Om Gandhi | Science Academy of Chicago | 7th Grade

THE PROTECTIVE POWER OF ANTIOXIDANTS

ABSTRACT

Antioxidants are natural molecules found in certain foods that help neutralize free radicals in our bodies. Free radicals are byproducts of metabolism and our environment. Internal factors such as inflammation and external factors such as pollution, UV exposure, and cigarette smoke can increase free radical production. Free radicals can damage cells all over the body and cause oxidative stress. Oxidative stress has been closely associated with heart disease, arthritis, stroke, cancer, dementia respiratory diseases, immune deficiency, Parkinson’s disease and other inflammatory conditions. Antioxidants serve as protection against the cell damage and may play a role in the prevention of various diseases.

Therefore, the purpose of this project is to find out which fruit, vegetable, and beverage have the most amount of antioxidants as well as to find out which acid will help best prevent oxidation. The results proved, based on the amount of enzymatic browning measured, that blueberries and blackberries, along with pineapples and oranges, have more antioxidants than any other fruit and vegetable. Also, green tea slowed more oxidation than the other two teas, herbal tea and black tea. Moreover, Ascorbic Acid (vitamin C) best prevented oxidation in a freshly cut apple slice. Lastly, all the fruits, vegetables, beverages and acids that were tested did have some degree of antioxidants in them.

INTRODUCTION

Oxygen is an essential element for life. The oxidative property of oxygen plays a vital role in various biological phenomena; being essential for life, oxygen can also create the damage within the cell by oxidative events. Oxygen is used by the cell to generate energy, and free radicals are formed as a consequence of ATP (adenosine triphosphate) production by the mitochondria. “Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the by-products resulting from the cellular redox process,” (Sen and Chakraborty, 2011). These reactive species play a dual role in human as both toxic and beneficial compounds. The delicate balance between their two opposite effects is undoubtedly a key aspect of life. At low or moderate levels, reactive species exert beneficial effects on cellular redox signaling and immune function, but at high concentrations, they produce oxidative stress, a harmful process that can damage cell function and structures. Free Radicals Free radicals are defined as molecules or molecular fragments containing one or more unpaired electrons. “This unpaired electron(s) are unstable and usually gives a significant degree of reactivity to the free radical,” (Sen and Chakraborty, 2011). Oxygen and nitrogen free radicals can be converted to other non-radical reactive species which are also dangerous for health. Free radicals can be produced from both endogenous and exogenous substances. The immune system cells, metabolic processes, inflammation, and stress are all endogenous substances in which free radicals can be produced from. Pollution, radiation, dietary factors, toxins and drugs are

all exogenous substance in which free radicals can be produced from. “Free radicals are also created from environmental pollution, certain industrial chemicals, and smoking,” (Bauer, 2005).

The relation between free radicals and molecular damage can be described by the concept of ‘oxidative stress’. The harmful effect of free radicals causing potential biological injury is termed oxidative stress and nitrosative stress. The term oxidative stress describes a harmful condition caused by the excess of ROS (reactive oxygen species) production and/or a decrease in antioxidant levels. Oxidative stress has been implicated in various diseases like cancer, cardiovascular diseases, neurological disorders, diabetes, and aging. “Oxidative stress causes different diseases via four critical steps: membrane lipid peroxidation, protein oxidation, DNA damage and disturbance in reducing equivalents of the cell; which leads to cell destruction, altered signaling pathways,” (Sen and Chakraborty, 2011). Similarly, overproduction of reactive nitrogen species is called nitrosative stress. Prolonged exposure to free radicals, even at a low concentration, may be responsible for the damage of biologically important molecules and potentially lead to tissue injury. Different mechanisms are involved in the pathogenesis of such diseases. Every biological molecule present in the human body is at risk of damage by free radicals. Such damaged cell molecules can impair cell functions or can lead to cell death, ultimately resulting in diseased states.

