the effects of resveratrol and quercetin on reactive oxygen species
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7/30/2019 The effects of Resveratrol and Quercetin on Reactive Oxygen Species
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Hunter Ross
The Effects of the Antioxidants, Resveratrol and Quercetin, onReactive Oxygen Species
IntroductionAntioxidants are widely accepted to be important to our health. Many foods we eat
such as fruits, vegetables, nuts, and even wine are heavily marketed and advertised
as containing antioxidant rich properties. But how do antioxidants keep us healthy
and prevent disease? Are these marketing tools assisting each of us in making
healthier decisions or simply making us spend more for nothing?We know that antioxidants are molecules that inhibit the oxidation of other
molecules. Oxidation reactions have the potential of producing free radicals, which
can cause damage to a cell and ultimately cell death through a series of chain
reactions. Antioxidants can prevent these chain reactions from proliferating by
removing the radical intermediate in addition to inhibition of oxidation reactions.
Antioxidants prevent the damaging effects by incorporating the free radical, such as
the hydroxyl radical, into the antioxidant molecule itself [1]. Oxidation reactions are
crucial for life, however they may also cause a lot of damage. If there is aninsufficient amount of antioxidants in a living organism to balance the byproducts of
oxidation reactions, this may cause oxidative stress and may damage or kill cells.
Our hope is to shed light on antioxidants and test the claims of their health benefits
on living cells.
Literature Review
Recent experiments in our molecular DNA lab have enhanced our understanding of
the effects of reactive oxygen species on DNA and living cells. These experimentsassisted in determining that reactive oxygen species (ROS) damage cells. Tolerance
to hydrogen peroxide conferred by a DNA repair protein argues in favor of the
hypothesis that ROS kill cells by attacking DNA. From additional experiments,
patterns indicated the high likelihood that the hydroxyl radical is the ROS
responsible for DNA damage induced by the addition of hydrogen peroxide.
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Knowing the damaging effects the hydroxyl radical has on DNA, we turned our
attention towards investigating the effects of antioxidants on ROS. The two
antioxidants chosen for comparison are quercetin (a flavonol), and resveratrol (a
polyphenol) (Figure 1). We decided to use these two as our model antioxidants
because they both are found in many fruits and vegetables we eat every day and
are also widely marketed in supplements for their antioxidant effects.
Due to resveratrol and quercetin being in so many of the foods we eat, they have
been implicated in many recent studies for their biological effects. Resveratrol, in an
article in Current Medicinal Chemistry, was called a star natural product and the
authors called for additional research into this amazing chemical [3]. In addition, a
study by Wang et al. in 2012 found that resveratrol decreased inflammatory signals
in epileptic rats [4]. Although the mechanism was admittedly not fully understood,
this indicated resveratrols widespread positive effect. Quercetin has also been in
recent research for its chemical benefits. A study by Chen et al. in 2012 showed that
quercetin in combination with pioglitizone showed a decrease in kidney damage in
diabetic rats [5]. Quercetin has also been in studies which highlight its role in
vascular physiology [6]. Lastly, a study investigating the in vitro effect of three red
wine polyphenols on reactive oxygen species indicated their positive effects but
were unable to get conclusive results of a positive or negative effect on
combinations of antioxidants [7]. Our goal is to find additional information on the
subject by investigating these two major antioxidants that has been in so much of
the recent literature.
Figure 1. Chemical Compounds Quercetin (a) and Resveratrol (b).
RationaleGiven the importance of DNA in our body, we should strive to protect its structure.
