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Food Research International 41 (2008) 124129

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Influence of milk and sugar on antioxidant potential of black tea

Vasundhara Sharma, H. Vijay Kumar, L. Jagan Mohan Rao

Plantation Products, Spices and Flavour Technology Department, Central Food Technological Research Institute, Mysore 570 020, India
Received 28 August 2007; accepted 23 October 2007

Abstract
Black tea is often consumed with milk and sugar. It is still unknown with certainty whether addition of milk and (or) sugar affects its
antioxidant activity. The present study is aimed to investigate the effect on antioxidant and radical scavenging activities of black tea on
addition of milk and sugar. Four types of black tea brew samples are prepared, viz., plain black tea (B), black tea with sugar (BS), black
tea with milk (BM) and black tea with milk and sugar (BMS). The freeze-dried solids of all the brew samples are evaluated for their
biological activities. The radical scavenging and antioxidant activities are estimated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH)
and b-carotene-linoleic acid model systems, respectively. It is observed that radical scavenging activity of B was highest followed by
BS and BMS. BM shows lowest activity. The antioxidant activity of black tea enhances and stabilizes with milk or sugar. The total polyphenol content is also determined and found to be higher in black tea sample compared to other samples. Both activities are compared
with that of butylated hydroxy anisole (BHA) as a standard antioxidant and found to be less in all cases.
! 2007 Elsevier Ltd. All rights reserved.
Keywords: Tea; Milk; Sugar; Polyphenols; Antioxidant activity; Radical scavenging activity

1. Introduction
Free radicals can have noxious effects on cells, and it is
believed that the damage caused by the free radicals is
reported in the etiology of several diseases. The radicals
are by products of various endogenous processes that can
be stimulated by external factors such as irradiation and
xenobiotics (Halliwell & Gutteridge, 1989). Antioxidants
protect organism against these radicals. It is also vital to
neutralise the destruction caused by the radicals with a sufficient supply of antioxidants.
Tea, the second most commonly consumed beverage in
the world and is rich source of flavonoids (Paquay et al.,
2000). Tea is produced predominantly in three forms i.e.,
green tea (without fermentation); oolong tea (partial fermentation) and black tea (fermented) and all are originating from Camellia Sinensis. Green tea, which is prepared
from the fresh tea leaf, contains mainly catechins, while
*

Corresponding author. Tel.: +91 821 2512352; fax: +91 821 2517233.
E-mail address: ljnatpro@yahoo.com (L. Jagan Mohan Rao).

0963-9969/$ - see front matter ! 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2007.10.009

black tea is composed of theaflavins and thearubigins as

major constituents, due to fermentation (i.e., an enzymatic
oxidation process) during manufacturing (Graham, 1992).
The main constituents in fresh tea leaves are (!)-epigallocatechingallate (EGCG), (!)-epigallocatechin (EGC),
(!)-epicatechingallate (ECG), (!)-epicatechin (EC), (+)gallocatechin (GC) and (+)-catechin (C). During the green
tea manufacturing process, some of the catechins undergo
isomerization at the C-2 position of flavan-3-ol; i.e., (!)
EGCG, (!)-EGC, (!)-ECG and (!)-EC get converted to
(+) GCG, (+) GC, (+)-CG and (+)-C, respectively.
Besides, tea contains three-xanthine alkaloids viz., caffeine
(major), theophylline and theobromine. These are also
important factors in determining the quality of green tea.
The way, the tea beverage is consumed varies among the
populations of different countries. In the United Kingdom,
Ireland, Canada and India, tea is consumed with a substantial amount of milk in it (Weisburger, 1997). But, in China
and Japan, tea is consumed without milk mostly. Milk contains various proteins, and interactions between polyphenols and proteins had been reported (Arts, Haenen, Voss,

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V. Sharma et al. / Food Research International 41 (2008) 124129

