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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
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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.
.
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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.
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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.
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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
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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
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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
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CHAPTER FOUR
4.0 Discussion - - - - - - - - 67
4.1 Conclusion - - - - - - - - 70
4.2 Suggestion for further research - - - - - 70
References - - - - - - - - - 71
Appendices - - - - - - - - - 80-88
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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
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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
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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
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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
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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
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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,
(Source: Nelson, 1981)
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
(Source: Nelson, 1981)
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
5 Oil + Surfactant 1: 1 Good
6 “ ” 3: 2 Good
7 Oil + Surfactant 2: 1 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.