Original Paper

Ann Nutr Metab 2005;49:189195

DOI: 10.1159/000087071

Received: October 30, 2004

Accepted: January 11, 2005
Published online: July 13, 2005

Addition of Milk Does Not Alter the

Antioxidant Activity of Black Tea
Vijayakumar C. Reddy G.V. Vidya Sagar D. Sreeramulu L. Venu
M. Raghunath
Division of Endocrinology and Metabolism, National Institute of Nutrition, Indian Council of Medical Research,
Hyderabad, India

Abstract
Tea is a polyphenol-rich beverage like wine and catechins are its chief polyphenols. Catechins have cardioprotective effects as they can scavenge free radicals and
inhibit lipid peroxidation. Epidemiological studies indicate an inverse relation between tea consumption and
the risk of cardiovascular and other chronic diseases.
Addition of milk to black tea has been reported to adversely affect its beneficial effects, but the data are not
unequivocal. Therefore, we assessed the effect of the addition of milk to black tea on its ability to modulate oxidative stress and antioxidant status in adult male human
volunteers. Although the area under the curve of plasma
catechins was lower on the consumption of tea with milk
compared to black tea, it did not affect the beneficial effects of black tea on total plasma antioxidant activity,
plasma resistance to oxidation induced ex vivo, and decreased plasma and urinary thiobarbituric acid reactive
substance levels. The results suggest that addition of
milk may not obviate the ability of black tea to modulate
the antioxidant status of subjects and that consumption
of black tea with/without milk prevents oxidative damage in vivo.
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Introduction

Oxidative stress is important in the development of

chronic degenerative diseases [1], and their pathologies
have long been associated with free radical damage [2].
Diet-derived antioxidants could therefore be important
in the protection against chronic diseases [3, 4]. Abundant epidemiological evidence indicates that consumption of black tea is associated with a reduced risk of coronary heart disease [5, 6] and stroke [7]. Catechins, the
main components of tea, have been shown to be responsible for the protective effect of tea in these epidemiological studies [8]. The suggested mechanism of the benecial effect of tea on cardiovascular diseases involves the
inhibition of lipoprotein oxidation in vivo by tea catechins [9] due to their ability to scavenge reactive oxygen
species and chelate metal ions [10, 11].
Tea, a rich source of polyphenols, accounts for over
half the dietary antioxidant intake, with a potential impact on antioxidant status in humans. Catechins, the
chief antioxidant constituents of green tea, are converted
to theaavin and thearubigin during its processing to
black tea [12]. In vitro studies have demonstrated that
green/back tea possesses considerable radical scavenging
potential [10, 13, 14], inhibits oxidation of low-density
lipoproteins (LDLs) [15] and plasma [16]. Indeed theaavins of black tea possess similar antioxidant potency as
catechins of green tea vis--vis inhibition of LDL oxidation in vitro [17]. However, their effects ex vivo and the

Dr. Vijayakumar C. Reddy

Division of Endocrinology and Metabolism, National Institute of Nutrition
Indian Council of Medical Research
Jamai Osmania PO, Hyderabad 500 007 (India)
Tel. +91 40 27008921/ext. 235, Fax +91 40 27019074, E-Mail challavkr@yahoo.com

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Key Words
Antioxidant activity
Antioxidant status
Catechins

Black tea
Milk and oxidative damage

Parameters

Mean 8 SE
(n = 9)

Age, years
Weight, kg
Height, cm
Body mass index
Systolic blood pressure
Diastolic blood pressure

