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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.
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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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