& Bast, 2001; Spencer et al., 1988). Tea extracts are powerful antioxidants being mainly due to the presence of the
flavonoids (Salah, Miller, Parganga, Tifburg, Bolwell &
Rice-Evanset, 1995; Zandi & Gordon, 1995). These compounds are believed to have physiological effects by acting
as free radical scavengers, which are generated by metabolic pathway within the body tissue (Zhao, Li, He, Cheng,
& Wenjuan, 1989) and also produced by environmental
pollution (Salah et al., 1995). Polyphenols present in
green tea as well as black tea possess antioxidant properties. Catechins, theaflavins and gallic acid contribute to
the antioxidant characteristics of tea (Vinson & Dabbargh,
1998).
Black tea is very often consumed with milk as mentioned above and it has been reported that there could be
possible non-covalent interactions between tea polyphenols
and polar milk proteins (Bartolome, Estrella, & Hernandez, 2000; Prigent et al., 2003), which may reduce/increase
the antioxidant potential of black tea polyphenols (Arts
et al., 2001).
However, other studies have shown milk to have no
effect on the antioxidant potential of black tea, in terms
of bioavailability and LDL oxidation (Holloman, Van
Het Hof, Tijburg, & Katan, 2000; Richelle, Tavazzi, & Offord, 2001; Van Het Hof, Kivits, Weststrate, & Tijburg
1998). Hence, it is still not clear with certainty the effects
of addition of milk/sugar/milk and sugar to tea composition on the antioxidant and free radical scavenging activities of black tea. An attempt was made in the present
study to find out these effects.
2. Materials and methods
2.1. Materials
Black tea (dust grade) was procured from the local market. FolinCiocalteus reagent, sodium bicarbonate, methanol, chloroform (Merck, India), linoleic acid, 1,1diphenyl-2-picrylhydrazyl radical (DPPH), b-carotene,
and butylated hydroxy anisole (Sigma, Bangalore, India),
tween-40 (Himedia, India) were obtained. The UV and visible absorbance of the samples were measured using Cintra
10 (GBC, Australia) UVVisible spectrophotometer.
2.2. Preparation of brews (extracts)
Four types brews were prepared viz., black tea (B),
black tea with sugar (BS), black tea with milk (BM), and
black tea with milk and sugar (BMS).
Black tea brew (B) was prepared by the addition of tea
powder (2 g) to boiling water (100 ml) and vigorous boiling
was continued for 2 min and tea brew (B) was filtered after
1 min, using filter paper.
Black tea with sugar brew (BS) was prepared by the
addition of tea powder (2 g) and sugar (4 g) to boiling
water (100 ml) and boiling was continued for 2 min. After
1 min the tea brew (BS) was filtered using filter paper.

Black tea with milk brew (BM) was prepared by the

addition of tea powder (2 g) and milk (40 ml) to boiling
water (60 ml) and boiling was continued for 2 min. Brew
(BM) was filtered after one more minute using filter paper.
Black tea with milk and sugar brew (BMS) was prepared
by the addition of tea powder (2 g), sugar (4 g) and milk
(40 ml) to boiling water (60 ml) and boiling was continued
for 2 min. After one more minute, tea brew (BMS) was filtered using filter paper.
All the four extracts (B, BS, BM, BMS) were then
freeze-dried and the yields of the extracts (tea brew powders) were presented in Table 1.
Freeze-dried tea brew powders (12.5, 98.58, 100.65,
194.97 mg of B, BS, BM and BMS, respectively) were dissolved in distilled methanol (25 ml) to prepare 500 ppm
solutions. Solutions of 200, 100, 50, 25 ppm were prepared
by serial dilution and the antioxidant activities were
studied.
2.3. Determination of total phenolic content
Samples were analysed for content of total phenolics
spectrophotometrically according to the FolinCiocalteu
method (Singleton & Rossi, 1965). Black tea extracts (B,
BS, BM, BMS) of appropriate amounts (100 mg,
788.64 mg, 805.20 mg and 1559.76 mg, respectively) were
introduced in test tubes and methanol: deionised water
(70:30) solution added, heated on a water bath maintained
at 70 "C for 10 min. The samples were cooled to room temperature and subjected to centrifugation. The supernatant
was mixed with saturated sodium carbonate (0.5 ml) and
FolinCiocalteus reagent (0.2 ml). The mixture was
diluted to 10 ml with deionised water; tubes were mixed
and incubated in dark for 60 min for the colour development. The absorbance of this solution measured at
765 nm. The total phenol content of each extractive was
estimated by comparison with a standard curve generated
from analysis of Gallic acid solutions and expressed as
mean (SD) mg of gallic acid equivalents per gram for
the extracts in triplicate.
2.4. Determination of radical scavenging activity using
DPPH model system
The 1,1-diphenyl-2-picryl hydrazyl radical (DPPH.)
used widely to evaluate the free radical scavenging ability
of various extracts (Jayaprakasha, Singh, & Sakaraih,
2001). The same method was adopted here to evaluate
Table 1
Yields of tea brew powders (B, BS, BM, BMS)
Sample

Yield (g)

