Evaluation of Antioxidant Potential of Monodora Myristica (African Nutmeg)

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
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.
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 AmorjiNike, Enugu state.
... Dr. Charles N. Ishiwu (Supervisor) .. Mr. Moses Ezenwali (Head of Department)
Date
Date
External Examiner August, 2010
Date
DEDICATION This work is dedicated to Almighty God for His faithfulness to me and for seeing me through, in this citadel of learning.
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. Ezesteven 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 wouldnt 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
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.
TABLE OF CONTENTS Title page Approval page Dedication Acknowledgement Abstract Table of content List of tables List of figure Abbreviation ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------i ii iii iv v vi-viii xi x xi
CHAPTER ONE 1.0 1.1 Introduction --------------------------------------------------------Significance of study ----------------------------------------------1 6
CHAPTER TWO 2.0 2.1 2.1.1 2.1.2 2.1.3 2.2 2.2.1 2.2.2 2.2.3 2.2.4 Literature Review -----------------------------------------------7 7 7 8 8 9 10 10 11 14
African nutmeg (Monodora myristica) ---------------------------Scientific classification ------------------------------------------
Habitat/ ecology of Mondora myristica ---------------------------Characteristics/morphology of monodora myristica ------------Oil Palm ---------------------------------------------------
Scientific classification ----------------------------------------------Origin and description of palm oil --------------------------------The Chemical composition of palm oil -------------------------Physical characteristics of palm oil products -----------------7
2.3 2.3.1 2.4 2.5 2.5.1 2.5.2 2.6 2.6.1 2.6.2 2.7 2.7.1 2.7.2 2.7.3 2.7.4 2.7.5 2.8 2.8.1 2.9
Palm kernel oil -----------------------------------------------------The chemical composition of palm kernel oil------------------Modern uses of palm oil and palm kernel oil------------------Lipid oxidation----------------------------------------------------Lipid oxidation pathway -----------------------------------------Mechanism of oxidation -----------------------------------------General antioxidant action --------------------------------------Mechanism of antioxidant action -----------------------------Antioxidant molecules -------------------------------------------General review of photochemistry of monodra myristica -Alkaloids ---------------------------------------------------------Flavonoids -------------------------------------------------------Glycosids -------------------------------------------------------Saponins ---------------------------------------------------------Tannins -----------------------------------------------------------Application of vegetable oils ----------------------------------Factors that cause oxidative rancidity in vegetable oil -----Nutritional signification -----------------------------------------
14 15 16 16 21 22 24 24 26 27 27 27 28 28 29 29 30 33
CHAPTER THREE 3.0 3.1 3.2 3.3 3.4 3.5 3.5.1 3.5.2 Materials and methods -------------------------------------------Equipment/apparatus ---------------------------------------------procedurement of raw materials --------------------------------Study design --------------------------------------------------------Sample preparation -----------------------------------------------Chemical analysis ------------------------------------------------Determination of acid value (Av) ------------------------------Determination of thiobarbituric acid number.-----------------8
34 35 35 37 37 38 38 38
3.6
Statistical analysis ------------------------------------------------
39
CHAPTER FOUR 4.0 4.1 4.2 4.3 Result and Discussion ------------------------------------------Changes in Acid value of Palm Kernel and palm oil -------Changes thiobarbituric acid value of palm kernel and palm oil--Effect of monodora myristica extract on the chemical indices of oil on storage -------------------------------47 40 46 46
CHAPTER FIVE 5.0 5.1 5.3 Summary and conclusion -------------------------------------------limitations--------------------------------------------------------------Future recommendation ---------------------------------------------48 51 51
References ------------------------------------------------------------------------Appendix I -------------------------------------------------------------------------Appendix II ------------------------------------------------------------------------Appenedix III ----------------------------------------------------------------------
53 62 63 65
LIST OFF TABLES Table 1: Table 2: Table 3: Table 4: Table 5: Table 6: Fatty acid composition of palm oil (palm oil) Fatty acid profile of palm kernel oil (palm kernel ) Acid value for palm kernel oil Acid value for palm oil Thiobarbituric acid value for palm kernel oil 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: AV: FFA: PV: PKO: PO: PUFA: ROS: SR: SS: TBA: Association of America Chemistry Society Acid value Free fatty acid Peroxide value Palm kernel oil Palm oil Polyunsaturated fatty acid Reactive oxygen specie Storage in room Storage in sun Thiobarbituric acid
12
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 mans 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 lowermolecular 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 1400s 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. 2. To carryout solvent extraction of Monodora myristica 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: Family:
Magnoliales Annonaceae
Genus :
Monodora
19
Species: Binomial name
myristica
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)
21
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.
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TABLE 1. Fatty acid composition of palm oil (PO)
% of total acids Acid 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:0 Range 0.1-1.0 0.9-1.5 0.1-0.3 4.2-5.1 Mean 0.2 1.1 0.1 4.5 Source: (Pantzaris and Ahmad, 2004).
41.8-46.8 44.0 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.
37.3-40.8 39.2 9,1-11.0 10.1 0.0-0.6 0.2-0.7 0.4 0.4
2.2.4 PHYSICAL CHARACTERISTICS OF PALM OIL PRODUCTS Palm oil is a semi-solid at room temperature (28C), the melting point range being from 3240C. The slip melting point method is commonly adopted for measuring this parameter. By the DSC method the fat melts completely at 3940C, when heated at 5C/min, from an oil cooled rapidly to 40C at 5C/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
25
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 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) Myristic (C14:0) Palmitic (C16:0) Capric (C10:0) Caprylic (C8:0) 48.2 16.2 8.4 3.4 3.3
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Stearic (C18:0) Oleic (C18:1) Linoleic (C18:2) Others (unknown)
2.5 15.3 2.3 0.4
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. .
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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 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:
28
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
29
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.
30
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:
31
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.
2.5.1
LIPID OXIDATION PATHWAY UNSATURATED FATTY ACIDS
