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Green tea catechins prevent low-density lipoprotein oxidation via their accumulation in low-density lipoprotein particles in humans
N U TR IT ION RE S EAR CH 3 6 ( 2 01 6 ) 1 6 –2 3
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Original Research
Green tea catechins prevent low-density lipoprotein oxidation via their accumulation in low-density lipoprotein particles in humans Norie Suzuki-Sugihara a , Yoshimi Kishimoto b,⁎, Emi Saita b , Chie Taguchi b , Makoto Kobayashi c , Masaki Ichitani c , Yuuichi Ukawa c , Yuko M. Sagesaka c , Emiko Suzuki a , Kazuo Kondo b, d a
Department of Nutrition and Food Science, Graduate School of Humanities and Sciences, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan b Endowed Research Department “Food for Health,” Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan c Central Reseach Institute, ITO EN, LTD., 21 Mekami, Makinohara, Shizuoka 421-0516, Japan d Institute of Life Innovation Studies, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma 374-0193, Japan
ARTI CLE I NFO
A BS TRACT
Article history:
Green tea is rich in polyphenols, including catechins which have antioxidant activities and are
Received 3 August 2015
considered to have beneficial effects on cardiovascular health. In the present study, we
Revised 29 October 2015
investigated the effects of green tea catechins on low-density lipoprotein (LDL) oxidation in vitro
Accepted 30 October 2015
and in human studies to test the hypothesis that catechins are incorporated into LDL particles and exert antioxidant properties. In a randomized, placebo-controlled, double-blind, crossover trial, 19 healthy men ingested green tea extract (GTE) in the form of capsules at a dose of 1 g total
Keywords:
catechin, of which most (>99%) was the gallated type. At 1 hour after ingestion, marked
Tea
increases of the plasma concentrations of (−)-epigallocatechin gallate and (−)-epicatechin
Epigallocatechin gallate
gallate were observed. Accordingly, the plasma total antioxidant capacity was increased, and
Lipoproteins
the LDL oxidizability was significantly reduced by the ingestion of GTE. We found that gallated
Low-density lipoprotein
catechins were incorporated into LDL particles in nonconjugated forms after the incubation of
Lipid peroxidation
GTE with plasma in vitro. Moreover, the catechin-incorporated LDL was highly resistant to
Humans
radical-induced oxidation in vitro. An additional human study with 5 healthy women confirmed that GTE intake sufficiently increased the concentration of gallated catechins, mainly in nonconjugated forms in LDL particles, and reduced the oxidizability of LDL. In conclusion, green tea catechins are rapidly incorporated into LDL particles and play a role in reducing LDL oxidation in humans, which suggests that taking green tea catechins is effective in reducing atherosclerosis risk associated with oxidative stress. © 2015 Elsevier Inc. All rights reserved.
Abbreviations: ANOVA, analysis of variance; EC, (−)-epicatechin; ECG, (−)-epicatechin gallate; EDTA, ethylenediamine-tetraacetic acid; EGC, (−)-epigallocatechin; EGCG, (−)-epigallocatechin gallate; EGCG3″Me, (−)-epigallocatechin 3-O-(3-O-methyl) gallate; GTE, green tea extract; LDL, low-density lipoprotein; TAC, total antioxidant capacity. ⁎ Corresponding author. Tel.: +81 3 5978 5810; fax: +81 3 5978 2694. E-mail address: [email protected] (Y. Kishimoto). http://dx.doi.org/10.1016/j.nutres.2015.10.012 0271-5317/© 2015 Elsevier Inc. All rights reserved.
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N U TR IT ION RE S EA RCH 3 6 ( 2 01 6 ) 1 6 –2 3
1.