Black
Black
Black
Black

0.54 0.02
4.26 0.20
4.35 0.25
8.42 0.55

tea
tea with sugar
tea with milk
tea with milk and sugar

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V. Sharma et al. / Food Research International 41 (2008) 124129

the black tea extracts (B, BS, BM, BMS). Black tea extracts
of different concentrations (25, 50, 100, 200 ppm) were prepared in different test tubes by dissolving in distilled methanol. The DPPH solution (0.1 mM) was freshly prepared
daily, stored in a flask covered with aluminum foil, and
kept in the dark at 4 oC between measurements. Four milliliter of a 0.1 mM methanol solution of DPPH was added
to these test tubes and shaken vigorously. The tubes were
then incubated in the dark at room temperature for
20 min. A DPPH blank sample was prepared without any
extract and methanol was used for the baseline correction.
Decrease in the absorbance at 517 nm was measured using
a UVVisible spectrophotometer. All experiments were
carried out in duplicate and repeated thrice. The percent
decrease in the absorbance was recorded for each concentration, and percent quenching of DPPH radical was calculated on the basis of the observed decrease in absorbance of
the radical. The radical scavenging activity was expressed
as the inhibition percentage and was calculated using the
following formula:
Radical scavenging activity %

control OD ! sample OD=control OD& ' 100

The radical scavenging activity of BHA was also measured

and compared with the various black tea extracts.
2.5. Antioxidant activity by b-carotene-linoleic acid assay
Black tea extracts (B, BS, BM, BMS) at different concentrations (25, 50, 100, 200 ppm) in distilled methanol
were incorporated into b-carotene-linoleic acid model system independently and the antioxidant activity was monitored spectrophotometrically at 470 nm (Farag, Badei,
Hewedi, & El-Baroty, 1989). The substrate suspension
was prepared by addition of b-carotene (4 mg dissolved
in 5 ml chloroform) into a covered round bottom flask containing tween 40 (600 mg) followed by the addition of linoleic acid (60 ll). The chloroform was removed
completely under vacuum using rota vapour at 40 oC.
The resulting solution was diluted with triple distilled water
(30 ml) and the emulsion was mixed well and further
diluted with oxygenated water (120 ml). The aliquots
(4 ml) of this reagent were transferred to different stoppered test tubes containing black tea extracts (1 ml) in
methanol at different concentrations. A control of methanol (1 ml) and emulsion (4 ml) was prepared. BHA solutions at 25, 50, 100 and 200 ppm were used as standard
for comparison. Zero adjustment was done using methanol. Absorbance of the samples was measured at a wavelength of 470 nm, immediately (t = 0), and subsequently
after every 30 min for 3 h (t = 180). The tubes were placed
in a water bath at 50 "C between the readings. Antioxidant
activity (AA) of the extract was evaluated in the terms of
photo oxidation of b-carotene using the following formula.
AA 1 ! A0 ! At =A00 At0 & ' 100

where
AA
A0
At
A00
At0

antioxidant activity
initial absorbence of the sample
absorbence of the sample after time t
initial absorbence of the control
absorbence of control after time t

3. Results and discussion

The brews were prepared as per the method commonly
used for the black tea beverage. The preparation of samples
for the activity studies was carried out in such a way that
the concentrations of various ingredients were similar to
the form in which black tea is normally consumed. The prepared brews were converted in to solid extracts for the
quantification as well as for the preservation for further
activity studies. Freeze drying was selected to obtain solid
extracts as this method was expected to cause minimum
changes in the properties of the material under study.
Activities of synthetic antioxidant were evaluations were
also carried out using BHA as an internal standard antioxidant BHA were determined under similar conditions at
different concentrations and activities of brew solids were
compared.
The polyphenol content in the four freeze dried tea samples (B, BS, BM, BMS) was determined. From Table 2 it
can be seen that the black tea extracts (B) possesses maximum amount of polyphenol (%) followed by (BS) and
(BMS). But the free total polyphenols were found to be
low in BM. This could be due to interaction between plant
phenolics and proteins either covalently or non-covalently.
Both ways may lead to precipitation of proteins, via either
multisite interactions (several phenolics bound to one protein molecule) or multidentate interactions (one phenolic
bound to several protein sites or protein molecules). This
could lead to the masking of polyphenols i.e., catechins
and the flavonoids on the milk proteins. (Arts et al.,
2002). The types of interaction depend on the molar ratio
of phenolic compounds to proteins (Haslam, 1989). The
total polyphenol content affected by the addition of milk
and sugar was shown in Fig. 1.
Radical scavenger reacts with DPPH, which is a stable
free radical and convert it to a,a-diphenyl-b-picryl hydrazine. The degree of discolouration indicates the scavenging
potentials of the extracts. Free radical scavenging potential
of tea extracts B, BS, BM, BMS at different concentrations
Table 2
Total polyphenol content in black tea extracts (B, BS, BM, BMS)
Sample particulars

