European Journal of Clinical Nutrition (2000) 54, 527±529
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In vivo antioxidant effect of green tea
H Sung1, J Nah1, S Chun1 H Park2, SE Yang1 and WK Min1*
1
Department of Clinical Pathology, Asan Medical Center and University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu,
Seoul 138-736, Korea; and 2Department of Clinical Pathology, Kangbuk Samsung Hospital and Sungkyunkwan University School of
Medicine, 108 Pyung-dong, Jongro-ku, Seoul 110-102, Korea
Objective: The object of this study was to investigate the in vivo antioxidant effect of green tea and dosage effect
of green tea on antioxidant effect.
Design: We tested 10 healthy subjects (aged 23 ± 25 y, ®ve women and ®ve men) with overnight fasting. The
total antioxidant capacity of plasma was measured at baseline and 60 min and 120 min after ingestion of 150 ml
green tea. Green tea was prepared by infusing 2.5 g of dried green tea leaves for 2 min at 80 C in 150 ml of water.
In the second week, they took 300 ml of tea (5.0 g of green tea leaves) and, in the third week, 450 ml of tea (7.5 g
of green tea leaves). The total antioxidant capacities of plasma were determined with a Total Antioxidant Kit
(Randox Laboratories Ltd, UK) using a Cobas Mira analyser (Roche Diagnostic Systems Inc., Switzerland). The
mean intra-assay coef®cient of variation was 1.2%.
Results: The total antioxidant capacity of plasma increased by 1.1% at 60 min and 2.1% at 120 min over baseline
value in subjects consuming 150 ml of green tea, which was statistically not signi®cant. However, total
antioxidant capacity of plasma after consuming 300 ml of green tea showed a signi®cant increase of 7.0%
after 60 min and 6.2% after 120 min (P < 0.0001), and after consuming 450 ml 12.0% after 60 min and 12.7%
after 120 min over baseline value (P < 0.0001).
Conclusions: Total antioxidant capacity of plasma was signi®cantly increased after taking green tea in amounts
of 300 and 450 ml. A positive increment according to green tea dosage was also observed.
Sponsorship: This work was funded by the Paci®c Corporation (Korea).
Descriptors: antioxidant effect; green tea; polyphenol; dosage
European Journal of Clinical Nutrition (2000) 54, 527±529
Introduction
Oxidative damage by oxygen free radicals are known to
be one of the mechanisms of chronic disorders such as
atherosclerosis or cancer (Halliwell & Gutteridge, 1989).
Oxygen free radicals are created through aerobic metabolism and are mostly removed by antioxidants in vivo
(Diplock, 1994). Three groups of antioxidants in vivo
prevent oxidative damage. Superoxide dismutase, glutathione peroxidase and metal-binding proteins are primary
antioxidants that prevent the formation of new free radicals.
Secondary antioxidants such as vitamins C and E, beta
carotene, uric acid, bilirubin and albumin prevent the
cascade of oxidative reactions by combining with free
radicals. Tertiary antioxidants such as DNA repair
enzyme and methionine sulphoxide reductase repair the
oxidative damage.
There are several reports that argue that long-term intake
of vitamin supplements of vitamins C and E or beta
carotene prevent chronic disorders caused by oxidative
damage (Barber & Harris, 1994; Gaziano et al, 1992;
*Correspondence: Dr WK Min, Department of Clinical Pathology, Asan
Medical Center and University of Ulsan College of Medicine, 388-1
Pungnap-dong, Songpa-gu, Seoul 138-736, Korea.
E-mail: [email protected]
Guarantor: WK Min.
Contributors: WKM was the lead investigator and the person mainly
responsible for all stages of the study. HS carried out the study and wrote
the paper. JN and SC supervised all aspects of the study. HP and SEY
assisted with the experimental design and the statisitical analysis of data.
All investigators were involved in preperation and subsequent revision of
the paper.
