although oil and water do not mix, a lecithin

molecule (phosphatidylcholine) holds them together. Lecithin is both lipophilic and

hydrophilic,, attracting both fat and water. The end of lecithin that contains a fatty acid is

attracted to the oil molecules and attaches to water molecule (Flack, 1996).

Lecithin acts as a bridge between water and oil or fat. It has the ability to keep fat-

like cholesterol particles in solution while they journey through the arteries and other

similar passages of fluid that carries fatty molecules in the body such as the lactiferous

ducts and sinuses in the breast. The action of lecithin as an emulsifier in the blood stream

occurs when the phospholipase enzymes cleave one or both of the fatty acids in the

lecithin. The partially degraded phospholipids is re-synthesized in the intestinal mucosa.

The hydrophilic or water-soluble, degraded phospholipids are transported out of the

breast via the milk.

The emulsifying action of lecithin is essential for the body‟s control of cholesterol

and triglyceride levels. Lecithin reduces large, dangerous cholesterol globules and

increases smaller, healthier HDL particles. In animal trials, lecithin administration for

atherosclerotic arteries resulted in an increase of phospholipids synthesis. The

phospholipids detached the deposited cholesterol and helped to remove obstruction. The

nutrients in the food we eat, the intermediates or breakdown products of metabolism, and

the by-products to be excreted are transported in the bloodstream. Fats, oils, lecithin

39

(phospholipids), and cholesterol are present in various forms such as chylomicroms.

VLDL, LDL, and HDL (Dreon, 1990).

HDL is synthesized mainly in the liver and contains cholesterol esters. A balance

of the various types of serum lipids is necessary. Once the serum lipids are out of

balance, as noted by high serum cholesterol, triglycerides, saturated fatty acids, low

HDL, and high LDL, injury to the vessel walls or deposits may eventually occur resulting

in the inhibition of the flow through the vessels. Supplying lecithin or phospholipids is of

particular importance to a healthy diet because of their effect on controlling serum lipids.

Soybean lecithin, for example is an excellent source of polyunsaturated fatty acids that

are oxidatively stable, in addition to the activity of the other individual component

present (DeMan, 1990)

In other for LDL cholesterol to cause damage to the surface vessel walls (arterial

as well as lactiferous sinus and intra-ductal walls), it has to be oxidized. If LDL

cholesterol is prevented from being oxidized, it does little or no damage. Lipid soluble

antioxidants, such as vitamin E, will help overcome this damage. Lecithin demonstrates

antioxidant activity and is synergistic with vitamin E. Since lecithin has the ability to

keep fat-like cholesterol particles in solution, they are unable to settle out and form

dangerous deposits on the walls of the ducts and sinuses. A build-up of these deposits

can contribute to the plugging of milk duct leading to the painful inflammation of the

breast-engorgement (Lawrence and Ruth, 1997). Lecithin enables fats, such as

cholesterol and other lipids to be dispersed in water content of breast milk which is then

removed from the body. The vital organs, breast, and arteries are thus protected from

fatty build-up (Kajiyama et al., 1996)

Lecithin helps to form a stable film barrier that prevents adhesion of food

products to one another. Direct incorporation, as in baked foods allows for better

machinability and minimized sticking to the mixing vessels. The best results are obtained

when the lecithin is surface-applied versus direct incorporation, such as on processed

cheese slices. Regulatory compliance should be reviewed when direct incorporation, is

practiced to comply with food and drug administration (FDA) standard when applied

directly to the product, such as processed cheese slice, lecithin helps form a stable

balance and prevents them from sticking. When used directly in products such as baked

40

foods, they enhance the cutting and shaping products and reduce sticking to mixing

vessels (Dashiell, 2003).

In pharmaceutical industries, its separating ability made it imperative as an

excipient used in drug formulations especially drug of insertion-suppositories, nasal

inhalants and drugs of gastrointestinal tract (GIT) (Novak, 1991). In bakeries, biscuits

and candy, manufacturers utilize the separating property of the lecithin in their general

production and this supports also the anti-spattering roles of lecithin (Vance, 2002).

1.7 Lecithin in agriculture

Lecithin is extensively used in agriculture by addition into animal feeds where it

supplies essential ingredients needed in animal ration. It is usually added in different

ratios to different animal feeds. It plays an important role in the ration of aquatic

animals; where it provides the phospholipids needed by aquatic species. Lecithin

improves feed processing, and adds to physicochemical characteristics required for feed

palatability to animals. Lecithin is a source of choline; some growth stimulating

compounds and inositol. It also serves as antioxidants for the highly unsaturated oils in

the feed as well.

Lecithin added to aquatic animals feed functions as growth enhancer, help for

effective utilization of triacylglycerol and cholesterol. It balances the omega-3- fatty acid

to other fatty acids ratio in reproductive processes, it provides fatty acids sources for

brood stock reproductive performance and fatty acid component of the ration, and it

enhances solublization of certain fats and retards leaching of water soluble components.

In extension-type feed production, lecithin provides necessary lubrication, allowing more

effective feed production and improves pellet integrity and stability. Addition of lecithin

helps prevent liver metabolism disorders and fish already suffering from fatty liver

degeneration make a rapid recovering. In piggery, the addition of lecithin up to 1.5% in

the piglet feed supplement results in increased weight gain in specific time as against feed

supplement without lecithin. Lecithin enhances fat utilization by pigs. However, report

has it that the emulsifying property of soybean has the potential of enhancing their

utilization of dietary fat by piglets.

