the effects of resveratrol and quercetin on reactive oxygen species

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

the effects of resveratrol and quercetin on reactive oxygen species

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