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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,


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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.
http://www.wtea.com/about-tea_health-benefits.aspxhttp://www.wtea.com/about-tea_health-benefits.aspx


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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.


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THEORETICAL FRAMEWORK


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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 orteacup

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.
http://en.wikipedia.org/wiki/Tea_infuserhttp://en.wikipedia.org/wiki/Tea_pothttp://en.wikipedia.org/wiki/Teacuphttp://en.wikipedia.org/wiki/United_Stateshttp://en.wikipedia.org/wiki/Canadahttp://en.wikipedia.org/wiki/Iced_teahttp://en.wikipedia.org/wiki/Iced_teahttp://en.wikipedia.org/wiki/Canadahttp://en.wikipedia.org/wiki/United_Stateshttp://en.wikipedia.org/wiki/Teacuphttp://en.wikipedia.org/wiki/Tea_pothttp://en.wikipedia.org/wiki/Tea_infuser


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Table A. Correlation coefficients between cardiovascular risk parameters and

body fat parameters by group

Bodyweight

Body fatratio

Visceralfat area

Cardiovasc

ular risk

parameter

Catechin/

control

r P r p r p

Systolic

blood

pressure

(mm Hg)*

Catechin 0.187 0.1678 0.105 0.4402 0.014 0.9169

Initial 130

mm Hg

Control 0.011 0.9338 0.145 0.2662 0.074 0.5698

Serum total

cholesterol

(mM)

Catechin 0.189 0.0359 0.068 0.4517 0.149 0.6251

Control 0.125 0.1794 0.068 0.4648 - 0.024 0.1095

Serum HDLcholesterol

(mM)

Catechin 0.001 0.9884 - 0.145 0.1103 - 0.115 0.2070

Control - 0.039 0.6777 - 0.007 0.9362 0.075 0.4217

Serum LDL

cholesterol

(mM)

Catechin 0.092 0.3109 0.095 0.2964 - 0.024 0.7919

Control 0.101 0.2783 0.180 0.0519 - 0.008 0.9329

Plasma

glucose

(mM)

Catechin 0.011 0.9050 - 0.021 0.8137 - 0.042 0.6427

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)


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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
http://en.wikipedia.org/wiki/Reducing_agenthttp://en.wikipedia.org/wiki/Thiolhttp://en.wikipedia.org/wiki/Ascorbic_acidhttp://en.wikipedia.org/wiki/Polyphenol_antioxidanthttp://en.wikipedia.org/wiki/Polyphenol_antioxidanthttp://en.wikipedia.org/wiki/Ascorbic_acidhttp://en.wikipedia.org/wiki/Thiolhttp://en.wikipedia.org/wiki/Reducing_agent


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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


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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.


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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.


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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?


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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 anti-

oxidant 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


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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 anti-

oxidant 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


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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.


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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.


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

VOLUME

in mL

TUBE

1

TUBE

2

TUBE

3

TUBE

4

TUBE

5

TUBE

6

TUBE

7

TUBE

8

DPPH

solution

2mL 2mL 2mL 2mL 2mL 2mL 2mL 2mL

Vitamin C

solution

0.10 0.20 0.40 0.60 0.80 1.00 1.10 1.20

Distilled

water

1.90 1.80 1.60 1.40 1.20 1.00 0.9 0.8

TOTAL

VOLUME

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.


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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 samplesm = slope of the line

b = absorbance of blankx = 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


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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 DPPHThe 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 t-

test 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:


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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

10Paraffin

11Containers

12Sample 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 anti-

oxidant level equal to the one given off by ascorbic acid at the same

wavelength


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f 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


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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%.


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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.

Figure 1. Vitamin C standardizat ion cu rve. The reagent is in con trol

and serves as the curve where the AEAC (Ascorbic Acid Equivalent

Ant iox idant Capci ty) wi l l be der ived. I t is a funct ion of co ncentrat ion as to the

absorbance.

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.0125 0.025 0.05 0.075 0.1

Absorbance

Concentration (mg/mL)

Concentration of Vitamin C in mg/mL


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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.

Figure 2. Anti -oxidant Act iv i ty o f Mi lk Tea versus Regular tea in mg /mL.

0

0.05

0.1

0.15

0.2

0.25

0.3

Average Abs of Green Teas Average Abs of Green Teas

w/ Milk

Absorbance

Samples


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Figure 3. Percent of Ant i -oxidant of mi lk tea versus regular tea.

FIGURE 4. EC50 level of Milk Tea vs. the Regu lar tea. The graph shows

the EC50level of the Milk Tea Samp les versus the Regular tea. The EC50levels

correspon d to th e amou nt of reagent (oxidant) in the sample.

0.0%

5.0%

10.0%

15.0%

20.0%

25.0%

30.0%35.0%

40.0%

45.0%

Average %AO in tea Average %AO in milk tea

%AO

Sample

65%

70%

75%

80%

85%

90%

EC50 of tea EC50 of milk tea

EC50

Sample


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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 milk s 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

BibliographyApel, K., & Hirt, H. (2004). eactive oxygen species: metabolism, oxidative stressand signal transduction.Annu. Rev. Plant Biology, 55, 373399.

Brand-Williams, W. C. (1995). Use of a free radical method to evaluate antioxidantactivity. Food Science and Technology, 28, 25-30.

Bondet, V. B.-W. (1997). Kinetics and mechanisms of antioxidant activity using theDPPH free radical method. Lebensmittel- Wissenschaft und -Technologie/FoodScience and Technology(60), 609-615.

Carmen Cabrera, P. R. (2006). Beneficial Effects of Green TeaA 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 itsactivity for the treatment of obesity. Phytomedicine 9 , 3-8.

Dulloo, A. G. (1999). Efficacy of a green tea extract rich in catechin polyphenolsand 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 ofClinical Nutrition, 63, 985990.

Kakuda, T. (2011). Neuroprotective effects of theanine and its preventive effects oncognitive 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-4,5-dihydroxybenzyl 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 ofNutrition, 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 oftea. European Heart Journal, 28(2), 219-223.

Masahito Shimizu, M. K. (2012). Nutraceutical Approach for Preventing Obesity-Related Colorectal and Liver Carcinogenesis. International Journal of Molecular

Science , 575-595.

Molly Lang, R. H. (2009). Epigallocatechin-gallate modulates chemotherapy-induced 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 andcardiovascular risks in humans. Obesity (Silver Spring) , 14731483.

Peterson, J. (2005). Major flavonoids in dry tea. Journal of Food Composition andAnalysis , 497-501.

Sa nchez-Moreno, C. L.-C. (1999). Free radical scavenging capacity and inhibitionof lipid oxidation of wines, grape juices and related polyphenolic constituents. FoodRes. Int. (32), 407-412.

Tea [Motion Picture]. Modern Marvels Television.

Unilever Nutrition Centre. (2000). A single dose of tea with or without milkincreases plasma antioxidant activity in humans. European Journal of ClinicalNutrition, 54, 8-92.

Unilever Research Vlaardingen. (1998). Bioavailability of catechins from tea: theeffect of milk. European Journal of Clinical Nutrition, 52(5), 356-359.

Yang, C. (2009, January). Antioxidative and anti-carcinogenic activities of teapolyphenols.Archives of Toxicology, 83 (1), p. 11.

Yang, J. D. (2003, October). Mechanisms of Cancer Prevention by TeaConstituents. The Journal of Nutrition , 3262S-3267S.


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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


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APPENDIX


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

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

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61

%AO

Samples

%AO in tea

%AO in milk

tea

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61

EC50

Samples

EC50 of tea

EC50 of

milk tea


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