39.482.58
70.982.59
16881.75
25.280.93
11583.10
79.082.16

results of the long-term human intervention studies are

equivocal at best [1820]. For example, Hertog et al. [5]
reported the reduced risk of coronary heart disease in
subjects consuming black tea, but did not observe such
benecial effects in subjects consuming tea with milk
[21]. They attributed this lack of effect to the consumption of tea with milk and the binding of tea catechins to
milk proteins reported earlier by Hasalam [22]. Indeed
Serani et al. [23] have shown that addition of milk decreases the antioxidant potential of black tea in humans.
However, subsequent studies did not show that addition
of milk impaired the bioavailability of black tea catechins
[24] or the plasma antioxidant activity [25]. Thus the effect of black tea consumption with or without milk on the
antioxidant status/oxidative damage in vivo remains to
be established unequivocally. The present study was designed to evaluate the effect of the addition of pre-boiled
milk to black tea on the bioavailability of tea catechins
and acute changes in plasma antioxidant activity and oxidative damage in human volunteers consuming a bolus
of black tea with or without milk.

Methods and Materials

Chemicals and Reagents. Catechin, 4-(dimethylamino)cinnamaldehyde, was purchased from Sigma Chemical Co. (St. Louis, Mo.,
USA). The black tea (Taj Mahal brand, India) powder was purchased from Brooke-Bond, India. All other chemicals used were of
analytical grade and procured locally. Single-toned, pasteurized
milk (4% total solids,1.75% protein and 3.5% fat), procured from
a local source, was boiled and added to black tea to a nal concentration of 20% v/v.
Nine, apparently healthy, adult male volunteers aged 2951
(mean 39.4) years gave their written, informed consent before participation in the study. Their anthropometric characteristics and
blood pressure were monitored and are presented in table 1. The
subjects were on their routine home diets and were asked to refrain

190

Ann Nutr Metab 2005;49:189195

from consuming avonoid-rich foods (tea, fruits, wine and green

leafy vegetables) and vitamin/mineral supplements for a week before the start of the study. The study protocol was approved by the
ethics committee of the National Institute of Nutrition, Hyderabad,
India.
Study Design
Black tea was prepared by boiling 7 g of black tea leaf powder
in 350 ml of tap water for 3 min and ltered. It was sweetened with
sugar and the volume was made up to 350 ml with boiled water.
Tea with milk was prepared as above excepting that 7 g of black tea
leaf powder was boiled for 3 min in 280 ml of water, ltered and
volume made up to 280 ml with boiled water. To this sugar and
70 ml of pre-boiled milk (4% total solids, 1.75% protein and 3.5%
fat) were added.
After an overnight fast, peripheral venous blood and urine samples were collected from the subjects before consuming 350 ml of
black tea (with sugar) as a bolus within a 3-min period. Subsequently, blood samples were collected 30, 60, 90, 120 and 180 min after
tea consumption, while urine samples were collected at 60, 120 and
180 min. The experiment was repeated on the same subjects with
350 ml of black tea with milk prepared as mentioned above, but
after a washout period of 7 days.
Sample Collection and Storage
Peripheral venous blood samples were collected in heparinized
tubes and placed immediately on ice for 30 min. They were centrifuged for 20 min at 1,500 g and the plasma was separated and stored
at 80 C until analysis. Urine samples were stored under toluene
in polythene bottles at 20 C.
Catechins in Tea
Catechins in tea were determined according to Kivits et al. [26]
but with slight modications. Briey, 100
l of tea (prepared for
subjects) was diluted to 1 ml with distilled water and catechins were
extracted twice with 2 ml of ethyl acetate each time. The pooled
ethyl acetate extract was evaporated to dryness under nitrogen and
the residue re-dissolved in 1 ml of methanol. This methanolic extract was processed as described by Kivits et al. [26] and read
against a standard curve prepared using pure catechin solution
(added to aluminum oxide and processed similar to the samples).
Parameters in Plasma
Catechins. Plasma catechins were determined spectrophotometrically according to Kivits et al. [26]. In brief, catechins were
extracted from plasma by solid-phase extraction using aluminum
oxide, followed by complexation with 4-(dimethylamino)cinnamaldehyde. The colored complex was measured spectrophotometrically at 637 nm. The plasma concentration of total catechins was
read from the standard curve prepared from human control plasma
spiked with known concentrations of catechin.
Total Antioxidant Activity. Total plasma antioxidant capacity
was measured using the ferric-reducing ability of plasma (FRAP)
assay [27]. The assay measures the change in absorbance at 593 nm
due to the reduction of ferric tripyridyl triazine complex to the bluecolored ferrous tripyridyl triazine by the electron-donating antioxidants in plasma. The absorbance at 593 nm was monitored
4 min after the addition of plasma to the FRAP reagent. The standard curve for the FRAP assay was prepared using ascorbic acid as
standard.

