EVALUATION OF ANTIOXIDANT POTENTIAL OF MONODORA MYRISTICA (AFRICAN NUTMEG)

BY

IBOLO MARTHA NNEKA

BC/2006/076

DEPARTMENT OF BIOCHEMISTRY

FACULTY OF NATURAL SCIENCES

CARITAS UNIVERSITY,

AMORJI-NIKE, ENUGU STATE.

AUGUST, 2010

1

TITLE PAGE

EVALUATION OF ANTIOXIDANT POTENTIAL OF MONODORA MYRISTICA (AFRICAN NUTMEG)

BY

IBOLO MARTHA NNEKA

BC/2006/076

A PROJECT SUBMITTED TO THE DEPARTMENT OF

BIOCHEMISTRY

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

AWARD OF BACHELOR OF SCIENCE (B.Sc.) DEGREE

IN BIOCHEMISTRY

AUGUST, 2010.

2

APPROVAL PAGE

We hereby certify that the research work in this report is solely the work of IBOLO

MARTHA NNEKA with Registration Number BC/2006/076 for the award of Bachelor of

Science Degree (B.Sc.) in the Department of Biochemistry, Caritas University Amorji-

Nike, Enugu state.

…………………………... ……………………………

Dr. Charles N. Ishiwu Date

(Supervisor)

………………………….. ……………………………

Mr. Moses Ezenwali

(Head of Department) Date

…………………………… ……………………………

External Examiner Date

August, 2010

3

DEDICATION

This work is dedicated to Almighty God for His faithfulness to me and for seeing me

through, in this citadel of learning.

4

ACKNOWLEDGEMENT

I give joyful thanks and praise to Almighty God who in spite of all odds, made this project

research a reality. He has always been faithful to me. My special appreciation goes to my

supervisor, Dr. Charles N. Ishiwu for his warmly guidance and supervision. His unreserved

kindness and understanding not only inspired me but also encouraged me throughout the

trying moments of this work. I also wish to express my indebtedness to my Honourable

HOD, Mr. Moses O.Ezenwali for his unrelentful assistance throughout my laboratory work

and who contributed in no small measure to the completion of this work.My sincere

gratitude goes to my most learned and zealous lecturers, Mrs. Oluchi Ajemba, Mr. Eze-

steven Peter, Dr. Ikpe, Mr. P. Ugwudike, and Mr. Omeh Yusuf.

My most profound gratitude goes to my loving and caring parents, Sir and Lady P. O. Ibolo

for their moral and financial support, whose love for education brought me to this citadel of

learning. My special thanks also goes to the managing director, Aniuzo international

limited (Palm Kernel Oil Mills Division), Emene, who source partly for the raw material

used for this research. My thanks also goes to my caring chancellor Rev. Sr. Kate Ikenga

for her advice and support to me. I thank my siblings: Blessing, Elizabeth, Anthony and

Christopher, who have been an everlasting source of love, strength and encouragement. I

wouldn’t forget to appreciate my colleagues, friends who have contributed inestimably

throughout the course of this work. In this respect, I am grateful to Patricia, Chidera, Kofi,

Chijioke, Tony, Chizoba, Peace, Modesta, and a host of others.

IBOLO MARTHA NNEKA

5

ABSTRACT

This work evaluates the antioxidant potential of Monodora myristica (African nutmeg).

Monodora myristica extract was obtained by solvent extraction using n-hexane and used as

treatment on freshly prepared crude palm kernel oil and palm oil. Equal volume of oil

samples were subjected to different concentration of extract treatment (0.2ml,0.4ml, 0.6ml,

0.8ml, 1.0ml using syringe. These oil samples were equally divided into two groups SS and

SR. Group SS was stored under the sun and group SR was stored in the room for three

weeks. These treated oil samples were analyzed on weekly basis at two different

parameters: Acid value (AV) of free fatty acid and thiobarbituric acid (TBA) value, using

standard methods. The main effect of extract was determined using ANOVA. For the two

varieties of oil, the acid value of free fatty acid increased significantly (P<0.05) as the

period extends for group SS without extract while those for group SR showed no significant

increase. But AV of oil samples treated with higher extract concentration decreased

significantly (P<0.05) for both groups SS and SR. TBA value also showed the same trend

of AV. Hence, monodora myristica extract yielded reducing effect in the oxidative level of

the oil varieties.

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TABLE OF CONTENTS

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

Approval page --------------------------------------------------------- ii

Dedication --------------------------------------------------------- iii

Acknowledgement --------------------------------------------------------- iv

Abstract --------------------------------------------------------- v

Table of content --------------------------------------------------------- vi-viii

List of tables -------------------------------------------------------- xi

List of figure -------------------------------------------------------- x

Abbreviation -------------------------------------------------------- xi

CHAPTER ONE

1.0 Introduction --------------------------------------------------------- 1

1.1 Significance of study ----------------------------------------------- 6

CHAPTER TWO

2.0 Literature Review ------------------------------------------------ 7

2.1 African nutmeg (Monodora myristica) ---------------------------- 7

2.1.1 Scientific classification ------------------------------------------ 7

2.1.2 Habitat/ ecology of Mondora myristica ---------------------------- 8

2.1.3 Characteristics/morphology of monodora myristica ------------- 8

2.2 Oil Palm --------------------------------------------------- 9

2.2.1 Scientific classification ----------------------------------------------- 10

2.2.2 Origin and description of palm oil --------------------------------- 10

2.2.3 The Chemical composition of palm oil -------------------------- 11

2.2.4 Physical characteristics of palm oil products ------------------ 14

7

2.3 Palm kernel oil ------------------------------------------------------ 14

2.3.1 The chemical composition of palm kernel oil------------------- 15

2.4 Modern uses of palm oil and palm kernel oil------------------- 16

2.5 Lipid oxidation----------------------------------------------------- 16

2.5.1 Lipid oxidation pathway ------------------------------------------ 21

2.5.2 Mechanism of oxidation ------------------------------------------ 22

2.6 General antioxidant action --------------------------------------- 24

2.6.1 Mechanism of antioxidant action ------------------------------ 24

2.6.2 Antioxidant molecules -------------------------------------------- 26

2.7 General review of photochemistry of monodra myristica -- 27

2.7.1 Alkaloids ---------------------------------------------------------- 27

2.7.2 Flavonoids -------------------------------------------------------- 27

2.7.3 Glycosids -------------------------------------------------------- 28

2.7.4 Saponins ---------------------------------------------------------- 28

2.7.5 Tannins ------------------------------------------------------------ 29

2.8 Application of vegetable oils ----------------------------------- 29

2.8.1 Factors that cause oxidative rancidity in vegetable oil ------ 30

2.9 Nutritional signification ----------------------------------------- 33

CHAPTER THREE

3.0 Materials and methods -------------------------------------------- 34

3.1 Equipment/apparatus ---------------------------------------------- 35

3.2 procedurement of raw materials --------------------------------- 35

3.3 Study design --------------------------------------------------------- 37

3.4 Sample preparation ------------------------------------------------ 37

3.5 Chemical analysis ------------------------------------------------- 38

3.5.1 Determination of acid value (Av) ------------------------------- 38

3.5.2 Determination of thiobarbituric acid number.------------------ 38

8

3.6 Statistical analysis ------------------------------------------------ 39

CHAPTER FOUR

4.0 Result and Discussion ------------------------------------------- 40

4.1 Changes in Acid value of Palm Kernel and palm oil -------- 46

4.2 Changes thiobarbituric acid value of palm kernel and palm oil--- 46

4.3 Effect of monodora myristica extract on the

chemical indices of oil on storage -------------------------------- 47

CHAPTER FIVE

5.0 Summary and conclusion -------------------------------------------- 48

5.1 limitations--------------------------------------------------------------- 51

5.3 Future recommendation ---------------------------------------------- 51

References ------------------------------------------------------------------------- 53

Appendix I -------------------------------------------------------------------------- 62

Appendix II ------------------------------------------------------------------------- 63

Appenedix III ---------------------------------------------------------------------- 65

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LIST OFF TABLES

Table 1: Fatty acid composition of palm oil (palm oil)

Table 2: Fatty acid profile of palm kernel oil (palm kernel )

Table 3: Acid value for palm kernel oil

Table 4: Acid value for palm oil

Table 5: Thiobarbituric acid value for palm kernel oil

Table 6: Thiobarbituric acid value for palm oil

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LIST OF FIGURES

Figure 1: African nutmeg seeds (Monodora myristica)

Figure 2: Africa Oil palm fruits (Elaeis guinesis)

Figure 3: Lipid oxidation pathway

Figure 4: Dried seed kernels of Afican nut meg.

Figure 5: Transverse section of palm fruit

Figure 6: n-Hexane extract of Monodora mystica

Figure 7: Crude palm oil

Figure 8: Crude palm kernel oil

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ABBRERVIATIONS

AOCS: Association of America Chemistry Society

AV: Acid value

FFA: Free fatty acid

PV: Peroxide value

PKO: Palm kernel oil

PO: Palm oil

PUFA: Polyunsaturated fatty acid

ROS: Reactive oxygen specie

SR: Storage in room

SS: Storage in sun

TBA: Thiobarbituric acid

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CHAPTER ONE

1.0 INTRODUCTION

Lipid oxidation is one of the major reasons that food deteriorate and is caused by the

reaction of fat and oil with molecular oxygen, leading to off-flavours that are generally

called rancidity(Basturk et al., 2007). Exposure to light, pro-oxidants and elevated

temperature will accelerate the reaction (Kubow, 2009). Lipid oxidation and resultant

flavour impairment has seriously limited the storage potential of most fat containing foods

(Ihekoronye and Ngoddy, 1985).

Rancidity covers a wide range of biological activities where the effect is to “make

things worse” and thus adversely affect man’s economy. Free radicals and microorganisms

are known to cause chemical characteristics that lead to oxidation and deterioration in

quality of vegetable oils derived from the seeds or fruits pulps of plants (Basturk et al,

2007). The keeping quality of the oils is basically dependent on their chemical

compositions, for instance, the percentages of the degree of unsaturation. Rancidity is

associated with off-flavour and odour of the oil. There are two causes of rancidity. One

occurs when oil reacts with oxygen and is called oxidative rancidity. The other cause of

rancidity is by the combination of enzymes and moisture. Enzymes such as lipase liberate

fatty acids from the triglyceride to form di and/or monoglycerides and free fatty acids and

such liberation of fatty acid is called hydrolysis, hence hydrolytic rancidity.

13

The oxidation of fats is an important deteriorative reaction with significant

commercial implications in term of product value. The initial oxidation products that

accumulate are hydroperoxides, which may subsequently break down to form lower-

molecular weight compounds such as alcohols, aldehydes, free fatty acids and ketones,

leading to autoxidative rancidity. The peroxide content present in alimentary fats attests to

its state of primary oxidation and thus its tendency to go rancid. Unsaturated fatty acids, in

fact, react with oxygen forming peroxides, which determine a series of chain reactions

whose end result is volatile substances having the characteristic smell of rancidness. These

reactions are accelerated by high temperatures and by exposure to light and oxygen (Yildiz

et al., 2002). The lower the peroxide and acid values, the better the quality of the

alimentary fats and their state of preservation.