Current literature indicates that antioxidant chemical compounds with aromatic rings
assist in the removal of the hydroxyl radical in a mechanism similar to that seen in
Figure 2 [1]. We hypothesize that antioxidants would assist in protecting cells from
DNA damage by quenching the unpaired electron. We further expect that if this
mechanism is correct then a chemical compound with a larger number of aromatic
rings will lead to a higher amount of cell survival. Results from an experiment like
this can ultimately identify compounds that would allow for greater DNA protection
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from ROS and promote better cell survival.JM109 E. Colibacterial cells lacking the RecA gene would be used across the entire
experiment. This would allow for similar conditions and to ensure that the RecA
gene is not aiding in an increase cell survival rate. Quercetin was chosen because itdiffers from resveratrol most significantly in that it contains three aromatic rings
where resveratrol has only two. Quercetin also contains five alcohol groups while
resveratrol contains three.Figure 2. Mechanism of Hydroxyl Radical and antioxidantSpecific Aims
Aim 1: The aim of this experiment is to determine if antioxidants assist in rescuingDNA from irreversible DNA damage caused by ROSs. We will test the extent of
protection offered by antioxidants against DNA damage induced by ROSs,
specifically hydrogen peroxide. To examine this, fraction of cell survival will be
measured in the presence of resveratrol, quercetin and a combination of both
antioxidants prior to exposing the cells to hydrogen peroxide. We expect a higher
rate of cell survival in cells treated with antioxidants when compared to the control,
indicating antioxidant effects to relieve the radical component of ROSs. In doing so,
we hope to find the mechanism of protection against ROSs provided by antioxidants
as well as compare the effects of the two different antioxidants.
Aim 2: Our second aim is to determine if antioxidant structural differences make a
difference in cell survival. To test this, we have chosen two antioxidants which vary
structurally.
MethodsAn in vivo experiment will be utilized with JM109 E. Coli bacteria. The JM109 cells
will be exposed to hydrogen peroxide at varying concentrations. These solutions will
then be exposed to either of two different types of antioxidants, a combination of the
two, or neither which will act as the control. The two antibiotics chosen for this
experiment are Quercetin and Resveratrol. Five tests will be performed for each four
different reaction conditions. Four different hydrogen peroxide concentrations will be
used (0mM, 0.3mM, 1mM, 3mM). Resveratrol was found to have an optimal
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concentration of 1mM. Quercetin will also be added at 1mM to keep the four
conditions similar. Both will be prepared in a 50:50 solution of DMSO and 95%
Ethanol. Samples will be serially diluted and seeded onto grid titration plates. After
24 hours of incubation at 37o C, the plates will be photographed and growth will be
analyzed. The data will be extrapolated from the seeded plates using a numerical
system assignment to the growth of the cell (ordinal data). Paired t-tests will be used
to calculate significance and p-values.
ConclusionThis experiment will be able to add efficacy to the claims made regarding the healthbenefits of antioxidants. This will be done by seeing an increase in cell survival
showing the positive effects of antioxidants on rescuing DNA from damage. Many
studies have looked at specific effects of antioxidants, however, our proposed
experiment will be able to directly state whether these two compounds, resveratrol
and quercetin, have an effect on cell survival. The effectiveness of the outcomes of
the experiment lies in the simplicity and direct measurements. We are confident in
obtaining our ultimate goal which is to determine whether the effects of these two
widely used antioxidants truly does in fact provide a benefit for cell health. Again, the
general nature of our experiment allows the takeaway message from this study to be
heard and utilized by every living person, not solely the scientific community. In
conclusion, this study will reveal the protective properties of these two antioxidants
with the overall goal of revealing true antioxidant health benefits in humans.
References1. Carrigan J Hayes, John K Merle, Christopher M Hadad, The chemistry of reactive
radical intermediates in combustion and the atmosphere, In: J.P. Richard, Editor(s),
Advances in Physical Organic Chemistry, Academic Press, 2009, Volume 43, Pages
79-134, ISSN 0065-3160, ISBN 9780123747495, 10.1016/S0065-3160(08)00003-8.
(http://www.sciencedirect.com/science/article/pii/S0065316008000038)
2. Hydroxyl Radical Reaction with trans-Resveratrol: Initial Carbon Radical Adduct
Formation Followed by Rearrangement to Phenoxyl RadicalDan-Dan Li, Rui-Min Han, Ran Liang, Chang-Hui Chen, Wenzhen Lai, Jian-Ping
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Zhang, and Leif H. SkibstedThe Journal of Physical Chemistry B 2012 116 (24), 7154-71613. He S, Yan X. From Resveratrol to Its Derivatives: New Sources of Natural
Antioxidant. Curr Med Chem. 2012 Dec 3. [Epub ahead of print] PubMed PMID:23210785.4. Wang SJ, Bo QY, Zhao XH, Yang X, Chi ZF, Liu XW. Resveratrol pre-treatment
reduces early inflammatory responses induced by status epilepticus via mTOR
signaling. Brain Res. 2012 Dec 1. doi:pii: S0006-8993(12)01821-5.
