CHAPTER I INTRODUCTION

BACKGROUND Tea is the second most consumed beverage in the entire world (MacFarlane, 2004). According to the History Channel, tea has been with us since 10th century B.C. It plays a big role in the Chinese, Indian and Western Culture. Tea comes from a plant called Camellia sinensis. Green tea, black tea and oolong tea all come from this plant. It is just that the leaves are processed differently. Green tea leaves are not fermented; they are withered and steamed. Black tea and oolong tea leaves undergo a crushing and fermenting process, says John Weisburger, PhD, senior researcher at the Institute for Cancer Prevention in Valhalla, N.Y The major subject of interest is the antioxidant found inside tea. These are the catechins. These antioxidants, collectively called polyphenols, have been purported to improve endothelial function (Habauzit, 2012), cut stroke risk, lower blood pressure, among others. (O'Riordan, 2012). Tea catechins is a most researched subject concerning the health potential of tea. Of all the catechins, EGCg

(epigallocatechin 3-gallate) has the most scientific attention, being singled out in a number of them as a key contributive element to the possible health effects of tea (Yang et al., 2009; Carmen Cabrera et.al., 2006; Yang J. D., 2003). A study conducted by Nagao,et.Al found out that tea lowers serum cholesterol levels,
1

reduces systolic blood pressure and reduces visceral fat area around the waist. Newer studies include anti-tumor properties as reported by Molly Lang et.Al. Recently, around the 1980s, a new type of tea was invented ---the milk tea. The origins of this tea are ambiguous, but a famous story of a man who was inspired by the famous cold coffee that originated in japan. He then added tapioca balls and milk to his tea thus, the milk tea. In the Philippines, The Manila Bulletin (Keyser, 2012) reports that Filipinos are gulping down milk tea as the preference nowadays for their daily fix of delicious beverage. It reports that Filipinos drink this because it is a sweet alternative to drinking traditional tea while still having all the benefits per se. The reason why people drink tea probably has no single answer. It has been with us for more than a thousand years. Legend has it that tea was discovered by the Chinese Emperor, Shan Nong, in 2737 B.C. The Emperor had a habit of boiling his drinking water. One day while he was in his garden a few tea leaves fell by chance into his boiling water which then gave off a rich, alluring aroma. The Emperor, upon drinking this brew, discovered it to be refreshing and energizing. He immediately gave the command that tea bushes to be planted in the gardens of his palace. Until the fifth century A.D., tea was primarily used as a remedy, due to the medicinal benefits attributed to it. This is probably the reason why people drink tea. It is refreshing, and appears to give a sense of better well-being and health.

SIGNIFICANCE OF THE STUDY This research aims to find out what the effect of milk is on green tea. This would lead to a clearer knowledge for tea drinkers and non-drinkers alike as to the nutritional implications of drinking milk tea over regular tea. This would also lead them to assess the primary reason for drinking milk tea as a tastier alternative for the regular tea.

RESEARCH QUESTION What is the effect of milk on the anti-oxidant capacity of green tea extracts as measured by DPPH assay at 520nm?

GENERAL OBJECTIVES To compare the anti-oxidant capacity of milk tea versus regular tea as measured spectrophotometrically by the DPPH assay at 520nm.

SPECIFIC OBJECTIVES To compute, and subsequently compare, the level of antioxidant (AO%) in a preparation of regular green tea versus a preparation of milk tea made using regular green tea with fresh whole milk. To determine the effective concentration (EC50) of the antioxidants present/remaining in both regular green tea and milk tea, and to ascertain which group contains more concentration in vitro.
3

THEORETICAL FRAMEWORK

CHAPTER II REVIEW OF RELATED LITERATURE

THE HISTORY OF TEA AND ITS CULTURE It has been said that tea is the 2nd most consumed beverage in the entire world (MacFarlane, 2004). In 2010 alone, The Food and Agriculture Oraganization of the United Nations noted that the production of tea exceeded 4.52 million tonnes. Tea has been consumed as far back as 10th century B.C. Tea contains catechins, a type of antioxidant. In a freshly picked tea leaf, catechins can compose up to 30% of the dry weight. Catechins are highest in concentration in white and green teas, while black tea has substantially fewer because of its oxidative preparation (Lee, 2002 and Peterson, et al. 2005). Tea has one of the highest polyphenol (a group of anti-oxidants of which catechins is a part) among common food and beverage products as reported by the U.S. Department of Agriculture. Tea catechins is a most researched subject concerning the health potential of tea The catechins in green tea are epicatechin (EC), epicatechin 3-gallate