Antioxidants Antioxidants are a class of molecules that are capable of

inhibiting the oxidation of another molecule. The body produces some antioxidants. In addition, the body’s natural antioxidant production can decline as one ages. Antioxidants are nature's way of providing the body’s cells with an adequate defense against attack by reactive oxygen species (ROS). Various benefits of antioxidants include: repairing damaged molecules, blocking metal radical production – “Some antioxidants have a chelating effect – they can grab toxic metals like mercury and arsenic, which can cause free radical formation, and "hug" them so strongly to prevent any chemical reaction from taking place,” (Divit, 2015). Providing a "shield effect" – Antioxidants, such as flavonoids, can act as a virtual shield by attaching to the body’s DNA to protect it from free radical attacks. Promoting cancer cells to "commit suicide" – Some antioxidants can provide anti-cancer chemicals that halt cancer growth and force some cancer cells to self-destruct (apoptosis). Different Types of Antioxidants If classified according to their solubility, antioxidants can be categorized as either soluble in lipids/fat (hydrophobic) or water (hydrophilic). Both of these are required by one’s body in order to protect cells. Lipid-soluble antioxidants are the ones that protect the body’s cell membranes from lipid peroxidation. Lipid peroxidation is the process in which free radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage. Some examples of lipid-soluble antioxidants are vitamins A and E, carotenoids, and lipoic acid. Water-soluble antioxidants are found in aqueous fluids, like the blood and the fluids within and around one’s cells (cytosol or cytoplasmic matrix). Some examples of water-soluble antioxidants are vitamin C, polyphenols, and glutathione. They can also be categorized as enzymatic and non-enzymatic antioxidants.

Enzymatic antioxidants are beneficial by breaking down and removing free radicals. They can flush out dangerous oxidative products by converting them into hydrogen peroxide, then into the water. Enzymatic antioxidants cannot be found in supplements, but instead are produced in one’s body. The main enzymatic antioxidants in our body are: Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GSHpx) and glutathione reductase. They are most abundant in our liver.

Non-enzymatic antioxidants are beneficial by interrupting free radical chain reactions. Some examples are carotenoids, vitamin C, vitamin E, plant polyphenols, and glutathione (GSH). Most antioxidants found in supplements and foods are non-enzymatic, and they provide support to enzymatic antioxidants by doing a "first sweep" and disarming the free radicals. This helps prevent our enzymatic antioxidants from being depleted. (Mercola, 2014)

Antioxidant Food Sources Fresh vegetables- Most of the vegetables you eat, especially the green leafy ones, are loaded with potent phytochemicals, which are plant compounds that act as antioxidants.

Fruits- Fresh berries like blueberries, blackberries, cranberries, and raspberries are the best antioxidants as they contain powerful phytochemicals that directly inhibit the DNA binding of certain carcinogens. Berries are also great sources of antioxidants like vitamin C, carotenes, and carotenoids, as well as nutrients like zinc, potassium, iron, calcium, and magnesium.

Herbs and spices- Aside from being an abundant source of antioxidants, these can have potential anti-cancer benefits. Herbs and spices differ mainly by source, as herbs typically come from the plant's leaves while spices come from the bark, stem, and seeds. Some of the best choices are ground cinnamon, oregano, turmeric, ginger, and garlic.

Organic green tea-This antioxidant-rich drink contains epigallocatechin-3-gallate (EGCG), a catechin polyphenol and one of the most powerful antioxidants. (Mercola, 2014)

Summary of Introduction Antioxidants are natural molecules found in certain foods that help neutralize free radicals in our bodies. Free radicals are byproducts of metabolism and our environment. Internal factors such as inflammation and external factors such as pollution, UV exposure, and cigarette smoke can increase free radical production. Free radicals can damage cells all over the body and cause oxidative stress. Oxidative stress has been closely associated with heart disease, arthritis, stroke, cancer, dementia respiratory diseases, immune deficiency, Parkinson’s disease and other inflammatory conditions. Antioxidants serve as protection against the cell damage and may play a role in the prevention of various diseases. The best source of antioxidants is plant- based foods including fruits and vegetables. To maximize antioxidant intake, people should eat at least 2-3 serving of fruits and vegetables daily.

PURPOSE AND HYPOTHESES

The purpose of this project is to find out which fruit and vegetable have the most amount of antioxidants as well as to determine which beverage out of green tea, black tea, and herbal tea have the most antioxidant effect. The purpose is also to find out whether Ascorbic Acid (vitamin C), Citric Acid, or Acetic Acid would help best prevent oxidation.

