FREE RADICAL SCAVENGING ACTIVITY OF AMERICAN DOGWOOD TREE

BARK

An Honors Thesis

Presented by

Emily Hinkle

Completion Date:
March 2020

Approved By:

_____________________________________________
Richard Schur, Honors Director

_____________________________________________
Madhuri Manpadi, Chemistry Department

_____________________________________________
Kevin Jansen, Biology Department

Drury University, 2020

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Abstract

Free radicals have been identified as a partial causative agent in many diseases, including

Parkinson’s disease and various types of cancer. Disease results partially from the body going

into oxidative stress when the number of antioxidants available to scavenge the generated free

radicals is too small. My research focuses on determining the antioxidant properties exhibited by

compounds isolated from the bark of an American Dogwood Tree (Cornus florida). The goal of

this project was to determine the effect of the dogwood compounds on nitric oxide and

superoxide radicals. The nitric oxide assay was performed using a Greiss-Ilosvay reaction with

ascorbic acid as a positive control. The superoxide assay was performed using curcumin as the

positive control.

Introduction

Several common medicines are derived from plants. Aspirin, commonly used to alleviate

pain, reduce fever, and prevent heart attack, is derived from the bark of the Willow Tree.1

Quinine, an anti-malarial drug, is derived from the bark of the Cinchona Tree.2 It is estimated

that 25% of modern pharmaceuticals are derived from plants, with some parts of the world still

relying on plant remedies passed down by oral tradition for medicine.3 My research focuses on

determining the antioxidant activity exhibited by compounds isolated from the bark of an

American Dogwood Tree (Cornus florida) on free radicals, including nitric oxide and

superoxide. The yearly mortality rate from cancer in the United States is 163.5 deaths per

100,000 patients.4 Part of the reason for the high mortality rate even with the advances in

treatment options is that some types of cancer resist traditional chemo- and radiotherapy. Due to

the great need for treatments that can be used on resistant mutations, novel cancer treatments are

in high demand. With successful completion of this line of research, we could provide a novel
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treatment for some types of cancer as well as a possible preventative agent for many types of

disease.

Free radicals are molecules that contain an unpaired electron and are thus extremely

reactive. The presence of free radicals in the body is necessary for certain processes, such as

active immune response and many signaling

pathways.5,6 Excess free radicals in the body can

lead to a condition called oxidative stress, which

results when the number of free radicals

Figure 1: Exhibits the mechanism of an antioxidant donating outweighs the number of antioxidants available to
an electron to a free radical.

“Antioxidants: Why do we need them?” Return 2 Health scavenge them. Antioxidants are stable molecules

that can donate an electron to a free radical to

stabilize it and prevent it from binding to other structures in the body as shown in Figure 1.

The body is constantly working to maintain homeostasis between free radical levels and

antioxidant levels, but certain factors such as aging, alcohol intake, and dietary choices increase

the number of free radicals in the body.7,8,9 When free radicals are not scavenged and quenched,

they bond with the nearest things to pair their lone electron. In the cell, they often bind with

lipids, DNA, or proteins which can lead to conditions such as breast cancer, diabetes mellitus

type II, and Crohn’s disease.6,10,11

Reactive oxygen species (ROS) are free radicals which include an oxygen molecule such

as nitric oxide, superoxide, and hydroxyl as shown in Figure 2. In the body, ROS are generated

through various processes vital to cell survival and proliferation such as cellular respiration.

Nitric oxide is produced in the body

when L-arginine is oxidized to citrulline

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by nitric oxide synthase.12 Superoxide is produced during the electron transport chain as

electrons are passed between protein complexes.10 ROS are necessary for cellular structure

maturation and play a vital role in phagocyte activity during the immune response to pathogenic

molecules. However, in chronic conditions where inflammation is constant, the overactivity of

phagocytes and granulocytes in an area of the body can cause overproduction of free radicals.