Although simple, procedures of acid value (AV) or peroxide value (PV)

determination are cumbersome, destructive to the sample, costly, require potentially

hazardous solvents, substantial personnel time, glassware and accurate preparation of

reagents and are dependent on a visual endpoint (Ismail et al., 1993; Van de Voort et al.,

1994).

Oxidation is concerned mainly with unsaturated fatty acids. Oxidative rancidity is of

special interest as it leads to the development of off-flavour that can be detected early on in

the development of rancidity (Basturk et al., 2007)

Some slight deterioration at least is to expected in any commercial oil-bearing material and

is, in fact, inherent in the process by which fat is formed (Morel,1997). In the living plants

and animals, fats, carbohydrates and proteins are synthesized in a complicated series of

14

steps with the aid of certain enzymes. These enzymes are capable of assisting the reverse as

well as the forward reactions and hence under proper conditions may promote the oxidation

and degradation of the very substances that, they have previously been instrumental in

synthesizing (Basturk et al., 2007)

Oils in general are known to be susceptible to oxidation and microbial attack. The

composition of the various oils determines the extent of oxidation and type of organisms

likely to thrive in them (Chow et al., 2000). Several studies have demonstrated that

environment factors affect not only the fatty acid composition of vegetable oil, but also,

although apparently indirectly, the spatial arrangement of those acids on the triacylglycerol

molecule (Tay et al., 2002). Triacylglycerol composition and structure are important in the

areas of nutrition, oil stability and possible physiological effects.

Palm oil is extracted from the mesocarp of the fruit of the oil palm, Elaeis

guineensis. crude palm oil (CPO) has a deep orange-red colour due to the high content of

carotenoids and is a rich source of vitamin E consisting of tocopherols and tocotrienols

(Nesaretnam and Muhammad, 1999). Both beta carotenes and vitamin E are well known

nutritional antioxidants.

Palm oil is known to support the growth of fungi and bacteria especially when it

contains moisture (Cornellus, 2001).. Their lipolytic enzymes are so active that even under

unfavorable conditions palm oil is seldom produced with a free fatty acid content (FFA) of

less than 2% and under favorable conditions of processing, the free fatty acid content of this

oil reaches 20%and higher. When the fruit is bruised, lipolytic action occurs and a near

maximum FFA (8-10%) is reached within 40 minutes. The FFA of unbruised fruits may

increase only 0.2% or less in the course of 4 days (Cornellus, 2001).

15

The exposure in the sun is made under radiations of weak temperatures, varying

daily, creating an environment favourable to the chemical and enzymatic reactions of

hydrolysis and oxidation (Tan et al., 2002).

This study is aimed at examining the oxidative and biodeteriogenic effects of free radicals

contaminating the oils from the varieties of the oil palm (Elaeis guineensis) and palm

kernel oil and the chemical components of the oils and the effect of solvent extract of ehuru

(African nutmeg).

Oil palm is indigenous to the Nigerian coastal area. It was discovered by European

explorers in the early 1400’s and was distributed throughout tropical Africa by humans who

practiced shifting agriculture about 5000 years ago. The palm plant originated from the

jungle forest of East Africa and about 5000 years ago, palm oil was used by the pharaohs

for cooking and lighting. The cultivation of oil palm is restricted to the eastern sub zones

where its growth is favoured environmentally and climatically. Besides, it is a major cash

crop in this region. The first oil palm plantation was established at Sumatra in 1911 and at

Malaysia in 1917. About this time it was simultaneously established in West Africa and

tropical America.

Over the years, a little attention was paid to the industrial use of palm kernel oil.

Nevertheless, recent studies have indicated that apart from their domestic uses that they can

be used as engine lubricants, as replacement for biodiesel if their properties are enhanced.

Although high in saturated fats, it is a different oil to extract from the nut or kernel of palms

which has a yellowish white colour and a pleasantly mild flavor similar to coconut oil in

fatty oil acid composition and properties.  Crude palm kernel oil (CPKO) is extracted from

palm kernels with palm kernel cake as a by-product. The physical and chemical properties

16

of the various palm oil products have been reviewed by Nesaretnam and Muhammad,

(1999).

Monodora myristica is a widespread and attractive small tree with very decorative

flowers appearing just before the leaves. The fruit is suspended on a long green stalk with

numerous seeds embedded in whitish sweet smelling pulp. The seed is oblong and pale

brown when fresh with a thin seed coat and hard kernel (Nesaretnam and Muhammad,

1999). The seed production is seasonal occurring between April to June. The fruits are

globular and ovoid; 3-4 inch long and about 3-5 inch diameter. The wood is hard. The seeds

are contained in a hard shell and have a very strong aroma . This plant is commonly called

Orchid flower tree in English, Ehuru Ofia in lgbo (Okafor, 2003). Monodora myristica is a

specie of calabash nutmeg, the edible seeds yield a nutmeg-flavoured oil which is used in

West Africa for cooking (Eggeling, 2002). Plants that belong to Annonaceae family are rich

in flavonoids and bioflavonoids and are known to have antioxidant activity (Shahidi et al.,

2009). Monodora myristica seed extract contains important pharmacological compounds,

alkaloids, flavonoids, and vitamins A and E as well as many important lipids; arhinolipids,

free fatty acids, glycolipids, phospholipids and sterols. The plant is widely used in

ethnomedicine, especially to relieve toothache as well as in the treatment of dysentery.

When roasted and ground, the seeds are rubbed on the skin for (unspecified) skin diseases

(Irvine, 2000). This suggests that the seeds of Monodora myristica plant could be

germicidal or antiseptic. The roasted ground seeds are chewed, then spat into the hand and

then rubbed across the forehead to relieve headache. The seeds are also crushed and used as

insecticide, while the root relieves toothache when crushed (Ogtinein unet al., 1999).

17

Monodora myristica seeds are also used for the treatment of constipation and as a stimulant

(Irvine, 2000). The essential oil from Monodora myristica seed is used in pharmaceutical

and dental preparation (Talalaji, 1999).

In this study, we have monitored characteristic parameter, namely acid value and

thiobarbituric acid value during storage of palm kernel oil and palm oil at different

environmental conditions treated with different concentration of seed extract of Monodora

myristica. Whereby, the acid value and thiobarbituric acid value, were assessed by the

conventional method and the UV-spectra were registered for each sample. Although

simple, procedures of acid value (AV) or peroxide value (PV) determination are

cumbersome, destructive to the sample, costly, require potentially hazardous solvents,

substantial personnel time, glassware and accurate preparation of reagents and are

dependent on a visual endpoint (Ismail et al., 1993; Van de Voort et al., 1994).

1.1 SIGNIFICANCE OF RESEARCH

The aim and objective of this research is to:

1. To carryout solvent extraction of Monodora myristica

2. To investigate the antioxidant effect of Monodora myristica extract on palm kernel

oil and palm oil at different environmental conditions.

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CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 AFRICAN NUTMEG

Figure 1: African nutmeg seeds (Monodora myristica)

2.1.1 Scientific Classification

Kingdom: Plantae

Phylum/Division: Spermatophytae

(unranked): Angiosperms

(unranked): Magnoliids

Order: Magnoliales

Family: Annonaceae

Genus : Monodora

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Species: myristica

Binomial name

Monodora myristica (African nutmeg)

2.1.2 Habitat/Ecology of Monodora myristica

Monodora myristica is tropically distributed. It is cultivated in East India, Malaysia, Sri

Lanka, West lndies and Africa. It could be propagated by stem culturing and budding

(Okafor, 2003). The Monodora species are also found in West Africa and are cultivated in

the southern parts of Nigeria. The trees are very common in Anambra, Abia, Delta, and

Enugu States.

Local Names

The plant is usually called Orchid flower and is also referred to as and called:

Ehuru - lgbo name

Ehinawosin - lkale name

Lakosin - Yoruba name

Uyenghen - Edo name

(Keay, 1989)

2.1.3 Characteristics/Morphology of Monodora myristica

Monodora myristica, commonly known as calabash nutmeg, ehuru, Jamaican nutmeg,

nuscade de Calabash, ariwo, airama, African nutmeg and African orchid nutmeg is a tropical

shrub of the Annonaceae or custard apple family of flowering plants (Okafor, 2003). The

flowers of Monodora myristica look very much like those of an orchid (hence the common

20

name of 'African orchid nutmeg'), and the fruit is a nearly spherical drupe about the same size

as an orange. The seeds and seed coats of the plant are used as a spice. The fruit contains a

number of these aromatic seeds embedded in a yellow pulp (Oguntinein et al., 1999).. The

seeds and their seed coat are removed and dried giving a heart-shaped spice some 3cm long

and 2cm broad at its widest part. Once dried these have an aroma reminiscent of nutmeg and

are sold whole to be grated as a nutmeg substitute (Talalaji, 1999).. At one time it was widely

sold as an inexpensive substitute for nutmeg, although this practice is less common today

outside its region of production (Nigeria). Calabash nutmeg has a nutmeg-like flavour with a

pungent overtone. The whole seed coat and seed is either ground and used as a seasoning for

West African soups or stews or is ground and used as a nutmeg-like flavouring in cakes and

desserts. As well as yielding calabash nutmeg the seed coat is often removed and the inner

true seed is sold as ehuru or ehiri (its name in igbo language, a Nigerian language).

2.2 OIL PALM

Figure 2: African Oil Palm fruits (Elaeis guineensis)

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2.2.1 Scientific Classification

Kingdom:Plantae

Family:Arecaceae

Subfamily:Arecoideae

Tribe:Cocoeae

Genus:Elaeis

Jacq. Species

Elaeis guineensis

Elaeis oleifera

2.2.2 ORIGIN AND DISCRIPTION OF PALM OIL

The oil palms (Elaeis) comprise two species of the Arecaceae, or palm family. They are

used in commercial agriculture in the production of palm oil . The African Oil Palm Elaeis

guineensis is native to west Africa, occurring between Angola and Gambia, while the

American Oil Palm Elaeis oleifera is native to tropical Central America and South

America. The generic name is derived from the Greek for oil, elaion, while the species

name refers to its country of origin.

The palm fruit takes five to six months to mature from pollination to maturity. The palm

of fruit is reddish, about the size of a large plum and grows in large bunches. Each fruit is

made up of oily, fleshy outer layer (the pericarp), with a single seed (the palm kernel), also

rich in oil. When ripe, each bunch of fruit weigh 40-50 kilogrammes.

22

Oil is extracted from both the pulp of the fruit (palm oil, an edible oil) and the kernel (palm

kernel oil, used in foods and for soap manufacture). For every 100 kilograms of fruit

bunches, typically 22 kilograms of palm oil and 1.6 kilograms of palm kernel oil can be

extracted (Mauro et al., 2001).

Palm oil is the reddish-orange oil extracted from the fruit and kennel of a palm tree (Elaeis

Guineensis), a native to tropical West Africa. It is the most widely produced vegetable oil

in the world. This edible oil contains a very high percentage saturated fat and used in

making soaps, margarine, and lubricants, besides being used in cooking.