Reddy/Vidya Sagar/Sreeramulu/Venu/
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Table 1. Anthropometric data and blood

pressure of human volunteers

Statistical Analysis
Data were analyzed statistically using the SPSS package (version 1.0) and are presented as mean 8 SE. Signicance of the differences in a given parameter on consumption of tea with or without milk was analyzed by Students t test at different time points.
The time course of differences in the values of a given parameter
were analyzed by one-way ANOVA (SPSS was set at p ! 0.05 signicance) followed by the post hoc least signicant difference
test.

Table 2. Plasma total catechin levels after

consumption of a bolus of 350 ml black tea
without/with milk (at 20%) at different time
points

Time
min

Tea without
milk,
mol/l

Tea with
milk,
mol/l

0
30
60
90
120
180
AUC

0.0580.003
0.0880.002***
0.3180.017**
0.4080.017
0.6780.013***
0.4680.014*
1.1480.07*

0.0580.002
0.0380.001
0.3880.015
0.4180.011
0.4280.013
0.4080.018
0.9580.04

Values given are mean 8 SE (n = 9).

AUC = Area under the curve.
* p < 0.05, ** p < 0.01 and *** p < 0.001
vs. tea with milk (by Students t test).

There was no signicant difference in the catechin content of black tea consumed by the subjects without and
with milk (203 and 196 mg, respectively). The time course
of changes in plasma catechin levels in the volunteers after consumption of black tea without and with milk is
given in table 2. Plasma catechin levels increased as early as 60 min after consumption of black tea with milk and
plateaued thereafter, whereas on consumption of black
tea without milk the peak value was reached 120 min after consumption. Further, the peak value of catechin as
well as the area under the curve for plasma catechin were
signicantly higher in subjects consuming black tea than
those consuming tea with milk. 60 min after tea consumption plasma catechin levels were signicantly higher in
subjects consuming black tea with milk, whereas at 120
and 180 min the values were higher on consumption of
black tea without milk.
The plasma antioxidant capacity, as determined by the
change in FRAP, increased with time after consumption
of black tea (g. 1). The FRAP values reached a maximum 60 min after consumption of black tea and plateaued thereafter. On the other hand, on consumption of

tea with milk the maximum level of FRAP was reached

at 3 h. While the mean difference in plasma FRAP levels
(compared to basal values) were signicantly different
between the two treatments at 60 and 120 min (p ! 0.05),
the values were comparable 180 min after tea consumption.
The relationship if any between the plasma catechin
levels and the FRAP activity was assessed by Pearson
correlation analysis. There was a signicant (p ! 0.001)
correlation between the two parameters in the subjects on
the consumption of tea without milk (r = 0.552), whereas
on consumption of tea with milk there was no signicant
correlation (r = 0.224). However, when both the treatment groups were considered together, there was a signicant (p ! 0.001) correlation between the two parameters (r = 0.377) albeit of lower magnitude.
The change in plasma lipid peroxidation (TBARS)
with time is shown in gure 2. Regardless of whether tea
was consumed with or without milk, plasma TBARS decreased as early as 60 min and the values decreased further with time until 180 min after the consumption of tea.
Similarly the urinary TBARS also decreased with time
and the decrease was steady until 180 min after the consumption of tea without or with milk (table 3). There were
no signicant differences between the 2 treatment groups
in both these parameters at any time point tested. Compared to the basal value, the TBARS levels were signicantly lower only at 120 and 180 min after the consumption of tea with or without milk.