41

Lecithin in fertilizers serves as conditioning and spreading agent. It is as well

incorporated into pesticides, where it functions for adhesion, antioxidants, biodegrading,

dispersing agent, and emulsifier,penetrating, spreading agent, stabilizer and viscosity

modifier. Precisely, lecithin performs the functions of emulsifier, wetting and dispersing

agent, caloric source, antioxidant, and surfactant,, source of choline,organically combined

phosphorous, and inositol lipotropic agent in animal feeds. Also, it enhances antibody

production, it is a milk replacer for calves, and for veal production, in mineral feeds

poultry feeds, fish feeds, pet food and feeds for fur-bearing animals.

1.8 Melon Seed Oil

Melon seed oil is obtained from the seed of melon fruit which is of the family

cucurbitacea. It contains most of the fatty acids esters of glycerol commonly called

triglycerides, which assist in supplying the world of edible oils and fats. The designation

fats and oils commonly distinguish substances that are respectively solid and liquid at

room temperature.

Melon seed oil has a long history of use in human nutrition, but it is extensively

used as illuminants and lubricants, and for soap making in ancient civilizations. The

advent of large-scale exploitation of mineral oils in the nineteenth century rapidly

reduced the role of melon oil in illumination and lubrication, and the introduction of the

synthetic detergent shortly before the World War II had a similar though, less severe

effect on the quantity of melon oil and fats used for soap making (Alut and Wenzt, 1983).

Melon oil is nowadays extracted by solvents method using soxhlet extractor.

When done, the oil and the solvent mixture are separated using separating funnel, after

which the oil is boiled over fire to evaporate any remaining solvent since those solvents

used are highly volatile. The fatty acids with the highest volume in melon oil are oleic

(cis-9-octadecanoic) acid. Melon oil contains up to 10 percent of octadecanoic acid, but

those that have 18-carbon chain-oleic, linoleic, and linolenic (cis, cis, cis,-9,12,15) octa-

decatrienoic acids are widely distributed in melon seed oil (Allen, 1999).

42

1.8.1 Characteristic/morphology of melon plant (Cucumis melo)

Melon plant is a runner, with a hairy green stem and green leaves that are shaped

like duck legs. It matures in 3-4 months. Depending on the variety, white or yellow

flowers are conspicuous on the plant towards pod-bearing stage. Melon plant is a legume

which bears light green netted pods almost up to the size of broad-leaved pumpkin pods.

The pods are the fruits which bear the seeds (Van der Vossen et al., 2004)

1.8.2 Habitat/Ecology of melon (Cucumis melo) plant:

Melon seed is a native of tropical countries, and Nigeria is one of largest

producers in the world (Njoku et al 1994). There are two major types in Nigeria – the

Bava (Adenopsis guinensis) and Egusi (Colocynthis citrulllus). Both have white seed

protected by a brown crown (Van der Vossen et al 2004). Melon seeds produce

important vegetable oil used for cooking, cosmetics, in the pharmaceutical industries and

as important staple oil in Southern Africa (Van der Vossen et al., 2004).

1.8.3 Distribution and Local names of Melon:

The melon or gourd family is of world-wide distribution in at least warmer

regions of the world, and various people have selected different types for domestication.

It is also the groups that are highly prized for both ornamental and economic purposes.

Squashes and pumpkins are of western hemisphere origin and cucumbers and melons are

from Africa and central America. Other species are grown in the tropics of Asia,

Polynesia and India.

Common names: (English) – melon.

Synonym: Cucumis melo.

Hausa – Egusi

Igbo – Egwusi

Yoruba – Egusi.

Abakaliki – Ahu

Opobo/Rivers – Obokobo

Nsukka – Ekeke

43

1.8.4 General profile of Cucumis melo

Kingdom Plantae – Plants

Subkingdom Tracheobionta – Vascular plants

Superdivision Spermatophyta – Seed plants

Dividion Magnoliophyta – Flowering plants

Class Magnoliopsida – Dicotyledons

Subclass Dilleniidae

Order Violales

Family Cucurbitaceae – Cucumber family

Genus Cucumis L – melon P

Species Cucumis melo L – cantaloupe P

Table 1.4: shows the classification of melon plant.

44

1.9 Rationale of the Study

Lecithin is a by-product of vegetable oil processing. Commercial lecithin is

especially obtained from soybean and other oil-bearing seeds. Soy-lecithin is the most

widely used in industries, and because of the pressure on other nutritional uses of

soybean, alternative sources of plant lecithin have continued to be of interest to many

researchers in many tropical countries that have oil-bearing seeds.

1.10 Research Objective

Nigeria is a major producer of melon seeds and most of the melon seeds are

utilized as constituents of soup, cake, sauce etc. This study looks at a possible extraction

of lecithin from melon seed oil in commercial quantities, its characterization and possible

industrial uses of lecithin.

1.11 Aims of the study

The aims of this work are:

(a) To determine the hull percentage yield of melon seed.

(b) To extract the melon seed oil.

(c) To determine the percentage yield of the melon seed oil.

(d) To characterize the extracted oil.

(e) To determine the physicochemical properties of melon seed oil.

(f) To determine the acute toxicity/lethality test.

(g) To extract lecithin from melon seed oil.

(h) To determine the lecithin percentage yield.

(i) To carry out lecithin solubility test.

(j) To determine the simple test for lecithin.

(k) To carry out Thin layer chromatography (TLC).

(l) To determine the antioxidant properties of lecithin.

(m) To prepare and characterize stable vit E SEDDSs

45

CHAPTER TWO

2.0 Materials and Methods

2.1 Materials

2.1.1 Chemicals/Reagents

All the chemicals used in this study were of analytical grade and were obtained

from Riedel de Haen (Germany), BDH Chemical Ltd (Poole England) Analar Scharlan

Chemie (Spain), Merck Germany and Burgoyne (India).