FREE RADICALS
OXYGEN
Insolubilization of proteins
HYDROGENPEROXIDES
Breakdown products Such as aldehydes, free
32
fatty acids, alchohol , and hydrocarbons Polymerization (dark colour) Source:
Oxidation of pigments, flavours and vitamins
(Ihekoronye and Ngoddy, 1985)
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
33
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 the double bond and so makes H removal easier.
Propagation During the propagation stage, the free radical reacts with oxygen to form peroxidecontaining 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).
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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: R +R RR ROR (Ihekoronye and Ngoddy, 1985)
RO + R
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
35
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 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
36
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 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 chainbreaking 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
37
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.
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 ncontaining 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
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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 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
39
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 antiinflammatory, anti-fungal, antimycolic, bacteriostatic and other biological activities. When ground in a powdering form, causes violent sneezing
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 40
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, 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
41
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 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
42
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, 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 150F (66C) 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).
43
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 xrays), 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.
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).
44
CHAPTER THREE 3.0 MATERIALS AND METHODS Maker Larle Ohaus scale corporation DHG 9101 Model Lemfield 801A Model Permagold Simax
3.1 Equipments / Apparatus weighing balance (triple beam balance) Analytical balance, Labouratory dry oven (hot air oven) Uv-Vis Spectrophotometer Water bath Volumetric flask Measuring cylinder, Beaker,
45
Cornical flask, burette Soxhlet extractor, Heating mantle, Extraction thimble, Condenser, pH meter Filter paper Mortar and pestle Hannah microprocessor Whatman Pyex
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 70C until use.The spices (dried samples) were estimated to have been in the market for 6-7days before purchase.
46
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.
Figure 4: African nutmegs: dried seed kernels, which have been removed from their fruit husk, seed covering and hard seed coat.
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Figure 5: Transverse section of palm fruit. 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
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The method of AOAC (1990) was used. The African nutmeg seeds were heated in an oven (hot air) at 105C (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 - 69C) using soxhlet apparatus. The whole filtrate was allowed to evaporate at room temperature leaving the oil.
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 cm 3 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.
49
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 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.
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3.6 STATISTICAL ANALYSIS All statistics were performed using MICROSOFT EXCEL version 2007 software.
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
51
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.
Table 3
ACID VALUE FOR PKO (MgKOH/g)
CONCN. OF EXTRAC T (ML) Control (0.00) 0.2 0.4 0.6 0.8 1.0
PRESTORAG E DAY
WEEK 1 SS
WEEK 1 SR
WEEK 2 SS
WEEK 2 SR
WEEK 3 SS
WEEK 3 SR
4.750.00 4.700.15 4.450.12 3.920.41 3.440.32 3.060.91
5.920.36 5.660.28 5.300.28 4.960.31 4.540.29 4.080.34
4.890.31 4.740.36 4.520.12 4.080.26 3.940.31 3.700.31
8.240.20 8.060.32 7.860.37 7.320.41 6.920.51 6.400,28
5.380.08 5.140.13 5.000.31 4.840.26 4.620.31 4.280.31
14.7210 14.680.31 13.560.48 13.180.26 13.060.26 13.100.13
9.700.51 7.740.26 7.220.21 6.260.21 6.180.26 5.990.31
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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; AV = Acid Value; PKO = Palm Kernel Oil; SD = Standard deviation SR = Storage in Room TBA = Thiobarbituric Acid PO = Palm Oil
Table 4: CONCN. OF PREWEEK 1
ACID VALUE FOR PO (MgKOH/g) WEEK 1 SR WEEK 2 SS WEEK 2 SR WEEK 3 SS WEEK 3 SR
STORAGE SS
EXTRACT DAY (ML) Control (0.00) 0.2 0.4 0.6 0.8 1.0 14.270.26 15.710.23 14.130.11 13.920.26 13.531.02 13.271.31 13.061.42 15.241.24 15.010.08 14.940.15 14.510.92 14.391.10 15.420.18 17.750.13 16.260.08 21.020.12 18.740.36 15.211.13 14.730.52 14.710.91 13.810.71 13.361.02 17.320.52 16.630.08 16.260.15 15.840.12 14.811.10 16.060.19 16.001.10 15.720.90 15.481.21 14.131.40 20.830.26 19.920.52 19.610.81 18.911.00 17.261.23 18.220.34 16.880.26 16.320.31 16.020.81 15.720.92
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
53
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).
Table 5: CONCN. OF PRE-
THIOBARBITURIC ACID VALUE FOR PKO WEEK 1 WEEK 1 SR WEEK 2 SS WEEK 2 SR WEEK 3 SS WEEK 3 SR
STORAGE SS
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
54
Table 6: CONCN. OF PRE-
THIOBARBITURIC ACID VALUE FOR PKO WEEK 1 WEEK 1 SR WEEK 2 SS WEEK 2 SR WEEK 3 SS WEEK 3 SR
STORAGE SS
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.
55
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).
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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.
57
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.
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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 (Koczo 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
59
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
60
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.
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.
61
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 1.
FUTURE RECOMMENDATIONS 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), 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
62
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.
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63
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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-alternativefuels-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.
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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.
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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 W (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).
74
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
n-1
Where x = qualitative index for each sample X = average mean; n = total number of qualitative index.
75
APPENDIX III
SAMPLES USED FOR THE STUDY
Figure: 6n- Hexane extract of monodora myristica