Antioxidant Activity of Tea with Milk

Ann Nutr Metab 2005;49:189195

Results

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Resistance to Oxidation. Plasma samples were subjected to peroxidation ex vivo by incubation in the presence of copper (CuSo4)
at a nal concentration of 5 mM, according to Simon and Mao [28].
The plasma lipid peroxidation was evaluated as thiobarbituric acid
reactive substances (TBARS) according to the method of Ohkawa
et al. [29]. The amount of TBARS in the sample was computed
from a standard curve prepared using malondialdehyde (obtained
by the hydrolysis of 1,1,3,3-tetraethoxypropane) as a standard. The
resistance of plasma to lipid peroxidation is inversely related to the
difference in TBARS levels in plasma incubated with and without
copper.
Plasma total cholesterol, triglycerides and uric acid were measured using appropriate kits purchased from Dr. Reddys Laboratories, Hyderabad, India, according to manufacturers instructions.
Urinary TBARS. Urinary TBARS were determined by the spectrophotometirc method reported previously by Ohkawa et al.
[29].

Table 3. Urinary TBARS (
mol/l) of subjects after ingestion of black tea without/
with milk (at 20%)

Time
min

Tea
without milk

Tea
with milk

0
60
120
180

1.3980.20a
1.1980.18a
0.9280.22b
0.7280.21b

1.7880.22a
1.3880.20a
0.8180.25b
0.6980.16b

Values are mean 8 SE (n = 9).

Values in a column with different superscripts are signicantly different (p < 0.05)
by one-way ANOVA.
No signicant differences at any time
point between tea consumed without or
with milk.

Table 4. Plasma resistance to oxidation in-

duced ex vivo with copper (TBARS) of subjects after ingestion of black tea without/
with milk
Time

Tea without
milk,
mol/l

Tea with
milk,
mol/l

0
60
120
180

7.7380.61a
5.2780.62b
5.1880.36b
5.0380.38b

7.4980.42a
5.3880.54b
5.0280.48b
5.1380.52b

Values are mean 8 SE (n = 9).

Values in a column with different superscripts are signicantly different (p < 0.05)
by one-way ANOVA.
No signicant differences at any time
point between tea consumed without or
with milk.

Resistance of plasma to oxidation induced ex vivo increased signicantly (p ! 0.05) by 60 min after consumption of tea without milk as evident from the signicantly
lower levels of TBARS formed (table 4) and the values
did not change any further. Addition of milk had no adverse effect on this parameter and there were no differences between the 2 treatment groups at any time point.

192

Fig. 2. Change in plasma lipid peroxidation over 180 min after

consumption of a single dose (350 ml) of black tea with or without
milk. Values are mean 8 SE (n = 9).

Ann Nutr Metab 2005;49:189195

Table 5. Plasma cholesterol, triglycerides

and uric acid after consumption of black tea
without/with milk at different time points
(values given are mean 8 SE)

Parameter

Without milk

With milk

Plasma total cholesterol (mmol/l)

0 min
5.8080.39
6.2480.41
60 min
5.3480.28
5.8080.34
120 min
5.7880.49
6.2780.39
180 min
5.3680.36
6.2980.39
Plasma triglycerides (mmol/l)
0 min
1.7580.13
60 min
1.7280.14
120 min
1.9880.16
180 min
1.8380.18

1.4580.12
1.6280.15
1.6280.13
1.6380.14

Plasma uric acid (mmol/l)

0 min
0.2580.03
60 min
0.2180.02
120 min
0.2580.03
180 min
0.2280.02

0.2680.02
0.2680.03
0.2580.02
0.2680.02

Uric acid levels in plasma did not change with time on

consumption of tea with or without milk (table 5). Also,
total cholesterol and triglyceride levels in plasma were
unchanged with time. Indeed, these parameters were
comparable in the subjects consuming black tea either
with or without milk (table 5) at all the time points
tested.

