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.
This week’s lab was the third and final step in a multi-step synthesis reaction. The starting material of this week was benzil and 1,3- diphenylacetone was added along with a strong base, KOH, to form the product tetraphenylcyclopentadienone. The product was confirmed to be tetraphenylcyclopentadienone based of the color of the product, the IR spectrum, and the mechanism of the reaction. The product of the reaction was a dark purple/black color, which corresponds to literature colors of tetraphenylcyclopentadienone. The tetraphenylcyclopentadienone product was a deep purple/black because of its absorption of all light wavelengths. The conjugated aromatic rings in the product create a delocalized pi electron system and the electrons are excited
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.
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
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
Vitamin C in the body acts as an antioxidant. Vitamin C loses electrons easily, a characteristic that allows it to perform as an antioxidant. In the body, antioxidants defend against free radicals. A free radical is a molecule with one or more unpaired electrons, which makes it unstable and highly reactive. By donating an electron or two, antioxidants neutralize free radicals and protect other substances from their damage. Figure 1 illustrates how vitamin C can give up electrons to stop free radical damage and then accept them again to become reactivated. This recycling of vitamin C is key to limiting losses and maintaining a reserve of
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
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
We are told to fill our plates with fruits and vegetables because, they contain a variety of colorful chemicals that are lumped together under the term “antioxidants.”
They are accessory pigment molecules that cascade light energy to primary pigments. Carotenoids absorb wavelengths in the blue and green region of the visible spectrum (400-550nm) and reflect wavelengths of 590-650nm so appear red-orange in colour. They are found in all plants and some photosynthetic bacteria. Carotenoids are separated into two groups, carotenes and xanthophylls. Carotenes (C40H56) are polyunsaturated hydrocarbons containing no oxygen and include pigments such as α-carotene, β-carotene, and lycopene. They give the orange colour to carrots and autumn leaves. Xanthophylls (C40H56O2) contain oxygen and include lutein and zeaxanthin. Carotenoids contain alternating carbon-carbon double bonds and single bonds, forming a conjugation system where electrons in the fourth outer shell are in p-orbitals which overlap. This overlapping produces a system of π-bonds with delocalised electrons. The delocalised electrons are free to move so are more easily lost because less energy is needed to raise them to an excited state. Shorter wavelengths towards the blue end of the spectrum with lower energies are absorbed because of the lower energy
The two major properties of vitamin C which make it an ideal antioxidant: First is the low one-electron reduction potentials of both ascorbate (282 mV) and its one-electron oxidation product, the ascorbyl radical (2174 mV) which is derived from the ene-diol functional group in the molecule. These low reduction potentials enable ascorbate and the ascorbyl radical to react with and reduce basically all physiologically relevant radicals and oxidants. For this reason, vitamin C has been said to be “at the bottom of the pecking order” (Carr and Frei, 1999). The second major property that makes vitamin C such an effective antioxidant is the stability of semidehydro-ascorbic acid or ascorbyl free radical(ASF) ,the species formed after the loss of one electron, with a half-life of 10−5 seconds and low reactivity of them (Carr and Frei, 1999;Packer, 2002; Padayatty et al., 2003).Once oxidized, ascorbate is turned into ascorbate free radical (AFR) a molecule that is relatively stable due to electron delocalization. Although AFR can donate another electron, it does not undergo further oxidation. Rather, it is reduced back to ascorbate via NADH-dependent
Green tea is made from the plant Camellia Sinensis’s leaves without fermentation (Suzuki, Miyoshi & Isemura, 2012) and is regarded as a popular and healthy drink in Asian countries because of its health-promoting potentials such as protecting against cancer and cardiovascular disease (Harvard Health Publications, 2012; Iwasaki et al., 2014). Lots of the positive effects are due to the antioxidative activity of (-)-epigallocatechin gallate (EGCG) which is a major component of green tea (Suzuki et al., 2012). And it draws researchers’ interests and attentions to further investigate. According to Suzuki et al., one example of the health advantages is that catechins in green tea bring an anti-obesity effect by stimulating lipid metabolism in liver (2012).
...ls and soybean oils. But, in other research, it said that coconut oils have the highest concentration of active alpha tocopherol. Nevertheless, the goal of anti oxidants is to minimize free radicals damage because uncontrolled free radicals reactions can cause damage to cells structures and functions and Vitamin E posses the ability.
Today people are becoming more aware of the advantages of using natural health products. Apart from it being a cost-efficient solution to maintain and attain healthier body, the products made from herbal and natural ingredients offer a myriad of benefits compared to those made from chemical and artificial ingredients. The followi...
This clearly shows that the fruits and vegetables play a vital role in maintaining our good health. Fruits and vegetables are seasonal and grown in different parts of our country. They need to be stored for longer period and transported to different places. It makes necessary for us to find out whether there is any damage to the useful contents of the fruits and vegetable during the storage and transportation or due to some preservatives or other factors and what kind of nutrients, minerals, etc. are present in their juices. With this idea in mind, the project has been undertaken.