Introduction
Various lines of research indicate that oxidized low-density lipoprotein (LDL) within the arterial wall promotes the development of atherosclerosis [1]. Protection against LDL oxidation is an effective strategy to prevent atherosclerosis [2], and growing evidence from epidemiologic studies has shown that dietary antioxidants contribute to the prevention of coronary heart disease [3,4]. Polyphenols are found in most foods and beverages of plant origin and are known to have antioxidant properties. Green tea is a large source of polyphenols among the Japanese population [5–7], and epidemiologic studies by the Japan Public Health Center–based Prospective Study group have revealed that the consumption of green tea can reduce the risk of all-cause mortality and the risk of mortality due to the 3 leading causes of death including heart disease, cerebrovascular disease, and respiratory disease [8]. Green tea extract (GTE) contains a number of catechins, including (−)-epigallocatechin gallate (EGCG), (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC), and (−)-epicatechin (EC) and has been known to have many beneficial properties that can help prevent atherosclerotic diseases via the regulation of obesity [9], hypertension [10], diabetes [11], and oxidative stress [12,13]. The molecular mechanism of the antiatherogenic activity of green tea catechins, particularly EGCG, which is the most abundant catechin, has also been determined in several studies [14,15]. Among the various antiatherogenic activities, the antioxidant effects are presumed to play a major role in mediating the cardioprotective role of green tea catechins. Our previous study reported that green tea catechins inhibited LDL oxidation when they were directly added to isolated LDL [16]. Moreover, we reported that the acute intake of green tea (5 g of green tea powder) successfully increased the resistance against LDL oxidation as well as the plasma catechin levels in healthy volunteers [17]. Nakagawa et al [18] reported that EGCG was incorporated into human plasma and could decrease the levels of plasma phosphatidylcholine hydroperoxide, a marker of oxidized lipoproteins, after a single oral intake of green tea catechins (254 mg of catechins containing 82 mg of EGCG). These findings prompted us to hypothesize that catechins, especially gallated catechins, may play a preventive role in LDL oxidation by being incorporated into LDL particles in plasma. It was recognized that catechins undergo glucuronidation and sulfation in the intestinal mucosa, liver, and kidney, and only 0.2% to 5.0% of the ingested doses were present in the circulating plasma after the ingestion of tea catechins [19–22]. Compared with other flavonoids like quercetin, catechins can be present in a free (nonconjugated) “active” form in human plasma [19]; however, no information regarding the incorporation of catechins into LDL is available. Thus, the aim of this study was to test our hypothesis that green tea catechins are incorporated into LDL particles and exert antioxidant properties. The protective mechanism of green tea catechins against LDL oxidation was investigated in vitro and in human studies by measuring the LDL oxidizability and catechin concentration both in plasma and in LDL.
2.
Methods and materials
2.1.
Reagents
Decaffeinated GTE powder (THEA-FLAN 90S), mainly composed of gallated catechins (as shown in Table 1), was obtained from ITO EN, LTD (Shizuoka, Japan). Purified catechins (EGCG, ECG, EGC, and EC), β-glucuronidase from Escherichia coli, and sulfatase type VIII from abalone entrails were purchased from Sigma-Aldrich (St Louis, MO, USA). The (−)-epigallocatechin-3O-(3-O-methyl) gallate (EGCG3″Me) was purchased from Nagara Science (Gifu, Japan). A Micro BCA Protein Assay Kit was obtained from Thermo Fisher Scientific (Rockford, IL, USA). Ethyl gallate was purchased from Tokyo Chemical Industry (Tokyo, Japan). Ethyl acetate, acetonitrile, and formic acid were purchased from Wako Pure Chemicals (Osaka, Japan). Green tea extract capsules containing 1 g of catechins and matching placebo capsules containing starch were formulated by the Sunsho Pharmaceutical (Shizuoka, Japan).
2.2.
Experimental design
To evaluate the effect of green tea catechins on LDL oxidizability and their ability to bind to LDL both in vitro and in vivo, we conducted human studies with healthy volunteers. These studies were approved by the Ethics Committee of Ochanomizu University in accordance with the principles of the Declaration of Helsinki. In addition, all participants were fully informed regarding the content and methods of this trial. Written informed consent was obtained prior to enrollment from each participant. Blood was drawn from an antecubital vein after fasting and was collected in tubes containing ethylenediamine-tetraacetic acid (EDTA) or clot accelerant for the separation of plasma and serum. Plasma samples were immediately prepared by centrifugation, and LDL was separated by single-spin density gradient ultracentrifugation at 417 000g for 40 minutes at 4°C [23]. The LDL protein concentration was determined using a Micro BCA Protein Assay Kit.
2.2.1.
Plasma loading with catechins (in vitro study)
Human plasma from healthy volunteers was incubated with purified catechins (EGCG, ECG, EGC, or EC, at 50 μmol/L) or GTE (3 μg/mL) for 20 minutes at 37°C, and LDL was immediately isolated [24,25]. The LDL oxidizability and catechins concentration in plasma or LDL were measured. Table 1 – Catechin composition of GTE (g/100 g) EGCG ECG Gallocatechin gallate (GCG) EGC Catechin gallate (CG) Gallocatechin (GC) EC Catechin (C) Abbreviation: ND, not detected.
53.6 12.5 2.8 0.4 0.4 0.1 ND ND
18 2.2.2.