2.1.2 Equipment/Instrument:

The equipment used for this study were provided in the Departments of Biochemistry,

Faculty of Pharmaceutical Science and Veterinary Medicine and the department of Food

Sciences and technology of the University of Nigeria, Nsukka.

2.1.3 Plant Material

The melon seeds were obtained from Kaduna main Market, Owulipa Itanabo

Market of Benue State and Umualor Town of Uzouwani Local Government Area of

Enugu State respectively.

2.1.4 Preparation of reagents for characterization

Normal Saline

A quantity, 9g of Nacl was dissolved in 100ml of distillated water. [

Wij’s Reagent:

A quantity, 2.0g of iodine trichloride was dissolved in 100ml of glacial acetic acid

and was mixed with 2.25g of iodine dissolved in 100ml glacial acetic acid. The solution

was made up to 250ml with glacial acetic acid.

0.1N KOH:

5.6g of KOH was dissolved in 100ml of distilled water.

0.53N HCl:

46

19ml of conc. HCl was dissolved in 100ml of distilled water

15% Potassium Iodide

A quantity, 15g of KI, was dissolved in 100ml distilled water

1.0% starch indicator

A quantity, 1.0g of starch was prepared with hot water into a gel and made up to

100ml with distilled water.

1M Tetraoxosulphate VI acid

A quantity, 30ml concentrated suolphuric acid was measured and diluted using

170ml distilled water and then made up to 1dm3

using distilled water

Saturated Potassium Iodide

A quantity, 5g was dissolved in 20ml of distilled water until the given volume

cannot dissolve more of the solute.

0.01M. Sodium thiosulphate (Na2S203.5H20)

A quantity, 10g of sodium thiosulphate was dissolved in distilled water and then

made up to 1dm3 using distilled water.

Acetic acid-chloroform

A quantity, 2:1 ratio of acetic acid chloroform mixture was mixed and stored in a

corked flask

TLC spray reagent – Concentrated tetraoxosulphate VI acid.

2.1.5 Extraction of Melon Seed Oil: The extraction of oil from melon seed oil was

carried out using the soxhlet extraction method. In this method, 300g of each variety was

ground and the solvent was added to the extracting flask in 2:1 volume ratio. Each

variety was made to extract for between 5 – 7 hours, at the end of which the solvent/oil

mixture was evaporated and the oil recovered from the solvent.

2.1.6 Determination of Oil Percentage Yield: A quantity 100g of the sample of the

coarse –milled melon seeds were each weighed into soxhlet and oil was extracted for six

(6) hours, at the end of which, the oil was concentrated and weighed

47

100

100

Yield

MlinYieldg

2.1.7 Characterization of the Oil: The oil was characterized for its physicochemical

properties-colour, solubility, viscosity, specific gravity, refractive index, acid value,

peroxide value, iodine value, and saponification values using the official and

recommended method of the American Oil Chemists Society (AOCS) 1990.

2.1.8 Physicochemical properties of the melon seed oil:

Acid Value: Generally, fat dissolves in an appropriate solvent, maximally exposing both

free and bound fatty acids. The acid value (AV) of a fat (or oil) is the number of

milligram of potassium hydroxides required to neutralize 1g of the fat (or oil) without

induced hydrolysis. The amount of the KOH consumed is a measure of the acidity of the

oil or fat. Using Cocks method 1996;

A quantity, 1.0g of the oil was weighed in a 250ml conical flask. This was

followed by the addition of 50ml mixture of 96% ethanol and diethylether and the content

warmed. The oil solvent mixture was then titrated with 0.1N KOH using four drops of

phenolphthalein and then it was swirled until a pink colour which persisted for one

minute was obtained.

Acid value is calculated using the formula:

W

INVvalueAcid

.56

Where

N = Normality of KOH solution

V = Volume (in ml) of KOH

W = Mass (in g) of the oil sample

Saponification value: When an oil is boiled with alkali such as KOH, it splits into

glycerol and the alkali salt of the component fatty acid, this process is known as

saponification. Complete hydrolysis of the fat takes place during refluxing of the fat.

The saponification value is the number of miligrammes of KOH required to neutralize the

fatty acid resulting from complete hydrolysis of Ig of fat. The saponification value gives

48

an indication of the nature of the fatty acid in the fat since only one mole of K+ reacts

with each fatty acid, indicating that the larger the saponification number, the more the

number of fatty acids liberated and the smaller the average chain length per gramme of

the fat (Pearson, 1976).

The method of Pearson (1876) was used. A quantity, I.0g of the sample was

placed into a round-bottom flask. This was heated gently for 30 minutes until

saponification is complete as indicated by the absence of an oil matter and appearance of

clear solution. A quantity, 3 drops of phenolphthalein indicator was added into the hot

soap solution and slowly titrated with 0.53N hydrochloric acid. This procedure was

carried out for the blank using the same quantity of KOH solution and under the same

conditions except that the sample was not added. The test and blank determination were

repeated twice and the average titre value obtained in each case. Saponification value

was calculated using the formula:

W

SBINvaluetionSaponifica

)(.56

Where

W = Mass (in g) of the oil.

N = Normality of hydrochloric acid

S = Volume (in g) of hydrochloric acid used in the test

B = Volume (in ml) of hydrochloric acid used in the blank

Iodine value: A quantity 0.5g portion of the oil was weighed and transferred into a 250

ml glass stoppered bottle. Then, 15 ml of Wijs solution was added to dissolve the oil and

a further 25ml of Wij‟s was added from a burette. The flask was closed and the content

mixed by manually shaking the flask. This was allowed to stand in the dark for 30

minutes.