(ECg), epigallocatechin (EGC), epigallocatechin 3-gallate (EGCg), catechin, and gallocatechin (GC). Of all the catechins, EGCg has the most scientific attention, being singled out in a number of them as a key contributive element to the possible health effects of tea (Yang et al., 2009; Carmen Cabrera et. al, 2006; Yang J. D., 2003).
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It would be necessary to point out at this moment that the group aims to research green tea in particular. It comes from the plant Camellia sinensis. Green tea, black tea and oolong tea all come from this plant. It is just that the leaves are processed differently. Green tea leaves are not fermented; they are withered and steamed. Black tea and oolong tea leaves undergo a crushing and fermenting process, says John Weisburger, PhD, senior researcher at the Institute for Cancer Prevention in Valhalla, N.Y. Tea is consumed differently around the world. Generally, tea is prepared by placing loose tea leaves, either directly or in a tea infuser, into a tea pot or teacup and pour freshly boiled water over the leaves. Modern tea is packaged in a tea bag. After a few minutes the leaves are usually removed again, either by removing the infuser, or by straining the tea while serving. In the United States and Canada, 80% of tea is consumed cold, as iced tea. The British prefer black tea, served in mugs with milk and perhaps sugar. (Tea) TEA AND ITS EFFECTS The studies on catechins (the antioxidants) in tea are overwhelming. Its touted benefits include reduction of obesity and lowering cardiovascular risk. One research conducted by a Japanese firm showed that the continuous ingestion of a GTE high in catechins led to a reduction in body fat, systolic blood pressure and LDL cholesterol, suggesting that the ingestion of this kind of extract contributes to a decrease in obesity and cardiovascular disease risks (Nagao T, 2007), as shown in the table below.
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Table A. Correlation coefficients between cardiovascular risk parameters and body fat parameters by group Body weight Cardiovasc Catechin/ ular risk control parameter Systolic blood pressure (mm Hg)* r Body fat Visceral ratio fat area P r p r p

Catechin 0.187

0.1678

0.105

0.4402

0.014

0.9169

Initial 130 Control mm Hg

0.011

0.9338

0.145

0.2662

0.074

0.5698

Serum total Catechin 0.189 cholesterol (mM) Control 0.125

0.0359

0.068

0.4517

0.149

0.6251

0.1794 0.9884

0.068 - 0.145

0.4648 0.1103

- 0.024 - 0.115

0.1095 0.2070

Serum HDL Catechin 0.001 cholesterol (mM) Control - 0.039

0.6777 0.3109

- 0.007 0.095

0.9362 0.2964

0.075 - 0.024

0.4217 0.7919

Serum LDL Catechin 0.092 cholesterol (mM) Control Plasma glucose (mM) 0.101

0.2783 0.9050

0.180 - 0.021

0.0519 0.8137

- 0.008 - 0.042

0.9329 0.6427

Catechin 0.011

Control

0.107

0.2489

0.013

0.8908

0.060

0.5176
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The effects of catechins on energy and fat metabolism have recently been examined in humans. Dulloo et al. reported that the ingestion of 270 mg/dL of catechins resulted in increased energy expenditure and lipid oxidation in 10 subjects (Dulloo, 1999). Chantre et al. reported that ingestion of 375 mg/dL of catechins tended to decrease waist circumference in 70 subjects (Chantre, 2002). Another particular study, although still in its infancy and has not been significantly proven in humans is the ability for the catechins in tea to inhibit apoptosis, which is cell death, in cholangiocarcinoma cells. It concluded that the green tea polyphenol EGCG (Epigallocatechin-gallate) sensitizes human

cholangiocarcinoma cells to chemotherapy-induced apoptosis and warrants evaluation as an adjunct to chemotherapy for the treatment of human cholangiocarcinoma. (Molly Lang, 2009) . There was even another study saying catechins in green tea possess anticancer properties against "cancer in various organs, including the colorectum and liver, and are known to exert anti-obesity, antidiabetic, and anti-inflammatory effects." "Branched-chain amino acids in green tea may prevent progressive hepatic failure in patients with chronic liver diseases, and might be effective for the suppression of obesity-related liver carcinogenesis. (Masahito Shimizu, 2012) A newer study conducted by Kakuda shows that catechins, particularly theanine, from tea extracts can protect the neurons in the brain from damage and preserve cognitive function. (Kakuda, 2011)

A LOOK AT ANTIOXIDANTS Although the topic of antioxidants is vast, the review of literature will limit only to the nature of antioxidants especially catechins, which is the point of emphasis. Metabolism involving oxygen such as respiration and other activities naturally generate natural by-products called reactive oxygen species or ROS. These are important in cell signaling and homeostasis (Apel & Hirt, 2004). However, during times of environmental stress such as UV or heat exposure, ROS levels can increase dramatically (Apel & Hirt, 2004). This results to cell damage or even cell death (apoptosis) and is cumulatively known as oxidative stress. Oxidative stress is closely related to these very reactive and unstable radicals (Fang, Yang, & Wu, 2002). It is their reactivity that is responsible for some human diseases like cancer and cardiovascular diseases and is able to cause oxidative damages to proteins, DNA, and lipids (Jacob & Burri, 1996). Antioxidants stop these reactions by removing free radical intermediates, and inhibit other oxidation reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid, or polyphenols. Catechines are a part of the polyphenols group.