Since palm oil has been consumed for its nutritional value and health benefits for more than

5,000 years, it is often said as nature's gift to the world. Today, it is the most widely

produced vegetable oil of the world. In some Asian countries, it is termed as ‘gold oil', for

its perfect balance of saturated and unsaturated fatty acids which do not adversely affect

cholesterol levels.

The purest form of palm oil is easily available in the tropical West Africa, Indonesia and

Malaysia, where it is widely cultivated. Besides being used in personal care products and

toiletries, it is also used to treat wounds and as a feedstock for biofuel.

 

2.2.3 The Chemical Composition Of Palm Oil

Palm oil consists mainly of glycerides made up of a range of fatty acids including

triglyceries, mono and diglycerides. The oil palm gives its name to the 16 carbon saturated

fatty acid palmitic acid found in palm oil; monounsaturated oleic acid is also a constituent

23

of palm oil while palm kernel oil contains mainly lauric acid. Besides this, it is the largest

natural source of tocotrienol, part of the Vitamin E family. It also contains high

concentration of Vitamin K and dietary magnesium.

Triglycerides constitute the major component, with small proportions of diglycerides and

monoglycerides. Palm oil also contains other minor constituents, such as free fatty acids

and non-glyceride components. This composition determines the oil's chemical and

physical characteristics (Cornelius, 2001)..

The fatty acid composition of crude Malaysian palm oil is given in table 1. About 50% of

the fatty acids are saturated, 40% mono-unsaturated, and 10% polyunsaturated. It contains

adequate amounts of n-6, 18:2 essential fatty acid. In its content of monounsaturated 18:1

acid, palm oil is similar to olive oil, which is as effective as the more polyunsaturated oils

in reducing blood cholesterol and the risk of coronary heart disease.

Crude palm oil contains approximately 1% of minor components: carotenoids, vitamin E

(tocopherols and tocotrienols), sterols, phospholipids, glycolipids, terpenic and aliphatic

hydrocarbons, and other trace impurities . The most important are carotenoids and vitamin

E, both of which possess important physiological properties. The iodine value is between

50 and 56.

24

TABLE 1. Fatty acid composition of palm oil (PO)

Source: (Pantzaris and Ahmad, 2004).

Since palm oil contains more saturated fats than canola oil, corn oil, linseed oil, soybean

oil, safflower oil, and sunflower oil, it can withstand extreme deep fry heat and is resistant

to oxidation.

  % of total acids

Acid Range Mean

12:0 0.1-1.0 0.2

14:0 0.9-1.5 1.1

16:0 41.8-46.8 44.0

16:1 0.1-0.3 0.1

18:0 4.2-5.1 4.5

18:1 37.3-40.8 39.2

18:2 9,1-11.0 10.1

18:3 0.0-0.6 0.4

20:0 0.2-0.7 0.4

25

2.2.4 PHYSICAL CHARACTERISTICS OF PALM OIL PRODUCTS

Palm oil is a semi-solid at room temperature (28◦C), the melting point range being

from 32–40◦C. The slip melting point method is commonly adopted for measuring this

parameter. By the DSC method the fat melts completely at 39–40◦C, when heated at

5◦C/min, from an oil cooled rapidly to −40◦C at 5◦C/min. The slip melting point is affected

by the content of free fatty acids and diacylglycerols. Thus crude oils have slightly higher

slip melting point than refined oils (Nyam et al., 2009).

Palm oil is produced from the fruit and kernel of the palm tree. The fruits are first

collected and pressed, yielding a rich, dark-red oil which is high in carotene (Pantzaris and

Ahmad, 2004). The oil thus obtained, is exposed to heat through processing and cooking

which turn its colour to pale creamy color. Conversion of crude palm oil to refined oil

involves removal of the products of hydrolysis and oxidation, colour and flavour. After

refining, the palm oil may be fractionated (separated) into liquid and solid phases by

thermo-mechanical means (controlled cooling, crystallization, and filtering).

2.3 PALM KERNEL OIL

Palm kernel oil (PKO) is obtained from processing the kernel from the fruit of the oil palm

tree (Elaies guineensis). Palm kernel oil has similar uses to coconut oil owing to their

similarity in composition (Pantzaris and Ahmad, 2004). Palm kernel oil (PKO) is gotten

26

from the kernel of the palm fruit and it is located inside the hard shell while the outer fleshy

mesocarp gives palm oil.

2.3.1 The Chemical Composition Of Palm kernel Oil

The major fatty acids in palm kernel oil are lauric acid (C12, 48%), myristic acid (C14,

16%) and oleic acid (C18, 15%) (Pantzaris and Ahmad, 2004).

The fatty acids mostly found in palm kernel oil are presented in Table 1 below.

 

Table 2. Fatty acid profile of palm kernel oil (PKO)

Type of fatty acid Percentage

Lauric (C12:0) 48.2

Myristic (C14:0) 16.2

Palmitic (C16:0) 8.4

Capric (C10:0) 3.4

Caprylic (C8:0) 3.3

Stearic (C18:0) 2.5

Oleic (C18:1) 15.3

Linoleic (C18:2) 2.3

Others (unknown) 0.4

27

Source: (Pantzaris and Ahmad, 2004).

Palm kernel oil, coconut oil, and palm oil are three of the few highly saturated vegetable

fats. Palm kernel oil, which is semi-solid at room temperature, is more saturated than palm

oil and comparable to coconut oil. Like all vegetable oils, these three palm-derived oils do

not contain cholesterol (found in unrefined animal fats)] although saturated fat intake

increases both LDL and HDL cholesterol.

2.4 Modern Uses of Palm Oil and Palm Kernel Oil

As much as 90% of the palm oil produced finds its way into food products, while remaining

10% is consumed by various industries. It is widely used preparing margarine, shortening,

and vegetable cooking oil. In many parts of the world, it is still consumed in its unrefined

state to obtain a distinctive colour and flavour. Palm oil is extensively used in preparing dry

cake mix used for baking biscuits, cakes and sponge cakes, soaps, sauces, fat substitutes,

etc. Recently, palm and kernel oils have been increasingly used as biodiesel fuel.

Palm kernel oil is a common cooking ingredient; its increasing use in the commercial food

industry throughout the world is buoyed by its lower cost, the high oxidative stability

(saturation) of the refined product when used for frying, and its lack of cholesterol and

trans-fatty acids, both viewed as being heart-healthy attributes. .

2.5 LIPID OXIDATION

Lipid oxidation and resultant flavour impairment has seriously limited the storage potential

of most oil containing food (Ihekoronye and Ngoddy, 1985) Lipid oxidation generally

28

occurs after a long induction period. Once started it is generally a very rapid reaction. Lipid

oxidation proceeds by a free radical mechanism.

A free radical is a compound with an odd number of unpaired electrons.

Two free radicals were formed. These radicals are very reactive and generally do not have

long life times (Morel, 1997).. They enter into three main types of reactions:

1. Abstraction:

2. Addition:

29

3. Combination:

Lipid oxidation follows three main steps:

Initiation

Propagation

Termination

1. Initiation involves the formation of free radicals.

Mechanism later:

In some cases may add oxygen directly to the double bond to form a biradical

30

Once the initial radicals have formed, the formation of other radicals proceeds rapidly..The

radicals can abstract H atoms from other lipids or react with other radicals to form alcohols

and ketones (Morel, 1997).

A.

B.

C.

31

Of great importance to the food industry is the splitting of C-C bonds and the formation of

aldehydes:

There can also be cleavage on the other side:

It is possible for two of the radicals formed to combine:

The change from the alcohol to the aldehyde is called a keto - enol shift. If the two free

radicals do not react with other molecules they may combine with each other.

32

2.5.1 LIPID OXIDATION PATHWAY

UNSATURATED FATTY ACIDS

FREE RADICALS

OXYGEN Insolubilization of proteins

HYDROGENPEROXIDES

Breakdown products

Such as aldehydes, free

fatty acids, alchohol Oxidation of pigments, flavours

, and hydrocarbons and vitamins

Polymerization

(dark colour)

Source: (Ihekoronye and Ngoddy, 1985)

33

Figure 3: lipid oxidation pathway

2.5.2 MECHANISMS OF OXIDATION

Autoxidation

This is a radical-chain process involving 3 sequences: Initiation, propagation and

termination.

1 – Initiation

In this stage, the molecule of unsaturated fatty acid loses a hydrogen atom leaving a free

radical which is required to start the propagation reaction (Ihekoronye and Ngoddy, 1985).

The reaction may be represented as follows:

RH R + H

In a peroxide-free lipid system, the initiation of a peroxidation sequence refers to the attack

of a ROS (with sufficient reactivity) able to abstract a hydrogen atom from a methylene

group (- CH2-), these hydrogen having very high mobility (Morel, 1997). . This attack

generates easily free radicals from polyunsaturated fatty acids. .OH is the most efficient

ROS to do that attack, whereas O2.- is insufficiently reactive.

This peroxidation process is inhibited by tocopherols, mannitol and formate. The presence

of a double bond in the fatty acid weakens the C-H bonds on the carbon atom adjacent to

34

the double bond and so makes H removal easier.

Propagation

During the propagation stage, the free radical reacts with oxygen to form peroxide-

containing free radicals (Ihekoronye and Ngoddy, 1985).

R + O2 ROO

These in turn reacts with another mole of unsaturated compound to produce hydrogen

peroxides and new free radicals capable of continuing the chain reaction. As a peroxyl

radical is able to abstract H from another lipid molecule (adjacent fatty acid), especially in

the presence of metals such as copper or iron, thus causing an autocatalytic chain reaction.

The peroxyl radical combines with H to give a lipid hydroperoxide (or peroxide). This

reaction characterizes the propagation stage (Morel, 1997).

 

The hydroperoxides formed are unstable and willdecompose either to stable derivatives or

will split into two or more free radicals (Ihekoronye and Ngoddy, 1985).

Termination

When two radicals interact to form stable non-radical products, termination occurs:

35

R + R RR

RO + R ROR (Ihekoronye and Ngoddy, 1985)

Termination (formation of a hydroperoxide) is most often achieved by reaction of a

peroxyl radical with a-tocopherol which is the main lipophilic "chain-breaking molecule" in

the cell membranes. Furthermore, any kind of alkyl radicals (lipid free radicals) L . can react

with a lipid peroxide LOO. to give non-initiating and non-propagating species such as the

relatively stable dimers (Morel, 1997).LOOL or two peroxide molecules combining to form

hydroxylated derivatives (LOH). Some bonds between lipid peroxides and membrane

proteins are also possible.

2.6 GENERAL ANTIOXIDANT ACTIONS

Primary antioxidants are compounds that are able to donate hydrogen atom rapidly to a

lipid radical forming a new radical, more stable than the initial one (Murray et a/., 1990).

Monodora mystrica contains polyphenols which act as antioxidant.

2.6.1 MECHANISM OF ACTION OF ANTIOXIDANTS

The principle mechanism of action have been proposed for antioxidant is chain-breaking

mechanism, by which antioxidants donate an electron to the free radical present in the

system (Murray et al., 1990). .

Electron Donation

36

Primary antioxidants are compounds that are able to donate hydrogen atom rapidly to a

lipid radical forming a new radical, more stable than the initial one (Murray et a/., 1990).