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Fig. 1. Change in plasma FRAP activity (expressed as
mol/l) over

180 min after consumption of a single dose (350 ml) of black tea
with/without milk. Values are mean 8 SE (n = 9). * p ! 0.05: black
tea with milk vs. black tea without milk.

Antioxidant Activity of Tea with Milk

Ann Nutr Metab 2005;49:189195

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Several in vitro studies have shown tea catechins to be

powerful antioxidants and inhibit peroxidation [30, 31].
However, most studies on the long-term ingestion of
green or black tea have produced varying results on the
inhibition of lipid peroxidation ex vivo or in vivo [16, 18,
19, 32]. Addition of milk to black tea has been reported
to adversely affect the plasma antioxidant activity of subjects compared to those consuming black tea [23]. However, data in this regard is scanty and equivocal. Whether or not populations which habitually consume tea with
milk, as is common in India, are deprived of its benecial
effects remains to be established.
In the present study, consumption of a single dose of
black tea without or with milk (203 and 196 mg total catechins, respectively) signicantly increased the plasma
catechins and the increase was comparable to the levels
reported by van het Hof et al. [24] in volunteers who consumed tea with milk. However, the nding that the peak
level as well as the area under the curve of plasma catechin were signicantly lower on consumption of tea with
milk appear to be in line with the reported adverse effects
of milk on the antioxidant effects of black tea in humans
[23]. That plasma catechin levels in the subjects were signicantly lower on the consumption of tea with milk than
black tea at all time points (except 60 min) appears to
suggest that addition of milk may adversely effect the absorption of tea catechins and this could be due to the
binding of tea catechins to milk proteins as reported earlier by Brown and Wright [33] and Siebert et al. [34].
Kimura et al. [35] did not observe any increase in total
plasma antioxidant activity in subjects consuming a single dose of tea polyphenol extract, while an increase in
FRAP has been reported on the consumption of wine
[36]. Therefore, we next assessed whether the decreased
bioavailability of catechins on the consumption of tea
with milk had any adverse effects on the antioxidant activity of plasma by its FRAP and resistance to copper-induced oxidation ex vivo.
As expected the plasma total antioxidant capacity
(FRAP) increased after ingestion of black tea without
milk, reaching a peak at 60 min and plateauing off thereafter. That addition of milk delaying the peak activity of
FRAP to 180 min is in line with the delayed increase in
FRAP on consumption of black tea with milk reported
earlier by Langley-Evans [37]. Not withstanding this, the
observation that FRAP activity was comparable at 180
min after consumption of tea with and without milk indicates that addition of milk may not adversely affect the

antioxidant potential of black tea in humans. Taken together with some earlier studies [25, 37] our results indicate that the addition of milk had no adverse effect on the
improvement in the antioxidant status of subjects brought
out by black tea.
The increased FRAP activity could be due to the increased plasma catechin levels and their ability to recycle
-tocopherol in vivo [38]. In line with this, we observed
a signicant correlation between plasma catechin levels
and the increase in plasma FRAP activity. But our nding that such a correlation was not seen in the subjects on
the consumption of tea with milk probably suggests that
the constituent of milk which is affected catechin bioavailability did not affect the antioxidant potential of
black tea as assessed by the plasma FRAP activity. That
a signicant correlation (although of lower magnitude)
was observed between the two parameters when both the
treatment groups were considered together may suggest a
denitive role of plasma catechins in the FRAP activity.
In addition to the comparable increase in the plasma
FRAP activity, we also observed that the capacity of plasma to resist oxidation induced ex vivo was enhanced signicantly in subjects on the consumption of black tea.
Acting as scavengers of free radicals and metal chelators
[39], tea catechins are known to inhibit oxidation of plasma lipids or LDLs. As such the increased resistance to
plasma oxidation ex vivo observed here could be due to
increased plasma catechins on consumption of tea. Interestingly, addition of milk did not adversely affect this
activity, and this could again be due to the fact that the
milk constituent which affected catechin absorption had
no effect on this parameter. Our results thus appear to
indicate that the addition of milk may not adversely affect the ability of black tea to improve the antioxidant
status of subjects.
Nakagawa et al. [40] and Hodgson et al. [41] reported
an increased resistance against serum lipid peroxidation
induced ex vivo in their acute study, while controlled
long-term intervention studies by Hodgson et al. [19] and
van het Hof et al. [42] showed no such benet. This discrepancy could be due to the fact that blood was collected
after an overnight fast in the later study and the known
half life of catechins in human blood is 6.9 h. Cherubini
et al. [32] did not observe protection of plasma against
lipid peroxidation on black tea consumption, probably
because peroxidation was induced by radical generator
2,2-azobis-(2-amidinopropane)hydrochloride, whereas
Nakagawa et al. [40] observed protection when copper
was used to induce oxidation. Thus the protective, anti-