THE MILK TEA Just as the origin of tea is ambiguous, so is its distinctly Asian sibling. The more famous story revolved around one man and his tea shop in the 1980s, who developed a beverage wherein Chinese tea was served cold. His supposed
9

inspiration came from cold coffee served in Japan during those times. Then, in 1988, his product development manager added on to this idea by putting tapioca balls (sago) into the iced drink. The bubble tea (milk tea as it is more commonly referred to locally) was then born. The other story states that another teashop owner by the name of Tu Tsong He Hanlin, added white fenyuan balls to his tea. The balls resembled pearls, earning it the nickname of pearl tea. Later on, the white balls were replaced with black ones, thus becoming bubble tea. Regardless of its origins, one thing remains certain: this drink has spread throughout the world, particularly in Asia. Now, this drink is consumed regularly by Filipinos, particularly by the youth. This is accentuated by the fact that Filipinos readily embrace and accept anything hip and fashionable. Bubble tea, or milk tea, is served in the Philippines much like it has been in Taiwan. Since there is no single shop that monopolizes the industry, most shops focus on differentiating their brand from others. Such tactics include addition of fruity flavors to the bubble tea, or other add-ons such as pudding or jelly. A study on the effects of this new craze is necessary due to the lack of research about the topic. Milk tea, just like many of the other crazes that have arrived in the Philippines, may or may not have a positive impact on our health. Its nutritional content and benefits are undocumented, despite the fact that its principal ingredients, milk and tea, have been around for centuries. Perhaps, mixing the two

10

would produce less-than-desirable effects? Maybe the two work antagonistically towards each other? The paper, then, aims to discover whether or not the addition of milk will reduce the antioxidant capacity of catechins. Here are a couple of research studies on how conflicting reports led to the pursuance of the topic by this group. An important reminder would be that although some of these studies were conducted more than 10 years ago, these studies were the only ones performed such that no study has succeeded in refuting or supporting the claims of the aforementioned research work thus making it the most up-to-date. Additionally, these researches were made outside the country, where milk tea is prepared differently.

THE FIRST STEP Back in 1998, a study by Unilever Research Labs in Netherlands conducted a study on the bioavailability of catechins in the blood after ingesting green or black tea and the effect of adding milk to it. The study was conducted on 12 humans who were given randomly green tea, black tea and black tea with semi-skimmed milk. Their blood was taken before and eight hours after consumption. The results showed that milk did not seem to affect the bioavailability of catechins in the blood and that they were still rapidly absorbed (Unilever Research Vlaardingen, 1998). The objective was accomplished and the results were unbiased. However, the question here is whether a correlation was made between bioavailability and anti-oxidant capacity of the catechins. These were not assessed.
11

Therefore, one cannot say that because a substance such as catechin has the same bioavailability with or without milk, its anti-oxidant capacity remains the same. The methodology was not flawed; however, it failed to determine the essential function of the catechin --- the anti-oxidant capacity.

A REFINEMENT The research was presumptively refined in 2000 by the Unilever Nutrition Center, a division arm of Unilever Labs by performing a similar study, this time using the FRAP (ferric reducting ability of plasma) to measure anti-oxidant activity of catechins. The test was noticeably different in that plasma concentration was measured up to two hours after ingestion of tea and that test subjects were put on a strict diet. The methodology here was very complicated and the value of FRAP required pre-determination of Vitamin C, uric acid and bilirubin which are interfering factors of the assay. (the aforementioned having reducing properties as well) . The results were the same and that the bioavailability was not affected by the addition of milk The experiment therefore is not very practical. It must be noted that the results were completely justified and that the study made mention that though milk does not abolish the anti-oxidant activity, the ability for these anti-oxidants to prevent in vivo damage remained to be established (Unilever Nutrition Centre, 2000).
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THE NO CAMPAIGN Dr. Verana Stangl, et al published in the European Heart Journal on September, 2006 that they have conducted a study on the effect of milk in tea as to the vascular benefits of black tea. They used human, rat and HPLC assays to affirm their research. It was shown that subjects given tea without milk showed markedly undisturbed flow mediated dilatation (FMD), which is the hallmark of endothelial function, in the brachial artery measured through ultrasound. Those subjects that had milk mixed with their tea showed only 10% of the dilatation. The results led the team to measure nitric oxide, the vasodilator, when tea (with and without milk) was ingested. The level of nitric oxide was measured as eNOS (Endothelial nitric oxide synthase) activity. Tea alone showed 400% eNOS activity while tea with milk completely eliminated nitric oxide activity. The researchers then decided to evaluate which of the individual milk proteins affect the production of nitric oxide. They assumed that casein, the major milk protein, binds with these catechins and therefore reduces its ability to cause catechin-dependent vasodilatation. The research concluded that catechins do bind to certain proline-rich proteins, such as casein, and that milk strikingly reduces the ability of catechins to mediate a vasodilatory effect, thus abolishing the cardiovascular benefits of tea (Mario Lorenz, 2006). The study was thorough and gave new insight to the effects of milk towards black tea.