10.1016/j.brainres.2012.11.027. [Epub ahead of print] PubMed PMID: 23211629.5. Chen P, Chen J, Zheng Q, Chen W, Wang Y, Xu X. Pioglitazone, extract of
compound Danshen dripping pill, and quercetin ameliorate diabetic nephropathy in
diabetic rats. J Endocrinol Invest. 2012 Nov 27. [Epub ahead of print] PubMed
PMID: 23211366.6. Chirumbolo S. Role of quercetin in vascular physiology. Can J Physiol
Pharmacol. 2012 Dec;90(12):1652-7. doi: 10.1139/y2012-137. Epub 2012 Nov 23.
PubMed PMID: 23210445.7. Kurin E, Muaji P, Nagy M. In vitro antioxidant activities of three red wine
polyphenols and their mixtures: an interaction study. Molecules. 2012 Dec
3;17(12):14336-48. doi: 10.3390/molecules171214336. PubMed PMID: 23208468.
Team MeddentTeam Members: Jennifer Nhan, Hunter Ross, Sam Walton
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Summary of Results: The Effects of the Antioxidants,
Resveratrol and Quercetin, on Reactive Oxygen Species
Methods
Various steps of the procedure conducted for this experiment were borrowed fromthe University of Utah Biology 3525 Molecular DNA laboratory 9: DNA damage invitro. The overall structure of our experiment was identical to lab 9 with regards thepreparation of cultured cells and reagents such as hydrogen peroxide and catalase.Modifications were made to incorporate the new reagents, resveratrol and quercetin.Cultures of JM109 were prepared and the cells were pelleted by centrifugation. Fivetests were performed for each four different reaction conditions, at four differenthydrogen peroxide concentrations. Reagents and reaction conditions were preparedaccording to table 1.Table 1.Control Resveratrol Quercetin Resveratrol + Quercetin60uL 50/50DMSO &Ethanol
50uL 50/50 DMSO& Ethanol, 10uL1mM Resveratrol
50uL 50/50 DMSO& Ethanol, 10uL1mM Quercetin
50uL 50/50 DMSO &Ethanol, 10uL 1mMResveratrol, 10uL 1mMQuercetin
After exposing the cells to the reaction protocols the different concentrations of
hydrogen peroxide (0mM, 0.3mM, 1mM, 3mM) were added. Catalase was added
after fifteen minutes to stop the reaction. Samples were then serially diluted and
seeded onto grid titration plates. After 24 hours of incubation at 37o Chttps://docs.google.com/the
plates were photographed and growth was analyzed for the various treatmentsThe data was extrapolated from the seeded plates using a numerical system
assignment to the growth of the cell (ordinal data). All five trials data was compared
for similarity. Paired t-tests were used to determine if any trials results different to a
significant degree. Mean values for each cell from all cumulative corresponding tests
and treatments were taken. This was then counted for total growth mean values
from all test and treatment groups. Values were normalized and plotted as shown in
Figure 1. Paired t-tests were used to calculate significance and p-values. P
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ResultsThe increase in fraction of cell survival in both resveratrol and quercetin treatments
indicate that both compounds have antioxidant properties that may quench theproduction of ROS such as hydrogen peroxide and more important the hydroxyl
radical (see figure 1 below).https://docs.google.com/ Quercetin alone has a greater
protective property for cells than resveratrol alone. An increase in cell survival was
also seen when a combination of resveratrol and quercetin was used although not
as great as quercetin alone. Both antioxidants appear to have properties that
increase fraction of cell survival, so the consumption of either of these antioxidants
may in turn decrease the rate of cell death caused by oxidative stress. Results seem
to be indicative that through a mechanism shown in figure 2 that both compounds
quench radicals in a similar way. However,https://docs.google.com/ Quercetin works
better to a significant degree (p
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