1. Introduction
Most people know about antioxidants and belive in them as preventers against cell damage, which in the most severe case can cause cancer. Almost all nutritions contain a certain amount of antioxidant – both chemical and/or biological. To measure the activity and amount of the antioxidants present in a sample, some distinctive but easy assays have been established. This paper will give a short overview of the ORAC (oxygen radical absorbance cacpacity) assay and compare it with other antioxidant assays.
Besides that, the paper introduces some preliminary results on antioxidant activity of the plant Apocynum venetum conducted by the author.
Fig. 1 on cover page from [9]
Table of Contents
1. Introduction 2
2. The ORAC assay – a brief introduction 4
3. Biochemical background of antioxidant activity 6
4. Comparison of ORAC with other antioxidant activity assays 7
5. Results in current research 8
6. Discussion and conclusions 9
References 10
2. The ORAC assay – a brief introduction
2.1 Theoretical background
The oxygen radical absorbance capacity (ORAC) assay is a method for measuring the total antioxidant activity in a biological sample. Biological samples include body fluids of animals and humans (serum, plasma, urine, saliva), plant extracts, agricultural and food products, and pharmaceutical products.[6]
The advantage of the ORAC assay is the wide range of applications as it can be used for both lipophilic and hydrophilic samples and compounds. Besides measuring the total antioxidant capacity, the assay can also qualitatively measure the amount of fast versus slow acting antioxidants in a sample.
The principle of the ORAC is based on the following scheme:
Fig. 2: Principal order of the ORAC assay[10]
The sample contains a certain amount of compounds with an antioxidant activity. In water soluble samples, fluorescein is used as the probe which is protected by the antioxidants.[3] After adding a certain amount of a free radical, the loss in fluorescence over time is measured until the whole fluorescence is eliminated and the scavenging activity of the antioxidant is vanished. By integrating the area under the kinetic curve relative to the blank, the concentration of all antioxidants present in the sample can be calculated. Trolox, a water soluble tocopherol derivative, is used as a standard to calculate the antioxidant activity of the sample in trolox equivalents (μmol TE/g).
2.2 Fluorescein reaction
Fluorescein belongs to the group of triphenylmethane dyes with a xanthene structure. Its fluorescence is based on the oxygen withdrawing groups and the intermittend double bounds shifting the wavelength towards the visible light range. Radicals can distubr this structure and erase the fluorescence by destructing one aromatic ring structure as seen in the reaction scheme.
Data from Table 1. confirms the theory that as the concentration of glucose increases so will the absorbance of the solution when examined with the glucose oxidase/horseradish peroxidase assay. Glucose within the context of this assay is determined by the amount of ferricyanide, determined by absornace, which is produced in a one to one ratio.1 Furthermore when examining the glucose standards, a linear calibration curve was able to be produced (shown as Figure 1). Noted the R2 value of the y = 1.808x - 0.0125 trend line is 0.9958, which is statistically considered linear. From this calibration curve the absorbance values of unknowns samples can be compared, and the correlated glucose concentration can then be approximated.
Fluorescence measurement provides very important information about the photochemistry of a particular molecule. The first part of this experiment was dealing with the fluorescence behavior of a Leucophor PAF. Information from both spectrophotometry and fluorimetry was used to measure the quantum yield as well as to explain why Leucophor PAF was use as commercial optical brightener. The second part of this experiment dealing with fluorescence quenching of quinine bisulphate solution (QBS) is the presence of sodium chloride.
The results of this experiment showed a specific pattern. As the temperature increased, the absorbance recorded by the spectrophotometer increased indicating that the activity of peroxidase enzyme has increased.At 4C the absorbance was low indicating a low peroxidase activity or reaction rate. At 23C the absorbance increased indicating an increase in peroxidase activity. At 32C the absorbance reached its maximum indicating that peroxidase activity reached its highest value and so 32 C could be considered as the optimum temperature of peroxidase enzyme. Yet as the temperature increased up to 60C, the absorbance decreased greatly indicating that peroxidase activity has decreased. This happened because at low temperature such as 4 C the kinetic energy of both enzyme and substrate molecules was low so they moved very slowly, collided less frequently and formed less enzyme-substrate complexes and so little or no products. Yet, at 23 C, as the temperature increased, enzyme and substrate molecules
In this experiment, column chromatography and thin layer chromatography were used to separate a mixture of fluorene and 9-fluorenone. These two methods were then compared, and the results were analyzed. In column chromatography, 0.1010 g of mixture was separated. During the separation, fluorene eluted first. This compound was white in color once dried with the rotary evaporator. A percent yield of 93.47% was calculated for fluorene. The product that eluted first was confirmed to be fluorene by the IR spectrum obtained and the experimental melting point. The IR spectrum RM-02-CC1 was the spectrum obtained for this compound. Aromatic carbon- hydrogen bonds, carbon-carbon double bonds and hydrogens attached to sp2 carbons were shown by peaks 3038
To determine the effects of two environmental factors, temperature and pH, on the enzyme peroxidase, a spectrophotometer was used to measure the absorbance of each reaction every twenty seconds for two minutes. The temperatures tested were 0°C, 23°C, 32°C, and 48°C; the pH levels tested were pH 3, pH 5, pH 7, and pH 9. The temperatures were kept constant by keeping the tubes at room temperature, or placing them in an ice bath, warmer, or a hot water bath. Peroxidase, hydrogen peroxide, guaiacol and a pH buffer were mixed together to produce a reaction for both the temperature and pH experiments.
This experiment requires four tubes with an enzyme solution, chelating agent and deionized water. Also a fifth tube that is the calibration tube for the spectrophotometer, which only has 5ml of dH2O. The calibration tube is used to level out the spectrophotometer to zero before each trial. The spectrophotometer was set at 540 nm, “since green is not a color seen with the conversion of catechol to benzoquinone.” The enzyme solution was made by using potato that was peeled so that the golden color of the skin wouldn’t react or interfere with the red color needed in the spectrophotometer. After it was peeled, it was cut into chunks to minimize excess heat created while it was blended. It was put in a chilled blender and 500ml of deionized water was added. Chilled, deionized water was used because it created a hypotonic environment that caused the cells from the potato to burst and release the catecholase. It was chilled
Enzyme peroxidase is essential in any cell metabolic reaction as it breaks down the harmful hydrogen peroxide to harmful products in the body. The report analyzed its effect on changes in temperatures by determining the optimum temperatures and the effects of its reversibility. Through the method of extracting the enzyme by blending it with potato tissue in phosphate buffer, the effects were analyzed on the effect of the dye guaiacol and the activity measured under different temperatures. The optimum temperature was obtained at 22.20C and above this temperature, the enzyme was denatured. Conclusively, increase in temperature increases
Strength The antioxidants are very strong. They are made very strong so that they don’t break or get ruined due to polymer oxidation in the
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