(African nut meg)
Figure: 7
Crude Palm Oil (CPO)
76
Figure: 8 APPENDIX IV Palm Oil Specifications
Crude palm kernel oil (CPKO)
STANDARD
PORAM
&MEOMA SPECIFICATIONS CODE PRODUCT DESCRIPTION FFA MNI IV MP DOBI R/Y 2.3OTSB1040 CRUDE PALM OIL (CPO) 5% 0.0025 2.4 OTSB1041 RBD PALM OIL AS PLAMITIC OTSB1042 RBD PALM OLEIN 0.10% 0.10% 50-55 33-39 56 24 0.10% 0.10% MIN MAX 48 44 0.20% 0.15% MAX MIN 3R 3R
COLOUR CL
PT
OTSB1043 RBD PALM STEARINE
3R
PALM FATTY ACID DISTILLATE OTSB1044 (PFAD)
70 % 1.0% MIN
70 % OTSB1045 PALM ACID OIL (PAO) 3.0% MIN OTSB1046 CRUDE PALM KERNEL OIL AS0.10% 0.10% 19
1R
77
LAURIC MAX (CPKO) 24 OTSB1047 RBD PALM KERNEL OIL 0.10% 0.10% 17-18 22 OTSB1048 RBD PALM KERNEL OLEIN OTSB1049 RBD PALM KERNEL STEARINE RBD PALM KERNEL FATTY ACID OTSB1050 DISTILLATE (PKFAD) OTSB1051 MARGARINE OTSB105 VEGETABLE GHEE OTSB1053 VEGETABLE SHORTENINGS OTSB1054 DOUGH FATS 0.10% 16.00% 33-40 YELLOW 0.10% 0.10% MIN MIN MIN 24 1R/10Y 1R/10Y
0.10% 0.10% 40-50 40 0.10% 0.10% 33-39 46-51
2.5R/25Y 3R
PORAM MEOMA FFA MNI IV MP DOBI R/Y SAP TFM PPM
PALM OIL REFINERS ASSOCIATION MALAYSIA MALAYSIAN EDIBLE OILS MANUFACTURERS ASSOCIATIO FREE FATTY ACID MOISTURE & IMPURITIES IODINE VALUE MELTING POINT DETERIORATION OF BLEACHIBILITY INDEX RED/YELLOW SAPONIFIABLE MATTER TOTAL FATTY MATTER PERMITTED ANTIOXIDANTS
78
http://www.oilpac.com/palmoilspec.htm SPECIFICATIONS Parameter % FFA % MVM Peroxide Value
Specifications 4.78 0.08 2.57
79