% Polyphenol mg/g (gallic acid

equivalent)

Black tea
Black tea
Black tea
Black tea
(BMS)

24.1 1.57
19.1 1.08
15.0 1.17
17.1 1.70

(B)
with sugar (BS)
with milk (BM)
with milk and sugar

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V. Sharma et al. / Food Research International 41 (2008) 124129

Comparison of % RSA at different concentrations

Total % polyphenol affected by the addition of Milk and Sugar

30

120
100

20

80

15

% RSA

% polyphenol

25

10
5
0

60
40

BS

BM

Black tea extracts samples

BMS
20

Fig. 1. Total polyphenol content of tea extracts affected by addition of

milk and sugar B = black tea extract; BS = black tea with sugar;
BM = black tea with milk; BMS = black tea with milk and sugar.

0
5 PPM

10 PPM

20 PPM

40 PPM

Concentrations in PPM

(5, 10, 20, 40 ppm) were evaluated using DPPH and is presented in Table 3. The data were analysed as per the above
design and the activities were separated using Duncans
New Multiple Range Test (p < 0.05). Statistical design
was two-factorial experiments with replicates. The activities of the samples in the same column followed by different
letters (ad) differ significantly. This indicated that there
was significant difference in the activity at different concentrations. From the experiments performed it was observed
that the radical scavenging activities of all the samples
increased with concentration of extract (B, BS, BM,
BMS) and standard antioxidant (BHA).
The results indicated that the radical scavenging activity
of extracts (B, BS, BM, BMS) is lower than that of BHA at
lower concentrations (5, 10 ppm), whereas, at higher concentrations (20, 40 ppm) the differences were lesser. The
radical scavenging activity of all the samples increased with
concentration. The activity of black tea (B) was found to be
more followed by BS, and BMS. The radical scavenging
activity of BM was found to be lesser compare to all the
extracts in all the cases. This was because the addition of
the milk to the black tea may mask the radical scavenging
activity capacity so that the radical scavengers do not reach
their optimum scavenging capacity due to the presence of
proteins (Arts et al., 2002). The non-covalent interactions
between phenolic compounds and proteins suggested to
be created by hydrophobic association, which may subsequently be stabilized by hydrogen bonding (Haslam,
1989; Murray, Williamson, Lilley, & Haslam, 1994). The
percentage of radical scavenging activity at different concentrations is shown in Fig. 2.

Black tea

Black tea + Milk

Black tea + Sugar

Black tea + Sugar + Milk

BHA

Fig. 2. RSA of tea brew extracts (B, BS, BM, BMS) at different
concentrations.

Antioxidant activity of the different tea extracts (B, BS,

BM, BMS) was evaluated using b-carotene-linoleic acid
model system. b-Carotene in the model system undergoes
rapid discolouration in absence of an antioxidant. Bleaching of b-carotene was a free radical mediated phenomenon
resulting from hydroperoxides formed from linoleic acid.
As b-carotene molecules lose their double bonds by oxidation, the compound loses its chromophore and also the
characteristic orange colour, monitored spectrophotometrically at 470 nm. The presence of different extracts hinders
the extent of b-Carotene bleaching by neutralizing the linoleate free radical. The data (Table 4) were analysed statistically as described under radical scavenging activity. The
activities of the samples in the same column followed by
different letters (ad) differ significantly. This indicated that
there is significant difference in the activity at different concentrations. From the experiments performed it was
observed that the antioxidant activities of all the samples
increase with concentration of extract (B, BS, BM, BMS)
and standard antioxidant (BHA). This effect of concentration on antioxidant activity becomes more pronounced as
the time allowed for oxidation increases. The antioxidant
activities of the tea extracts were found to be lower than
that of BHA when compared at same concentrations (5,
10, 20 and 40 ppm). The antioxidant activities of BS, BM

Table 3
Radical scavenging activity* (%) of black tea extracts (B, BS, BM, BMS)
Concentrations (ppm)

BS

BM

BMS

BHA

5
10
20
40

11.4 1.18a
25.0 0.41b
55.2 0.91c
73.7 0.79d

14.1 0.53a
16.2 0.59b
53.8 1.13 c
58.9 0.99d

11.0 1.43a
24.0 0.67b
33.5 0.88c
45.0 0.86d

25.1 0.70a
26.0 1.25b
39.4 1.15c
50.2 0.88d

50.2 0.59a
69.6 0.79b
83.0 1.04c
94.8 0.88d

Different superscripts in the same column indicate that activities differ significantly.
*
DPPH radical assay.

