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Original Paper

Ann Nutr Metab 2005;49:189195 DOI: 10.1159/000087071

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

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] . Abun-dant epidemiological evidence indicates that consump-tion of black tea is associated with a reduced risk of coro-nary heart disease [5, 6] and stroke [7] . Catechins, the main components of tea, have been shown to be respon-sible for the protective effect of tea in these epidemio-logical studies [8] . The suggested mechanism of the ben-e cial effect of tea on cardiovascular diseases involves the inhibition of lipoprotein oxidation in vivo by tea cate-chins [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 im-pact on antioxidant status in humans. Catechins, the chief antioxidant constituents of green tea, are converted to thea avin 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 thea a-vins of black tea possess similar antioxidant potency as catechins of green tea vis--vis inhibition of LDL oxida-tion in vitro [17] . However, their effects ex vivo and the

Key Words Antioxidant activity Antioxidant status Catechins Black tea Milk and oxidative damage

Abstract Tea is a polyphenol-rich beverage like wine and cate-chins are its chief polyphenols. Catechins have cardio-protective effects as they can scavenge free radicals and inhibit lipid peroxidation. Epidemiological studies indi-cate 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 ad-versely affect its benefi cial effects, but the data are not unequivocal. Therefore, we assessed the effect of the ad-dition of milk to black tea on its ability to modulate oxi-dative 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 benefi cial ef-fects of black tea on total plasma antioxidant activity, plasma resistance to oxidation induced ex vivo, and de-creased 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 dam-age in vivo.

Copyright 2005 S. Karger AG, Basel

Received: October 30, 2004 Accepted: January 11, 2005 Published online: July 13, 2005

Dr. Vijayakumar C. ReddyDivision of Endocrinology and Metabolism, National Institute of NutritionIndian Council of Medical ResearchJamai Osmania PO, Hyderabad 500 007 (India)Tel. +91 40 27008921/ext. 235, Fax +91 40 27019074, E-Mail challavkr@yahoo.com

2005 S. Karger AG, Basel02506807/05/04930189$22.00/0

Accessible online at:www.karger.com/anm

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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 bene cial effects in subjects consuming tea with milk [21] . They attributed this lack of effect to the consump-tion of tea with milk and the binding of tea catechins to milk proteins reported earlier by Hasalam [22] . Indeed Sera ni et al. [23] have shown that addition of milk de-creases 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 ef-fect 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 de-signed 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 ox-idative damage in human volunteers consuming a bolus of black tea with or without milk.

Methods and Materials

Chemicals and Reagents. Catechin, 4-(dimethylamino)cinnam-aldehyde, was purchased from Sigma Chemical Co. (St. Louis, Mo., USA). The black tea (Taj Mahal brand, India) powder was pur-chased 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 concen-tration of 20% v/v.

Nine, apparently healthy, adult male volunteers aged 2951 (mean 39.4) years gave their written, informed consent before par-ticipation 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

from consuming avonoid-rich foods (tea, fruits, wine and green leafy vegetables) and vitamin/mineral supplements for a week be-fore 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 sam-ples were collected from the subjects before consuming 350 ml of black tea (with sugar) as a bolus within a 3-min period. Subsequent-ly, 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 centri-fuged 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 modi cations. Brie y, 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 ex-tract 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 spectrophoto-

metrically 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)cinnam-aldehyde. The colored complex was measured spectrophotometri-cally 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 blue-colored ferrous tripyridyl triazine by the electron-donating anti-oxidants in plasma. The absorbance at 593 nm was monitored 4 min after the addition of plasma to the FRAP reagent. The stan-dard curve for the FRAP assay was prepared using ascorbic acid as standard.

Table 1. Anthropometric data and blood pressure of human volunteers

Parameters Mean 8 SE(n = 9)

Age, years 39.482.58Weight, kg 70.982.59Height, cm 16881.75Body mass index 25.280.93Systolic blood pressure 11583.10Diastolic blood pressure 79.082.16

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Antioxidant Activity of Tea with Milk Ann Nutr Metab 2005;49:189195 191

Resistance to Oxidation. Plasma samples were subjected to per-oxidation ex vivo by incubation in the presence of copper (CuSo 4 ) at a nal concentration of 5 m M , 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 mea-sured using appropriate kits purchased from Dr. Reddys Labora-tories, Hyderabad, India, according to manufacturers instruc-tions.

Urinary TBARS. Urinary TBARS were determined by the spec-trophotometirc method reported previously by Ohkawa et al. [29] .

Statistical Analysis Data were analyzed statistically using the SPSS package (ver-

sion 1.0) and are presented as mean 8 SE. Signi cance of the dif-ferences in a given parameter on consumption of tea with or with-out 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 sig-ni cance) followed by the post hoc least signi cant difference test.

Results

There was no signi cant difference in the catechin con-tent 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 af-ter consumption of black tea without and with milk is given in table 2 . Plasma catechin levels increased as ear-ly 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 af-ter consumption. Further, the peak value of catechin as well as the area under the curve for plasma catechin were signi cantly higher in subjects consuming black tea than those consuming tea with milk. 60 min after tea consump-tion plasma catechin levels were signi cantly 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 maxi-mum 60 min after consumption of black tea and pla-teaued 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 signi cantly different between the two treatments at 60 and 120 min (p ! 0.05), the values were comparable 180 min after tea consump-tion.