N U TR IT ION RE S EAR CH 3 6 ( 2 01 6 ) 1 6 –2 3
Human study 1
We conducted a randomized, placebo-controlled, doubleblind, 2-way crossover trial (Fig. 1A). Participants in the study were recruited by public offering on our Web site. Nineteen healthy male volunteers ranging from 25 to 53 years of age were enrolled in the study. The sample size was estimated from previous studies with a similar design. Based on the power calculations made using G*power 3 software [26], the number of participants (n = 19) was sufficient for detecting a difference in LDL lag time with at least 80% power with P < .05 using a 2-tailed test. The participants were asked to complete 2 sessions in which GTE capsules containing 1 g of catechins or matching placebo capsules were ingested in a randomized order. Test days were separated by at least 2week washout periods. In the 12 hours before the tests, participants were not permitted to eat or drink except for water. Blood samples were collected from the participants before and 1 hour after consumption. The LDL oxidizability, serum total antioxidant capacity (TAC) value, and catechins concentration in plasma were measured.
2.2.3.
Human study 2
In order to confirm the amount of catechins in the LDL fraction after the intake of GTE, we recruited 5 healthy female volunteers (ranging from 22 to 30 years of age) and conducted
an additional human study with GTE capsules containing 1 g of catechin (Fig. 1B). The LDL oxidizability and catechins concentration in LDL were measured.
2.3.
Measurement of LDL oxidizability
Low-density lipoprotein oxidizability was determined by lag-time assay as described previously [16,27]. The LDL samples (final concentration of protein, 70 μg/mL) were oxidized by 2,2-azobis-4-methoxy-2,4-dimethylvaleronitrile. The kinetics of LDL oxidation were determined by monitoring the absorbance of conjugated dienes at 234 nm using a DU800 spectrophotometer (Beckman Coulter, Brea, CA, USA) at 4-minute intervals at 37°C. The lag time for the start of LDL oxidation was defined as the time interval between the initiation and intercept of the 2 tangents drawn to the lag and propagation phases of the absorbance curve at 234 nm.
2.4.
Determination of catechin levels in plasma and LDL
Sample preparation and extraction was carried out by the method of Unno et al [28] and Nakagawa et al [29] with some modifications. Each plasma (200 μL) or LDL fraction (80 μL) was mixed with 10 μL of 10% (wt/vol) ascorbic acid solution, and the mixture was stored at −80°C until analysis. To determine the
A. Randomized, placebo-controlled, double-blind, crossover trial (Human study-1)
B. Additional study (Human study-2)
Enrollment
Enrollment
Assessment for eligibility (n=19)
Randomization (n=19)
Study period-1
Placebo (n=13)
GTE (n=6)
Assessment for eligibility (n=5)
GTE (n=5)
Analyzed (n=5) LDL lag-time LDL catechin concentration
Washout (2 weeks)
Study period-2
Placebo (n=6)
GTE (n=13)
Analyzed (n=19) LDL lag-time TAC Plasma catechin concentration
Fig. 1 – Flowchart of the study design. Human study 1 was conducted with 19 healthy men in a randomized, placebo-controlled, double-blind, crossover method (A), and human study 2 was additionally conducted with 5 healthy women to confirm the amount of catechins in the LDL fraction (B).
total amounts of catechins, including free (nonconjugated) and conjugated forms as well as their metabolites, β-glucuronidase (250 units) and sulfatase (20 units) were added to the plasma samples. The mixture was incubated at 37°C for 45 minutes. To determine the nonconjugated form of catechins, the samples were analyzed without these enzymes. For the extraction of plasma catechins, the mixture was mixed with 300 μL of 0.1 mol/L phosphate buffer containing 0.5 mM EDTA-2Na (pH 3.5) and 40 μL of ethyl gallate (2.5 mg/L) solution containing 0.5% (wt/vol) ascorbic acid and 0.01 mg/mL EDTA-2Na as an internal standard. Then, 500 μL of ethyl acetate was added to the mixture, and this was vortexed. After centrifugation at 21 000g for 10 minutes at 4°C, the supernatants obtained were collected. This extraction was repeated 3 times, and the supernatants obtained were combined, evaporated, and dissolved in 200 μL of 6% (vol/vol) acetonitrile containing 0.094% (wt/vol) formic acid. The solution was centrifuged at 21 000g for 10 minutes at 4°C, and the supernatants were analyzed by ultraperformance liquid chromatography (UPLC)-/MS/MS using the method of Spáčila et al [30]. The UPLC–MS/MS system used included the Acquity UPLC (Waters, Milford, MA, USA) and the Acquity TQD Mass Spectrometer (Waters). The data were acquired and processed using MassLynx software version 4.1 (Waters). The analytical column was C18 BEH (100 mm × 2.1 mm i.d.; 1.7 μm; Waters). The concentration of catechins in the LDL was normalized to the protein content in the fraction.
2.5.
Measurement of serum TAC
Serum TAC was determined by a 2,2′-azinobis-(3-ethylbenzothiazoline6-sulfonic acid) radical scavenging assay (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions [31]. The values were quantified as millimolar Trolox equivalents.
2.6.