Discussion

oxidant effects of tea catechins appear to be specic for

the species of the free radicals involved. On the other
hand, as reported by McAnlis et al. [18], the lack of protection against LDL oxidation after consumption of black
tea could be due to the very low levels of tea avonoids
(50 mg) ingested.
We next assessed whether this increased plasma antioxidant capacity was reected in the reduced oxidative
stress of the subjects on the consumption of tea with or
without milk. As expected, there was a signicant timedependant decrease both in the plasma and urinary
TBARS on the consumption of black tea, indicating a
decrease in the oxidative stress of subjects consuming tea
without milk. A similar decrease in plasma and urinary
lipid peroxidation has been reported earlier after the consumption of green tea, fruit juice and carrot, respectively
[4345]. The benecial effects of black tea in reducing
cardiovascular disease risk have been attributed to its catechins, which inhibit oxidation of plasma lipids in vivo
by acting as chain-breaking antioxidants and as metal
chelators [4547].
Not withstanding that milk appeared to affect catechin
absorption, it was interesting that addition of milk had
no adverse effect on the reduction of oxidative stress (lipid peroxidation/TBARS in plasma and urine) by black
tea. These observations are in agreement with those of
Miura et al. [48] but differ from those of Serani et al.
[23] who showed that addition of milk inhibited the benecial antioxidant effects of black tea.
In the present study, the levels of plasma uric acid,
another known antioxidant, were not changed signicantly on consumption of tea with/without milk and these

results are consistent with those of van het Hof et al. [42].
The plasma uric acid levels appeared to be slightly higher
after consumption of tea with milk than black tea and this
could be due to the contribution of uric acid present in
milk [49]. Taken together with the available literature, it
appears that the benecial effects of tea may be dependent on the amounts of catechin consumed/bioavailable.
Many epidemiological studies have reported an association between tea consumption and the reduction of
serum lipids and cholesterol [5052]. However, there was
no change in plasma cholesterol and triglycerides in the
present study and this lack of effect could be due to the
acute nature of this study. Nevertheless, our results are
consistent with those of Nakagawa et al. [40] and van het
Hof et al. [42].
It is thus evident from the present studies that black
tea has potent antioxidant properties in vivo. It is indeed
of interest that addition of milk to black tea may not adversely affect the antioxidant capacity of black tea or its
ability to prevent in vivo oxidation by improving the antioxidant status of subjects, although it may affect the
absorption/bioavailability of tea catechins.

Acknowledgements
The authors acknowledge the participants for their enthusiastic
cooperation and interest in the study. We are grateful to Dr. B. Sivakumar, Director, NIN, and Dr. Kamala Krishnaswamy, former
Director NIN, for their keen interest in the study. We acknowledge
the technical assistance of Mrs. Anitha Chauhan.

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