The Relation Between Single/Double Or Repeated Tea Catechin Ingestions

Home , Catechin, Epicatechin gallate

European Journal of Clinical Nutrition (2002) 56, 1186–1193 ß 2002 Nature Publishing Group All rights reserved 0954–3007/02 $25.00 www.nature.com/ejcn ORIGINAL COMMUNICATIONS The relation between single=double or repeated tea catechin ingestions and plasma antioxidant activity in humans

M Kimura1, K Umegaki1, Y Kasuya2, A Sugisawa1 and M Higuchi1*

1National Institute of Health and Nutrition, Tokyo, Japan; and 2Asahi Soft Drink Co. Ltd., Beverage Research and Development Laboratory, Ibaragi, Japan

Objective: The purpose of this study was to investigate the effects of single=double or repeated intake of a normal amount of tea catechin on plasma catechin concentrations and antioxidant activity in young women. Design: First, after an overnight fast, ﬁve healthy subjects were given water or single=double dose(s) of tea polyphenol extract (164 mg tea catechins containing 61% epigallocatechin gallate in 190 ml water). Blood samples were taken before and 30, 60 and 180 min after the ingestion. Second, 16 healthy subjects ingested the tea polyphenol extract three times a day at mealtimes for 7 days followed by withdrawal of tea polyphenol extract for 7 days. Blood samples were taken before and after ingestion, and 7 days after the withdrawal of tea catechin. Subjects were prohibited from drinking any beverages containing polyphenols or antioxidant supplements during the study period. Catechin and other antioxidant concentrations in the plasma were measured, and changes in antioxidant activity were evaluated by ferric reducing ability of plasma assay. Results: Single=double ingestion of tea polyphenol extract did not cause an increase in the antioxidant activity. There was no also change in antioxidant activity after the ingestion of tea polyphenol extract for 7 days. Plasma-free epigallocatechin gallate concentration remained at the pre-study level; however, the plasma FRAP value decreased signiﬁcantly at 7 days after the withdrawal of tea polyphenol extract. Decreases in endogenous antioxidants in the plasma, including vitamin C and bilirubin, were also observed 7 days after withdrawal of tea polyphenol. Conclusions: The results suggest that continuous daily intake of tea catechins affects the concentrations of endogenous antioxidants in the plasma and has the potential to maintain total antioxidant activity. European Journal of Clinical Nutrition (2002) 56, 1186 – 1193. doi:10.1038=sj.ejcn.1601471

Keywords: tea catechin; antioxidant activity; FRAP assay; epigallocatechin gallate; young women