After standing, 20ml of 15% potassium / Iodide (KI) solution was added and the

bottle stoppered and shaken thoroughly and the sides of the bottle and the stopper were

washed with 100ml of recently boiled and cooled water. Then, the solution was titrated

with a standard 0.1N sodium thiosulphate (Na2S2O3) solution, the reagent being added

with constant shaking until the yellow colour of the iodine has almost disappeared.

Before continuing the titration 2ml of 1% starch indicator was added, when the blue

49

colour had almost disappeared. The bottle was stoppered and shaken vigorously for the

remaining iodine in the organic layer to pass in to the water layer. Two blank

determinations with the same quantities of reagents were carried out at the same time and

under the same conditions. Iodine value was calculated using the formular,

M

VVMvalueIodine

)(69.12 1

Where

M = Molarity of Na2S2O3

M = Mass of oil in grammes

V = Volume of Na2S2O3 used for the blank

V1 = Volume of Na2S503 used for the sample

Perodixe value: In the presence of reactive oxygen species, (ROS) unsaturated fatty

acids undergo oxidation at the double bonds. The combination with oxygen results in the

formation of peroxides, volatile aldehydes, ketones, and acids. The peroxide value is

therefore a test of the degree of deterioration of the fat or oil. It is determined by

subjecting KI at room temperature to the oxidant effect of peroxides. The iodine thus

liberated is titrated with sodium thiosulphate (Pearson 1976).

A quantity 1.0g of the oil was added 2ml of 0.2N KI solution and was swirled to

dissolve the sample. This was be followed by saturated sample that was weighed out and

dissolved in 25ml of Glacial acetic acid –chloroform in 0.2N KI solution. These were

allowed to stand for one minute in the dark. The addition of 35ml distilled water

followed during which a pink colour due to KI disappeared. A starch indicator (2 drops),

were added and the solution turned blue-black. This indicates the presence of peroxide.

The resulting solution was titrated with 0.2N Na2S2O3 solution until the blue –black

colour turned colourless. The process was repeated for the sample and the blank. In the

blank, when 2 drops of starch indicator were added, the solution did not turn blue black

and this indicates the absence of peroxide. The average titre was then taken in each case.

The peroxide value is given by the formula;

W

NBSvaluePeroxide

)(1000

50

Where,

S = Volume (in ml) of Na2S2O3 used in the sample

B = Volume (in ml) of Na2S2O3 solution used in the blank

N = Normality of sodium thiosulphate solution

W = Weight (in g) of the oil.

2.1.9 Acute toxicity/lethality (LD50) Test

Determination of the acute toxicity was carried out using the method described by

Lorke (1983). Three groups of adult albino mice consisting of three animals per group

were used, the extracted oil was injected orally in doses of 10,100 and 1000 mg/kg body

weight for the first three groups to determine the toxic range. The result of this first test

was used as a basis for selecting the subsequent doses of 1600, 2900 and 5000 mg/kg

body weight injected orally for the 4th

group of mice. Animals were fed with the normal

rat feed and water and were observed for any death and changes in general behaviour.

2.1.10 Extraction of Lecithin from Melon Seed Oil: Lecithin was extracted by

measuring 70 ml of melon seed oil into a cleaned and dried conical flask, and heated up

to 700C. At this temperature, 2% of water and some drops of H2O2 was added and stirred

for 1 hour. This is called degunmming of the oil. The presence of H2O2 is to help in

drying the lecithin yield, after which, the lecithin was de-oiled by stirring with acetone.

2.2 Determination of Lecithin Parentage Yield

The total lecithin extract from each of the three regional varieties of Cucumis

melo were weighed and recorded in percentage

1

%100

oilofvolumetotal

yieldlecithinofwtyieldpercentage

2.2.1 Physicochemical Properties of Lecithin

The lecithin extract was characterized for colour, solubility in water and organic

solvents, simple test for it was investigated, thin layer chromatography was done, the

51

antioxidant property-oil stability test was also determined, after which a stable vitamin E

SEDDSs formulation was prepared and characterized.

2.2.1.1 Lecithin Solubility Test: Lecithin is an organic compound that is soluble in

acetone, chloroform, petroleum ether at 40-700C in pure form. However, the solubility

test was carried out using 1g of lecithin in 2ml of water and/or organic solvents. The

melon seed oil lecithin extract was insoluble in methanol, water and ethanol. The table

showing the degree of solubility of lecithin is shown in chapter three.

2.2.1.2 Simple Test for Lecithin

1. A quantity 1g of lecithin was dissolved in 2ml of warm water.

2. It was also identified in the liquid sample by addition of KOH and testing for

choline

3. 2ml HCL was added for the precipitation of the fatty acids, after specification

with KOH and filtering

4. The filterate was tested for phosphate as follows:-

(a) Lecithin solubility in water, ether, ethanol, chloroform, methanol and acetone was

carried out.

(b) The phospholipids was precipitated by adding 2ml saturated calcium chloride

(Cac12) to an emulsion of lecithin in water

(c) 3 ml of lecithin was saponified with 3ml of 1% sodium carbonate (Na2CO3) for 10

minutes.

(d) The addition of 2ml hydrochloric acid (HCL) to the soap, precipitates the fatty

acids.

(e) The mixture was filtered and 1ml of 5% ammonium molybdate was added and a

brilliant yellow precipitate indicates the presence of phosphate. The degree of

phosphorus content is shown in chapter three.