13

However, the study was conducted largely in vivo. In fact, the determination of total catechins after centrifugation with milk gave no indication of the methodology used to measure the catechins. The study was also based solely on endothelial function. Catechins do play a major role in cardiovascular function, but it also has other roles such as insulin regulation as conducted on a separate study by Anderson, Polansky from the J Agric Food Chem 50:7182-6 (2002). It is important to note that green tea was NOT studied because the population target only included those people drinking black tea (which is commonplace to consume with milk) whereas green tea is almost always consumed without milk. However, the growing trend of the milk tea has changed this dynamic. Hence, the group aims to improve the study by applying the scenario into the modern, Philippine setting of consuming tea (which is similar to consuming a fruit shake) instead of the Western style. Between the researches, there is a gap of knowledge that needs to be filled. How can one research say that catechins remain almost completely bioavailable even with milk and another study claiming that the milk protein, chiefly casein, binds with the catechins, and therefore abolishing its effects? Does abolishing its endothelial effect mean all its other anti-oxidative properties are also abolished? Did former tests detect bound and unbound catechins as one, thereby reporting them as bioavailable altogether?

14

THE INFORMATION GAP There seems to be a commonality in the problems encountered in these researchesmethodology. What is the best way to measure anti-oxidative capacity? Until now, the assay largely been used was the FRAP assay. But that was subject to interfering factors and calibration curves, though standardized, cannot be completely accurate because results rely heavily on individual values. In short, though in vivo testing provides a clear indication of how these catechins function, it does not tell us its capacity to do so. If the question is whether or not milk affects tea and that the binding of protein to catechins does affect its anti-oxidative capacity, shouldnt the determination of all these be performed in vitro? The

research performed by Dr. Stang et al did perform such in vitro test, but no methodology was put in place for the in vitro determination. Dr. Aruna Prakash, PhD, Fred Rigelhof and Eugene MIller, PhD of Medallion Labs conducted an experiment on DPPH and how it measures the antioxidant capacity of certain fruits, vegetables, etc. DPPH is a stable free radical with characteristic absorption at 515 nm and antioxidants react with DPPH and convert it to 2,2-diphenyl-1-picrylhydrazine. The DPPH radical has been widely used to test the free radical-scavenging ability of various natural products and has been accepted as a model compound for scavenging free radicals in tea. In the DPPH radical-scavenging method, a compound with high antioxidant potential effectively traps the radical, thereby preventing its propagation and the resultant
15

chain reaction. The degree of discoloration from violet to yellow indicates the scavenging potential of the antioxidant extract, which is due to the hydrogen donating or radical scavenging ability (Brand-Williams, 1995). High % DPPH radical scavenging activity would imply a high activity. The samples were extracted in 80% methanol for 4 hours at 35 degrees Celsius. DPPH was added and Trolox agent was used as a standard. The absorbance was measured at 517nm and compared. The methodology provided was quick and simple. The research however has not implicated anything other than the fact that DPPH is a relatively accurate way to measure anti-oxidant capacity and that no correlation with health benefits were made. The study also does not include the measurement of tea. The study claims that DPPH is able to measure all kinds of polyphenols (a broad group of anti-oxidants of which catechin is a member). The mechanism of an anti-oxidant is that it binds to free radicals to prevent oxidative damage to tissues and cause effects such as cell aging, liver disease, and cancer, among others. Therefore, in order to measure anti-oxidant capacity, a substance that acts a free radical must be used as a reagent upon which the antioxidant acts upon. The reaction must then be quantified. The groups research aims to bridge the information gap by utilizing the DPPH assay. This assay is rapid, simple and requires no pre-treatment of samples and measures the anti-oxidant activity directly as a scavenger free radical. While assays such as the FRAP rely on the reduction of iron, this assay is much more refined, owing to the fact that the reagent becomes the radical itself---which is what
16

anti-oxidants act upon. This reagent also binds to both soluble and insoluble forms of flavonoids and is more accurate in determining anti-oxidant capacity. There is reason to believe that it is necessary to perform this kind of assay on this much-debated issue to further resolve conflicting reports on the effect of milk in tea. We are linking the studies made by Dr. Stangl and using the methodology proposed by Dr. Prakash, et al. The issue may be resolved by using the correct sample (green tea alone, milk and green tea combined) with the correct proportion as well. British tea customarily has 10-15% milk and is drunk like coffee mixed with creamer. The type of consumption of tea this group aims to study is the more current and growing fad especially here in the Philippines, which is milk mixed with tea (and is taken cold), with additives such as tapioca balls and sugar (of which will not be entertained in the study).