Introduction in food, are widely consumed. Scalbert and Williamson Oxidative damage to important biomolecules, including (2000) reported that flavonoids made up approximately lipoprotein and DNA, is considered to accompany arterio- two-thirds of the total polyphenol intake in their cohort, sclerosis, carcinogenesis and acceleration of aging. This with the total intake being approximately 1 g=day. Tea con- oxidative damage may be inhibited by daily intake of anti- tains abundant flavonoids, and tea consumption has been oxidants (Ames, 1983). Flavonoids, which are antioxidants reported to have beneficial effects on antioxidant function (Ho et al, 1992; Salah et al, 1995; van het Hof et al, 1998). The majority of flavonoids in green tea, which is widely consumed in Asia and the Middle East, are catechins. Rice-Evans (1999) reported that tea catechins, particularly epigallocate- *Correspondence: M Higuchi, National Institute of Health and Nutrition, chin gallate (EGCg), have more powerful antioxidant activity 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan. on a molar basis than vitamins C or E in vitro. E-mail: [email protected] and [email protected] Although there have been many in vitro studies on the Guarantor: M Higuchi. Received 18 July 2001; revised 5 March 2002; antioxidant activity of various foods, there have been only a accepted 11 March 2002 few in vivo studies (Leenen et al, 2000; van het Hof et al, Tea catechin and plasma antioxidant activity M Kimura et al 1187 1999). In vitro studies have demonstrated the powerful anti- (Osaka, Japan). All solvents were of HPLC grade, and all oxidant activity of tea catechins (Unno et al, 2000; Lotito & other chemicals were of reagent grade. Fraga, 1998; Zhu et al, 1999; Yang & Koo 2000; Rice-Evans, 1999), but the potential health benefits are unlikely to be realized until they are substantiated in vivo. It is therefore Analytical methods important to evaluate the effect of tea catechins in humans in vivo. (1) Measurement of plasma antioxidant activity by FRAP Several previous studies have investigated the effects of a assay. Plasma antioxidant activity was measured by FRAP single ingestion of tea or tea catechin in humans (Benzie assay as described previously by Benzie and Strain (1996). et al, 1999; Leenen et al, 2000; Pietta et al, 1998; Serafini et al, Briefly, ferric to ferrous ion reduction at low pH in plasma 1996). The peak concentrations of catechin in human forms a colored ferrous-2,4,6-tri(2-pyridyl)-s-triazine com- plasma have been reported to occur at between 30 and plex. FRAP reagent (240 ml) was warmed to 37C and a 60 min after ingestion. Some of these reports have shown reagent blank (M1) reading was taken at 593 nm (DU600; slight increases in antioxidant activity after the ingestion. Beckman, Tokyo, Japan). Twenty-four ml of milli-Q water These increases were induced by ingestion of larger-than- and 8 ml plasma sample were added to 240 ml FRAP reagent. usual amounts of green tea leaf-extract. However, the effects Absorbance (A) was read after 4 min. The change in of repeated normal tea catechin ingestions on antioxidant absorbance (DA 593 nm) from M1 to the A reading was activity remain to be clarified. calculated for each sample as standardized by DA 593 nm Therefore, the purpose of this study was to examine the of standard solutions (iron (II) sulfate heptahydrate). We effect of daily tea catechin consumption on the changes of confirmed that the intra- and inter-assay variations for this plasma catechin concentration and antioxidant activity, as method were both 1.2%. We conducted the in vitro assay to determined by the ferric reducing ability of plasma (FRAP; define the effective concentration of tea catechins (EGCg or Benzie & Strain, 1996), after single=double ingestion or tea polyphenol extract) on antioxidant activity using repeated ingestion of normal amounts of highly purified this assay. The FRAP value increased in a concentration- catechins rich in EGCg. In addition, we observed the dependent manner; however, more than 2000 nM of changes of other plasma antioxidant concentrations, includ- catechin was needed to obtain a linearly significant increase ing vitamin C, a-tocopherol, uric acid and bilirubin. in the FRAP value (data not shown).

Methods (2) Measurement of catechin concentration of plasma. Subjects Plasma catechin concentrations were analyzed using HPLC All subjects were non-smoking healthy female university equipped with an electrochemical detector (ECD; Umegaki students who were recruited from a dietitian program and et al, 2001). Brieﬂy, plasma (200 ml) was mixed with 16% were fully apprised of the details of the study. All subjects ascorbic acid (10 ml) in a micro tube. For the analysis of total gave their informed consent to participate in the study and catechins (free and conjugated forms), the plasma sample were instructed to maintain their habitual patterns of phy- was added to a 20 ml mixture of b-glucuronidase (500 units) sical activity and diet throughout the entire study period. and sulfatase (25 units), and 100 ml of 0.2 M sodium acetate Food intake was recorded for 2 days by the subjects and buffer (pH 5) containing 0.5 mM EDTA, and then incubated checked before the study commenced. The study protocol for 45 min at 37C. For the analysis of free catechins, 20 mlof was approved by the Ethical Committee of the National water was added instead of b-glucuronidase and sulfatase, Institute of Health and Nutrition (Japan). and the incubation was omitted. The reaction mixture was added to 350 ml of acetonitrile with 20 ml of ethyl gallate solution (10 mM) as an internal standard, vigorously mixed for 5 min, and then centrifuged at 10 000 g for 15 min. The Materials catechins in the supernatant were extracted using a Bond