The relationship if any between the plasma catechin levels and the FRAP activity was assessed by Pearson correlation analysis. There was a signi cant (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 signi cant correlation (r = 0.224). However, when both the treat-ment groups were considered together, there was a sig-ni cant (p ! 0.001) correlation between the two param-eters (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 de-creased as early as 60 min and the values decreased fur-ther 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 con-sumption of tea without or with milk ( table 3 ). There were no signi cant differences between the 2 treatment groups in both these parameters at any time point tested. Com-pared to the basal value, the TBARS levels were signi -cantly lower only at 120 and 180 min after the consump-tion of tea with or without milk.

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

Timemin

Tea withoutmilk, mol/l

Tea withmilk, mol/l

0 0.0580.003 0.0580.00230 0.0880.002*** 0.0380.00160 0.3180.017** 0.3880.01590 0.4080.017 0.4180.011

120 0.6780.013*** 0.4280.013180 0.4680.014* 0.4080.018AUC 1.1480.07* 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).

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Resistance of plasma to oxidation induced ex vivo in-creased signi cantly (p ! 0.05) by 60 min after consump-tion of tea without milk as evident from the signi cantly lower levels of TBARS formed ( table 4 ) and the values did not change any further. Addition of milk had no ad-verse effect on this parameter and there were no differ-ences between the 2 treatment groups at any time point.

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.

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).

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

Timemin

Teawithout milk

Teawith milk

0 1.3980.20a 1.7880.22a

60 1.1980.18a 1.3880.20a

120 0.9280.22b 0.8180.25b

180 0.7280.21b 0.6980.16b

Values are mean 8 SE (n = 9).Values in a column with different super-

scripts are signi cantly different (p < 0.05) by one-way ANOVA.

No signi cant 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 sub-jects after ingestion of black tea without/with milk

Time Tea withoutmilk, mol/l

Tea withmilk, mol/l

0 7.7380.61a 7.4980.42a

60 5.2780.62b 5.3880.54b

120 5.1880.36b 5.0280.48b

180 5.0380.38b 5.1380.52b

Values are mean 8 SE (n = 9).Values in a column with different super-

scripts are signi cantly different (p < 0.05) by one-way ANOVA.

No signi cant differences at any time point between tea consumed without or with milk.

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 5.8080.34120 min 5.7880.49 6.2780.39180 min 5.3680.36 6.2980.39

Plasma triglycerides (mmol/l)0 min 1.7580.13 1.4580.12

60 min 1.7280.14 1.6280.15120 min 1.9880.16 1.6280.13180 min 1.8380.18 1.6380.14

Plasma uric acid (mmol/l)0 min 0.2580.03 0.2680.02

60 min 0.2180.02 0.2680.03120 min 0.2580.03 0.2580.02180 min 0.2280.02 0.2680.02

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.

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Discussion

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 sub-jects compared to those consuming black tea [23] . How-ever, data in this regard is scanty and equivocal. Wheth-er or not populations which habitually consume tea with milk, as is common in India, are deprived of its bene cial 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 cat-echins, respectively) signi cantly increased the plasma catechins and the increase was comparable to the levels reported by van het Hof et al. [24] in volunteers who con-sumed tea with milk. However, the nding that the peak level as well as the area under the curve of plasma cate-chin were signi cantly 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 sig-ni cantly 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 ab-sorption of tea catechins and this could be due to the binding of tea catechins to milk proteins as reported ear-lier 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 sin-gle 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 ac-tivity of plasma by its FRAP and resistance to copper-in-duced 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 there-after. 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 in-dicates that addition of milk may not adversely affect the

antioxidant potential of black tea in humans. Taken to-gether with some earlier studies [25, 37] our results indi-cate 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 in-creased plasma catechin levels and their ability to recycle -tocopherol in vivo [38] . In line with this, we observed a signi cant correlation between plasma catechin levels and the increase in plasma FRAP activity. But our nd-ing 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 bio-availability did not affect the antioxidant potential of black tea as assessed by the plasma FRAP activity. That a signi cant correlation (although of lower magnitude) was observed between the two parameters when both the treatment groups were considered together may suggest a de nitive role of plasma catechins in the FRAP activi-ty.

In addition to the comparable increase in the plasma FRAP activity, we also observed that the capacity of plas-ma to resist oxidation induced ex vivo was enhanced sig-ni cantly 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 plas-ma 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. Inter-estingly, 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 af-fect 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 bene t. This dis-crepancy 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-

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oxidant effects of tea catechins appear to be speci c for the species of the free radicals involved. On the other hand, as reported by McAnlis et al. [18] , the lack of pro-tection 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 anti-oxidant capacity was re ected in the reduced oxidative stress of the subjects on the consumption of tea with or without milk. As expected, there was a signi cant time-dependant 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 con-sumption of green tea, fruit juice and carrot, respectively [4345] . The bene cial effects of black tea in reducing cardiovascular disease risk have been attributed to its cat-echins, 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 (lip-id 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 Sera ni et al. [23] who showed that addition of milk inhibited the ben-e cial antioxidant effects of black tea.

In the present study, the levels of plasma uric acid, another known antioxidant, were not changed signi cant-ly 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 bene cial effects of tea may be depen-dent on the amounts of catechin consumed/bioavail-able.

Many epidemiological studies have reported an asso-ciation 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 ad-versely affect the antioxidant capacity of black tea or its ability to prevent in vivo oxidation by improving the an-tioxidant 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. Si-vakumar, 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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