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CHAPTER III METHODOLOGY

STUDY DESIGN The research design is comparative. It deals only with the comparison of the anti-oxidant activity of milk tea and regular tea using percentage values and the EC 50 Method. The design is also experimental in nature.

STUDY POPULATION Inclusion Criteria The tea to be selected must be green tea (Twinnings green tea extract). The milk added will be fresh whole milk (Nestle). Exclusion Criteria Only fresh whole milk from Nestle, and Twinnings green tea were used included in the sampling and experimentation. Sampling Procedure The sampling procedure was done by determining a desired statistical power of the trial, and the effect size. The desired effect was 0.5 (medium effect), and the power was arbitrarily set at the default 0.8. Sample Size The Sample size, at medium effect (0.5) and power at 0.8, was found to be 64.
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MANEUVERS Data Collection Technique The materials included are the green tea (in the form of a tea bag) and the milk; the milk tea was independently prepared by the researchers. This was done to eliminate bias; all the milk and tea used were of the same caliber. Procedure for Data Collection This procedure was based on an experiment performed by Libag, et. al, of the Department of Chemistry Our Lady of Fatima University involving the antioxidants found in plants like the Kamias and Calachuchi. Tea Preparation The purchased tea bags were submerged in absolute methanol for 30 minutes. This amount of time is sufficient to extract the antioxidants present in the tea bag. Methanol was chosen for the primary reason that it is also the solvent of the reagent and it possesses excellent antioxidant extraction properties. Two (2) mL of the resulting solution was used for the assay.

The milk tea samples were treated similarly; all were put through an immersion procedure in fresh whole milk for ten dips. This allowed contact with milk and the relatively short amount of time was chosen to ensure antioxidants dont diffuse out of the bag and into the milk. Afterwards, the same procedure was followed as per the regular tea samples.
19

Standardization of Vitamin C Calibration Curve The purpose of this step is to establish a calibration curve as a basis to determine whether tea has anti-oxidant capacity or not by checking whether the assay is valid by using a standard as a positive control. Ascorbic Acid or Sodium Ascorbate (in salt form) is a well accepted standard (Bondet, 1997; Kim, 2002; Sa nchez-Moreno, 1999). Another purpose for this curve is for it to serve as a basis to derive the antioxidant capacity. Generally, it should be presented in terms of the number of DPPH molecules reduced by one mole of substrate. Since tea extracts cannot be computed for its molar value, the equivalent anti-oxidant per gram of extract was used; hence this procedure. A vitamin C (sodium ascorbate) preparation from a tablet such as Fern-C was made at initial concentrations of 0.25mg/mL. The DPPH reagent prepartion was patterned on the procedure of Molyneux. 10mg of DPPH was dissolved in 250mL 80% ethanol to produce a concentration of 100uM. The blank contained the DPPH reagent alone.

20

Figure 1. Table showing the preparation of Vitamin C solutions for standardization

VOLUME in mL DPPH solution Vitamin C solution Distilled water TOTAL VOLUME

TUBE 1 2mL

TUBE 2 2mL

TUBE 3 2mL

TUBE 4 2mL

TUBE TUBE 5 2mL 6 2mL

TUBE TUBE 7 2mL 8 2mL

0.10

0.20

0.40

0.60

0.80

1.00

1.10

1.20

1.90

1.80

1.60

1.40

1.20

1.00

0.9

0.8

4mL

4mL

4mL

4mL

4mL

4mL

4mL

4mL

The extracts from the green tea and milk tea preparation were prepared at 0.125mg/mL. This was done by taking 2.5microliter of extract and adding to 1.9 mL water to achieve 0.125mg/mL. A 2mL DPPH solution was added to 2mL of the extract solution for all the 8 control groups designated 1 to 8. The same was performed on the experimental group. The handling of all solutions required the use of semi-automated pipettes with preset calibrations and disposal tips. In this way, interferences were avoided and cross-contamination of samples eliminated.