For example, in patients with Crohn’s disease in inflammatory bowel disease, it has been shown

that the increased inflammation leads to tissue damage caused by nitric oxide.11

Although ROS are vital to cellular survival and mounting an effective immune response,

they have also been linked to many disease processes and certain behaviors can increase their

concentration in the body. Some ROS are generated endogenously, such as those caused by

mental stress, invading pathogens, and inflammation among other factors. Other ROS are

generated exogenously and brought into the body such as those from cigarette smoke, alcohol

intake, and metabolism of heavy metals.12,13,14 Each of the processes that increase the amount of

ROS in the body move the level of free radicals in the body closer to being larger than the

number of available antioxidants. In some cases, free radicals initiate the disease pathway; in

other cases, the pathogenesis of a disease is worsened significantly by excess free radicals.

Free radicals have been linked to many disease processes, including cancer and diabetes

mellitus type II. Diabetes mellitus type II is most often identified in overweight and obese adults,

characterized by insulin resistance which renders cells unable to absorb glucose from the

bloodstream. Although the disease itself is initiated by chronic high blood sugar, leading to

overstimulation of insulin receptors, the disease’s pathogenicity has been linked to ROS. Chronic

hyperglycemia, which is common in type II diabetics, causes increased free radical production
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and results in increased lipid peroxidation which worsens ischemia and macrovascular

complications.6

Many types of cancer have also been shown to have a free radical component.9,10,15

Hydrogen peroxide, an intermediate to the more harmful hydroxyl radical, and superoxide were

both shown to be greatly increased in breast cancer patients, especially in stage 2 patients who

exhibited a 93% increase.10 Additionally, in stage 2 breast cancer patients, malondialdehyde, a

marker indicating increased lipid peroxidation, showed a 42% increase as compared to healthy

patients.10 It has also been shown that ROS can initiate alternate signaling pathways which aid in

survival of tumors, metastasis, vascularization, and proliferation in cancerous cells. Increased

ROS production within malignant tumors themselves has been linked to ischemia of surrounding

tissue and increased metabolic activity of cancer cells.

Although free radicals in high levels can be detrimental, some level of free radicals is

necessary for the body to function. There has also been research into the threshold at which free

radicals become harmful to the body. However, the variety of pathways that bring free radicals

into the body makes determining a universal level that is “safe” incredibly difficult. A study by

Zastrow et al. concluded that the amount of ROS must be greater than that of the secondary lipid

oxygen species generated by lipid peroxidation to be at a safe level for the body. Additional

research is being undertaken in the area of a “universal free radical ground state” with the hope

that the level of free radicals in the body may one day be an available metric for healthcare

providers to use.16

Antioxidants are necessary to quench free radicals after their functions are fulfilled to

prevent them from binding to structures within the cell and causing damage. Enzymatic

antioxidant production is carried out in the body by the enzymes; superoxide dismutase, catalase,
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glutathione peroxidase, and glutathione reductase. Non-enzymatic metabolic antioxidant

production is carried out by lipoid acid, glutathione, l-arginine, coenzyme Q10, melatonin, and

uric acid among others. Nutrient antioxidants are those which cannot be synthesized within the

body, rather must be taken in through food or supplements and include vitamin E, vitamin C,

carotenoids, omega-3 and omega-6 fatty acids among others.12

Although antioxidants can quench free radicals, in donating an electron to the free

radical, the antioxidant itself becomes oxidized and must be regenerated before it can quench

another free radical.12 Certain products can increase the antioxidant production of the body such

as coriander and monoterpenes from essential oils of citrus fruits.17,18 Other products act as

antioxidants themselves, such as mentha, anise, seaweed preparation complex, and simple

sugars.17,19,20,21 Additionally, vitamins A, C, and E all have antioxidant properties. Exogenous

antioxidants that act as radical scavengers in the body have different functions. For example,

vitamin E is a fat-soluble vitamin which allows it to safeguard the cell membrane from lipid

peroxidation by free radicals.12 Vitamin C is capable of regenerating the antioxidant form of

vitamin E and is water-soluble, which renders it valuable for quenching ROS before initiation of

lipid peroxidation.12,22

An increased need for novel drugs has resulted in a rise in natural product discoveries

that exhibit antioxidant properties over the past decade. Studies such as those into the medicinal

properties of perillyl alcohol, seaweed complex preparation, and Grangea maderaspatana have

shown great promise in the search for natural free radical scavengers.18,20,27 The bark of the

Dogwood Tree has been used historically as a substitute for quinine as an antimalarial agent.