Received 24 August 1999; revised 26 December 1999; accepted
11 January 2000
Rimm et al, 1993; Stampfer et al, 1993; Verlangieri et al,
1985). Not only vitamin supplements but also drinks that
contain high levels of antioxidants could prevent chronic
disorders (Bushman, 1998; Hertog et al., 1993). Red wine
is one of the best known such drinks (Maxwell et al, 1994;
Whitehead et al, 1995), and the green tea favored by
Orientals also contains high level of antioxidants. A few
epidemiologic studies have reported that the incidence of
coronary heart disease and cancer decreased with intake of
green tea (Bushman, 1998; Kohlmeier et al, 1997; Stoner
& Mukhtar, 1995) and that the oxidation of low density
lipoprotein was suppressed with green tea in vitro (Luo
et al, 1997). However, the studies dealing with in vivo
increase of antioxidant effects of green tea are rare (Benzie
et al, 1999; Pietta et al, 1998; Sera®ni et al, 1996). As there
is no report that explain the increment in antioxidant effect
according to the volume of green tea, the authors carried
out this study to elucidate the dosage effect of green tea on
antioxidants in vivo.
Methods
Human subjects
This study was approved by the Ethical Committee of the
Asan Medical Center. The study subjects were 10 healthy
adult volunteers (®ve women and ®ve men). All of them
were free of clinical, biochemical or haematological manifestations of cardiovascular, hepatic, renal or endocrine
disorders. Before the study they were instructed to avoid
drinking green tea for one week, otherwise to keep to their
normal diet. They came to the laboratory in the morning
after overnight fasting.
Antioxidant effect of green tea
H Sung et al
528
Measurement of total antioxidant capacity
The Total Antioxidant Status kit (Randox Laboratories Ltd,
UK) was applied to a Cobas Mira (Roche Diagnostic
System Inc., Switzerland) chemistry analyser.
The assay principle is: ABTS (2,20 -azino-di-2-ethylbenzthiazoline sulphonate) is incubated with a peroxidase
(metmyoglobin) and H2O2 to produce the radical cation
ABTS‡. This has a relatively stable blue-green colour,
which is measured at 600 nm. Antioxidants in the added
sample cause suppression of this colour production to a
degree which is proportional to their concentration (Miller
et al, 1993,1997). The assay was calibrated against atocopherol analogue Trolox (Roche) and results were
expressed as millimoles per litre of Trolox activity.
To measure the accuracy and reproducibility of the kit,
the Randox Total Antioxidant Control (Randox Laboratories Ltd, UK) was used and the intra-assay coef®cient of
variation was 1.2% and inter-assay coef®cient of variation
2.4%.
In vitro study
Tea infusions were prepared with commercially available
tea (tea bag). A tea bag containing 2.5 g of tea leaves was
dipped into 150 ml of boiling tapwater. The mixture was
allowed to stand for 2 min. This tea was subjected to
measurement of antioxidant concentration.
In vivo study
The study subjects ingested 150 ml of green tea made from
2.5 g of commercial green tea leaf for 1 week, 300 ml from
5.0 g for the following week and 450 ml from 7.5 g in the
third week. Heparinized blood (5 ml) was taken at baseline
(after fasting), and 1 h and 2 h after ingesting green tea with
vacuum tubes (sodium heparin tube, Becton Dickinson
Vacutainer System). Freshly drawn samples were centrifuged at 3000 rpm for 5 min to separate the plasma,
which was analysed immediately or frozen at ÿ70 C
until analysis.
Statistics
Data are expressed as mean s.d. The means of antioxidants before and after ingesting green tea were analysed
statistically with a paired t-test using a statistical package
programme from MedCalc1 (MedCalc1, Mariakerke,
Belgium).
Results
The mean ( s.d.) concentration of total antioxidant of
2.5 g of green in 150 ml tap water was 2.44 0.13 mmol=l.