Biological organs contain many polyunsaturated fatty acids (PUFA), such as linoleic, lenic

and arachidonic acid, mainly in the form of ester with cholesterol. These PUFA can

undergo lipid peroxidation that can be interrupted by the primary antioxidant by the

donation of electrons.

The whole process can be depicted as follows.

RH + O2 (Singlet oxygen) - - - - -ROOH

ROOH + ~ e ' ~- - - - - RO. + HO- + ~ e ' ~

ROOH + ~ e ' ~- - - - ROO. + H' + ~ e ' ~

ROO.+a-TO. - - - - Non radical products.

RH = Polyunsaturated fatty acid (PUFA)

ROOH = PUFA hydroperoxide

RO. = Alkoxyl radical

ROO. = Peroxyl radical

a - TO. = Tocopheryl radical

Metal Chelation

This can be accomplished by deactivation of high-energy species, absorption of UV light,

scavenging of oxygen and thus reducing its concentration (Omenn et al, 1996). Chelation

of metal catalyzes free radical reaction or inhibits peroxidase. The ability of antioxidant to

chelate transition metal ions can be followed spectroscopically. High molecular weight

proteins bind directly or indirectly to redox active metals and thus inhibit the production of

37

metal-catalyzed free radicals. Some low molecular weight compounds, such as

polyphenols, in addition to their ability to donate hydrogen atom and thus act as chain-

breaking antioxidant, can also chelate transition metal ions and hence inhibit free radical

formation (Omenn et al., 1996).

2.6.2 ANTIOXIDANT MOLECULES

Antioxidants are a group of substances, which when present at low concentrations, in

relation to oxidizable substrates, significantly inhibit or delay oxidation and oxidative

processes, while often being oxidized themselves (Kanner et al., 1999). The application of

antioxidants are widespread, in industries theyare used in preventing polymer from

oxidative degradation, rubber and plastic from losing strength, gasoline from autooxidation,

synthetic and natural pigments from discolouration and as additives to cosmetics, food

(especially food with high fat content) beverages and baking products

(Kanner et a/, 1999).

Vitamin E - The Tocotrienols: Super Anti-Oxidants

Vitamin E is one of the most important phytonutrients in edible oils. It consists of eight

naturally occurring isomers, a family of four tocopherols (alpha, beta, gamma and delta)

and four tocotrienols (alpha, beta, gamma and delta) homologues. While most Vitamin E

supplements on the market today are composed of the more common tocopherols,

tocotrienols are believed to be a  much more potent antioxidant than tocopherols.

Tocotrienols are naturally present in most plants, however they are found most abundantly

in palm oil extracted from palm fruits.

38

2.7 GENERAL REVIEW OF PHYTOCHEMISTRYOF MONODORA MYRISTICA

2.7.1 Alkaloids

They are a group of basic secondary plant substance, which usually possesses an n-

containing heterocyte. Alkaloids exist in plants as salts, amine or n-oxides. Dicotyledonous

plants are the real producers of alkaloids (Evans, 1989). They appear in large members and

in many variation in these plants. They are bitter to taste, so when present in plants, insects

and predators tend to move away from such plants. They also protect the plant from the

effect of singlet oxygen (Bonner and Varner, 1965). Alkaloids at high concentration,

produces a variety of toxic effects on animals. Their pharmaceutical and medicinal

importance can be seen to act on the cardiovascular system and some have been resorted to

be antihypertensive. Alkaloids also contribute to liver disease and hepatocellular tumor

(Antoniodes and Owen, 1982). Alkaloids of Catharanthus roseus are used in cancer

chemotherapy.

2.7.2 Flavonoids

The origin of the names is from a Latin word "FLAVUS" meaning yellow. They are a

series of related water soluble phenolic glycosides having in common a basic structural

unit. The CI5 skeleton of flavones. The flavones are sap-soluble (Bonner and Varner,

1965). The phonetic compound contributes to the colour of soft fruits, which are scarlet,

crimson and purple anthocyamins e.g. cyamidin-3-rutinoside. They are widely distributed

in nature but are more common in the higher plants and in young tissues, where they occur

39

in the cell sap. Flavonoids contribute to the taste and flavour of foodstuffs (Bonner and

Varner,..1965). Flavonoids when consumed in certain quantity could lead to serious

disorder in the system.

2.7.3 Glycosides

These are the products obtained after condensation of sugar with different types of organic

hydroxyl compounds. These are referred to as the cardiac-active or cardio-tonic glycosides

examples include amygdalin (Stryer, 1975). In small doses, glycosides promote mild gastric

irritation causing a reflux from the bronchioles. This can be attributed to its wide

usage but in larger dose, they lead to vomiting (Evans, 1989). A larger number of

glycosides and their aglycone have antimicrobial activities.

2.7.4 Saponins

Saponins are useful in the production of soft drinks, beers, confectioneries, shampoos,

soaps, fibre extinguishers and beverages and this is attributed to its foaming ability (Liener,

1972). They are quite toxic when injected into the bloodstream and are harmless when

taken by mouth since the sarsaparilla is rich in saponins but is used in the preparation of

non-alcoholic beverages (Evans, 1989). The highest sapogenin concentration occurs in the

reproductive parts of the plants, the seeds containing 18% trigonenin (Bonner and Varner,

1965). Saponin have some medicinal properties, since it has beenreported to have anti-

inflammatory, anti-fungal, antimycolic, bacteriostatic and other biological activities. When

ground in a powdering form, causes violent sneezing

40

2.7.5 Tannins

The word "tannin" signifies substances present in plant extracts, which are able to combine

with protein of animal hides, prevent their Putrefaction and the conversion to leather

(Evans, 1989). Those tannins are responsible for the taste qualities of wines, tea and coffee.

They are astrigent and styptic (i.e. the dry sensation felt in the mouth). Tannins

due to their antiseptic properties prevent fungal attacks (Bonner and Varner, 1965; Evans,

1989). They also have tumorigenic and carcinogenic effects.

2.8 APPLICATIONS OF VEGETABLE OILS

Many forest trees produce seeds that contain fatty oils; these can be processed into

vegetable oils for use in cooking, food industry and soap-making, and also as fuel.

Producing fixed oils is a simple process and can be done locally, with locally made

equipment. In the first stage, the oil is extracted from the seeds by dry expression or by

boiling the crushed raw material in water.

Vegetable oils also provide inputs to the more complex detergent industry, which uses fatty

alcohol derivatives of lauric oils, which currently come mainly from palm kernels -

primarily coconut (Cocos nucifera) and African oil palm (Elaeis guineensis), with smaller

amounts from wild stands of babassu palm (Orbignya sp.) (De Silva and Atal, op. cit.).

Palm oil is processed to produce edible fats (margarine), soaps and candles and is used in

pharmacy and cosmetics and as an important raw material in oleochemistry (fat chemistry).

Palm kernel oil (PKO) is more unsaturated and hence can be hydrogenated to a wider range

of products which could be used either alone or in blends with other oil for biscuit dough,

41

filling creams, cake icing, ice cream, imitation whipping cream, substitute chocolate and

other coatings, sharp melting and melting margarines etc. Lauric oil (CNO, PKO) is very

important in soap making and a good soap must contain at least 15% lauric acids for quick

lathering while soap made for use in sea water is based on virtually100% lauric oils. Mostly

palm kernel oil are now used for the manufacture of short chain fatty acids, fatty alcohols,

methyl esters, fatty amines, for use in detergents, cosmetics and many other cosmetic

products but less consideration is given it for other purpose. Monodora myristica seed are

used as condiment in West Africa, a decoction of the seed is used to treat guinea worm

infection. The seeds are used as a remedy for constipation, when mixed with palm oil.

Roasted and powdered seeds of the plant are very effective in curing stomach ache. The

seeds are rubbed on the forehead to cure headache (Gill, 1992).

2.8.1 FACTORS THAT CAUSE OXIDATIVE RANCIDITY IN VEGETABLE OIL

Many factors can affect the tendency of an oil to become rancid. The first is too much

exposure to air. Since oxidative rancidity is the most likely kind of rancidity to affect your

food oils, you always want to keep those oils in bottles that are tightly capped. (A tightly

capped bottle will help prevent your oil from being unnecessarily exposed to oxygen.)

The next factors are heat and light. Since both of these factors can also speed up the

rancidity process, protection from heat and light are also important when it comes to your

food oils. With respect to light, your best bet is to purchase oils in bottles made from darker

(tinted) glass (usually dark brown or dark green glass). You'll also want to store your oils in

a cabinet that is lightproof. With respect to heat, many oils are best kept in the refrigerator

42

where the temperature remains continuously low. (I will explain in a moment why I do not

believe refrigeration is necessary for extra virgin olive oil, but why I still believe it is very

important to store this oil in a cool spot.) Protecting your food oils from light and heat is a

moment-by-moment process. For example, it means paying attention to the spot you place a

bottle of oil when using it in a recipe. You never want to place it directly next to or above a

stove that is turned on due to the increased risk of damage from heat. You also want to take

the trouble of capping the bottle whenever you are not pouring oil from it.

The chemical composition of an oil is also a key factor in the risk of rancidity. Here the

basic principles involve saturated and unsaturated fat. The more saturated fat contained in

an oil, the less susceptible it is to rancidity. The greater the amount of unsaturated fat in an

oil, the more likely it is to become rancidity. Since the healthiest plant oils are all highly

unsaturated, they are especially susceptible to rancidity.

Some unsaturated oils, like extra virgin olive oil, are a little less susceptible to rancidity

because a larger amount of their unsaturated fat falls into a special category called

"monounsaturated." Extra virgin olive is about 75% monounsaturated, which is somewhat

unusual for a plant oil. Plant oils usually have more polyunsaturated fat than

monounsaturated fat, and that is one reason why they are particularly susceptible to

rancidity. While the highly monounsaturated nature of extra virgin olive oil doesn't mean

that you can forget about the issue of rancidity, it does mean that this unique oil is a little

more stable than oils that have much fewer monounsaturates.

Both omega-3 and omega-6 fatty acids are always polyunsaturated. When it comes to plant

oils, if you are trying to make sure that your diet contains an ample supply of omega-3s,

43

you are always at the greatest risk for rancidity. Flaxseed oil, for example, contains about

15 grams of alpha-linolenic acid per ounce. Alpha-linolenic acid is a polyunsaturated

omega-3 fatty acid not found in a wide variety of foods, and it's the basic building block for

all other omega-3 fatty acids. Many food scientists look upon the alpha-linolenic acid found

in flaxseeds oil as the most delicate part of its composition that needs to be protected from

oxidative rancidity. In a case like flaxseed oil, where the chemical composition of the oil

places it at great risk for rancidity, it's best to avoid any type of heating at temperatures

above 150°F (66°C) and to store the oil in the refrigerator.

Free radicals

Every cell has chemical reactions involving the oxidation and reduction of molecules.

These reaction or redox pathways can lead to the production of free radicals. A free radical

is any chemical species capable of independent existence possessing one or more unpaired

electrons. Biological free radicals are thus highly unstable molecules that have electrons

available to react with various organic substrates (Sahart, 2001).

