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Oxidative stress is a concern for human physiology and food industries; it has been linked to cause many diseases such as, cardiovascular disease, atherosclerosis cancer, neurodegenerative disorders, diabetes, cataract ageing and DA damage. In food industry; oxidative stress is the big cause of oxidative rancidity and spoilage of fruits, vegetable and beverage. For this reasons there has been so much research conducted to understand better free radicals and the role of antioxidants in scavenging free radical. There is much interest being shown in the Mediterranean diet due to the discovery of its health benefits. Different methods so far have been developed to measure antioxidant activity present in different products. In this project, antioxidant activity of phenolic compound from olive and olive oil extract will be examined; using known methods (DMPD) N<N-dimeththyl-p-phenylenediamine and (FRAP) Ferric Reducing Ability of Plasma will
Free radicals are chemicals with unpaired electron such as superoxide, hydroxyl radical, hydrogen peroxide, peroxyl radical, nitric oxide and nitrogen dioxide. They are produced by oxygen and nitrogen (reactive oxygen species) and (reactive nitrogen species) respectively. They do not live long and have half lives in milli-, micro- or nanoseconds. Free radicals are not stable; therefore they go about reacting with any biological molecule in the body to achieve stability. In doing so, much damage is brought to the tissue and leads to etiology of number of human diseases.
Free radicals became present in our bodies through normal human metabolic processes and through different physiochemical situations such as, exposure to environmental pollutant, radiation, ultraviolet light cigarette smoke and other toxic substances.
Dangers of free radical
There is no biological substance not subject to free radical damage. They cause damage to the lipids present in the membrane by causing lipid peroxidation (LP). This process leads to the abstract of hydrogen atom from methylene group CH2 resulting into unpaired electron on carbon atom ( .CH). This radical becomes stable by causing molecular rearrangement thus resulting into conjugated diene. When this react with oxygen, lipid peroxyl radical (LOO) is produced; this in turn removes another hydrogen atom from the lipid producing lipid hydroperoxidess (LOOH) and causes more LP. Production of toxic substances such as malondialdehyde MDA, 4_hydroxynonenal (4-HNE), Isoprostanes and different 2-alkenals are due to lipid peroxidation : free radicals affects carbohydrates by removing the hydrogen bond from carbon atom and the result is carbon centred radicals . Breakage of chain in the hyaluronic acid is the result of this carbon radical. The DNA is affected by the additional of extra base or abstraction of the hydrogen atom from the sugar moiety. This damage to the DNA leads to the mutation of DNA and over time; the changes in genetic material can lead to many human cancer. This can be supported by different research which shows a connection of free radicals with tumour suppressor genes and proto-oncogenes in human cancer.
Proteins are inactivated by free radicals and the inactivated proteins build up in the body lead to different disease such as cataract and Lipofuscin in Alzheimer's disease.
The activation of Lipid Peroxidation, protein oxidation and protein nitration leads to different neurodegenerative Disorders. Increased levels of 4-hydroxynonenal (4-HNE) is a characteristic of Alzheimer's disease (AD) as a result of LP. 4-HNE leads to neurotoxic action. Another complication of LP is the destruction of membrane ion-motive ATPases (sodium/ potassium ATPase and calcium ATPase), glucose transporters and glutamate transporters; this then causes death to the neuron.
Research shows the involvement of free radicals in the start and complication of Diabetes. Constant increased levels of plasma glucose (hyperglycemia) in the diabetic results into oxidative stress. This is because of auto- oxidation of glucose, non -enzymatic glycosylation and polyol pathway. Free radicals such as superoxide and hydroxyl are produces from auto- oxidation of glucose. These radicals with any biological substance and can stop vasodilatation when they react with nitric oxide in blood vessels. Superoxide and hydroxyl radicals leads to advanced end product of glycation AGEs like midozoles and pyrroles. Crosslinking AGEs- protein with macromolecules causes abnormalities in the tissue.