The plasma antioxidant concentrations of 10 volunteers
after ingesting 150 ml of green tea increased to
1.33 0.06 mmol=l at 1 h and 1.34 0.06 mmol=l at 2 h
after ingestion from 1.31 0.06 mmol=l at baseline. There
was an increment of 1.1% (paired t-test P > 0.05) at 1 h and
2.1% (P > 0.05) at 2 h without signi®cance. After ingestion
of 300 ml of green tea, plasma antioxidant concentrations
increased from basal 1.29 0.10 mmol=l to 1.38 0.11 mmol=l at 1 h and 1.37 0.11 mmol=l at 2 h. They
were also signi®cantly higher than that of baseline (7.0% at
1 h P < 0.0001, 6.2% at 2 h P < 0.0001). After ingestion of
450 ml of green tea, plasma antioxidant concentrations
increased from basal 1.33 0.06 mmol=l to 1.49 0.07 mmol=l at 1 h and 1.50 0.05 mmol=l at 2 h. They
European Journal of Clinical Nutrition
Figure 1 Changes in total antioxidant capacity according to volume of
green tea. Values are expressed as mean ‡ s.d. *Statistically signi®cant
differences compared with basal values (0 hour), P < 0.0001.
were also signi®cantly higher than that of baseline (12.0%
at 1 h P < 0.0001, 12.7% at 2 h P < 0.0001; Figure 1).
Discussion
After taking 150 ml of green tea, the total antioxidant
capacity of the plasma increased by 1.1% at 60 min and
2.1% at 120 min over baseline value in subjects consuming
150 ml of green tea, which was statistically not signi®cant.
However, the total antioxidant capacity of plasma after
consuming 300 ml of green tea showed a signi®cant
increase of 7.0% after 60 min and 6.2% after 120 min,
and after taking 450 ml 12.0% after 60 min and 12.7%
after 120 min over baseline value. With these ®ndings we
could assume that antioxidant effect of green tea is sustained for at least 2 h.
Yang et al (1998) measured (ÿ)-epigallocatechin-3gallate(EGCG), (ÿ)-epigallocatechin (EGC), and (ÿ)-epicatechin(EC), which are the main polyphenols with an
antioxidant effect in green tea, and reported a maximum
plasma concentration time (Tmax) of 1.5 ± 2.5 hours. Sera®ni et al (1996) reported that after taking 300 ml of green
tea total radical antioxidant parameter (TRAP) showed the
highest increment of 40% at 30 min after ingestion and
decreased thereafter, reaching pre-ingestion values at
80 min, which was different from the current study.
Benzie et al (1999) reported that there was 4% increase
of ferric reducing=antioxidant power (FRAP) after 40 min
after taking 400 ml of green tea, which returned to basal
value after 2 h. The differences between this study and
other reports in the degree of increment in antioxidant
capacity, time to peak increase, and dosage effect could
be due to the methods of measuring and the ethnic or
individual diversity in absorption and metabolism of green
tea.
Whitehead et al (1995) reported the increment of 18% at
1 h and 11% at 2 h of serum antioxidant capacity (SAOC)
after ingesting 300 ml of red wine. Although it is dif®cult to
assess and compare exact natures of two studies, as the
measuring methods were different, ingestion of 300 ml of
red wine showed antioxidant effect similar to ingestion of
450 ml of green tea. Green tea is considered to have high
value as favorite food or drinks as it does not contain
alcohol.
Antioxidant effect of green tea
H Sung et al
The objective of this study was to investigate the
increase or increment in antioxidant capacity with taking
and subsequent absorption of green tea. The roles of each
component of green tea in the increase in antioxidant
capacity still need further investigation.
Weisburger (1999) reported that some of the protective
health effects by taking green tea were reducing the
oxidation of low density lipoprotein, thus lowering the
risk of coronary artery disease and reducing oxidized
metabolites, which could result in lowering the incidence
of cancer. Ji et al (1997) reported in their epidemiological
studies that males who consumed 300 g of tea in a month
and females 200 g showed signi®cantly lower prevalence of
carcinomas of colon, rectum and pancreas. And there were
reports that carcinomas of esophagus (Gao et al, 1994), and
stomach (Yu et al, 1995) were lower in those taking tea.