Many free radicals are generated from naturally occurring processes such as oxygen

metabolism and inflammatory processes. For example, when cells use oxygen to generate

energy, free radicals are created as a consequence of ATP production by the mitochondria

(Sahart, 2001). Exercise can increase the levels of free radicals as can environmental

stimuli such as ionizing radiation (from industry, sun exposure, cosmic rays, and medical x-

rays), environmental toxins, altered atmospheric conditions (e.g. hypoxia and hyperoxia),

ozone and nitrous oxide (primarily from automobile exhaust). Lifestyle stressors such as

cigarette smoking and excessive alcohol consumption are also greater levels of oxidative or

nitrosative stress.

44

2.9 NUTRITIONAL SIGNIFICANCE

Hydrolytic rancidity caused by the release of free fatty acids from glycerides, is

significantly important in terms of flavour production but is of little consequence in terms

of nutrition as the fats are enzymically hydrolysed in the small bowl before they are

absorbed by the body. (Hydrolytic rancidity gives strong cheeses like stilton their sharp

burning taste).

Oxidative rancidity leads to the formation of both unpalatable and toxic compounds. Three

distinct classes of substance occuring in oxidised fat have been shown to be toxic:

a) Peroxidised fatty acids  (peroxidised fatty acids destroy both vitamin A and E in foods).

b) Polymeric material (under normal food processing conditions these appear in small

enough quantities to be insignificant).

c) Oxidised sterols (thought to be involved in the causation of artherosclerotic disease).

45

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Equipments / Apparatus Maker

weighing balance (triple beam balance) Larle ®

Analytical balance, Ohaus scale corporation

Labouratory dry oven (hot air oven) DHG 9101 Model

Uv-Vis Spectrophotometer Lemfield

Water bath 801A Model

Volumetric flask Permagold

Measuring cylinder, Beaker, Simax

Cornical flask, burette

Soxhlet extractor, Heating mantle, Pyex

Extraction thimble, Condenser,

pH meter Hannah microprocessor

Filter paper Whatman

Mortar and pestle

46

3.2 Procedurement Of Raw Materials

The selected indigenous spice, African nutmeg (Monodora myristica) was purchased

from ogbete main market in Enugu state of Nigeria and identified by Dr. B.C. Ndukwu (a

plant taxonomist) of department of plant science and biotechnology, University of Port

Harcourt and was furtherly authenticated by Dr Charles N. Ishiwu of Department Of Food

Science And Technology, University of Nigeria, Nsuka and also a senior lecturer in

Biochemistry Department of Caritas University, Enugu state.

One purchase, the spices (seeds) were collected into sterile glass desiccators and stored in

an oven maintained at 70ºC until use.The spices (dried samples) were estimated to have

been in the market for 6-7days before purchase.

The varieties of vegetable oil used palm kernel oil and palm oil were processed and

obtained from Aniuzo International Limited ( Palm Kernel oil Mills Division), Emene in

Enugu state and Anieke Palm oil Mills, Ubulu-uku in Delta state. These were done in large

quantity to minimize chances of variation and to maintain experimental homogeneity in

sample seletion.

.

47

Figure 4: African nutmegs: dried seed kernels, which have been removed from their fruit

husk, seed covering and hard seed coat.

Figure 5: Transverse section of palm fruit.

48

3.3 STUDY DESIGN

This study was conducted on two different varieties of vegetable oil (palm kernel oil

and palm oil). Each of the varieties was divided into two groups A and B. Group A was

exposed to adverse tropical condition under the sun, while Group B was kept indoors.

There were 6 samples of equal volume for each group of each variety of oil (five(5) of the

samples contain different concentrations of extract African nutmeg, and one(1) contains no

extract). Therefore, there were total of 12 samples for each variety.The study was

conducted for 0, 1, 2, and 3 weeks. Each sample was analyzed in triplicates and the mean

was used in final content calculation. All experimental procedures were carried out

simultaneously under the same condition used for storage.

3.4 SAMPLE PREPARATION

The method of AOAC (1990) was used. The African nutmeg seeds were heated in

an oven (hot air) at 105ºC (for easy extraction of the oil ). They were then weighed in the

digital weighing balance and grounded using spiral grinder. The freshly collected seeds of

the Monodora myristica were sun dried and powdered using a pistle and mortar. The

powder was defatted with n-hexane (65 - 69°C) using soxhlet apparatus. The whole filtrate

was allowed to evaporate at room temperature leaving the oil.

49

3.5 CHEMICAL ANALYSIS

3.5.1 Determination Of Acid Value(AV)

The free fatty acid content of a fat/oil is the number of milligrammes of KOH

required to neutralize lg of FFA present in fat/oil sample.The acid value is the number of

mg of KOH necessary to neutralize the free acid in lg of sample. The acid values

(MgKOH/g) of the oil samples were determined according to Polish Standard (PN-EN ISO

660:2005). Weighed samples of around 20 g were dissolved in 100 cm3 of ethanol: diethyl

ether mixture (1:1, v/v) and titrated with 0.1 N potassium hydroxide solution using

phenolphthalein as an indicator. Analyses were carried out in triplicate the acid value is the

mg KOH used to neutralize 1.0 g of each oil sample. Results were used as reference data

for model building.

The acid value is given by T – B x 5.61/W0.1M KOH contains 5.6mg/ml or 5.6g/l where

T=Titre value of the sample; B=Titre value of a blank. The blank was provided as a control

by titrating 2.5ml of the neutral alcohol without sample. The free fatty acid (FFA) is

normally determined as oleic acid where by the acid value = 2 x FFA.

NaOH may be used and a generalized formula may be used (for palm oil and fractions):

25.6 x MNaoH x V/W where V= Volume of NaOH solution used in ml; W=Weight of

sample

3.5.2 Determination Of Thiobarbituric Acid Number (TBA)

The thiobarbituric acid value TBA was determined by modification of method described by

Odo and Ishiwu, (1999), the PORIM Test Method. Thiobarbituric acid value TBA is the

50

intensity of pink pigment formed between 2-tiobarbituric acid and the oxidized lipid

measured optically in a colorimeter. This has been found to increase as oxidation advanced.

Malonaldehyde is probably involved in the reaction (Odo and Ishiwu, 1999)

. 10g of the sample is added into 50ml distilled water in a distillation flask. 2.5ml of 4M

HCl is added to raise the pH to about 1.5. Then antibumping granules are added and the

distillation kit is set up. The mixture is heated in a heating mantle such that 50ml distillate

is collected in 10minutes from the time boiling started. 5ml of the distillate and 5ml of TBA

reagent (0.288g/100ml) of glacial acetic acid were added into a stoppered tube and heated

in a boiling water bath for 35minutes. Blank determination was made using 5ml of distilled

water and 5ml reagent. The tubes were cooled in running water and the reading of the

absorbance against blank was taken at 538nm.

3.6 STATISTICAL ANALYSIS

All statistics were performed using MICROSOFT EXCEL version 2007 software.

51

CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

The mean acid value (AV) for free fatty acid and thiobarbituric acid (TBA) values of tested

oil sample are shown in Table 3 - 6, respectively. Data for the tested oil samples were

obtained by measuring samples from the same producer in triplicate. Mean values in Table

3 - 6 are followed by lists those pairs of weeks, between which statistically significant

difference exists.

Tables 3 and 4 respectively showed the Acid Values (AV) of free fatty acid, while tables 5

and 6 showed the thiobarbituric acid values (TBA) of crude palm kernel oil and palm oil

stored with varying concentration of 0.2%-1.0% of n-hexane extract of Monodora myristica

seed, stored in the sun and in the room. The trend observed above for AV was also the same

with that of TBA in all the storage conditions only that the AV values were higher than that

of TBA. Ihekoronye and Ngoddy (1985) reported that the AV of any lipid were both

measure of hydrolytic rancidity and that the lower their values, the slower was the rate of

hydrolytic rancidity. Hence crude palm oil stored with varying concentration of 0.2%-1.0%

extract of Monodora myristica seeds were less prone to oxidative rancidity. This showed

that the extracts at varying concentration demonstrated high antioxidant activity. However

the antioxidant activity was higher as concentration of Monodora myristica extract

increases at both environmental conditions.

52

Table 3 ACID VALUE FOR PKO (MgKOH/g)

CONCN.

OF

EXTRAC

T (ML)

PRE-

STORAG

E DAY

WEEK 1

SS

WEEK 1

SR

WEEK 2

SS

WEEK 2

SR

WEEK 3

SS

WEEK 3

SR

Control

(0.00) 4.75±0.00 5.92±0.36 4.89±0.31 8.24±0.20 5.38±0.08 14.72±10 9.70±0.51

0.2 4.70±0.15 5.66±0.28 4.74±0.36 8.06±0.32 5.14±0.13 14.68±0.31 7.74±0.260.4 4.45±0.12 5.30±0.28 4.52±0.12 7.86±0.37 5.00±0.31 13.56±0.48 7.22±0.21

0.6 3.92±0.41 4.96±0.31 4.08±0.26 7.32±0.41 4.84±0.26 13.18±0.26 6.26±0.21

0.8 3.44±0.32 4.54±0.29 3.94±0.31 6.92±0.51 4.62±0.31 13.06±0.26 6.18±0.26

1.0 3.06±0.91 4.08±0.34 3.70±0.31 6.40±0,28 4.28±0.31 13.10±0.13 5.99±0.31

Changes in mean ± SD acid value content of Palm kernel oil stored in sun and Palm kernel

oil stored in the room

KEY

SS = Storage in Sun; SR = Storage in Room

AV = Acid Value; TBA = Thiobarbituric Acid

PKO = Palm Kernel Oil; PO = Palm Oil

SD = Standard deviation

53

Table 4: ACID VALUE FOR PO (MgKOH/g)

CONCN.

OF

EXTRACT

(ML)

PRE-

STORAGE

DAY

WEEK 1

SS

WEEK 1

SR

WEEK 2

SS

WEEK 2

SR

WEEK 3

SS

WEEK 3

SR

Control

(0.00)

14.27±0.26 15.71±0.23 15.42±0.18 17.75±0.13 16.26±0.08 21.02±0.12 18.74±0.36

0.2 14.13±0.11 15.24±1.24 15.21±1.13 17.32±0.52 16.06±0.19 20.83±0.26 18.22±0.34

0.4 13.92±0.26 15.01±0.08 14.73±0.52 16.63±0.08 16.00±1.10 19.92±0.52 16.88±0.26

0.6 13.53±1.02 14.94±0.15 14.71±0.91 16.26±0.15 15.72±0.90 19.61±0.81 16.32±0.31

0.8 13.27±1.31 14.51±0.92 13.81±0.71 15.84±0.12 15.48±1.21 18.91±1.00 16.02±0.81

1.0 13.06±1.42 14.39±1.10 13.36±1.02 14.81±1.10 14.13±1.40 17.26±1.23 15.72±0.92

Changes in mean ± SD acid value content of Palm oil stored in sun and Palm oil stored in

the room.

We can note that unsaturated fatty acid content drops with the time of addition of extract

then is stabilized from the first week. This fall is felt much in the case of the crude palm

kernel oil. It would be due to the fact that when the oil samples are exposed to the sun and

in the free air, their unsaturated fatty acids fix oxygen and oxidize (Blumenthal and Stier,

1991).