EGCg, gallocatechin gallate (GCg), epicatechin gallate (ECg), Elute cartridge (C18; size 500 mg=3 ml), then analyzed by epigallocatechin (EGC), and epicatechin (EC) were pur- HPLC-ECD (Coulochem II, ESA Inc., Bedford, MA, USA). chased from Funakoshi Co. Ltd (Tokyo, Japan). b-glucuro- The analytical conditions were as follows: column RP18 nidase (type X-A) and sulfatase (type VIII) were obtained (IRICA Instruments Inc., Kyoto, Japan); mobile phase, from Sigma Chemical Co. (St Louis, MO, USA) and 2,4,6-tri 50 mM phosphoric acid – 0.05 mM EDTA – 14% acetonitrile (2-pyridyl)-s-triazine was obtained from Fluka Chemicals (pH2.5); ﬂow rate, 1 ml=min; column temperature, 35C; (Switzerland). As a highly puriﬁed tea catechin, Polyphenon detector, guard cell, þ 250 mV; analytical cell, 7 150 mV 100S (tea polyphenol extract) was supplied by Mitsui Norin for electrode 1 and þ 150 mV for electrode 2. The results Co. (Tokyo, Japan). The tea polyphenol extract contained were shown as the free and conjugated EGCg concentrations 61% EGCg, 6% GCg, 4% ECg, 14% EGC, and 15% EC. Other or free and conjugated total catechin (EGCg þ EGC þ reagents were purchased from Wako Pure Chemical Ind. GCg þ ECg þ EC) concentrations.

European Journal of Clinical Nutrition Tea catechin and plasma antioxidant activity M Kimura et al 1188 (3) Other measurements. The analysis of uric acid, biliru- fuged at 1500 g for 15 min to prepare plasma, which was bin and total-cholesterol was entrusted to SRL Ltd, Tokyo, divided into several portions for each analytical item, and Japan. The concentrations of vitamin C and a-tocopherol in stored at 780C until assay. plasma were analyzed by HPLC methods (Umegaki et al, 1995). Experiment 2: the effects of 7-day tea catechin ingestion and withdrawal in humans. After overnight fasting, 16 Experimental protocol (Figure 1) healthy subjects consumed the tea polyphenol extract Experiment 1: acute consumption effects of tea catechin in (164 mg in 190 ml water) three times a day at mealtimes humans. As depicted in Figure 1, five healthy subjects for 1 week (days 0 – 6). Subjects were then withdrawn from received three treatments on three separate days over an the tea polyphenol extract on days 7 – 13 of the study. Their interval of at least 6 days as follows: treatment 1 — after an venous blood samples were taken before ingestion on day 7 overnight fast, the subjects were given 190 ml plain water (after ingestion) and on day 14 (after withdrawing). From (water ingestion); treatment 2 — after an overnight fast, the 30 h before until the conclusion of the study, subjects were subjects were given 164 mg tea polyphenol extract in 190 ml not allowed to consume antioxidant supplements or any water (a single ingestion); treatment 3 — after an overnight beverages containing polyphenols (green tea, black tea, fast, the subjects were given 164 mg tea polyphenol extract coffee or red wine). Blood samples were prepared in a in 190 ml water twice (a double ingestion). These treatments manner similar to that in experiment 1 (Figure 1). were given in random order. In the case of double ingestion, the subjects received the same fluid 2 h after the first ingestion. The subjects abstained from all beverages containing tea flavonoids for 30 h before each treatment. After over- Statistical analysis night fasting, blood samples were taken before and at 30, 60, Data are presented as the individual group means plus and 180 min after the fluid ingestion. Blood samples were standard error (SEM). Comparisons within the same group collected into heparinized tubes, and immediately centri- were made by repeated one or two-factor ANOVA followed

Figure 1 Study design. Experiment 1: acute effects of tea polyphenol extract ingestion. Subjects were given water or a single=double dose of tea polyphenol extract (164 mg catechins=190 ml water) and blood samples were taken before and at 30, 60, 180 min after ingestion. Experiment 2: the effects of a 7-day tea polyphenol extract ingestion. Subjects ingested tea polyphenol extract (164 mg tea catechins=190 ml water) at mealtime for 7 days and then ingested no tea polyphenol extract for 7 days. Blood samples were taken before and after ingestion and after withdrawal.