Statistical analyses
All data are presented as means ± SE. Statistical analyses were performed using analysis of variance (ANOVA), followed by a post hoc test. When appropriate, repeated-measures ANOVA was used. Results were considered significant when P < .05. The statistical analyses were performed using the GraphPad Prism 5 software package (GraphPad Software, La Jolla, CA, USA).
3.
Results
3.1. Comparison of the effect of 4 major catechins on LDL oxidation lag time in vitro After the incubation of EGCG, EGC, ECG, or EC with plasma in vitro, the oxidizability was determined in each isolated LDL by lag-time assay. As shown in Fig. 2, the LDL oxidation lag time was prolonged the most by ECG, second-most by EGCG, and third-most by EC, and was unchanged by EGC at 50 μmol/L. In our preliminary experiment, the concentration of each catechin remaining after preincubation was higher in plasma with EGCG and ECG than in plasma with EGC and EC (data not shown).
19
LDL lag time (min)
N U TR IT ION RE S EA RCH 3 6 ( 2 01 6 ) 1 6 –2 3
Fig. 2 – Comparison of the effect of 4 major catechins on LDL oxidation lag time in vitro. Plasma was incubated with 4 major catechins (EGCG, ECG, EGC, or EC), and then LDL was isolated. The LDL oxidizability was measured by a lag-time assay. Values are means ±SE (n = 5). ***P < .001 and **P < .01, indicating a significant change in lag time compared with before intake by Dunnett test after 1-way repeated-measures ANOVA.
3.2. Human study 1: LDL oxidizability and plasma catechins concentration after GTE intake Nineteen healthy participants consumed GTE capsules containing 1 g of catechins or placebo capsules in a randomized order. Before this study, we performed a pilot trial to determine the time to the peak concentration of plasma catechins and found that EGCG and ECG appeared transiently in the plasma and reached their highest level at 1 hour after the GTE intake. As a result, the LDL oxidation lag time was significantly prolonged after the ingestion of GTE compared with that before the GTE intake (31.5 ± 1.4 minutes vs 38.6 ± 1.5 minutes; P < .001; Fig. 3A). This finding indicated that GTE could reduce LDL oxidizability in human participants as well as in vitro. We also found that GTE significantly increased the serum TAC value 1 hour after intake (P < .001; Fig. 3B). In contrast, no significant changes in either LDL lag time or TAC were observed after the ingestion of the placebo. The plasma catechins and caffeine concentration are shown in Table 2. The baseline concentrations of each catechin were at low levels as they were measured in the morning after fasting. (−)-Epigallocatechin gallate and ECG, 2 major components of GTE, and EGCG3″Me, a metabolite of EGCG, were markedly increased in the plasma 1 hour after GTE intake. The total concentration of catechins reached 2752.2 ± 456.7 μg/L; these consisted mostly of gallate-type catechins. The increased catechins were largely found in nonconjugated forms (73.8%). There were no significant changes in the concentrations of catechins after the intake of the placebo.
3.3. Concentration of catechins in LDL isolated after incubation of GTE with plasma in vitro To examine the possibility that the increased plasma catechins accumulate in the LDL fraction after GTE intake, GTE was preincubated with plasma in vitro, and then LDL was isolated from the plasma by ultracentrifugation. (−)-Epigallocatechin gallate and ECG were detected both in the plasma (EGCG: 1033.5 ± 153.3 μg/L and ECG: 251.8 ± 31.0 μg/L) and in the LDL
20
A
B
LDL lag time (min)
TAC (mM Trolox equivalent)
N U TR IT ION RE S EAR CH 3 6 ( 2 01 6 ) 1 6 –2 3
Fig. 3 – Low-density lipoprotein oxidizability and TAC before and after GTE intake in placebo-controlled study. After overnight fasting, 19 healthy volunteers consumed GTE capsules containing 1 g catechins or placebo capsules. The blood samples were collected before and at 1 hour after intake. A, The LDL oxidizability was measured by a lag-time assay. B, The serum TAC was determined by an ABTS radical scavenging assay. Values are means ± SE (n = 19). ***P < .001, indicating a significant change in LDL lag time or TAC compared with before intake by Bonferroni/Dunn test after 2-way repeated-measures ANOVA. ABTS indicates 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid).
fraction (EGCG: 64.8 ± 10.6 ng/mg LDL protein and ECG: 16.6 ± 2.9 ng/mg LDL protein; Table 3). These abundance ratios in both plasma and LDL were similar to the abundance rations in GTE (EGCG/ECG ≈ 4:1).
consisted largely of gallate-type catechins. The percentage of nonconjugated forms was 56.6%.