The first hypothesis is that the amount of antioxidants in vegetables such as broccoli and kale will be greater than fruits such as berries. The second hypothesis is that herbal tea will have more of an antioxidant effect compared to green tea and black tea. The third hypothesis is that Ascorbic Acid (vitamin C) will help best prevent oxidation. Lastly, the fourth hypothesis is that all the fruits, vegetables, beverages, and acids that are tested will have some degree of antioxidants in them.

VARIABLES AND MATERIALS

The independent variable is the solution that the apple slice was dipped into. Each solution is different as it consists of different fruits/vegetables/acids mixed with 100 ml of water. The dependent variable is the amount of oxidation occurring and measured in the apple slices. The control variables are: time, the type of apples used, and environment that the apple slices were kept in. Each reagent was given the same amount of time and condition to fight against oxidation.

The materials used for this project are: 50 grams of blueberry, two apples, water, 50 grams of orange, herbal tea, a spoon, 50 grams of kale, black tea, glass bowls, 50 grams of cucumber, green tea, paper towels, 50 grams of blackberry, citric acid powder, a measuring cup, 50 grams of broccoli, vinegar, measuring scale, 50 grams of tomatoes, Ascorbic acid, a camera, 50 grams of pineapples, tongs, a timer, rubber gloves, a lab coat, a blender

PROCEDURE

Part 1: Preparing Samples 1. To prepare the pineapple juice samples, measure 50 grams of pineapple using a measuring scale. Then, through the measuring cup, measure 100 ml of water. 2. Mix 100 ml of water and 50 grams of pineapple in a blender. Then, pour the juice from the blender into a bowl and label the bowl. 3. Repeat the same process to make fruit and vegetable juices of kale, broccoli, cucumber, tomato, blackberry, orange, and blueberry. 4. To prepare the samples for tea, first boil water using an electric kettle. Then, once the water is boiled, pour the water into three different bowls and label them. Place the tea bags into the three different labeled bowls for about two minutes. 5. For the acid solutions, dissolve Vitamin C powder and Citric Acid powder into 100 ml of water. Use 100 ml of vinegar to represent Acetic Acid.

Part 2: Mixing the Apple Slices with the Reagents 1. Take two whole apples and cut them into fifteen slices. Then, place an untreated apple slice on a paper towel and label the towel, "Apple Baseline." 2. Using tongs, drop the fourteen cut slices into different labeled solution bowls for one minute. After a minute, place the dipped apple slices onto paper towels and glue labels above every slice.

Part 3: Observations and Data Collections 1. Create a color- coded Rating Scale (from 0 to 10) to use as a reference when the independent observers note the amount of browning on the apple slices. Then, before they start observing, train the observers based on the code. 2. Let four independent observers record the observations after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, and 96 hours. Take pictures and use the average of their findings to calculate final results.

RESULTS AND DISCUSSION

Enzymatic browning occurs as a result of oxidation. The browning scale of 0 means no browning while 10 indicates dark brown. Since enzymatic browning occurs as a result of oxidation, if there is no browning, there is no or very little oxidation. The lower the score, the more amount of antioxidants in the reagent since antioxidants prevent oxidation from occurring.

According to the experimental data outcomes, the amount of oxidation occurring in the blackberry and blueberry reagent was at 5/10 at 96 hours, which is less than any other solution. Orange and pineapple were at 5.5/10 at 96 hours with broccoli and kale at 6/10 at 96 hours. At the same time, the apple baseline was at 10/10.

The results also demonstrate that green tea has more of an antioxidant effect than herbal tea and black tea as green tea was at 6.5/10 while the other teas were at 8 and 7 respectively. The foods which least slowed down enzymatic browning were cucumber, tomato, and herbal tea as they were at 6.5, 6.5, and 8 respectively. Apple slices dipped in Ascorbic acid (vitamin C) slowed down oxidation on freshly-cut apple slices more than Acetic acid and Citric acid as Vitamin C was at 5.5/10 while the others were at 8.5 and 7, respectively.