European Journal of Clinical Nutrition Tea catechin and plasma antioxidant activity M Kimura et al 1189 by post-hoc test. Pearson’s correlation coefficient was used to increase (180 min: free concentration, 85Æ 32 nM, conju- assess the relationships among plasma antioxidant ability, gated concentration, 264Æ 16 nM). The changes in total catechin concentrations, vitamin concentrations and other plasma catechin concentrations were similar manner to the plasma parameters. These statistical analyses were performed changes in EGCg concentrations after ingestion (60 min: free using Stat View software (Version 5, Abacus Concepts Inc., concentration 190Æ 2 nM, conjugated concentration Berkeley, CA, USA). 460Æ 7 nM; 180 min: free concentration 120Æ 2 nM, conjugated concentration 560Æ 7 nM). A double ingestion of tea polyphenol extract dose-dependently increased plasma cate- Results chin concentrations (180 min: free EGCg concentration Experiment 1 259Æ 31 nM, conjugated EGCg concentration 330Æ 45 nM, free total concentration 342Æ 40 nM, conjugated total con- Subjects. Five healthy subjects (age, 21Æ 0 y; height, centration 881Æ 107 nM). Neither EGCg nor total catechin 161Æ 2 cm; weight, 50.9Æ 1.6 kg; BMI, 20.4Æ 1.7 kg=m2) suc- concentration changed after water ingestion. cessfully completed each of the three treatments: water ingestion and single and double tea polyphenol extract ingestion. Their daily intakes of energy, protein, fat, carbo- Changes in total antioxidant activity (FRAP assay) and hydrate, vitamin C, vitamin E and catechin were antioxidant concentrations in plasma. Figure 3 illustrates 6780Æ 497 kJ, 64.0Æ 4.2 g, 60.5Æ 8.0 g, 203.2Æ 19.2 g, the effects of water or tea polyphenol extract ingestions on 135Æ 30 mg, 12.1Æ 2.6 mg and 675Æ 268 mg, respectively. plasma FRAP activity expressed as an absolute change from baseline. The FRAP value did not increase at 60 min when free EGCg concentration was maximal, as shown in Figure 2, Changes in plasma catechin concentrations. Figure 2 and also did not increase at 180 min when conjugated EGCg shows plasma free and conjugated EGCg concentrations concentration was highest in the plasma after a single dose before and at 30, 60 and 180 min after a tea polyphenol of tea polyphenol extract. No significant differences in these extract ingestion. Free EGCg concentration increased imme- concentrations were observed among the three treatments. diately after ingestion, while conjugated EGCg concentra- Uric acid, vitamin C and a-tocopherol=total cholesterol con- tion did not change over the first 30 min. Both free and centrations did not change after each ingestion and did not conjugated EGCg concentrations increased similarly from differ significantly among the three treatments (data not 30 to 60 min after ingestion (60 min: free concentration, shown). 142Æ 28 nM, conjugated concentration, 154Æ 19 nM). Thereafter, the free EGCg concentration gradually decreased, while the conjugated EGCg concentration continued to Expertment 2 Subjects. Sixteen subjects (age, 21Æ 0 y; height, 159Æ 3cm; weight, 49.3Æ 2.3 kg; BMI, 19.5Æ 1.0 kg=m2) completed the study successfully. Their daily intakes of energy, protein, fat, carbohydrate, vitamin C, vitamin E and catechin were 6530Æ 300 kJ, 60.9Æ 2.5 g, 52.1Æ 4.3 g, 198.0Æ 11.3 g,

Figure 2 Acute effects of tea polyphenol extract ingestion: changes in Figure 3 Experiment 1: change in plasma FRAP activity over 180 min conjugated and free EGCg concentration in plasma over 180 min after after a single dose of water, a single dose and a double dose of tea water, a single and double tea polyphenol extract ingestion. polyphenol extract.