54

CONCN.

OF

EXTRACT

(ML)

PRE-

STORAGE

DAY

WEEK 1

SS

WEEK 1

SR

WEEK 2

SS

WEEK 2

SR

WEEK 3

SS

WEEK 3

SR

Control

(0.00)

3.12±0.08 4.14±0.22 4.00±0.26 6.92±0.84 5.50±1.10 9.10±0.81 6.30±0.75

0.2 3.06±0.01 4.09±0.16 3.86±0.29 6.69±1.04 5.32±1.24 8.68±0.50 6.00±0.35

0.4 3.08±0.05 4.12±0.05 3.51±0.33 6.21±1.13 5.01±1.21 8.53±0.13 5.54±0.26

0.6 3.19±0.06 3.81±0.08 3.06±0.36 5.90±1.19 4.78±1.26 8.30±0.26 5.08±0.10

0.8 2.30±0.12 3.15±0.13 2.87±0.13 5.42±0.91 4.43±1.00 7.83±0.26 4.80±0.13

1.0 2.02±0.17 2.72±0.19 2.31±0.21 4.91±0.75 3.90±1.20 7.35±0.26 4.22±0.13

Table 5: THIOBARBITURIC ACID VALUE FOR PKO

Changes in mean ± SD TBA value content of Palm kernel oil stored in sun and Palm kernel

oil stored in the room.

From the values deduced from thiobarbituric acid TBA evaluation, the trend is similar to

that of acid value

55

Table 6: THIOBARBITURIC ACID VALUE FOR PKO

CONCN.

OF

EXTRACT

(ML)

PRE-

STORAGE

DAY

WEEK 1

SS

WEEK 1

SR

WEEK 2

SS

WEEK 2

SR

WEEK 3

SS

WEEK 3

SR

Control

(0.00)

5.20±0.35 6.57±0.26 5.50±0.21 9.20±0.19 6.61±0.20 10.11±0.17 7.00±0.11

0.2 5.01±0.22 6.29±0.13 5.35±0.29 8.92±0.19 6.36±0.17 9.888±0.15 6.72±0.08

0.4 4.90±0.13 6.01±0.21 5.10±0.31 8.41±0.21 6.10±0.34 9.52±0.11 6.51±0.14

0.6 4.72±0.52 5.74±0.41 4.81±0.26 7.00±0.26 5.90±0.26 9.07±0.13 6.32±0.14

0.8 4.49±0.34 5.42±0.32 4.52±0.26 6.81±0.16 5.71±0.21 8.77±0.25 6.01±0.21

1.0 4.11±0.26 5.10±0.19 4.15±0.21 6.38±0.19 5.20±0.34 8.41±0.27 5.52±0.19

Changes in mean ± SD TBAvalue content of Palm oil stored in sun and Palm oil stored in

the room.

For the two varieties of oil, the acid value of free fatty acid increased significantly

(P<0.05) as the period extends for group SS without extract while those for group SR

showed no significant increase. But AV of oil samples treated with higher extract

concentration decreased significantly (P<0.05) for both groups SS and SR. TBA value also

showed the same trend of AV. Hence, monodora myristica extract yielded reducing effect

in the oxidative level of the oil varieties.

56

From the study, it is evident that the extract of seeds of Monodora myristica has

promising antioxidant activity. In the present study, the percentage (%) yield of the extract

was found to be 18.9%, which is relatively low when compared to a previous study on the

plant. In a previous study (Esuoso et al., 2000) reported that Monodora myristica seed

extract had a yield of 34.7-68.8%. The possible difference in the yield could be as a result

of geographical and climatic factors, which has been found to affect plant constituents, or

time of collection of the seed, method of storage, the variety of the parent plant and the

nature of the soil on which it is planted (Alam et al., 1982).

In the fatty acid content of palm kernel oil and palm oil, stored in the sun, values in the

same column, bearing different superscripts differ significantly P<0.05.hence there was

significant increase in oxidation which also reduced as the extract increased.

Monodora myristica seed has been found to contain a lot of secondary plant metabolites

namely: alkaloids, carbohydrates, flavonoids, glycosides, proteins, saponins, and tannins.

Alkaloids at high concentration, has been found to produce a variety of toxic effects on "

animals. They also protect the plant from the effect of singlet oxygen (Bonner and Varner,

1965). These plant constituents are known to be biological active, eliciting a variety of

actions such as antioxidant effects (Bauer et al., 1996). It can also be concluded that the

antioxidant activity of the extract could be attributed to flavonoids, which are fourid in

antioxidant plant such as Aspidium cacutarium (Ghoghari et al., 2006), Phyllanthus debilis

klein ex Willd (Kumaran and Karunakaran, 2006) as well in Tephrosia purpurea (Jain et al.,

2006).

57

4.1 Changes In Acid Value of PKO and PO

The variation of the percentage of free fatty acid of crude palm kernel oil and palm

oil placed in the sun is shown in Table 3-4. At equal volume of oil, the little decrease in

free fatty acid content was observed during the first 3 weeks. Whereas,the group kept in the

dark room showed significant decrease in the free fatty acid content. But the higher the

concentration of extract treatment in each sample, the lesser the free fatty acid value.

Higher value of percentage free fatty acid content was observed in crude palm oil.

It would be due to the fact that when PKO and PO are exposed to the sun and in the free air,

their unsaturated fatty acids fix oxygen and oxidize (Blumenthal and Stier, 1991). The acid

value of an oil may be used as a measure of quality. However, the acid value of the oil must

not be too high, as this denotes an excessively high content of free fatty acids, which causes

the oil to turn sour. Discoloration may also occur. Palm kernel oil should have an acid value

of at most 0.1 - 1.0% [1], or 5%. Oils and fats spoil by readily becoming rancid. Rancidity

is promoted by light, atmospheric oxygen and moisture and leads to changes in odor and

taste.

4.2 Changes in Thiobarbituric acid value of PKO and PO

The variation of the thiobarbituric acid number of crude palm kernel oil and pam oil placed

in the sun is shown in Table 5-6 . At equal volume of oil, the signicant decrease in content

was observed during the first 3 weeks of storage at different environmental conditions. But,

the group kept in the dark room showed significant decrease than those kept under the sun.

But the higher the concentration of extract treatment in each sample, the lesser the TBA

value. Higher value was observed in crude palm oil.

58

It would be due to the fact that when PKO and PO are exposed to the sun and in the free air,

their unsaturated fatty acids fix oxygen and oxidize (Blumenthal and Stier, 1991).

Most any food can technically become rancid. The term particularly applies to oils.

Oils can be particularly susceptible to rancidity because their chemistry which makes them

susceptible to oxygen damage. When food scientists talk about rancidity, they are often

talking about a specific type of rancidity involving oxygen damage to foods, and this type

of rancidity is called "oxidative rancidity." During the process of oxidative rancidity,

oxygen molecules interact with the structure of the oil and damage its natural structure in a

way that can change its odour, its taste, and its safety for consumption.

Spices contain phenols and essential oils, which are inhibitory to microorganisms

(Nakatani, 1999). It was reported that fat and proteins bind or solubilize phenolic

compounds thereby reducing their availability for antimicrobial activity (McMance and

Widdowson, 1993; McNeil and Schmidt,1993).

4.3 Effect Of Monodora Myristica Extract On The Chemical Indices Of Oil On

Storage

The main objective of this study was to evaluate the antioxidant potential of

monodora myristica. Two different varieties of oil were treated with monodora myristica

extract and tested to determine the antioxidant potentials of monodora myristica using

standard methods. The analysis results demonstrated that the extract treatment of the oil

samples enhanced antioxidation.

59

CHAPTER FIVE

5.0 SUMMARY AND CONCLUSION

The study results favoured the highest concentration of treatment and storage of the

tested oil samples at the different environmental conditions.

Recently, the determination of PV in commercial oils was assessed by the modern Infrared

Spectroscopy (IR) (Yildiz et al., 2002); which can also be extended to the determination of

AV in butter. The theoretical principle of IR had been reported earlier (Koczoñ et al., 2001,

2003, 2006) and the technique finds application in the food analysis (Ismail et al., 1993;

Chippie et al., 2002; Guillen and Cabo, 2002; Tay et al., 2002; Van de Voort et al., 2004)

and significantly less number for monitoring of chemical changes in foods (Quilitzsch et

al., 2005).

Fats and oils are quite unstable substances. When stored for any considerable length

of time, especially when the temperature is high and the air has free access to them, they

deteriorate and spoil. In this respect different fats differ markedly. Some spoil very much

more rapidly than others. Among the various fats, spoilage takes the form of rancidity. The

fat acquires a peculiarly disagreeable odor and flavor. A vast amount of scientific research

has been carried on to determine the cause and nature of rancidity, but investigators are far

from agreement on the subject. For present purposes it is sufficient to point out that

spoilage of a fat, usually identical with rancidity, is accompanied by partial splitting of the

fat into glycerin and fatty acids. The glycerin disappears, or at any rate is unobjectionable,

but the fatty acids remain dissolved in the fat, give it an acid reaction, and contribute to its

objectionable rancid flavor. The rancidity of a given parcel of fat is not necessarily the

60

result of long storage under unfavorable conditions. The fat may have been spoiled and

rancid from the moment of its production. This will inevitably be true when the materials

from which it was produced have undergone decomposition. Thus the fat obtained from

putrefying carcasses will be rancid and so will the oil expressed from fermented cottonseed.

In other words, to obtain a sound and sweet fat, the raw material must be sound and sweet;

it must be worked up speedily before it has had time to decompose; and this must be done

under clean and sanitary conditions. The fat thus obtained must be stored under favorable

conditions and its consumption cannot be too long delayed. These conditions it is difficult

to obtain in many of the less civilized portions of the world, especially in the tropics, where

many fat- and oil-yielding raw materials are produced. Hence fats and oils made at the

source of the raw materials may be less sound than those produced at or near the place of

consumption.

All oils are fats, but not all fats are oils. They are very similar to each other in their

chemical makeup, but what makes one an oil and another a fat is the percentage of

hydrogen saturation in the fatty acids of which they are composed. The fats and oils which

are available to us for culinary purposes are actually mixtures of differing fatty acids so for

practical purposes we'll say saturated fats are solid at room temperature (20C) and

unsaturated fats we call oils are liquid at room temperature. For dietary and nutrition

purposes fats are generally classified as saturated, monosaturated and polyunsaturated, but

this is just a further refinement of the amount of saturation of the particular compositions of

fatty acids in the fats.

Connoisseurs of good edible palm oil know that the increased FFA only adds ‘bite’ to the

oil flavour. At worst, the high FFA content oil has good laxative effects. The free fatty acid

61

content is not a quality issue for those who consume the crude oil directly, although it is for

oil refiners, who have a problem with neutralization of high FFA content palm oil.

Oxygen is eight times more soluble in fats than in water and it is the oxidation

resulting from this exposure that is the primary cause of rancidity. The more

polyunsaturated a fat is, the faster it will go rancid. This may not, at first, be readily

apparent because vegetable oils have to become several times more rancid than animal fats

before our noses can detect it. An extreme example of rancidity is the linseed oil (flaxseed)

that we use as a wood finish and a base for oil paints. In just a matter of hours the oil

oxidizes into a solid polymer. This is very desirable for wood and paint, but very

undesirable for food.