2.2.1.3 Thin layer chromatography (TLC): Thin layer chromatography is fast and

gives a high resolution and compact spot due to very fine particle size of the stationary

phase media. The thin layer plates were prepared by applying aqueous slurry of the

52

chosen medium (silica gel) onto a clean glass plate using a spreader. The most active

form of silica gel was produced and layered on the chromatographic plate, after which it

is allowed to dry in an oven at 100-1100C for about 2hrs. It is worthy to note that the

plate was about 2mm thick with a size of 20cmx20cm. The choice of the solvent used to

run the chromatogram was based on the nature of the lipid desired. For lecithin, 90ml of

n-hexane was mixed with 10ml of diethyl ether and 1ml of glacial acetic acid. This

solvent was poured into the chromatographic tank and plates were spotted 2cm from the

origin and placed in the tank. The tank was then covered with glass that had been air

tight with grease. Development was allowed to continue by the solvent moving upward

by capillary action until the solvent front had traveled the required distance. The plate

was then removed and the solvent front was marked with a pencil before allowing the

plate to dry. The dry plate was sprayed with concentrated sulphuric acid (H2SO4) and the

relative mobility (Rf) of the lecithin was viewed and calculated.

2.2.1.4 Antioxidant Property of Lecithin –Oil Stability Test

The active oxygen method (AOM) employed a specially designed apparatus

conforming to the AOCS specification. In this method, approximately 20ml oil was

placed in an aeration tube in a water bath (97.80C) while a controlled flow of dried

filtered air was bubbled at 2.33ml/second through the sample. Oil sample was withdrawn

periodically and examined for peroxide value according to AOCS standard method.

Results are reported in hours for samples to reach peroxide value of 100ml Eq/kg of oil.

In the test which involves 0.01g -0.05g concentrations of lecithin each in 20ml oil, for

pure lecithin (control) and lecithin extract respectively. At the end of every 1 hour, each

test tube‟s lecithin/oil mixture was tested for peroxide value. A graph of the lecithin

concentration against the peroxide values of the control and test showed the antioxidant

property of lecithin.

2.2.1.5 Preparation of Stable Vit. E SEDDSs: A series of vitamin E SEDDSs were

prepared with fixed concentration of vitamin E, (0.0155mg), varying concentrations of

melon seed oil and tween 80. Vitamin E was dissolved in the amount of melon seed oil

and the tween 80 were accurately weighed and added to vitamin E/oil solution. The

53

mixture was stirred using magnetic stirrer until a state of isoropicity was reached. The

mixture was put in a conical flask and warmed in a water bath at 370C for 30 minutes.

After the said time, the solution was titrated against with warm distilled water. The

titration and agitation of the oil, vitamin E and surfactant mixture will continue until a

stable white emulsion is obtained. At this end-point, the titre will be calculated and the

emulsion characterized.

54

2.2.1.6 Stable Vit E SEDDs Composition

S/No Vit E

(Mg)

Melon seed

oil (mg)

Tween 80

(mg)

Distilled

water (mg)

Total (mg)

1 0.0155 0.6340 0.0767 0.1209 0.2765

2 0.0155 0.5440 0.0658 0.0799 0.2355

3 0.0155 0.4530 0.0548 0.0799 0.1955

4 0.0155 0.3660 0.0432 0.0799 0.1754

5 0.0155 0.0272 0.0329 0.0568 0.1324

Table 2.1: The stable Vit E SEDDSs formulations contain synthetic Vit E, Melon

seed oil, surfactant (tween 80) and distilled oil/water emulsion.

55

2.2.1.7 Characterization of SEDDSs

The primary means of self-emulsification assessment is visual evaluation. The

efficiency of self-emulsification could be estimated by determining the rate of

emulsification, droplet-size distribution and turbidity measurement. However, this stable

Vitamin E SEDDSs was characterized of the following:-

(1) Visual assessment/Appearance: This helps to provide important information about

the self emulsifying and micro emulsifying property of the mixture and about the

resulting dispersion.

(2) Droplet/particle Size: This is a crucial factor in self-emulsification performance

because it determines the rate and extent of drug release as well as the stability of

the emulsion. Photon correlation microscopy, microscopic techniques or a Coulter

Nanosizer are mainly used for the determination of the emulsion droplet/particle

size which was determined using a photomicrograph with a suitable clean slide and

was viewed with 40mm objectives microscope.

(3) Turbidity Determination: This is to identify efficient self-emulsification by

establishing whether the dispersion reaches equilibrium rapidly and in a

reproducible time. The method which used the fuller‟s earth standard was used in

the determination. This was performed on the SEDDSs which have passed the

visual observation test (marked good). In the turbidity measurement, the amount of

scattered light (when an incident light is subjected to strike small particles) is

measured and used in turbidity calculations as per the Rayleigh‟s theory. Light

scattering by colloids conforms to Rayleigh‟s theory, which predicts that light

scattering or measured turbidity (r) in a simplified equation can be given by r =

knv2 in which k is a machine constant, v is particle volume and n is the number of

particles. The turbidity measurements may be reasonable compromise when

dissolution of drug in SEDDSs cannot be measured due to poor solubility of drug.

(4) pH Measurement:- Generally, SEDDSs formulations are checked of their pH

range. This is usually done so as to ascertain the degree of bioavailability of the

incorporated drug. It is a common knowledge that the pH of the human stomach is

acidic, and the charge of the oil droplets in conventional SEDDSs is negative due to

56

the presence of free fatty acids. Hence, some droplets of 0.1N hydrochloric acid are

sometimes used to make the pH of SEDDSs acidic to facilitate absorption and

bioavailability in the stomach.

