Evaluation Of Antioxidant Potential Of Monodora Myristica (African Nutmeg)

Project and Seminar Material for Biochemistry

Evaluation Of Antioxidant Potential Of Monodora Myristica (African Nutmeg)


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.

Chapter One

1.0 Introduction

1.1 Background of the Study

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.

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 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).

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 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).

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).

1.2 Statement of the Problem

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).

1.3 Objectives of the Study

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.

1.4 Research Question

The following questions will guide this study;

  1. What is the solvent extraction of Monodora myristica?
  2. What is the antioxidant effect of Monodora myristica extract on palm kernel oil and palm oil at different environmental conditions?

1.5 Significance of the Study

The findings of this study will enable the dignitaries in the biochemistry field understand the solvent extraction of Monodora myristica, and the antioxidant effect of Monodora myristica extract on palm kernel oil and palm oil at different environmental conditions.

Additionally, subsequent researchers will use it as literature review. This means that, other students who may decide to conduct studies in this area will have the opportunity to use this study as available literature that can be subjected to critical review. Invariably, the result of the study contributes immensely to the body of academic knowledge with regards to the evaluation of antioxidant potential of monodora myristica (African nutmeg).

1.6 Scope of the Study

The study will evaluate the antioxidant potential of monodora myristica (African nutmeg). The study will further narrow down to the solvent extraction of Monodora myristica, and the antioxidant effect of Monodora myristica extract on palm kernel oil and palm oil at different environmental conditions.

1.7 Limitation of the Study

Like in every human endeavour, the researcher encountered slight constraints while carrying out the study. Insufficient funds tend to impede the efficiency of the researcher in sourcing for the relevant materials, literature, or information and in the process of data collection. More so, the researcher simultaneously engaged in this study with other academic work. As a result, the amount of time spent on research will be reduced.

1.8 Definition of Terms

  • 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

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 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 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.

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. Palm Oil Tester 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, Palm Oil Tester 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 Palm Oil Tester matches that of AOCS/MPOB reference methods, with the advantages that Palm Oil Tester 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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