Antioxidants are often added to fat-containing foods in order to retard the

development of rancidity due to oxidation.  Natural anti-oxidants include flavonoids,

polyphenols, ascorbic acid (vitamin C) and tocopherols (vitamin E). 

Synthetic antioxidants include butylated hydroxyanisole (BHA), butylated hydroxytoluene

(BHT), propyl 3,4,5-trihydroxybenzoate also known as propyl gallate and ethoxyquin. 

The natural antioxidants tend to be short-lived, so synthetic antioxidants are used when a

longer shelf life is preferred. 

62

The effectiveness of water-soluble antioxidants is limited in preventing direct oxidation

within fats, but is valuable in intercepting free radicals that travel through the watery parts

of foods. 

A combination of water-soluble and fat-soluble antioxidants is ideal, usually in the ratio of

fat to water.

In addition, rancidification can be decreased, but not completely eliminated, by

storing fats and oils in a cool, dark place with little exposure to oxygen or free radicals,

since heat and light accelerate the rate of reaction of fats with oxygen. (Oxidative rancidity

or autooxidation is a chemical reaction with a low activation energy consequently the rate

of reaction is not significantly reduced by cold storage).

5.1 LIMITATIONS

There were limitations to the present study which were barriers in achieving ideal

experimental conditions.

The current study was conducted on limited parameters of tested intervals and constant

temperature, the ranges for the interval for test at the tropical environmental conditions may

have been varied. Therefore the present study did not show the significant stability of the

tested oil samples.

5.2 FUTURE RECOMMENDATIONS

1. PalmOilTester which is a fast, user-friendly and reliable testing system for crude and

refined palm oil is recommended as it enables the determination of acidity (FFA),

63

DOBI & Carotene content, the values of Peroxide (PV), anisidine (AnV) and iodine

(IV) in few minutes.With its simplicity, PalmOilTester is ideal to performe analysis

during every production stages in palm oil industry to monitor the quality of oil in

real time, from the oil mill to the refinery plant, during the acceptance and storage

phases, as well as during trading of finished products.Several comparative studies

have demonstrated that the analytical accuracy of PalmOilTester matches that of

AOCS/MPOB reference methods, with the advantages that PalmOilTester is easier

to use and outputs results much faster.

2. The use of apparatus called Rancimat is recommended to calculate  effect of

antioxidant  on oil and fat, though other methods like used for determination of

rancidity are Peroxide value ( Primary Oxidation) and Anisidine value( Secondary

Oxidation)   in fat or oil .Peroxide value provides the extent of rancidity present in

the oil.Peroxi de value is found by formation of iodine when oil or fat are

reacts with iodine ion.  Totox  value are also used to check the quality of oil and fat .

Totox  Value = Anisidine value + 2x Peroxide value. Thiobarbituric acid value also

provides useful information on oxidative level of rancid oil.

64

BIBLIOGRAPHY

Akpanabiatu, M. I. Ekpa, O. D. ,  Mauro, A. and Rizzo, R. (2001). Nutrient Composition of

Nigerian Palm Kernel from Dura and Tenera Varieties of the Oil Palm (Elaeis

guineensis). Food Chem. 72:173-177.

Aubourg, S. (1999). Recent advances in assessment of marine lipid oxidation by using

fluorescence. Journal of American Oil Chemistry society. 76: 409-419.

Aubourg, S. (2001). Fluorescence study of the pro-oxidant effect of free fatty acids on

vegetable oils. Journal of science Food Agriculture. 81:385-390.

Banso, A. and Ayodele, P.O. (2005). Effect of two tropical spice extracts on the growth of

fungi in fruit juices. Nigerian Journal of Applied Arts and Sciences (NIJAAS) 1: 35-

42.

Banso, A. and Sani, A . (2003). Antimicrobial effects of leaf extract of Ricinus communis.

African Scientist. 4(3): 129-133.

Basturk, A., Javidipour, I., and Boyaci, I. H. (2007). Oxidative stability of natural and

chemically interesterified cottonseed, palm and soybean oils. Journal of Food

Lipids. 14 (2):170-188.

Bonner, J. and Varner, J. E. (1965). Plant Biochemistry. Academic Press, London. pp. 252-

703.

Chow, M. C. and Honley, C. C. (2000). Surface active properties of palm oil with respect to

the processing of palm oil. Journal of Oil Palm Research. 12(1): 107-116.

65

Cornelius, J. A. (2001) Comparison of Traditional and Industrial Palm Oil. Oil Palm News;

No.28.Tropical Development and Research Institute, London.

Coursey, D. G. (1963). The Deterioration of Palm Oil during Storage. Journal of West

African Science Association. 7:101-13.

Cutler, R. G. (1984). Antioxidants, aging, and longevity. In Free Radicals in Biology,

Academic, Orlando, Florida. I: 371-428.

Damenn, G. S., Goodman, G. E., Thornquist, M. D., and Cullen, M. R. (1996). Free

radicals and Antioxidants. Bio. Med., 334: 11 50-11 55.

Demarco, E., Savarese, M., Parisini, C., Battimo, I., Falco, S., Sacchi, R. (2007). “Frying

performance of a sunflower/palm oil blend in comparison with pure palm oil”.

European Journal of Lipid Science and Technology 109: 237.

Egan, H., Kirk, R. S. and Sawyer, R. (1981). Pearson’s Chemical Analysis Food. 8 th ed.

Churchill Livingstone, New York, Pp 507-546.

Eggeling, W. J. (2002). The Indigenous Trees of the Uganda protectorate (Revised and

enlarged by Ivan R. Dale). Government printer, Entebbe Uganda, Crown Agents

for the Colonies, London. Pp. xiii and 491.

Ekeke G.I, Uwakwe A.A. and Nwaoguikpe, R.N. (2000). Edible legumes as nutritionally

beneficial antisickling agents Nig. J. Biochemistry Mol. Biol. 16: 200-201.

Ekpa, O. D. an kpe, U. J. (1996). Effect of Coconut Oil Concentration on the Melting point

profile and Free Fatty Acid Formation of Palm Oil. Nigerian Journal 0f Chem. Res.

1 8-12. 

66

Ekpa, O. D.,  Fabara, E. P. and Morah, F. N. I. (1994) Variation in Fatty Acid Composition

of Palm Oils from two Varieties of the Oil Palm (Elaeis guineensis). J. Sci. Food

Agric. 64: 483-486.    

Ekpa, O. D., Akpanabiatu, M. I., Amelio, M. and Rizzo, R. (2001). A Comparative Study

of the Triglyceride and Fatty Acid Compositions of Palm Oil from Plantations in

South-Eastern Nigeria. Global J. Pure & Applied Sci. 7:61-65.

Ekpa, O. D., Akpanabiatu, M. I., Amelio, M. and Rizzo, R. (2001). A Varietal Differences

and Polymorphism in Palm Oil: A Case Study of Palm Oil Blended with coconut

Oil. Global J.  Pure & Applied Sci. 7:277-283. European Journal of Lipid Science

and Technology :Lipid - Fett Volume 109 Issue 4, Pages 373 – 379 Special Issue:

Palm oil Published Online: 15 Mar 2007 Copyright © 2010 WILEY-VCH Verlag

GmbH & Co. KGaA, Weinheim

Esuoso, K. O., Lutz, H., Bayer, E., Kutubuddin, M. (2000). Unsaponifiable lipid

constituents of some underutilized tropical seed oil. Journal of Agricultural

Food.Chem. 48(2): 231-234.

Evans, R. W. (1989). Pharmacognosy (13'~ ed); Baillere Tindall, London. pp. 527-665.

Ghoghari, A. M., Bagul, M.S., Anandjiwala, S., Chauhan, M. G. and Rajani, M. (2006).

Free radical scavenging activity of Aspidium cicutarium rhizome. J. Natural

Remedies. 6(2): 1 31 -1 34

Gibon, V. (2007). Palm oil refining. European Journal of Lipid Science and Technology

109(4).

67

Gill LS (1992). Ethnomedical uses of plants in Nigeria. University of Benin Press, Benin

city, Nigeria. p. 165, 248.`

Goh, S.H., Choo, Y.M., Ong, A.S. (2005). Minor constituents of palm oil. Journal of

American Oil Chem Soc 1985;62:237-40.

Ihenkoronye A.I. and Ngoddy P.O. (1985). Tropical Fruits and Vegetables. In: Integrated

food Science and Technology for the Tropics. Macmillan Publisher Ltd, London. pp

63-75.

Irvine, F. R. (2000). Woody Plants of Ghana with special reference to their uses. Oxford

University Press, London pp. 13-23.

Ismail, A.A., Van de Voort, G. Emo and Sedman, J. (1993). Rapid quantitative

determination of free fatty acids in fats and oils by Fourier transform infrared

spectroscopy. Journal of American Oil Chem Soc., 4: 335-341.

Jacobsberg B. (2000). Palm oil characteristics and quality. In: O. S. Chai, and A.

Awalludin, (Eds). Proceedings of the 1st MARDI Workshop on Oil Palm

Technology. Kuala Lumpur, Malaysia: Malaysia Agricultural Research and

Development Insitute (MARDI), 1974:48-70.

Jain, A., Singhai, A. K. and Dixit, V. K. (2006). In vitro evaluation of Tephrosia purpurea

pers for antioxidant activity. Journal of Naturalremedies. 6(1): 162-1 64.

Kanner, P. H., Guyton, K. E., and Gorospe, M. (1999). Oxidative stress and the

molecular biology of antioxidant defense (J. G. Scandolios, (Ed). Cold Spring

Harbor Laboratory Press, New York, pp. 247-272.

68

Kapoor, I. P. S., Bandana Singh, Gurdip Singh, Carola S. De Heluani, M. P. De Lampasona

and Cesar, A. N. C. (2009). Chemistry and in Vitro Antioxidant Activity of Volatile

Oil and Oleoresins of Black Pepper (Piper nigrum) Journal of Agricultural and

Food Chemistry;57 (12), 5358-5364

Kubow, S. (2009). Lipid Oxidation Products in Food and Atherogenesis. Nutrition Reviews

51(2).

Kuku, F. O. (1976). Some Mould-induced Changes in Palm Kernels. Nig Stored Products

Res Inst Tech Rep 9:62-72.

Kumaran, A. and Karunakaran, J. (2006). Antioxidant activity of polyphenols from

phyllantus debilis klein ex Willd. Journal NaturalRemedies. 6(2): 141 -146

Matthäus, Bertrand (2007). "Use of palm oil for frying in comparison with other high-

stability oils". European Journal of Lipid Science and Technology 109: 400.

Mielke, S. (2001). Present and future position of palm and palm kernel oils in world supply

and trade. Journal of American Oil Chemistry Society.,1985;62:193-97.

Morel, C. 0. (1997). Mechanisms of Fatty Acid Oxidation. http:llwww.atozofhealth.com.

Downloaded on the 1lth June 2005.