European Journal of Clinical Nutrition Tea catechin and plasma antioxidant activity M Kimura et al 1190 151Æ 24 mg, 8.4Æ 1.4 mg and 431Æ 75 mg, respectively, extract ingestion for 7 days was the highest among the three before the experiment. The tea polyphenol extract did time points (P < 0.05 vs before and vs after withdrawal, not contain vitamin C, therefore during the experiment respectively; Figure 4a). On the other hand, there was no vitamin C intake (98Æ 18 mg=day) was lower than before difference in the free EGCg concentration between before the experiment. and after the ingestion. However, free EGCg concentration after withdrawal was signiﬁcantly reduced (P < 0.05 vs after Changes in plasma catechin concentrations. Figure 4 ingestion; Figure 4b). The changes in plasma total catechin shows the changes in fasting plasma EGCg concentrations. concentrations were similar to the changes in EGCg concen- Æ The conjugated EGCg concentration after tea polyphenol trations (free concentration: before ingestion, 27 2nM, after ingestion, 21Æ 3 nM, after withdrawal, 15Æ 3 nM; conjugated concentration: before ingestion, 190Æ 27 nM, after ingestion, 803Æ 72 nM, after withdrawal, 224Æ 16 nM, respectively).

Changes in total antioxidant activity (FRAP assay) and antioxidants in plasma. Figure 5 shows the changes in mean levels of plasma total antioxidant activity as determined by FRAP assay. The FRAP value after tea polyphenol extract ingestion for 7 days was not significantly different from the level before the ingestion. The FRAP value after withdrawal was significantly lower than the level before or that after the 7 day ingestion (P < 0.001). Table 1 shows the levels of uric acid, bilirubin and antioxidant vitamins. Lower levels of uric acid were observed after tea polyphenol extract ingestion and after withdrawal compared to those before the ingestion (P < 0.05, respectively). Bilirubin and a-tocopherol=total cholesterol concentrations after ingestion slightly increased compared with those before the ingestion, but these increases were not statistically significant. Although the vitamin C concentration remained at the initial level throughout tea polyphenol extract ingestion, it was dramatically reduced after the withdrawal (D30.6Æ 8.2 mM vs before the ingestion, D24.1Æ

Figure 4 Experiment 2 (the effects of 7-day tea polyphenol extract Figure 5 Experiment 2 (the effects of 7 day tea polyphenol extract ingestion and withdrawal): change of the plasma EGCg concentrations ingestion and withdrawal): change in the antioxidant activity using the after overnight fasting. *P < 0.05, ***P < 0.001. FRAP assay. ***P < 0.001.

European Journal of Clinical Nutrition Tea catechin and plasma antioxidant activity M Kimura et al 1191 Table 1 Experiment 2 (the effects of 7-day tea polyphenol extract ingestion and withdrawal): the meanÆ s.e.m. levels of uric acid, bilirubin, vitamin C and a-tocopherol concentration before ingestion, after ingestion of tea polyphenol extract for 7 days and following withdrawal for 7 days

Result

Before ingestion After ingestion After withdrawal Before ingestion Before ingestion After ingestion (n ¼ 16) (n ¼ 16) (n ¼ 16) vs after ingestion vs after withdrawal vs after withdrawal

Uric acid (mM) 270Æ 11 251Æ 11 247Æ 12 * * NS Bilirubin (mM) 10.3Æ 0.7 11.2Æ 0.8 8.7Æ 0.5 NS NS * Vitamin C (mM) 80.8Æ 5.4 74.3Æ 6.7 50.2Æ 4.7 NS * * a-toc=T-cho (mM=mM)a 1.78Æ 0.07 1.92Æ 0.05 1.82Æ 0.05 NS NS NS

NS, not signiﬁcant. *P < 0.05. aa-tocopherol=total cholesterol.