4. 3.4. Human study 2: Catechins concentration in LDL after GTE intake Finally, we determined the catechins concentration in the LDL fraction after GTE intake in an additional human study. The plasma catechins and caffeine concentrations are shown in Table 4. As a result of the present human study 1, the LDL oxidation lag time was significantly prolonged after the ingestion of GTE compared with that before intake (28.7 ± 1.8 minutes vs 33.0 ± 2.5 minutes; P < .05; Fig. 4). (−)-Epigallocatechin gallate and ECG, 2 major components of GTE, were markedly increased in the plasma (Table 4) and in the LDL fraction (Table 5) at 1 hour after GTE intake. The total concentration of catechins in the LDL fraction reached 202.9 ± 32.4 ng/mg LDL protein; these catechins
Discussion
It is well established that the increase in the susceptibility of LDL to oxidative processes accelerates atherosclerosis formation. Green tea catechins have been known to protect human LDL against oxidative attack [17,18], but the mechanisms by which the catechins inhibit LDL oxidation after ingestion have been poorly understood. In this study, we observed that oral acute intake of GTE resulted in increases in the LDL catechins level that effectively reduced the LDL oxidizability in healthy participants. The ability of catechins to prevent LDL oxidation is considered to be due to their radical-trapping effects and also their function as hydrogen donors to α-tocopherol radicals [32]. Moreover, the presence of the catechol structure of the B ring is known to be a prime determinant of the
Table 2 – Concentration of catechins and caffeine in plasma before and after GTE intake in human study 1 Placebo Before (μg/L) Total (free + conjugated) EGCG 27.2 ECG 27.5 EGCG3″Me 3.6 Caffeine 506.1 Total catechins 58.3 Free (nonconjugated) EGCG 7.4 ECG 2.0 EGCG3″Me 0.0 Caffeine 589.9 Total catechins 9.4
GTE After 1 h (μg/L)
After − before (μg/L)
Before (μg/L)
After 1 h (μg/L)
After − before (μg/L)
Free/total (%)
± ± ± ± ±
8.6 10.6 3.6 103.5 21.2
26.9 11.7 3.0 532.6 41.6
± ± ± ± ±
7.4 5.8 3.0 121.8 15.4
−0.3 −15.8 −0.6 26.5 −16.7
25.4 24.8 4.7 481.4 54.9
± ± ± ± ±
6.8 8.7 3.4 113.1 17.0
1913.1 681.2 157.9 432.7 2752.2
± ± ± ± ±
324.5 131.0 31.2 92.4 456.7
1887.7 656.4 153.2 −48.7 2697.3
– – – – –
± ± ± ± ±
2.1 1.2 0.0 149.1 2.5
11.1 2.6 2.1 535.9 15.9
± ± ± ± ±
2.8 1.3 2.1 154.9 4.5
3.7 0.6 2.1 −54.0 6.5
17.7 2.8 4.0 452.5 24.5
± ± ± ± ±
5.6 2.8 2.8 87.0 9.5
1486.2 424.9 105.2 413.9 2016.0
± ± ± ± ±
238.8 80.1 20.3 100.2 320.6
1468.5 422.1 101.2 −38.6 1991.5
77.8 64.3 66.1 – 73.8
Values are means ± SE (n = 19).
21
Table 3 – Concentration of catechins in LDL isolated after incubation of GTE in vitro
EGCG ECG
Concentration in plasma (μg/L)
Concentration in LDL (ng/mg LDL protein)
1033.5 ± 153.3 251.8 ± 31.0
64.8 ± 10.6 16.6 ± 2.9
Values are expressed as means ± SE (n = 5).
prevention of LDL oxidation in vitro [16]. Our previous study was in line with these theories; it demonstrated that the coincubation of green tea catechins with LDL led to the prolongation of the oxidation lag time, with the magnitude of the effect of the catechins ordered as follows: ECG > EC > EGCG > EGC [17]. With regard to the in vivo situation, however, it remains to be discussed whether the structural difference affects the stability of green tea catechins in circulating plasma and/or the affinity of these catechins to LDL. In the present study, we found that gallated catechins showed a greater ability to resist LDL oxidation than non–gallated catechins after plasma loading in vitro (ECG > EGCG > EC > EGC). Our preliminary data also indicate that the stability of each catechin after preincubation was higher in gallated types. In support of our data, Ishii et al [33] reported that the galloyl moiety participated in the interaction of EGCG with human serum albumin, which was of critical importance to the stability of EGCG. In order to clarify the relationship between orally ingested gallated catechins and the oxidative susceptibility of LDL, we conducted studies of humans who ingested GTE. Green tea extract is an aqueous extract from green tea leaves and is composed mostly of EGCG and ECG. In a randomized, placebocontrolled, double-blind, crossover design, GTE intake was found to reduce the susceptibility of LDL to oxidation and increase TAC in healthy Japanese men. After 1 hour, significant increases of plasma total catechins were observed, and approximately 74% of those catechins were detected in nonconjugated forms, a finding
Table 4 – Concentration of catechins and caffeine in plasma before and after GTE intake in human study 2 Before (μg/L)
Total (free + conjugated) EGCG 34.3 ± 14.0 ECG 78.5 ± 1.3 EGCG3″Me 0.0 ± 0.0 Caffeine 1098.4 ± 366.9 Total catechins 112.8 ± 14.1 Free (nonconjugated) EGCG 30.6 ± 19.7 ECG 31.9 ± 19.5 EGCG3″Me 0.0 ± 0.0 Caffeine 1115.6 ± 349.7 Total catechins 62.5 ± 32.4
After 1 h (μg/L)
1585.4 ± 393.2 472.0 ± 105.9 33.9 ± 14.8 1024.6 ± 316.8 2091.3 ± 512.1
After − before (μg/L) Free/total (%) 1551.1 393.5 33.9 −73.8 1978.4
– – – – –
998.6 ± 245.2 968.0 62.4 278.9 ± 50.1 247.0 62.8 6.5 ± 3.2 6.5 19.1 985.8 ± 300.3 −129.8 – 1283.9 ± 297.9 1221.5 61.7
Values are expressed as means ± SE (n = 5).