The potential protective health effects of 12% increase in
antioxidant capacity, temporary or sustained, still need
further investigation.
In Korea, it is recommended by tea manufacturers that
the tea bag containing 2 ± 5 g of green tea be put into 20 ±
150 ml of hot water and repeated two to three times (Kim,
1996).
Polyphenols of green tea exist as catechins in unprocessed leaf form and do not change when processed to dried
green tea (Graham, 1992; Hara, 1994). Catechins have
strong antioxidant effect and they are known to lower
low density lipoprotein (LDL) oxidation in vitro (de Whalley et al, 1990; Luo et al, 1997). The in vivo effects are
controversial in that some argue that it suppress the oxidation of LDL (Ishikawa et al. 1997), while others report that
there are no signi®cant effects (van het Hof et al, 1997,
1999).
The Total Antioxidant Status measuring kit of Randox1
used in the current study had the advantages that it could
determine the total neutralizing capacity of oxygen free
radicals, including albumin, and that it could be applied to
chemistry analysers easily. The kit showed high accuracy
and reproducibility with an intra-assay coef®cient of variation of 1.2% and inter-assay coef®cient of variation of
2.4%. The Randox Total Antioxidant Status kit was
reported to show a speci®city less desirable in measuring
antioxidant (Cao & Prior, 1998). We tried to overcome this
problem by measuring antioxidant capacity in the same
subjects before and after green tea.
In conclusion, signi®cant increment in antioxidant
effects in vivo were observed after ingesting 300 and
450 ml of green tea, and the increased antioxidant effects
lasted for 2 h. Positive dosage effects were also observed.
References
Barber DA & Harris SR (1994): Oxygen free radicals and antioxidants: a
review. Am. Pharm. NS34, 26 ± 35.
Benzie IFF, Szeto YT, Strain JJ & Tomlinson B (1999): Consumption of
green tea causes rapid increase in plasma antioxidant power in humans.
Nutr. Cancer 34, 83 ± 87.
Bushman JL (1998): Green tea and cancer in humans: a review of the
literature. Nutr. Cancer 31, 151 ± 159.
Cao G & Prior RL (1998): Comparison of different analytical methods for
assessing total antioxidant capacity of human serum. Clin. Chem. 44,
1309 ± 1315.
de Whalley CV, Rankin SM, Hoult JR, Jessup W & Leake DS (1990):
Flavonoids inhibit the oxidative modi®cation of low density lipoproteins by macrophages. Biochem. Pharmac. 39, 1743 ± 1750.
Diplock AT (1994): Antioxidant and free radical scavengers. In: Free
Radical Damage and its Control, ed. CA Rice-Evans & RH Burdon, pp
113 ± 130. Amsterdam: Elsevier.
Gao YT, McLaughlin JK, Blot WJ, Ji BT, Dai Q & Fraumeni JF Jr (1994):
Reduced risk of esophageal cancer associated with green tea consumption. J. Natl. Cancer Inst. 86, 855 ± 858.
Gaziano JM, Manson JE, Buring JE & Hennekens CH (1992): Dietary
antioxidants and cardiovascular disease. Ann. NY Acad. Sci. 669,
249 ± 258 (discussion 258 ± 259).
Graham HN (1992): Green tea composition, consumption, and polyphenol
chemistry. Prev. Med. 21, 334 ± 350.
Halliwell B & Gutteridge JMC (1989): Free radicals, ageing and disease.
In: Free Radicals Biology and Medicine, ed. B Halliwell & JMC
Gutteridge, pp 416 ± 508. Oxford: Clarendon Press.
Hara y (1994): Antioxidative action of tea polyphenols: Part 1. Am.
Biotechnol. Lab. 12, 48.
Hertog MG, Feskens EJ, Hollman PC, Katan MB & Kromhout D (1993):
Dietary antioxidant ¯avonoids and risk of coronary heart disease: the
Zutphen Elderly Study. Lancet 342, 1007 ± 1011.