Evaluation of Antioxidant Potential of Monodora Myristica (African Nutmeg)

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EVALUATION OF ANTIOXIDANT POTENTIAL OF MONODORA MYRISTICA (AFRICAN NUTMEG)

IBOLO MARTHA NNEKA BC/2006/076

EVALUATION OF ANTIOXIDANT POTENTIAL OF MONODORA MYRISTICA (AFRICAN NUTMEG)

IBOLO MARTHA NNEKA BC/2006/076

A PROJECT SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY

External Examiner August, 2010

CHAPTER ONE 1.0 1.1 Introduction --------------------------------------------------------Significance of study ----------------------------------------------1 6

African nutmeg (Monodora myristica) ---------------------------Scientific classification ------------------------------------------

Statistical analysis ------------------------------------------------

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

Species: Binomial name

2.2 OIL PALM

Figure 2: African Oil Palm fruits (Elaeis guineensis)

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

37.3-40.8 39.2 9,1-11.0 10.1 0.0-0.6 0.2-0.7 0.4 0.4

Stearic (C18:0) Oleic (C18:1) Linoleic (C18:2) Others (unknown)

2.5 15.3 2.3 0.4

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

Lipid oxidation follows three main steps: Initiation Propagation Termination

There can also be cleavage on the other side:

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

LIPID OXIDATION PATHWAY UNSATURATED FATTY ACIDS

Breakdown products Such as aldehydes, free

fatty acids, alchohol , and hydrocarbons Polymerization (dark colour) Source:

Oxidation of pigments, flavours and vitamins

(Ihekoronye and Ngoddy, 1985)

Figure 3: lipid oxidation pathway 2.5.2 MECHANISMS OF OXIDATION

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

2.6.1 MECHANISM OF ACTION OF ANTIOXIDANTS

2.7 GENERAL REVIEW OF PHYTOCHEMISTRYOF MONODORA MYRISTICA

3.4 SAMPLE PREPARATION

3.5 CHEMICAL ANALYSIS

ACID VALUE FOR PKO (MgKOH/g)

4.750.00 4.700.15 4.450.12 3.920.41 3.440.32 3.060.91

5.920.36 5.660.28 5.300.28 4.960.31 4.540.29 4.080.34

4.890.31 4.740.36 4.520.12 4.080.26 3.940.31 3.700.31

8.240.20 8.060.32 7.860.37 7.320.41 6.920.51 6.400,28

5.380.08 5.140.13 5.000.31 4.840.26 4.620.31 4.280.31

14.7210 14.680.31 13.560.48 13.180.26 13.060.26 13.100.13

9.700.51 7.740.26 7.220.21 6.260.21 6.180.26 5.990.31

Table 4: CONCN. OF PREWEEK 1

ACID VALUE FOR PO (MgKOH/g) WEEK 1 SR WEEK 2 SS WEEK 2 SR WEEK 3 SS WEEK 3 SR

Table 5: CONCN. OF PRE-

Table 6: CONCN. OF PRE-

Changes in Thiobarbituric acid value of PKO and PO

SAMPLES USED FOR THE STUDY

Figure: 6n- Hexane extract of monodora myristica

Crude Palm Oil (CPO)

Figure: 8 APPENDIX IV Palm Oil Specifications

Crude palm kernel oil (CPKO)

OTSB1043 RBD PALM STEARINE

PALM FATTY ACID DISTILLATE OTSB1044 (PFAD)

0.10% 0.10% 40-50 40 0.10% 0.10% 33-39 46-51

PORAM MEOMA FFA MNI IV MP DOBI R/Y SAP TFM PPM

http://www.oilpac.com/palmoilspec.htm SPECIFICATIONS Parameter % FFA % MVM Peroxide Value

Specifications 4.78 0.08 2.57

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