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V. Sharma et al. / Food Research International 41 (2008) 124129

Table 4
Antioxidant activity* (%) of black tea extracts (B, BS, BM, BMS)
Concentrations (ppm)

BS
a

5
10
20
40

14.4 0.36
30.7 0.70b
38.6 0.15c
45.9 1.05d

BM
a

BMS
a

35.0 1.47
43.7 1.47b
55.7 1.14c
62.0 0.95d

40.1 1.09
47.3 1.01b
53.5 0.77c
59.6 1.13d

BHA
a

39.2 0.08
44.9 1.00b
55.8 0.29c
59.4 0.42d

70.0 1.51a
73.8 1.04b
76.1 0.65c
82.8 1.49d

Different superscripts in the same column indicate that activities differ significantly.
*
b-Carotene-linoleic acid assay.

Comparison of antioxidant activity at different concentrations

90

% Antioxidant activity

80
70
60
50
40
30
20
10
0
5 PPM

10 PPM

20 PPM

40 PPM

Concentrations in PPM
Black tea

Black tea + Milk

Black tea + Sugar

Black tea + Milk + Sugar

BHA

Fig. 3. Antioxidant activity of tea brew extracts (B, BS, BM, BMS) at
different concentrations.

and BMS, were more or less similar when compared at

same concentrations but those of B were lower in most
cases. Also the antioxidant activity of BHA remained stable on increase in time allowed for oxidation whereas that
of tea samples decreased as the time of oxidation increased.
This was particularly noticeable at lower concentrations (5
and 10 ppm). However, the antioxidant activity of the BS,
BM and BMS was fairly constant in the range of 5065% at
20 and 40 ppm concentrations. Hence, an addition of milk
or, sugar or, milk with sugar increased as well as stabilized
the antioxidant activity (Fig. 3).
The radical scavenging activity of the black tea (B) was
higher than that of BS, BM and BMS, while the different
effect was observed for antioxidant activity in the case of
b-carotene-linoleic acid model system. The reason could
be that the DPPH was long-lived nitrogen radical, which
bears no similarity to the highly reactive and transient peroxy radicals involved in b-carotene and linolate model.
Many antioxidants that react quickly with peroxyl radicals
might react slowly or may even be inert to DPPH (Huang,
Ou, & Prior, 2005). In the present study also, this could be
the cause for the difference between both activities.
4. Conclusions
The aim of the present study is to investigate the effect of
antioxidant activity of usual tea compositions with milk

and sugar. Four types of tea brew samples were prepared,

viz, plain black tea (B), black tea with sugar (BS), black tea
with sugar (BS) and black tea with milk and sugar (BMS)
by using freeze drying method. Freeze drying was chosen as
the method to obtain solid extracts with minimum changes
in the properties of the material during process. Loss of
solid was observed in respect of BS (6%), BM (8.6%),
and BMS (3.9%). This may due to retention of the brew
solids/extracts on the deposed tea waste during the filtration of brew.
The total polyphenol content was also determined and
found to be higher in black tea (B) sample compared to
various samples. It was also observed that in radical
scavenging (DPPH) assay the activity of black tea sample
(B) was found to possess the highest activity followed by
black tea with sugar, black tea with milk and sugar. The
addition of milk to black tea shows less activity. In antioxidant (b-carotene-linoleic acid) assay the addition of
milk or sugar increases and stabilizes the antioxidant
activity.
In conclusion, the effect of antioxidant activity of usual
tea compositions with milk and sugar were found to be
lower than that of BHA when compared at same concentrations (5, 10, 20, 40 ppm). The radical scavenging (DPPH
assay) activity of black tea (B) was found to be higher followed by BS, and BMS. The activity of BM was found to
be low compare to all the extracts in all the cases. However,
in the case antioxidant activity (b-carotene-linoleic acid)
assay, the activity of the BS, BM and BMS is fairly constant in the range of 5065% at 2040 ppm concentrations.
Hence, the addition of milk or, sugar or, milk with sugar
increases as well as stabilizes the antioxidant activity,
although radical scavenging activity decreases marginally.
The interactions of tea components with sugar and/or
milk, which altered the different activities on addition of
sugar, milk and milk and sugar, needed to be investigated
to understand the exact mechanisms.
Acknowledgements
The authors are thankful to Director, Central Food
Technological Research Institute, Mysore, Head, Plantation Products, Spices and Flavour Technology, and Head,
Human Resource Development Department, Central Food
Technological Research Institute, Mysore for giving the
opportunity to carry out this work.

V. Sharma et al. / Food Research International 41 (2008) 124129

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