During the Civil War, quinine supplies were blockaded by the Union, and the soldiers in the

South turned to boiling the bark of the abundant Dogwood Tree to make a tea which was served
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to malaria-infected soldiers. Recently, it was discovered that the anti-malarial property of

Dogwood comes from the molecule chincodine, which is similar in structure to quinine.23

Another study determined that the bark of the American Dogwood Tree exhibits moderate

antiplasmodial activity as well as antileishmanial activity.24 The promising work on the historical

medical use of Dogwood and antioxidant activity of other natural products provides a basis for

our study of the American Dogwood Tree (Cornus florida) bark as a potential cancer treatment.

The main goal of this project was to test the antioxidant properties of the Dogwood

compounds. Free radicals can be produced in the body as a byproduct of normal cell metabolism

and are used in some cellular processes. However, these free radicals can become toxic to the

cell that produced them or the surrounding tissue when their production or use is not controlled

effectively by the cell. Free radical toxicity stems from the unstable molecules’ attempt to bind to

other molecules to attain stability. Superoxide and nitric oxide radicals have been shown to cause

initiation and increase the metastatic potential of tumors.25 It has also been noted that free

radicals are involved in many pathological processes and are a cause of oxidative stress which

can lead to diseases of its own.26

Methods

In our study, a sample from the bark of one Dogwood Tree (Cornus florida) was broken

into smaller pieces and soaked in 90% ethanol overnight, then filtered by rotary evaporation

(Buchi, Model Number 10023432). The resulting crude extract was further purified into

individual compounds based on the difference in adsorption with silica gel by flash

chromatography (Teledyne, Model Number 213L20077) to isolate individual cytotoxic

compounds. The resulting compounds are those that were tested for their antioxidant effects on

free radicals, including nitric oxide, superoxide, and DPPH using various assays. If the
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compounds are effective antioxidants, the compounds could be a potential therapy for preventing

initiation and metastasis of cancers as well as other pathological processes.6,9,11,15

Figure 3: Shows the mechanism of color generation in response to nitrite presence in a

sample due to an azo compound being generated by the combination of sulfanilamide
with nitrite and then the resulting compound combining with the
naphthylethylenediamine solution.

Promega Greiss Reagent System, 2019

Figure 4: Shows the mechanism of the Greiss reaction if nitrite is not present, which
results in a lack of color and low absorbance.

Promega Greiss Reagent System, 2019.

The nitric oxide assay

was performed with sodium

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nitroprusside to generate

nitric oxide following the

procedure described by Patel

et al. and Sreejayan et al.27,28

Sodium nitroprusside, which

had been diluted to a

concentration of 10 m using deionized water, was added to each test vial in the amount of 2 ml.

Phosphate buffer solution at pH 7.4 was added to each vial in the amount of 0.5 ml. The test

compounds were added to their respective vials in the amount of 0.5 ml. The vials were

incubated at 25° C for 150 minutes. During the last 15 minutes of each incubation, Greiss reagent

1 and 2 (obtained from Promega G2930) were brought to room temperature. At the end of the

150 minute incubation, 50 l

of the incubated mixture was

added to each well of a 96-

well plate. Next, 50 l of

sulfanilamide solution

(Greiss reagent 1) was added

to each well and mixed. The plate was incubated at room temperature for 5 minutes. After the

incubation, 50 l of the NED solution (Greiss reagent 2) was added to each well, and the plate

was covered to prevent light exposure and incubated for 5 minutes. The plate was read at 550 nm

on a spectrophotometer (Tecan, Model Number RS-232C). In a later run, to produce higher

absorbance levels, the incubated radical mixture was added to a 1.5 ml cuvette instead of the 96-

well plate after the 150-minute incubation. Greiss reagent 1 was added in the amount of 0.5 ml
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and the mixture was incubated for 5 minutes. After the incubation, Greiss reagent 2 was added

and the cuvettes were allowed to incubate for 5 minutes protected from light and were then read

at 550 nm. The reaction for the nitric oxide assay is shown in Figure 3. Ascorbic acid in 1M and

200 um concentrations were used as the positive control.