9.6 mM vs after the ingestion, respectively). The bilirubin sporadic tea catechin intake is insufficient to increase anti- concentration also significantly decreased after the withdra- oxidant activity. wal (D2.6Æ 0.9 mM vs after the ingestion, P < 0.05). FRAP Green tea is one of the most popular beverages in Japan. values were positively correlated with bilirubin and vitamin Generally, Japanese individuals usually consume around C concentrations (r ¼ 0.44, 0.66, P < 0.05, respectively). 100 – 200 mg catechins from 2 – 3 cups of green tea at each mealtime, which is provided by 2 – 3 g of tea leaves. Thus, the daily tea catechin intake of the Japanese is presumed to be Discussion approximately 500 mg. It is not clear whether such contin- The object of this study was to assess whether a normal level uous daily intake of catechins induces antioxidant effects. of tea catechin intake causes an elevation of antioxidant We therefore used a 7 day tea polyphenol extract ingestion activity, which we presumed to reflect the concentration of followed by withdrawal to examine the effects of daily endogenous antioxidants. The results showed that single consumption of green tea on antioxidant activity. (164 mg catechin) or double (328 mg catechin) ingestion of We found that continuous 7 day intake of tea polyphenol tea polyphenol extract did not induce an increase in anti- extract did not increase antioxidant activity in the plasma; oxidant activity (Figure 2). There was also no change in the however, a significant decrease in antioxidant activity antioxidant activity after 7 day tea polyphenol extract inges- occurred 7 days after the withdrawal of the intake. In our tion; however, there was a significant decrease of the activity, preliminary study, in which we investigated continuous tea as estimated by FRAP values, in the plasma after the with- polyphenol extract ingestion in elderly women, we obtained drawal of tea polyphenol extract for 7 days (Figure 4). similar results (data not shown). Previous studies have indi- Benzie and Strain (1996), Leenen et al (2000) and Serafini cated that when catechins are administered, they are et al (1996) have shown that a single dose of black or green absorbed from the intestinal tract and exist as conjugated tea causes an increase in FRAP value or TRAP (total plasma and=or methylated forms in the plasma (Piskula & Terao, antioxidative capacity) value and that the peak plasma 1998; Da Silva et al, 1998; Okushio et al; 1999). It has also catechin concentration occurs at 30 – 60 min after ingestion. been suggested that conjugation and methylation of the In contrast to our experiment, subjects ingested a larger- phenolic moiety led to a decrease in its antioxidative activ- than-normal amount of tea catechin(s) (640 – 1500 mg vs a ity, via the suppression of scavenging chain-initiating normal value of 100 – 300 mg) in these reports. In the present oxygen radicals or chelating transition metal ions (Nanjo study, plasma free total catechin and free EGCg concentra- et al, 1996; Rice-Evans et al, 1996). On the other hand, Baba tions were 680 and 350 nM, respectively at 3 h after ingestion et al (2000) and Koga and Meydani (2001) reported that of 164 mg tea polyphenol extract. Consistent with our various conjugated forms of catechin enhanced antioxida- results, Pietta et al (1998) and Nakagawa et al (1997) reported tive activity. Consequently, it is not clear whether free or that less than 2% of a given dose of EGCg was absorbed, and conjugated catechins are more effective at enhancing anti- suggested that the ingestion of one cup of green tea contain- oxidant activity. In this study, FRAP values were not influ- ing about 150 mg of total catechin could assure a plasma enced by the obvious changes of conjugated and free EGCg total catechin concentration in the range of 400 – 600 nM. concentrations in plasma induced by continuous tea poly- Our in vitro study revealed that the detectable range of tea phenol extract ingestion. Antioxidant activity was not sig- catechins was > 2000 nM using FRAP assay. If we can pre- nificantly different between normal catechins intake and 7 sume that the detectable range of catechin concentration in day ingestion of tea polyphenol extract (approximately vitro may be within a range similar to that in vivo, it may be 500 mg=day). However, antioxidant activity after withdrawal plausible that elevated catechin concentration after inges- of tea polyphenol extract significantly decreased by about tion of a normal amount of tea catechin did not increase the 64 mM when compared to the values before and after the FRAP value. This would suggest that a normal amount of ingestions.

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