21

During the experiment, the DPPH reagent was protected from light, and all reagents in lyophilized form were prepared using an analytical balance to ensure exact measurements. The solutions were placed in a cuvette and read at 520nm in a spectrophotometer at the Velez College laboratory. The absorbances were collected. To minimize and keep stray light (extraneous light) from interfering with absorbances, the reading was done in the dark and the cuvettes used did not contain scratches because this can interefere with the reading. The absorbance of the Vitamin C curve was constructed and the slope was taken to form a regressive function and an equation to calculate the antioxidant mg/mL was formed as:

where y = absorbance of samples m = slope of the line b = absorbance of blank x = antioxidant activity in mg/mL To represent the results in percentage, the equation below was used to convert the mg/mL to percent:

where Ao = antioxidant present Since the research is about resolving the anti-oxidant capacity remaining in tea versus milk added to tea, another computation was necessary to show the ability of anti-oxidants to inhibit DPPH (which acts as
22

the free radical). This method is known as the EC50. This is the ability of the substrate (in this case the catechins) to cause 50% loss of DPPH activity. Percent inhibition was expressed as EC values of 0.125mg/mL of crude extracts. The computation EC values, designated as C will be as follows: C ) x 100,

Where Ao = absorbance of control (DPPH only) Ac = absorbance of solution containing the extract and DPPH The values were then tabulated to compare the controls (tea only) with the samples (milk and tea) as to the EC50 values and as to the antioxidant value in mg/mL shown earlier. In order to determine the significance of the values, an independent ttest was performed. It is a not parametric test that tests the null hypothesis that two populations are the same. The variables needed are the standard deviations, means, and sample sizes from both populations. The formula is as follows:

where:

23

Data Collection Tools 1 Spectrophotometer 2 Cuvettes 3 Test tubes 4 Pipettes 5 Regular Tea 6 Milk Tea 7 DPPH reagent 8 Distilled water 9 80% methyl alcohol 10 Paraffin 11 Containers 12 Sample cups
13 Vitamin C tablet (Fern-C)

OPERATIONAL DEFINITION OF TERMS a Regular tea - tea prepared only by infusing GREEN tea extract (in tea bags) with water; no additives b Milk tea regular green tea that is added with milk c Antioxidant -a substance that inhibits oxidation or reactions promoted by oxygen, peroxide or free radicals, as in this case, the DPPH reagent serves as the free radical d Antioxidant Capacity - the amount of antioxidant that is able to bind or inhibit the DPPH e Ascorbic Acid Equivalent Antioxidant Capacity the anti-oxidant level measured as a function of Vitamin C; it is simply the level of antioxidant level equal to the one given off by ascorbic acid at the same wavelength

24

EC50- the method of measuring the antioxidant capacity wherein an antioxidant can inhibit a radical to 50% of its activity.

g DPPH stands for 2,2-diphenyl-1-picrylhydrazyl; serves as the free radical to which antioxidant binds to

VARIABLES The independent variable would be the regular tea. Nothing was added to the test system. This served as the control group. The dependent variable was the milk tea, unto which fresh milk was added to the test system. This served the experimental group.

DATA ANALYSIS The first set of raw data, which is the absorbance of the Vitamin C solutions were taken then presented graphically. The slope was then computed for so that the mg/mL of antioxidants could be taken. A regressive equation was used then the percent antioxidant was then computed for subsequently based on the individual absorbances the 64 samples gave. The results of the computations were presented in a graph. The final set of data was computed for using the formula in percent inhibition determination. The results were again presented in a graph. From the tabulated data, the mean %AO and standard deviation from both sets were computed. Smiths statistical package was then used to compute for the
25

t value. The EC50 values were used to confirm that the %AO values were correct. The higher the %AO in solution, the lower the EC50 remaining in sample. The computed t-value will then be compared against the p-value of 0.05. If the p-value is lesser than or equal to the computed t, then the null hypothesis will be rejected.

TESTABLE HYPOTHESES Null: Milk will not significantly reduce the anti-oxidant capacity of green tea.
Alternative: Milk will significantly reduce the anti-oxidant capacity of green tea.

STATISTICAL TEST The statistical test employed for the experiment was the independent T-test; the values are interval in nature and are independent samples, namely, the regular tea and milk tea. The software utilized was the free software Smiths statistical package available online.

LEVEL OF SIGNIFICANCE The level of significance arbitrarily chosen for the hypothesis test was 5%.

26

Chapter IV RESULTS AND DISCUSSION

During the experiment, the DPPH reagent was protected from light, and all reagents in lyophilized form were prepared using an analytical balance to ensure exact measurements. Once the DPPH was added to the tea mixture, the color changed from purple to yellow, indicating that the reaction was valid and that the reagent was functioning.

Concentration of Vitamin C in mg/mL

0.035 0.03 0.025 Absorbance 0.02 0.015 0.01 0.005 0 0.0125 0.025 0.05 Concentration (mg/mL) 0.075 0.1

Figure 1. Vitamin C standardization curve. The reagent is in control and serves as the curve where the AEAC (Ascorbic Acid Equivalent Antioxidant Capcity) will be derived. It is a function of concentration as to the absorbance.