LDL lag time (min)
N U TR IT ION RE S EA RCH 3 6 ( 2 01 6 ) 1 6 –2 3
Fig. 4 – LDL oxidizability before and after GTE intake in human participants. After overnight fasting, 5 healthy volunteers consumed GTE capsules containing 1 g catechins. Blood samples were collected before and at 1 hour after intake. The LDL oxidizability was measured by a lag-time assay. Values are means ± SE (n = 5). *P < .05, indicating a significant change in lag-time compared with before intake by paired t test.
that was in line with a previous report suggesting that EGCG appeared mostly in nonconjugated forms [34]. On the other hand, other flavonols such as quercetin, rutin, and epicatechin have been reported to appear predominantly in the conjugated form [35–37]. In plasma, the nonconjugated form of catechins has been believed to be an important factor in antioxidant activity, because the chemical structure of catechins determines their antioxidant activities, and catechin metabolites have fewer antioxidant activities than nonconjugated catechins [38]. We therefore speculated that nonconjugated EGCG could accumulate in LDL after GTE intake and could exert a protective effect against oxidation. In addition, methylated EGCG was also detected in plasma after the intake of GTE. Although some reports demonstrated that O-methyl EGCG showed higher bioactivity than EGCG [39], O-methyl EGCG might not be greatly involved in the antioxidant effect of GTE intake due to the small amount of it present in plasma.
Table 5 – Concentration of catechins and caffeine in LDL before and after GTE intake in human study 2 Before After 1 h After − before (ng/mg LDL (ng/mg LDL (ng/mg LDL Free/total protein) protein) protein) (%) Total (free + conjugated) EGCG 10.7 ± 7.9 ECG 10.8 ± 6.8 EGCG3″Me 0.0 ± 0.0 Caffeine 41.6 ± 10.0 Total 21.5 ± 8.9 catechins Free (nonconjugated) EGCG 21.0 ± 13.8 ECG 31.9 ± 5.4 EGCG3″Me 0.0 ± 0.0 Caffeine 37.9 ± 9.5 Total 53.0 ± 18.5 catechins
145.9 ± 27.5 56.9 ± 5.1 0.0 ± 0.0 43.6 ± 11.2 202.9 ± 32.4
135.3 46.2 0.0 2.1 181.5
– – – – –
108.8 ± 24.9 46.8 ± 6.3 0.0 ± 0.0 43.6 ± 12.3 155.6 ± 30.4
87.8 14.9 0.0 5.8 102.7
64.9 32.2 – – 56.6
Values are expressed as means ± SE (n = 5).