DATA

OXIDATION ON APPLE SLICES (FRUIT, VEG, AND TEA)

Sample 30 min

1 hour

2 hours

4 hours

8 hours

12 hours

24 hours

48 hours

96 hours

Apple Baseline

0 1 2 3.5 4.5 5.5 6 9 10

Blueberry 0 0 0 0 0.5 1 2 3.5 5

Orange 0 0 0 0.5 1 1.5 2 4 5.5 Kale 0 0 0 0.5 1 1.5 3 4.5 6 Cucumber 0 0 0.5 1 1.5 2 3.5 5 6.5 Blackberry 0 0 0 0 0.5 1 2 3.5 5 Broccoli 0 0 0.5 1 1.5 2.5 3.5 4.5 6 Tomatoes 0 0 0.5 1 1.5 2 3.5 5 6.5 Pineapples 0 0 0 0.5 1 1.5 2.5 4 5.5 Herbal Tea 0 0.5 1 1.5 2 3 5 6.5 8 Black Tea 0 0.5 1 1.5 2 2.5 4 5.5 7 Green Tea 0 0 0.5 1 1.5 2 3.5 5 6.5

OXIDATION ON APPLE SLICES (ACIDS) Sample 30 min 1 hour 2 hours 4 hours 8 hours 12 hours 24 hours 48 hours 96 hours Ascorbic Acid 0 0 0.5 1 1.5 2 3.5 4.5 5.5 Citric Acid 0 0 1 1.5 2 2.5 4 5.5 7 Acetic Acid 0 0.5 1.5 2.5 3.5 4 5.5 7 8.5 Apple Baseline 0 1 2 3.5 4.5 5.5 7.5 9 10

Rating Scale of Enzymatic Browning (Oxidation) Color-Code 10- Fully Dark Brown 9- Brown 8- Close to Brown 7- Fully covered light brown 6- Close to fully light brown 5- Half covered light brown 4- Close to half covered light brown 3- Some light brown patches 2- 1 or 2 very light brown patches 1- Barely or no browning 0- No browning present

EXPERIMENTAL ERRORS Potential experimental errors are possible. One error could be that the observations were based on visual assessments which can introduce subjective variability. A small number of observers were used for this study. Another possible error is the variability of the size of the apple slices. More possible errors also may include: an inexact amount of substrate used in the solution, color tainting by the substrate solution, extrapolation of the results to effect on the human body.

PRACTICAL APPLICATION There are many fruits and vegetables, some low and some high in levels of antioxidants. This project helps people identify which plant- based foods have the most amount of antioxidants in order to assess which foods have optimal health benefits. Antioxidants help the human body repair cell damage caused by free radicals, so consuming fruits and vegetables that have high antioxidants may prevent future diseases. Plants have naturally occurring antioxidants in varying degree. This experiment also practically allows one to measure and quantify the degree of antioxidant capabilities of these natural substances.

CONCLUSION As stated in the first hypothesis, the amount of antioxidants in broccoli and kale will be greater than any other fruit or vegetable. The experimental data did not support this hypothesis. The results proved, based on theamount of enzymatic browning measured, that blueberries and blackberries, along with pineapples and

oranges, have more antioxidants than any other fruit and vegetable. The second hypothesis was also proven incorrect, as it stated that herbal tea would slow the most amount of enzymatic browning in freshly cut apple slices. As per the results, green tea slowed more oxidation than the other two teas, herbal tea and black tea. The third hypothesis, which stated that Ascorbic Acid (vitamin C) would help best prevent oxidation in a freshly cut apple slice (apple slice represents human body’s cells and tissues), is supported by the results. The final hypothesis of this project was also proven correctly. All the fruits, vegetables, beverages and acids that were tested indeed did have some degree of antioxidants in them. According to this experiment and its results, consuming blueberries and blackberries, along with oranges, pineapples, and food enriched with Vitamin C provide the most amount of antioxidants which is beneficial for overall health and well-being.

ACKNOWLEDGEMENTS In the successful accomplishment of this project, I would like to primarily thank my family members, Hetal Gandhi, Sneha Gandhi, Shreeya Gandhi, and Mayank Shah, who helped me carry out this project successfully and for their valuable advice and immense support.I would also like to express my deep gratitude and sincere thanks to my honorable science teacher, Mr. J. Linardakis, whose valuable guidance and wisdom has served as a major contributor towards the completion of this project. Last, but not least, I would like to especially thank my principal and assistant principal, Mr. Taysever and Mr. Akif.

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