ANTIOXIDANTS - FACT OR FAD?
FALL, 2013
Overview
Fruits and vegetables contain a variety of antioxidants that include phenolic compounds,
vitamins C and E, carotenoids, chlorophylls and flavonoids. The type and concentration
of antioxidants present in a particular fruit or vegetable will vary from species to species
and from strain to strain. It will also vary with a whole host of external factors ranging
from how the plant is grown and harvested on the farm to how it is packaged and stored
in the market. Since many antioxidant compounds are pigments, a good rule of thumb
for determining the initial antioxidant content of a fruit or vegetable is to look for deep,
rich colors (greens, reds, yellows, blues). Unfortunately, antioxidant contents are also
impacted by the way you store and cook your produce when you get it home, so simply
looking for dark colors in the marketplace will not tell you how much antioxidant you
actually manage to consume.
In this laboratory exercise, you will compare the antioxidant properties of two
commercial drinks that have made a claims (implicit or explicit) regarding their
antioxidant capacities.
Scientific Background
Oxidation – Reduction Reactions are those in which electrons move from an electron
donor to an electron acceptor. At the end of the reaction, the donor is oxidized, and the
acceptor is reduced. These oxidation and reduction reactions play critical roles in many
cellular processes, perhaps most famously in the electron transport systems that transfer
electrons and energy as part of respiration and photosynthesis. A typical oxidationreduction reaction is shown below. In this case, NADH is the electron donor and oxygen
is the electron acceptor. In many biological reactions, the transfer of electrons is also
associated with the transfer of hydrogen atoms.
NADH +
H+ +
Electron donor
½ O2

Electron acceptor
H2O
+
Reduced oxygen
NAD+
Oxidized NADH
Antioxidants The basis for antioxidant activity also lies in oxidation-reduction
chemistry. Reactive oxygen species (ROS) such as the superoxide anion (O.2-), the
hydroxyl radical (.OH) and hydrogen peroxide (H2O2) are the natural byproducts of our
own oxygen-requiring metabolism. They are highly reactive because they have at least
one oxygen atom with an unpaired free electron. As a result, they tend to steal electrons
from other molecules (they oxidize them) in order to fill out their own electron pair.
ROS play important roles in our cells’ abilities to grow, proliferate and to fight infections.
Nevertheless, they can also do serious damage to our DNA, proteins, cell membranes,
and lipids when they oxidize them. Excess ROS have even been associated with
increased susceptibility to cancer and cardiovascular disease. Because of this, our cells
contain many enzymes that destroy ROS as they are produced. Antioxidants, which are
chemicals that can inactivate ROS by reducing them before they have a chance to do
oxidative damage (see below), are found naturally in many plants and plant products, and
have become increasingly popular as food additives in the United States.
Antioxidant +
Electron donor
ROS

Electron acceptor
Reduced ROS + Oxidized Antioxidant
But do these food additives work? Well, that depends on how you define ‘work’. As
summarized in the figure below, which is based on information from Harold E. Seifried,
Darrell E. Anderson, Evan I. Fisher and John A. Milner. A review of the interaction
among dietary antioxidants and reactive oxygen species. The Journal of Nutritional
Biochemistry, Volume 18, Issue 9, September 2007, Pages 567-579, the overall health of
our cells depends on a delicate balancing act between processes that generate ROS and
those that remove them.
Factors that increase
ROS
Factors that decrease
ROS
So… if our cells can inactivate free radicals by themselves, does eating antioxidants do
more harm than good? The answer is far from simple, and the jury is still out. Some
studies have linked the consumption of antioxidants to decreased rates of cancer and
cardiovascular disease, while others report just the opposite. It is likely that these
questions will be clarified as scientists learn more about the interactions between our own
antioxidants, ROS, and a whole host of modifying factors including underlying diseases,
the types and amounts of antioxidants consumed in our diet, and the interaction of those
antioxidants with pharmaceuticals. For now it is safe to say that eating foods rich in
antioxidants is still a good and healthy thing to do – but you should talk to your physician
before consuming over-the counter antioxidant food additives.
Antioxidant Assay Procedures
Step 1. Prepare your Drink Samples for Analysis
i.
ii.
iii.
iv.
Add 2 mL of your first drink to 2 mL of methanol.
Repeat for your second drink
Repeat for V8 Berry Splash, which will serve as your positive control.
You will use these prepared samples in steps 2 and 3 below.
Step 2. Analyze total Phenolic Content
Phenolics are chemical compounds that contain an –OH group attached to an aromatic
hydrocarbon group. They can serve as important antioxidants in our diets. The Folin
Ciocalteu reagent in this assay (procedure in appendix) reacts with phenolic compounds
such as phenolic acids, flavonoids and tannins that are commonly found in plants.
Phenols. (2008, January 22). In Wikipedia, The Free Encyclopedia.
Retrieved 18:41, February 6, 2008, from
http://en.wikipedia.org/w/index.php?title=Phenols&oldid=1862313
34
You will be preparing triplicate test tubes for each of your samples, the positive
control and the negative control for this analysis. Thus, you should have 12 tubes
total.
i.
ii.
iii.
iv.
v.
vi.
vii.
Prepare the 12 test tubes by adding 1.8 mL of Folin-Ciocalteu reagent to each.
Add 40 uL of your first prepared drink sample (from Step 1) into the first three
test tubes.
Add 40 uL of your second prepared drink sample (from Step 1) into the next three
test tubes.
Add 40 uL of your positive control sample (from Step 1) into the third set of three
test tubes.
Add 40 uL of methanol into the final three test tubes. This will serve as your
negative control.
Let all 12 tubes stand at room temperature for 5 minutes.
Add 1.2 mL of 7.5% (w/v) Sodium Bicarbonate to each tube.
viii.
ix.
Let sit at room temperature for 1 hour.
Read absorbance at 765 nm.
3. Perform ABTS Assay for Total Antioxidants
The ABTS (2,2’-azinobis-(3-ethylbenzothiazline-6-sulfonic acid)) assay is based on the
ability of antioxidants to interact with the ABTS•+ radical and reduce it. Since the
unreduced radical absorbs light at 734 nm, any interaction with an antioxidant will
decrease the amount of light it can absorb at that wavelength. In other words, the higher
the amount of antioxidants in your test substance, the lower the absorbance value.
Image of ABTS from Sigma-Aldrich Catalog
Making this reagent (procedure in appendix) requires a bit more effort than the total
phenolic technique that follows, but it has the advantage of being a very short assay once
the reagents are ready to go. Also, it assays directly for antioxidant activity. The other
two techniques simply look for specific types of antioxidants.

As before, you will be making three tubes each of your 2 drink samples, your positive
control sample, and your negative control sample (methanol).
i.
In twelve separate test tubes, add 3.9 mL of ABTS solution to 100 L of each
your various samples. Mix vigorously.
ii.
Let stand at room temperature for at least 6 minutes.
iii.
Read the absorbance at 734 nm
More Fun Stuff
For great information regarding the antioxidants present in many different foods (fruits,
vegetables, oils and even breakfast cereals!), try The Berry Doctor’s Journal and the
ORAC report (http://berrydoctor.com/broadcast/2007/USDA-ORAC.htm ,
http://www.ars.usda.gov/Services/docs.htm?docid=15866 )