Murray, P. E., Vaya, J. and Aviran, M. (1990). Nutritional Antioxidant: Mechanisms .

of Action, Analyses of Activities and Modern Application. Bentham Science

Publishers Ltd. New York, pp. 99-110.

Naczk, M., J. Pink, R. Zadernowski and D. Pink, (2002). Multivariate model for the

prediction of total phenolic acids in crude extracts of polyphenols from canola and

rapeseed meals: A preliminary study. Am. Oil Chem. Soc., 8: 759-762.

69

Nakatani N (1999). Antioxidative and antimicrobial constituents of herbs and spices. In:

Spices, Herbs and Edible Fungi. G. Charalambous (Ed). Elsivier, London. pp. 251-

271.

Nasirullah, (2005). Physical refining: electrolyte degumming of nonhydratable gums from

selected vegetable oils. Journal of Food Lipids 12(2).

Nesaretnam, K and Muhammad, B. (1993). Nutritional properties of palm oil. Selected

Readings on the Palm Oil and its Uses. p. 57-67

Nielsen S. and Suzanne S. (2002). Introduction to chemical analysis of foods. Daryaganj,

Indian, Pp 181-202

Nwachukwu, N. (2000). Nutritional and Antinutritional substances in some selected

indigenous spices PhD Thesis, FUTO, Nigeria.

Nyam, K.L., Tan, C.P., Lai, O.M., ., Long, K., Che Man, Y.B.(2009). Physicochemical

properties and bioactive compounds of selected seed oils  LWT - Food Science and

Technology. 42 (8):1396-1403

Odo, F. O. and Ishiwu, C. N. (1999). Experimental procedures for food and water analysis.

Enugu: Computer edge. Pp 25-35.

Oguntimein, B., Ekundayo, O., Laasko, I. and Hitunen, R. (1999). Constituents of the

essential oil of Monodora tenuifolia. Flav. And Fragr. J., 4: 193-1 95.

Ojiako, O. A. and Akubugwo, E. I. (1997) An Introductory Approach to Practical

Biochemistry CRC Publications, Owerri, Pp. 132.

70

Okafor, J. C. (2003). Some useful Tropical Plans in Health Care Delivery, Forestry

Commission HQ, Enugu. pp. 3-9.

Okiy, D. A. (1979) Aspects on Quality of Palm Oil. NIFOR Newsletter No. 18 Pp 7-8.

Omenn, G. S., Goodman, G. E., Thornquist, M. D. and Cullen, M. R. (1996). Free radicals and Antioxidants. Bio. Med., 334: 11 50-11 55.

Palm Oil Research Institute of Malaysia, (PORIM). (1990). The PORIM Test Method.

Palm Oil Research Institute of Malaysia.

 Pantzaris T.P and Mohammed J. A. (2000): Palm Kernel oil article. Palm Oil Research

Institute of Malaysia (PORIM), 2000.

Pantazari TP, Ahmad MJ (2004). Palm Kernel oil. Palm oil processing technology report.

Res. Instit. of Malaysia. p.220.

Pearson, D. (1976) The Chemical Analysis of Foods. 7th Edition, Edinburgh (Churchill

Livingstone).

Quilitzsch, R., M. Baranska, H. Schulz and E. Hoberg, (2005). Fast determination of carrot

quality by spectroscopy methods in the UV-VIS, NIR and IR range. J. Applied Bot.

Food Qual., 79: 163-167.

  Renuka, D., Jayalekshmy R. (2007). Antioxidant efficacy of phytochemical extracts from

defatted rice bran in the bulk oil system  Food Chemistry 104 (2), pp. 658-664.

Rutowski, A. (1983). Traditional Palm Oil Processing in Western Africa. Fette Seifen

Anstrichmittel 85:262 – 267.

Sahart, M. H. (2001). Free radicals and Antioxidant. Biol. Med., 28:1685-1 696.

71

Shahidi, F. and Zhong, Y. (2009). Measurement of antioxidant activity in food and

biological systems ACS Symposium Series 956, pp. 36-66.

Simeh, A., Tengku A.and Tengku M. A. (2001). "The Case Study on the Malaysian Palm

Oil" Oil palm - the backbone of economic growth Global Oils and Fats business

magazine. 6(2): 6-8.

Stevenson, T. ( 2006). "Malaysia targets alternative fuels market". The Daily Telegraph

(London).http://www.telegraph.co.uk/finance/2952784/Malaysia-targets-alternative-

fuels-market.html. Retrieved 22 September 2009.

Stryer, L. (1988). Biochemistry 3rd ed. W. A. Freeman and Company, New York pp. 331

and 470.

.Talalaji, S. J. (1 999). Essential oil from Monodora myristica grown in Ghana. West

African Pharmacist, 4: 64-65.

Tan, B. K., Ohih, F. C. (1997). Malaysian palm oil chemical and physical characteristics.

PORIM Technology 1981;3. New findings on palm oil. Nutr Rev 1987;45:205-07.

Tan, C.P., Selamat, J. and Yusoff, M. S. (2002). Comparative studies of oxidative stability

of edible oils by differential scanning colorimeter and oxidative stability index

methods. Food Chemistry.76(3), pp.378-385.

Tay, A., Singh, S.S., Krishnan, A. and Gore, J.P. (2002). Authentication of olive oil

adulterated with vegetable oils using fourier transform infrared spectroscopy.

Lebensm. Wiss. u. Technol., 35: 99-103.

72

Van de Voort, F.R., Ismail, J. Sedman, J. Dubois and Nicodemo, T. (1994). The

determination of peroxide value by Fourier transform infrared spectroscopy. J. Am.

Oil Chem. Soc., 9: 921-926.

Yamane, K., Kawasaki, K.,Sone, K., Hara, T., Prakoso, T. (2007). Oxidation stability of

biodiesel and its effects on diesel combustion and emission characteristics.

International Journal of Engine Research 8(3).

Yesil-celiktas, O. (2009). Influence of supercritical carbon dioxide and methanolic extracts

of rosemary on oxidation and sensory properties of wheat germ oil. Journal of Food

Quality 8: 885-898.

Yildiz, G., R.L. Wehling and Cuppett, S. L. (2002). Monitoring PV in corn and soybean

oils by NIR Spectroscopy. Journal of American Oil Chemistry Society., 11: 1085-

1089

73

APPENDIX I

PREPARATION OF REAGENT AND SAMPLES

CRUDE FAT CONTENT OF MONODORA MYRISTICA

Sample: Monodora myristica (minced) 90g

Reagents: n-hexane (750ml).

(Extract concentration at equal volume of oil: 0.2, 0.4, 0.6, 0.8, 1.0 (ml))

ACID VALUE DETERMINATION (FREE FATTY ACID (FFA))

Sample: palm kernel oil (1g for each concentration)

Palm oil (1g for each concentration)

Reagents: Ethanol 95%v/v

Potassium hydroxide solution (0.1N)

Phenolphthalein indicator

Fat solvent- (ethanol and ether in 1:1 ratio)

Oxalic acid (0.1N)

Acid value =MgKOH/g

THIOBARBITURIC ACID DETERMINATION

Sample: palm kernel oil (10g for each concentration)

Palm oil (10g for each concentration) Reagents: HCl (4M)

Thiobarbituric reagent (0.288g/100ml of glacial acetic acid); Distilled water.

74

APPENDIX II

CALCULATIONS

CRUDE FAT CONTENT

%Fat = C-A × 100

B

Where A= Weight of empty flask

B= weight of the sample

C= Weight of flask + oil after drying

ACID VALUE

The acid value is the number of milligrams of potassium hydroxide required to neutralize

the acidity of one gramme of the oil or fat.

Acid value = 5.61× T (MgKOH/g)

Where T= volume of the milliliters of 0.1N required.

W= weight in grams of the oil sample

5.61 = Equivalent weight of KOH ( constant).

75

W

THIOBARBITURIC ACID NUMBER

TBA= 7.8 × O.D. mg malonaldehyde/kg

Where O.D= optical density (absorbance unit)

7.8= constant (k)

Wavelength = 538nm (Odo and Ishiwu , 1999).

Average for each parameter qualitative index was calculated thus;

Mean X= X/n

Where X= sum of the number of acid

n= total number of acid values

Standard deviation of qualitative index for each sample at different concentration of extract

and different environmental condition was calculated using the formular,

S.D= ∑(x- X) 2

Where x = qualitative index for each sample

X = average mean; n = total number of qualitative index.

76

n-1

APPENDIX III

SAMPLES USED FOR THE STUDY

Crude Palm Oil (CPO)

Crude palm kernel oil (CPKO)

 APPENDIX IV

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Figure: 6 n- Hexane extract of monodora myristica (African nut meg)

Figure: 7

Figure: 8

Palm Oil Specifications

 STANDARD PORAM

&MEOMA SPECIFICATIONS  

CODE PRODUCT DESCRIPTION FFA MNI IV MP DOBICOLOUR

R/Y

CLOUD

PT

OTSB1040 CRUDE PALM OIL                 (CPO) 5% 0.0025    2.3-

2.4   

OTSB1041 RBD PALM OIL AS PLAMITIC 0.10% 0.10% 50-55 33-39   3R  

OTSB1042 RBD PALM OLEIN 0.10% 0.10%56

MIN

24

MAX  3R  

OTSB1043 RBD PALM STEARINE 0.20% 0.15%48

MAX

44

MIN  3R  

OTSB1044

PALM FATTY ACID DISTILLATE

                                       (PFAD)

70 %

MIN1.0%          

OTSB1045 PALM ACID OIL                   (PAO)70 %

MIN3.0%          

OTSB1046

CRUDE PALM KERNEL OIL AS

LAURIC                                                                    

(CPKO)

0.10% 0.10%19

MAX    1R  

78

OTSB1047 RBD PALM KERNEL OIL 0.10% 0.10% 17-1824

MIN  1R/10Y  

OTSB1048 RBD PALM KERNEL OLEIN 0.10% 0.10%22

MIN

24

MIN  1R/10Y  

OTSB1049 RBD PALM KERNEL STEARINE              

OTSB1050RBD PALM KERNEL FATTY ACID

DISTILLATE                     (PKFAD)             

OTSB1051 MARGARINE 0.10% 16.00%   33-40   YELLOW  

OTSB105 VEGETABLE GHEE              

OTSB1053 VEGETABLE SHORTENINGS 0.10% 0.10% 40-50 40   2.5R/25Y  

OTSB1054 DOUGH FATS 0.10% 0.10% 33-39 46-51   3R  

 

PORAM PALM OIL REFINERS ASSOCIATION MALAYSIA

MEOMA MALAYSIAN EDIBLE OILS MANUFACTURERS ASSOCIATION

FFA FREE FATTY ACID

MNI MOISTURE & IMPURITIES

IV IODINE VALUE

MP MELTING POINT

DOBI DETERIORATION OF BLEACHIBILITY INDEX

79

R/Y RED/YELLOW

SAP SAPONIFIABLE MATTER

TFM TOTAL FATTY MATTER

PPM PERMITTED ANTIOXIDANTS

                   

                     

 

http://www.oilpac.com/palmoilspec.htm

 SPECIFICATIONS

Parameter Specifications

% FFA 4.78

% MVM 0.08

Peroxide Value 2.57

80