Free radicals are associated with ageing this is due to, ROS production and damaged mitochondria DNA. Atherosclerosis result when LPL has been oxidised and peroxides are produced. The free radical then damages the arterial wall which leads to the inflammation of arterial wall then atherosclerosis develops.
Benefit of free radicals
Despite the effect free radicals causes to the body, it is important to understand that they are produced in normal human metabolic processes to perform essential roles. They are produced in the mitochondria during the process of oxidative phosphorylation; this is an important process because it produces more ATP for the body. Immune system component such as macrophage and cytotoxic lymphocytes destroys pathogens and cancer cell by releasing free radicals. They are used by Cytochrome P450 in the liver to detoxify xenobiotics. They are used by oxygenese to produce components such as prostaglandins and leukotrienes. They are used in apoptosis of damaged cells (TPA et al 2004)
Antioxidants are molecules capable to stop detrimental effect caused by free radicals; they are present in the body and others we get from the diet. Antioxidant can be enzymic and non enzyimic. Those of enzymic nature include superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, thioredoxin,thiols and disulfide bonding. Non enzymic antioxidant includes vitamin E (α- Tecopherol), vitamin C (ascorbic acid), carotenoids, flavonoids phenol compounds, α- lipoic acid and glutathione. Antioxidant protect from the harmful effect caused by free radicals in three different ways. Preventative antioxidant such as superoxide dismutase stops the creation of (ROS). Interceptive antioxidant like vitamin C, vitamin E, glutathione, some thiol compounds, carotenoids and flavonoids. The third action of antioxidant is to repair the damage caused by free radical and this involves repair enzymes (TPA et al 2004) such as methionine-S-sulfoxide reductase A (MSRA) and methionine-R-sulfoxide reductase B (MSRB) (Taungjaruwinai at al 2009)
Antioxidant have proved to be effective in neutralising free radicals, the population is now being encouraged to use natural product such as tea which contains good amount of antioxidant. According to research this has proved to reduce many human cancers. There is now medication being develop to help prevent and treat diseases such as Alzheimer's disease, Parkinson's disease, stroke, atherosclerosis, diabetes and cancer (TPA et al 2004)
Research has shown a lower incidence of Coronary Heart Disease in the Mediterranean basin compared non Mediterranean countries. This difference has been attributed to the Mediterranean diet which is rich in legumes, grain, fresh fruit and vegetable and olive oil which serves as the main fat component in this diet.
Olive oil was not worldly recognised; of the total vegetable oil it contributed to approximately 4% and the world production of about 2,00,000 tones / year. The oil is now receiving much attention due to its health benefit. Non Mediterranean countries such as united state of America, Canada, Australia, South America, and Japan have started producing the oil so the world production is increasing.
Olive oil comes from Drupes of Olea Europea L tree. This tree grows best at 30 to 45 degree Celsius. The main production about 95% comes from Mediterrean countries; of this percentage 75% is from Spain, Italy, Greece and Maghrebian countries.
Olive oil contains Oleic acid and linolic acid; linolic acid is the main monounsaturated fatty acid and ranges from 56 to 84 % of the total fatty acid. Linolic acid is the main essential fat acid and amounts to 7 - 10 %. Studies have shown MFA lowers cholesterol levels in the plasma. Other studies show that MFA increases the level of HDL better than polyunsaturates (PUFA) when they replace carbohydrates in the diet.
In addition to the above fatty acids, olive oil contains: vitamin such as alpha and gamma tecopherols,B-carotene and chlorophylls (are responsible for the colour of the oil), phytosterols, pigment, teicpenic acid, flavonoid such as luteolin and quercetin,squalence and phenolic compounds (Francesco et al 2002).
In this research the main interest will be the examination of antioxidant activity of this component of olives and olive oil phenolic compounds. Phenolic compound contains hydroxytyrosol ( 3,4-digydroxyphenylethanol), tyrosol,oleuropein and its aglycon caffeic acid, vanillic acid, syringic acid protocatechuic acid and p- hydroxyphenylacetic acid.
The hydroxytyrosol is the main strong antioxidant present in olive; they stabilise free radicals by donation hydrogen (Francesco et al 1998).