(5) Viscosity Determination:- The viscosity of SEDDSs is its ability to form lubricant

films. It is the ratio of shear stress to shear rate or simply the flow resistance to

SEDDSs. The kinematics viscosity is directly observed in capillary tube

viscometers. Viscosity is a function of molecular size and orientation since it

increases with increase in saturation range and chain length of fatty acids. The

viscosities of the formulations were determined with Gallenkamp universal torsion

viscometer after 5% volume/volume distilled water dilution. The result obtained

showed that the viscosity of the Vitamin E SEDDSs formulations increases as the

concentrations of the oil/surfactant ratios increase.

57

CHAPTER THREE

RESULTS

3.1 Percentage Yield of the Oil.

Table 1: shows the variety and percentage oil yield of the three regional varieties of the

Nigeria. Cucurbitaceous seeds Cucumis melo (melon)

Variety Percentage yield of the oil

Benue 46.00%

Enugu 41.67%

Kaduna 46.30%

The percentage yield of oil is high in Kaduna (46.30%) when compared to that in Benue

(46.00%) and Enugu (41.67%) which is the smallest of the three varieties.

58

3.2: The hull percentage yield of the melon seeds

Table 2 shows the hull percentage yield of the three regional varieties of Cucumis melon.

(melon)

Variety Hull percentage yield per 100g of melon

seed

Benue 34.00%

Enugu 31.50%

Kaduna 36.00%

The hull percentage yield of the melon seeds is high in Kaduna variety (36.00%) when

compared to Benue variety (34.00%) and Enugu variety (31.50%).

59

3.3: Physical properties of the three varieties of the Cucumis melo (melon).

Table 3 shows the physical properties of the three varieties of the Cucumis melo (melon).

Variety Colour Viscosity Specific gravity Refractive

index

Odour

Benue Yellow 368.47 0.945 0.0080 Odourless

Enugu Yellow 290.19 0.896 0.0075 Odourless

Kaduna Yellow 358.01 0.956 0.0118 Odourless

The specific gravity of the oil was found to be between 0.896-0.956, while the refractive

index was 0.0075-0.0118. The colour was yellow and odourless in all the samples.

60

3.4: Chemical Properties of the three regional varieties of Cucumis melo. (melon).

Table 4 Showed the Chemical Properties of the three regional varieties of Cucumis melo.

(melon).

Variety Acid value

(mgKOH/g)

Iodine value

(Wijs)

Saponification

(mgKOH/g)

Peroxide

value

Ester value

(mgKOH/g)

Benue 1.112 119.9 181.0 8.00 180.0

Enugu 0.561 116.5 195.0 16.0 194.0

Kaduna 1.122 129.0 123.0 8.00 122.0

Ester value is the difference between the saponification value and the acid value.

The acid value was found to be between 0.561 – 1.22, the iodine value was between

116.5 – 129.0. The saponification value was found to be between123.0 – 195.0, the

peroxide value was between 8.0 – 16.0, while the ester value was found to be 122.0 –

194.0.

61

3.5 Percentage Yield of Lecithin

Table 5 shows the percentage yield of lecithin from the three regional varieties of

Cucumis melo (Melon).

Regional variety Percentage yield of lecithin

Benue (B) 0.49%

Enugu (E) 0.55%

Kaduna (K) 0.70%

The percentage yield varied varied from 0.49 – 0.70%.

62

3.6 Phosphate Test of the three Regional Varieties of Cucumis melo (Melon)

Table 6 shows the result of the phosphate test of the lecithin extract.

Regional variety Phosphate test

Benue (B) ++

Enugu (E) +

Kaduna (K) ++

Key + - dull yellow

++ - faint yellow

+++ - bright yellow three regional varieties of Cucumis melo (melon)

There were varying amounts of lecithin in the three varieties of the melon seed oil.

63

3.7 Solubility of lecithin in water and organic solvents

Table 7 shows the solubility of the lecithin extract in water and organic solvent.

Lecithin extract Acetone Chloroform Pertroleum ether Methanol Water

Benue ++ ++ ++ + +

Enugu ++ ++ ++ + +

Kaduna +++ +++ +++ ++ +

Std lecithin +++ +++ +++ +++ +++

Key +: Insoluble

++: Slightly soluble

+++: Soluble

The lecithin dissolved very well in all the solvents and was comparable to the standard

lecithin. However, they are insoluble in water and methanol.

64

3.8 Result of the Acute Toxicity Test

Table 8 shows toxicological studies result of melon seed oil

Dosage mg/kg body weight = usedanimalsofNumber

animalsDeadofNumber

S/No Dosage No of Death Recorded

1 10 0/3

2 100 0/3

3 1000 0/3

4 1600 0/1

5 2900 0/1

6 5000 0/1

The result shows that up to the dosage of 5000mg/kg. Body weight, no death was

recorded and that means the oil is not toxic.

65

3.9 Isotropicity /Stability Test

Table 9 Shows the isotropicity test of the stable vitamin E SEDDSs formulations

S/no Oil/surfactant mixture Concentrations (Ratio) Remark

1 Oil/surfactant mixture 1: 9 Bad

2 “ ” 1: 4 Bad

3 Oil + Surfactant 1: 2 Good

4 “ ” 2: 3 Good


6 “ ” 3: 2 Good


8 “ ” 4: 1 Bad

9 Oil + Surfactant 9: 1 Bad

10 “ ” 10: 0 Bad

The result shows that formulations 1,2,8,9 and 10 described as „‟bad‟‟ could not attain

equilibrium, and therefore did not form good emulsions. The ones marked good-

formulations 3,4,5,6 and 7 attained the isotropicity test and form „‟good‟‟ emulsions

called Vit E SEDDSs.