Ji BT, Chow WH, Hsing AW, McLaughlin JK, Dai Q, Cao YT, Blot WJ &
Faumeni JF Jr (1997): Green tea consumption and the risk of pancreatic
and colorectal cancers. Int. J. Cancer 70, 255 ± 258.
Kim JT (1996): Drinking method of tea. In: Science and Culture of Tea,
ed. JT Kim, pp 271 ± 284. Seoul: Bolimsa (in Korean).
Kohlmeier L, Weterings KG, Steck S & Kok FJ (1997): Tea and cancer
prevention: an evaluation of the epidemiologic literature. Nutr. Cancer
27, 1 ± 13.
Luo M, Kannar K, Wahlqvist ML & O'Brien RC (1997): Inhibition of
LDL oxidation by green tea extract. Lancet 349, 360 ± 361.
Maxwell S, Cruickshank A & Thorpe G (1994): Red wine and antioxidant
activity in serum. Lancet 344, 193 ± 194.
Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V & Milner A (1993): A
novel method for measuring antioxidant capacity and its application to
monitoring the antioxidant status in premature neonates. Clin. Sci.
(Colch.) 84, 407 ± 412.
Miller NJ Johnston JD, Collis CS & Rice-Evans C (1997): Serum total
antioxidant activity after myocardial infarction. Ann. Clin. Biochem. 34,
85 ± 90.
Pietta P, Simonetti P, Gardana C, Brusamolino A, Morazzoni P &
Bombardelli E (1998): Relationship between rate and extent of catechin
absorption and plasma antioxidant status. Biochem. Mol. Biol. Int. 46,
895 ± 903.
Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Colditz GA &
Willett WC (1993): Vitamin E consumption and the risk of coronary
heart disease in men. N. Engl. J. Med. 328, 1450 ± 1456.
Sera®ni M, Ghiselli A & Ferro-Luzzi A (1996): In vivo antioxidant effect
of green and black tea in man. Eur. J. Clin. Nutr. 50, 28 ± 32.
Stampfer MJ, Hennekens CH, Manson JE, Colditz GA, Rosner B & Willett
WC (1993): Vitamin E consumption and the risk of coronary disease in
women. N. Engl. J. Med. 328, 1444 ± 1449.
Stoner GD & Mukhtar H (1995): Polyphenols as cancer chemopreventive
agents. J. Cell Biochem. Suppl. 22, 169 ± 180.
van het Hof KH, de Boer HS, Wiseman SA, Lien N, Westrate JA &
Tijburg LB (1997): Consumption of green or black tea does not increase
resistance of low density lipoprotein to oxidation in humans. Am. J.
Clin. Nutr. 66, 1125 ± 1132.
van het Hof KH, Wiseman SA, Yang CS & Tijburg LB (1999): Plasma and
lipoprotein levels of tea catechins following repeated tea consumption.
Proc. Soc. Exp. Biol. Med. 220, 203 ± 209.
Verlangieri AJ, Kapeghian JC, el-Dean S & Bush M (1985): Fruit and
vegetable consumption and cardiovascular mortality. Med. Hypotheses
16, 7 ± 15.
Weisburger JH (1999): Tea and health: the underlying mechanisms. Proc.
Soc. Exp. Biol. Med. 220, 271 ± 275.
Whitehead TP, Robinson D, Allaway S, Syms J & Hale A (1995): Effect of
red wine ingestion on the antioxidant capacity of serum. Clin. Chem. 41,
32 ± 35.
Yang CS, Chen L, Lee MJ, Balentine D, Kuo MC & Schantz SP (1998):
Blood and urine levels of tea catechins after ingestion of different
amounts of green tea by human volunteers. Cancer Epidemiol. Biomarkers Prev. 7, 351 ± 354.
Yu GP, Hsieh CC, Wang LY, Yu SZ, Li XL & Jin TH (1995): Greentea consumption and risk of stomach cancer: a population-based
case ± control study in Shanghai, China. Cancer Causes Control 6,
532 ± 538.
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