The superoxide assay was performed by mixing 100 l of 156 m nitro blue tetrazolium

chloride, 100 l of 468 m nicotinamide adenine dinucleotide hydrate, 10 l of test compound,

and 10 l of 60 m PMS in each well of a 96 well plate. The plate was incubated at room

temperature for 5 minutes and the absorbance was measured on a plate reader at 560 nm. All

reagents were made fresh in 100 mM phosphate buffer saline at pH 8 before each run. Curcumin

in 10 m, 25 m, and 50 m concentrations were used as the positive controls.

Results

Results of the nitric oxide standard assay exhibited the expected baseline values (see Fig.

4) when it is run with the Dogwood samples.

Figure 5: Shows the percentage scavenging values resulting from the nitric oxide assay.

Results of the superoxide assay (see Fig. 5) exhibited the need for further research into

possible causes of negative radical scavenging values.

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Figure 6: Shows the percentage scavenging values from the superoxide assay.

Discussion

Although the work would have been more complete if there had been more time to finish

running the assays with Dogwood samples, the current results set the baseline for what the

controls should look like. Much of our time was spent troubleshooting the assays. The initial

blank values in the nitric oxide assay were low due to the sodium nitroprusside being an old

bottle. After a new bottle was ordered, the absorbance produced by the blank wells increased,

which indicated a greater amount of free radical production. We worked to further increase the

number of free radicals produced to allow a more accurate measurement of the dogwood

compounds’ free radical scavenging ability. The ascorbic acid control was not stable as we

thought it would be, which we learned in the second run of the nitric oxide assay. The ascorbic

acid stock was made fresh each time going forward and the free radical scavenging exhibited by

the ascorbic acid increased. Finally, in order to further increase the blank values to allow a more

accurate measurement of the Dogwood compounds’ free radical scavenging activity, the assay

was run in a cuvette as opposed to a plate. The cuvette allowed a larger amount of sample to be

tested, which increased the number of free radicals measured.

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In the superoxide assay, the values were negative so much of our troubleshooting

centered on figuring out why. The NADH we started with was an old bottle, so ordering a new

bottle produced higher blank values, which indicated greater free radical production.

Additionally, we discussed waiting to mix in the PMS with the rest of the assay until directly

before analysis in the plate reader. Although in future studied, PMS likely will be added directly

before analysis, we did not have time to determine if it would have increased the blank values.

Another improvement might have been changing the concentration of the Dogwood samples to

allow a larger amount to be added to samples. There was only a small amount of each Dogwood

sample, so we added only 1 l to each well and such a small amount could have resulted in any

number of errors such as the compound getting stuck on the side of the well or being measured

inaccurately by the pipette.

Areas of Future Study

Future study will likely focus on running the nitric oxide assay with the Dogwood

compounds. The Dogwood compounds were not able to be run in the nitric oxide assay due to

the extensive troubleshooting that was necessary for the assay to produce acceptable baseline

data. Additionally, more work will need to be done on the superoxide assay to determine why the

results were negative values. The Dogwood compounds will eventually be run in an assay for

2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and hydrogen peroxide as well to determine their

effect on these compounds. Further free radical assays could provide insight into the scope at

which Dogwood compounds are effective antioxidants. Another important consideration will be

whether the Dogwood compounds can achieve significant scavenging activity in an assay run

with cells.
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Acknowledgements

Special thanks to Drs. Madhuri Manpadi and Kevin Jansen for their support and guidance

of this project as well as Erik Way and Andrew Ryan for their work on the project.
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