66

3.10 Physicochemical properties of the stable Vit E SEDDSs Formulations

Table 10 shows the physicochemical properties of the vitamin E. SEDDSs formulations

Formulation

number

Colour Viscosity

(Cps)

Concentrations

of

oil/surfactant

ratios

Visual

observation

pH Mean

turbidity

unit

(Mg/L)

Mean

droplet

size (μm)

1 Yellow - 1: 9 Bad - - -

2 White - 1: 4 Bad - - -

3 White 195 1: 2 Good 6 2400±0.2 42.0 ± 0.2

4 White 196 2: 3 Good 6 4400±0.3 24.2 ± 0.1

5 White 197 1: 1 Good 6 5000±0.1 72.4 ± 0.2

6 White 196 3: 2 Good 6 4000±0.2 36.5 ± 0.1

7 White 195 2: 1 Good 6 4600±0.1 48.0 ± 0.3

8 Yellow - 4: 1 Bad - - -

9 Yellow - 9: 1 Bad - - -

10 yellow - 10: 0 Bad - - -

3.10 Physicochemical properties of the Stable Vit E SEDDSs Formulations

The viscosity of formulation number (5) is high when compared to other good

formulations. Similarly, the pH of the good formulation is acidic. The mean turbidity of

formulation number (5) and its mean droplet size are high with 5000mg/L and 72.4+

0.2μm respectively when compared to other good formulations.

67

CHAPTER FOUR

4.0 DISCUSSION

The studies on the extraction of melon seed oil and lecithin was carried out to

ascertain the possible effect of soil type and location on various oils/ lecithin parameters.

The percentage yield of the oil was 46. 30%. This amount is promising and agrees with

earlier findings (Ononogbu; 2002) and (Orasenaya, 2006). Again, the high yield of the oil

suggests that the oil from melon seed may have some industrial and pharmaceutical

applications. The Production would be feasible and this may likely compete with the

imported soybean oil which is more expensive. The use of n-hexane as a solvent in the

extraction of total lipid has remained the best solvent if the oil is targeted for industrial

use. This is because; n-hexane oil-extract has toxicological acceptability, relative

selectivity for triglycerides and ease of recovery, (Njoku et al, 1998).

The results of the physicochemical properties of melon seed oil are shown in

Table 3.3 and Table 3.4. From the result, Melon seed oil has a yellow colour and specific

gravity of .896-956 and refractive index of 0.0075-0.0118. The viscosity of the oil is

290.19 -368.47. The saponification value of the oil was between 181-1123 mg KOH/g,

which falls within the range of values obtained for some vegetable oils 188-225 mg

KOH/g (Aremu et al, 2006). The iodine value of melon seed oil (116.5-129.0 Wijs), is

comparable to values earlier obtained for Arachis oil and Cotton seed oil respectively

(Pearson 1976). In view of the fact that oils generally present with iodine values of above

100. Wijs (Duel, 1951; Simpson and Corner- Orgarzally, 1986), melon seed oil could

therefore be categorized as semi-drying oil, thus, making it suitable for utilization in

certain industrial formulatory and dietary purposes, (Ibiyemi et al, 1992).

The toxicity test carried out with the extracted oil using albino mice indicated that

there was no significant (P<0.05) modification in the general behaviour of the animals to

which the oil was administered up to 5000mg/ kg body weight of the animals (Table 8).

The result of this study suggests strongly that melon seed oil is safe and edible. The result

equally provides support for its continued wide usage and acceptability of melon seed

68

and oil as essential part of the staple food in Nigeria, Southern Africa and Asia

(Orasenaya,2000; Ononogbu,2002).

Lecithin was extracted from the melon seed oil, with the yield

of(1.17%>1.02%>0.72%) obtained for Kaduna, Benue and Enugu respectively. This was

confirmed by the thin layer chromatography and solubility test carried out with the

extracts and standard lecithin. The cause of the low yield is not clear, though it may

however be attributable to the handling manner, method of extraction of the melon seed.

Nevertheless, the study produced (0.70g), (0.55g) and (0.49g) per 100ml of the oil for

Kaduna, Benue and Enugu respectively.

The result of the antioxidant property shows that it has antioxidant effect and can

be a good source of antioxidants (preservatives) needed in industries for protection of oils

from rancidity.

Evaluation of SEDDSs for isotropicity / stability/ phase separation Test: The ten batches

of SEDDSs formulations consisting of melon seed oil to surfactant (Tween 80) of the

various stated ratios based on the visual observation for isotropicity/stability test showed

50% :50% success and failures respectively. This then serves as a selection process for

thermodynamically stable SEDDSs which can then be assessed for other performance

parameters applicable to SEDDSs. In this study, the SEDDSs formulations of serial

numbers 1,2,8,9 and 10 were shown to have failed the isotropicity test, and were

remarked as bad concentration ratios.

However, the five batches of melon seed oil/surfactant formulations did not show

any degree of phase separation, and were said to be isotropically stable. These batches

were added 0.0155mg of vit E which after agitation and titration with distilled water at

37oc maintained the stability of the SEDDSs formulations over a long period of

time(Attama et al.,2003).

Physicochemical Properties of the Stable Vit E SEDDSs formulations: The white

colour of the stable Vit E SEDDSs formulation marked good relative to the yellow colour

of the formulations marked bad confirmed that the SEDDSs formulations are emulsions.