27

The spectrophotometric readings are of interval measurements. A zero reading does not indicate the absence of the anti-oxidant. Therefore, the experiment converted these readings into percentage values using the equation of the slope which is y=mx+b. In this manner, the values were then representing measurements that have meaningful zeroes. The statistical tool used when predicting the anti-oxidant level of the tea samples from the values set up in the Vitamin C standardization curve was the simple linear regression as shown in Figure 1. This is used to determine the Ascorbic Acid Equivalent Anti-oxidant Capacity. Simply put, the formula equates the absorbance of the tea samples to that of Ascorbic Acid and deriving the level of anti-oxidant from that equation.

0.3 0.25 Absorbance 0.2 0.15 0.1 0.05 0 Average Abs of Green Teas Average Abs of Green Teas w/ Milk

Samples

Figure 2. Anti-oxidant Activity of Milk Tea versus Regular tea in mg/mL.

28

45.0% 40.0% 35.0% 30.0% %AO 25.0% 20.0% 15.0% 10.0% 5.0% 0.0% Average %AO in tea Average %AO in milk tea Sample

Figure 3. Percent of Anti-oxidant of milk tea versus regular tea.

90% 85% EC50 80% 75% 70% 65% EC50 of tea Sample EC50 of milk tea

FIGURE 4. EC50 level of Milk Tea vs. the Regular tea. The graph shows the EC50 level of the Milk Tea Samples versus the Regular tea. The EC50 levels correspond to the amount of reagent (oxidant) in the sample.
29

As shown in Figures 2 and 3, the levels of antioxidant in regular tea proved to be greater than the ones present in the milk tea for all samples. In Figure 4, the percent inhibition of milk tea samples or EC50 are greater than those of regular tea. The interpretation of this means that a greater amount of antioxidant in the milk tea is required to inhibit the DPPH reagent than that of the regular tea. This means that the antioxidant in regular tea is more capable of causing the reagent to be inhibited or inactivated; thus, it has greater antioxidant capacity. The findings agree with the %AO computation. The results of the independent t-test on the %AO show that at a 5% level of significance, the p value comes out at 0.00390600. This is lower than the 0.05 or 5% level of significance set for the experiment; therefore, the null hypothesis (Ho) was rejected. Milk significantly affects the anti-oxidant capacity of regular tea. This study is consistent with the one performed by Dr. Stang et. al which was aimed at determining if the antioxidants in the tea were still able to affect endothelial function in the sense that the antioxidants were still active. This however is very different from the studies done by the Uniliver Research Labs in the 90s where the studies showed complete bioavailibity of the antioxidants in tea despite the milk. It seems then that bioavailability and antioxidant capacity are not synonymous. Bioavailability may be seen as merely counting the antioxidants while antioxidant capacity can be seen as determining whether these antioxidants still have its properties to bind to oxidizing agents, DPPH in the case of this study.

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In whatever preparation of tea, for as long as there is milk added to it, it will depress antioxidant capacity. It must be noted that the study used tea bags extracted with methanol and immersed directly with milk. This preparation is optimal, since the full concentration of antioxidants were extracted and made to react with milk. Commercial preparations of milk tea have considerably less concentrations of tea per serving since the extraction process isnt done with methanol and contain many other ingredients. It is also dubious as to whether an entire teabag is utilized per serving, viewed in terms of the economy of such preparation. In other words, since the study showed that the antioxidant capacity is much lower in such controlled and ideal conditions, it is very unlikely to assume that the trend will be different in other studies and commercial preparations.

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Chapter V CONCLUSION AND RECOMMENDATIONS

The findings of this research are in line with experiments using other methodologies; milk will affect the antioxidant capacity of green tea. The reason as to why this occurs has not yet been proven. It is believed that the milk proteins (particularly casein) bind to the antioxidants of tea therefore rendering it incapable of binding to free radicals such as the reagent used in the experiment. So if one were to take milk tea for the touted health benefits found in conventional tea, one may have to think twice. Plus, milk tea sold commercially isnt really just milk with tea; they usually contain sugar, cream, cheese, tapioca balls and other ingredients. It would be recommended that for those seeking to get the benefits of the antioxidants of tea, one should consume it conventionally. It must be noted, however, that the research only demonstrated milks effect on antioxidant capacity. Other factors were not included in the study, namely: the additional effect/s of adding sugar and other ingredients to the milk tea, and the actual in vivo effect. The actual in vivo effect is much harder to quantify, as it includes factors such as intestinal absorbance, first-pass metabolism, etc. Further research may concentrate more on these effect/s. Another area for further study would be to explore other kinds of tea like oolong, earl grey tea, etc. to see if it produces similar results. Further study may also lead to the identification of the exact interaction of milk and tea at a molecular level to prove this effect.
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REFERENCES