22
N U TR IT ION RE S EAR CH 3 6 ( 2 01 6 ) 1 6 –2 3
To identify whether gallated catechins could bind to the LDL fraction, the concentration of catechins in the LDL fraction was measured after plasma loading with GTE. The EGCG and ECG concentrations in the LDL fraction were significantly increased, suggesting that gallated catechins were incorporated into the LDL in vitro. To support these data, the catechin levels in the LDL fraction after the intake of GTE were determined in an additional study involving 5 volunteers. As expected, the ingestion of GTE containing 1 g catechins caused a significant increase of catechins in the LDL fraction as well as a reduction of the susceptibility of LDL to oxidation. To our knowledge, only one published study evaluated the plasma and lipoprotein levels of catechins after repeated tea consumption and found that the accumulation of catechins in LDL particles was not sufficient to improve the resistance of LDL to oxidation [40]. Generally, catechins and proteins have strong interactions through hydrogen bonding and/or hydrophobic attractions. Catechins were reported to be localized mainly in the proteinrich fraction of plasma after repeated green tea consumption, and they were distributed in lipoprotein, with the high-density lipoprotein amount being the highest, the LDL amount the second highest, and the very low-density lipoprotein amount the lowest [40]. Low-density lipoprotein particles contain not only proteins but also phospholipids. Catechins, particular gallated catechins, also interact with the trimethylammonium group of phosphatidylcholine, which is the predominant phospholipid in humans [41]. Because the trimethylammonium group of phosphatidylcholine is a hydrophilic group, it might be located on the surface of LDL particles. In the present study, the amount of total catechins ingested was higher than in the previous study. High levels of EGCG in the nonconjugated form were detected in the LDL fraction, a finding that supports our hypothesis that EGCG directly contributed to the protection against LDL oxidation via its interaction with proteins and phosphatidylcholine. Importantly, the hypocholesterolemic activity of green tea catechins has also been reported in experimental animals [42–44] and humans [45]. The increase of plasma cholesterol concentration is an independent risk factor for atherosclerosis [46]. These previous reports, combined with our present results, suggest that green tea may prevent the progression of oxidized LDL–triggered atherosclerosis by means of the synergistic action between the cholesterol-lowering effect and the antioxidative property in humans. There are several potential limitations to our study. The LDL lag time was the only marker of LDL oxidation that we determined. Human study 2 included a limited number of participants (n = 5), but a marked accumulation of gallated catechins was observed after GTE intake in the LDL in all participants. The effect of a lower GTE intake must be evaluated. However, the determination of catechin levels not only in plasma but also in LDL enabled us to precisely evaluate the stability and distribution of each catechin in human blood and the contribution of each to the reduction of LDL oxidizability. In conclusion, after the ingestion of GTE, sufficient amounts of gallated catechins were accumulated in the LDL fraction and led to reduced oxidation of LDL in human participants. These results are consistent with our hypothesis that green tea catechins prevent LDL oxidation through a direct mechanism, at least in part. Taking green tea and/or gallated catechins could be effective for reducing the atherosclerosis risk associated with oxidative stress.
Acknowledgment This study was supported, in part, by JSPS KAKENHI (Grant No. 2510977) and by ITO EN, LTD. N.S. is supported as a research fellow of the Japan Society for the Promotion of Science. The authors are grateful to the volunteers for participating in the study. The authors declare that there is no conflict of interest associated with this manuscript.
REFERENCES
[1] Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989;320:915–24. [2] Steinberg D. The LDL, modification hypothesis of atherogenesis: an update. J Lipid Res 2009;50:S376–81 [Suppl.]