The viscosity result in the Centipoints obtained showed the range of 165-167Cps.

This relates well to the thinly viscous emulsions produced where the external phase

medium is a gel like the vit E added. The thinly viscous emulsions have a viscosity range

69

of 120-200Cps(Taha et al.,2004). The concentration of the oil/surfactant ratio is a strong

determinant of colour, viscosity, visual observation remark, mean turbidity, stable as well

as mean droplet size. Also, the droplet/ particle size indicated that increasing the

concentrations of Tween 80 in the SEDDSs formulations resulted in an improved

emulsion with smaller particle size. Such a decrease in droplet size might be the result of

more surfactant being available to stabilize the oil-water interface.

The result of the study showed that as the concentrations of the oil/surfactant

ratios increase uniformly, other parameters of SEDDSs outside the particle size also

increase. The study also revealed that because the drug delivered was vit E which has

antioxidant, and which blends well with the natural melon seed oil which was also very

rich in antioxidant vitamins. These mixtures proffered a powerful synergism which

results in the vit E SEDDSs remaining stable over months(Attama et al., 2003).

The mean turbidity unit in mg/L of 500±0.1 and 2400±0.2 were recorded as

highest and lowest respectively. Also, the mean droplet particle size range 24.2±0.1 -

72.4±0.2 was recorded by this study and it compared well with a arrange of 30±4.04 -

411±8.08 reported by (Taha et al., 2004 and Taha, 2009).

Pharmaceutically, lecithin and some vegetable oils are used as excipients and they

play extensive roles in great numbers of drug manufacture. Also, the melon seed oil

played a vital role in the self-emulsifying drug delivery systems (SEDDSs) which

enhances the bioavailability and absorption of most lipophilic drugs. The result of the

appearance, pH, viscosity, particle size, turbidity and stability of SEDDSs showed that

melon seed oil, though it has not been investigated, it is not irrelevant to mention here

that it is a novel pharmacopoeial vegetable oil for it compared very well with the known

ones like cotton seed, soyabean oils, etc.

Precisely, outside the good nutritional status of melon seed oil, it has a high

potential of being a cheap and local source of lecithin in Nigeria, Africa, and Asia. The

promotion of cultivation of melon seeds should be improved to help increase oil, cake,

and lecithin production in Nigeria, so as to help motivate cottage industries lessen over-

dependence on fossil fuel, save foreign reserve spent on soybean lecithin importation, and

improve our food, paint, textile, plastic and pharmaceutical industries and agriculture.

Lecithin extraction from melon seed oil produces appreciable quantity, but for the effect

70

of temperature and choice of solvent. Most Organic solvent like chloroform/methanol,

petroleum ether etc is able to extract melon seed oil but not appreciable lecithin. The

basic cause of low lecithin yield was the discarding of the sludge that generally settle at

the bottom of the flask during extraction with Chloroform/methanol as the extracting

solvent. Care should also be taken not to use high temperatures during extraction for it

diminishes lecithin yield as well as elevate the oil temperature close to the frying

temperature thereby making lecithin extraction very difficult.

Apparently, fresh melon seeds form the farm are not good for lecithin extraction

for they contain much water, and melon seed oil extracted from fresh seeds are prone to

fungal deterioration. Conversely, very old seeds from harvesting time are not also good

since they do not yield quality lecithin. Maximal lecithin yield from melon seeds oil is

obtained from melon seeds grown in rocky soil, which should be harvested and more than

half the seed‟s water content are dried.

Certainly, it is to the interest of a Biochemist, Pharmacist, Cosmetist and other

food processors to know the acid values, peroxide value, iodine value, specific gravity

and colour of lecithin before using it for their various purposes. This is to avoid getting

wrong results in their targeted usage.

4.1 CONCLUSION

The present study shows that the melon seed oil contains appreciable quantity of

lecithin. There is also a variation in the contents of the melon seed oil with respect to

changes in soil nutrients, agronomical methods as well as changes in geographical

locations as regards relief. It also exposes the industrial, agricultural and health uses of

lecithin.

4.2 SUGGESTION FOR FURTHER RESEARCH

(1) Melon Seed Oil formulations as fertility improver

(2) The Application of Melon Seed Oil in making skin Care Products

(3) Melon extracts as anti-aging formulations.

71

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APPENDICES

Figure A.1: Melon fruit in the farm intercropped with cassava.

81

Figure A.2: Melon seed during drying in the sun

82

Figure A.3: Shows the three regional varieties of lecithin extract

Key: K = Kaduna B = Benue E = Enugu

K B E

83

Hexane-extracted crude oil

Filtered

Filtered crude oil

H20/Heat to 700C

Hydrated phosphatides

Centrifuge

Separated lecithin sludge

H202

Bleached lecithin

Dried

Lecithin

Figure A.4: Flow chart of lecithin extraction from melon seed oil

84

Figure A.5: Graph of Standard lecithin and refined soybean oil

85

Figure A.6: Graph showing the antioxidant property of lecithin –oil stability test

86

Figure A.7: Photomicrograph of oil/surfactant ratios of 1:1 1:3: 2:1 2: 3 3:2 ( x 400)

87

Figure A.8: Shows the TLC Chromatogram of the three lecithin extracts from three

Nigerian regional varieties of Cucurbitaceous seeds (Melon) Cucumis melo. The

chromatogram shows that the Rf of the Kaduna lecithin > Benue lecithin > Enugu

lecithin.

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EXTRACTION, CHARACTERIZATION AND INDUSTRIAL USES … WORK MICHO... · extraction, characterization and industrial uses of lecithin from three varieties of cucumis melo (melon seed)