Bibliography Apel, K., & Hirt, H. (2004). eactive oxygen species: metabolism, oxidative stress and signal transduction. Annu. Rev. Plant Biology , 55, 373399. Brand-Williams, W. C. (1995). Use of a free radical method to evaluate antioxidant activity. Food Science and Technology , 28, 25-30. Bondet, V. B.-W. (1997). Kinetics and mechanisms of antioxidant activity using the DPPH free radical method. Lebensmittel- Wissenschaft und -Technologie/Food Science and Technology (60), 609-615. Carmen Cabrera, P. R. (2006). Beneficial Effects of Green Tea A Review. Journal of the American College of Nutrition , 25 (2), 79-99. Chantre, P. L. (2002). Recent findings of green tea extract AR25 (Exolise) and its activity for the treatment of obesity. Phytomedicine 9 , 3-8. Dulloo, A. G. (1999). Efficacy of a green tea extract rich in catechin polyphenols and caffeine in increasing 24-h energy expenditure and fat oxidation in humans. Am J Clin Nutr , 10401045. Fang, Y., Yang, S., & Wu, G. (2002). Free radicals, antioxidants, and nutrition. Nutrition , 18, 872879. Jacob, R., & Burri, B. (1996). Oxidative damage and defense. American Journal of Clinical Nutrition , 63, 985990. Kakuda, T. (2011). Neuroprotective effects of theanine and its preventive effects on cognitive dysfunction. Pharmacological Research , 64 (2), 162168. Keyser, A. B. (2012). It's A Tea Thing! Manila: Manila Bulletin. Kim, J.-K. N.-O. (2002). The first total synthesis of 2,3,6-tribromo-4,5dihydroxybenzyl methyl ether (TDB) and its antioxidant activity. Bull. Korean Chem. Soc , 23 (5), 661-662. Lee, K. W. (2002). Antioxidant Activity of Black Tea vs. Green Tea. The Journal of Nutrition , 132 (4), 2248-2251. MacFarlane, A. (2004). The Empire of Tea. The Overlook Press.

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Mario Lorenz, N. J. (2006). Addition of milk prevents vascular protective effects of tea. European Heart Journal , 28 (2), 219-223. Masahito Shimizu, M. K. (2012). Nutraceutical Approach for Preventing ObesityRelated Colorectal and Liver Carcinogenesis. International Journal of Molecular Science , 575-595. Molly Lang, R. H. (2009). Epigallocatechin-gallate modulates chemotherapyinduced apoptosis in human cholangiocarcinoma cells. Liver International , 29 (5), 670677. Nagao T, H. T. (2007). A green tea extract high in catechins reduces body fat and cardiovascular risks in humans. Obesity (Silver Spring) , 14731483. Peterson, J. (2005). Major flavonoids in dry tea. Journal of Food Composition and Analysis , 497-501. Sa nchez-Moreno, C. L.-C. (1999). Free radical scavenging capacity and inhibition of lipid oxidation of wines, grape juices and related polyphenolic constituents. Food Res. Int. (32), 407-412. Tea [Motion Picture]. Modern Marvels Television. Unilever Nutrition Centre. (2000). A single dose of tea with or without milk increases plasma antioxidant activity in humans. European Journal of Clinical Nutrition , 54, 8-92. Unilever Research Vlaardingen. (1998). Bioavailability of catechins from tea: the effect of milk. European Journal of Clinical Nutrition , 52 (5), 356-359. Yang, C. (2009, January). Antioxidative and anti-carcinogenic activities of tea polyphenols. Archives of Toxicology , 83 (1), p. 11. Yang, J. D. (2003, October). Mechanisms of Cancer Prevention by Tea Constituents. The Journal of Nutrition , 3262S-3267S.

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35

36

BUDGET

ITEMS

ESTIMATED PRICE

DPPH reagent

6,150.00

Fresh milk

30.00

Twinnings Green tea

100.00

Methanol

40.00

Distilled Water

25.00

Vitamin C

12.00

Bondpaper

100.00

Folder

6.00

TOTAL

6,463.00

37

APPENDIX

38

70.0% 60.0% 50.0% %AO 40.0% %AO in tea 30.0% 20.0% 10.0% 0.0% 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 Samples %AO in milk tea

FIGURE 1. Antioxidant output of the 64 samples of milk tea and regular tea

100% 90% 80% 70% 60% EC50 50% 40% 30% 20% 10% 0% 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 Samples EC50 of tea EC50 of milk tea

FIGURE 2. EC50 levels of all 64 samples of milk tea and regular tea

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