Among the method developed to measure antioxidant activity, Ferric Reducing Ability of Plasma (FRAP) is one of the most efficient methods of assessing antioxidant activity. It is quick, reproducible and gives recognized index of antioxidant ability of biological fluids. In the presence of chemical reluctant or biological antioxidants; the ferric (FeIII-TPTZ) is reduced to a ferrous
(FeII-TPTZ) blue in colour. The results are collected by comparing the change in absorbance at 593 of the sample with change in absorbance of known concentration containing ferrous ions. (Iris et al 1996)
in the DMPD (N,N-dimethyl-p-phenylenediamine) method, a coloured DMPD radical is produced in the presence of an oxidant solution. When an antioxidant solution is added to the DMPD radical, a stable DMPD radical is produced and the solution is decolourised. This method is fast and effective (Vincenzo et al 1999). So far I have used the DMPD method and the following is the procedure
DMPD Assay method
3.116g anhydrous sodium acetate
0.72 acetic acid
0.3375g ferric acid
500ml volumetric flask
100mls volumetric flask
25mls volumetric flask
5mls volumetric flask
2mls plastic cuvette
Making5mls DMPD stock solution
Weigh 104.5mg of DMPD directly into 5mls volumetric flask
Make up to the volume with deionised water
Making 500mls acetate buffer
Weigh out 3.116g of sodium acetate and 0.72mls of acetic acid
Add to 500mls volumetric flask
Fill it up to the volume with deionised water
Making 25mls ferric chloride solution
Weigh0.3375g of ferric chloride directly into 25mls volumetric flask
Make up to the volume with deionised water
Method for DMPD working solution
Collect 1ml of DMPD (100mM) solution
Add to 100mls volumetric flask
Make up to the volume (100mls) with acetate buffer
Add 0.2ml of ferric chloride solution
Now collect 2mls of this solution and put it in a 2msl plastic cuvette
Measure the absorbance at 250 - 850 nm
In the above graphs, the absorbance of the DMPD radical is reducing while the concentration of Trolox in figure 1 and ascorbic acid in figure 2 increases. Free radical DMPD decolourises in the presence of oxidant Trolox and ascorbic acid they have donate an electron to stabilise the radical thus the reduction in absorbance being seen,
Project work plan 2010/2011
Week number 11
Week commencing 6th Dec
8th Dec: 3 hours in the laboratory doing practical work and analysis
9th Dec: 5 hours reading and writing
Week number 12
Week commencing 13th Dec
15th Dec: 3 hours in the laboratory practical and analysis work
16th Dec: 5 hours reading and writing
Week number 16
Week commencing 3rd Jan
5th Jan: 4 hours in the laboratory doing practical and analysis
6th Jan: 4 hours reading and writing
Week number 17
Commencing on 10th Jan
12 Jan: 4 hours reading and writing
13th : 4 hours in the laboratory doing practical work and analysis.
Week number 18
Commencing on 17th Jan
19th Jan : 4 hours reading and writing
20th Jan :4 hours practical work and analysis
Commencing 31st Jan
2nd Feb : 4hours in the laboratory doing practical and analysis work
3rd : reading and writing approximately 4 Hours
Week number 21
Commencing on 7th Feb
9th Feb: reading and writing approximately 6 hours
10th: in the laboratory approx 2 hours
Week number 22
Commencing on 14th Feb
16th : in the laboratory about 2 and half hours practical and analysis work
17th : reading and writing for about 6 hours
Week number 23
Commencing on 21 Jan
23rd Jan: 6 hours reading and writing
24th : 2 in the Laboratory practical and analysis
Week number 24
Commencing on 28th Mar
2rd Mar: in the laboratory practical and analysis work approx 2 and half hours
3rd Mar: about 6 hours reading and writing
Week number 25
Commencing on 7th Mar
9th Mar: writing for about 6 hours
1oth Mar: practical and analysis about 3 hours
Week number 26
Commencing on 14th Mar
16th Mar: practical and analysis work approximately 4 hours
17th Mar: writing for about 6 hours