. [3] Hertog MG, Feskens EJ, Kromhout D. Antioxidant flavonols and coronary heart disease risk. Lancet 1997;349:699. [4] Huxley RR, Neil HA. The relation between dietary flavonol intake and coronary heart disease mortality: a meta-analysis of prospective cohort studies. Eur J Clin Nutr 2003;57:904–8. [5] Fukushima Y, Ohie T, Yonekawa Y, Yonemoto K, Aizawa H, Mori Y, et al. Coffee and green tea as a large source of antioxidant polyphenols in the Japanese population. J Agric Food Chem 2009;57:1253–9. [6] Taguchi C, Fukushima Y, Kishimoto Y, Saita E, Suzuki-Sugihara N, Yoshida D, et al. Polyphenol intake from beverages in Japan over an 18-year period (1996-2013): trends by year, age, gender and season. J Nutr Sci Vitaminol (Tokyo) 2015;61:338–44. [7] Fukushima Y, Tashiro T, Kumagai A, Ohyanagi H, Horiuchi T, Takizawa K, et al. Coffee and beverages are the major contributors to polyphenol consumption from food and beverages in Japanese middle-aged women. J Nutr Sci 2014;3:e48. [8] Saito E, Inoue M, Sawada N, Shimazu T, Yamaji T, Iwasaki M, et al. Association of green tea consumption with mortality due to all causes and major causes of death in a Japanese population: the Japan Public Health Center-based Prospective Study (JPHC Study). Ann Epidemiol 2015;25:512–518 e3. [9] Nagao T, Hase T, Tokimitsu I. A green tea extract high in catechins reduces body fat and cardiovascular risks in humans. Obesity (Silver Spring) 2007;15:1473–83. [10] Khalesi S, Sun J, Buys N, Jamshidi A, Nikbakht-Nasrabadi E, Khosravi-Boroujeni H. Green tea catechins and blood pressure: a systematic review and meta-analysis of randomised controlled trials. Eur J Nutr 2014;53:1299–311. [11] Tsuneki H, Ishizuka M, Terasawa M, Wu JB, Sasaoka T, Kimura I. Effect of green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and on glucose metabolism in healthy humans. BMC Pharmacol 2004;4:18. [12] Hirano-Ohmori R, Takahashi R, Momiyama Y, Taniguchi H, Yonemura A, Tamai S, et al. Green tea consumption and serum malondialdehyde-modified LDL concentrations in healthy subjects. J Am Coll Nutr 2005;24:342–6. [13] Ohmori R, Kondo K, Momiyama Y. Antioxidant beverages: green tea intake and coronary artery disease. Clin Med Insight Cardiol 2014;8:7–11. [14] Zheng Y, Morris A, Sunkara M, Layne J, Toborek M, Hennig B. Epigallocatechin-gallate stimulates NF-E2–related factor and heme oxygenase-1 via caveolin-1 displacement. J Nutr Biochem 2012;23:163–8. [15] Naito Y, Yoshikawa T. Green tea and heart health. J Cardiovasc Pharmacol 2009;54:385–90. [16] Hirano R, Sasamoto W, Matsumoto A, Itakura H, Igarashi O, Kondo K. Antioxidant ability of various flavonoids against
N U TR IT ION RE S EA RCH 3 6 ( 2 01 6 ) 1 6 –2 3
[17]
[18]
[19] [20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
DPPH radicals and LDL oxidation. J Nutr Sci Vitaminol (Tokyo) 2001;47:357–62. Ohmori R, Iwamoto T, Tago M, Takeo T, Unno T, Itakura H, et al. Antioxidant activity of various teas against free radicals and LDL oxidation. Lipids 2005;40:849–53. Nakagawa K, Ninomiya M, Okubo T, Aoi N, Juneja LR, Kim M, et al. Tea catechin supplementation increases antioxidant capacity and prevents phospholipid hydroperoxidation in plasma of humans. J Agric Food Chem 1999;47:3967–73. Miyazawa T. Absorption, metabolism and antioxidative effects of tea catechin in humans. Biofactors 2000;13:55–9. Nakagawa K, Okuda S, Miyazawa T. Dose-dependent incorporation of tea catechins, (−)-epigallocatechin-3-gallate and (−)-epigallocatechin, into human plasma. Biosci Biotechnol Biochem 1997;61:1981–5. Lee MJ, Maliakal P, Chen L, Meng X, Bondoc FY, Prabhu S, et al. Pharmacokinetics of tea catechins after ingestion of green tea and (−)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability. Cancer Epidemiol Biomarkers Prev 2002;11:1025–32. Lee MJ, Wang ZY, Li H, Chen L, Sun Y, Gobbo S, et al. Analysis of plasma and urinary tea polyphenols in human subjects. Cancer Epidemiol Biomarkers Prev 1995;4:393–9. Chung BH, Segrest JP, Ray MJ, Brunzell JD, Hokanson JE, Krauss RM, et al. Single vertical spin density gradient ultracentrifugation. Methods Enzymol 1986;128:181–209. Lourenco CF, Gago B, Barbosa RM, de Freitas V, Laranjinha J. LDL isolated from plasma-loaded red wine procyanidins resist lipid oxidation and tocopherol depletion. J Agric Food Chem 2008;56:3798–804. Wu CH, Lin JA, Hsieh WC, Yen GC. Low-density-lipoprotein (LDL)–bound flavonoids increase the resistance of LDL to oxidation and glycation under pathophysiological concentrations of glucose in vitro. J Agric Food Chem 2009;57: 5058–64. Faul F, Erdfelder E, Lang AG, Buchner A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 2007;39:175–91. Nakayama S, Kishimoto Y, Saita E, Sugihara N, Toyozaki M, Taguchi C, et al. Pine bark extract prevents low-density lipoprotein oxidation and regulates monocytic expression of antioxidant enzymes. Nutr Res 2015;35:56–64. Unno T, Sagesaka YM, Kakuda T. Analysis of tea catechins in human plasma by high-performance liquid chromatography with solid-phase extraction. J Agric Food Chem 2005;53:9885–9. Nakagawa K, Nakayama K, Nakamura M, Sookwong P, Tsuduki T, Niino H, et al. Effects of co-administration of tea epigallocatechin-3-gallate (EGCG) and caffeine on absorption and metabolism of EGCG in humans. Biosci Biotechnol Biochem 2009;73:2014–7. Spáčila Zdeněk, Nováková Lucie, Solich Petr. Comparison of positive and negative ion detection of tea catechins using tandem mass spectrometry and ultra high performance liquid chromatography. Food Chem 2010;123:535–41. Miller NJ, Rice-Evans CA. Factors influencing the antioxidant activity determined by the ABTS.+ radical cation assay. Free Radic Res 1997;26:195–9.
23
[32] Zhu QY, Huang Y, Tsang D, Chen ZY. Regeneration of alpha-tocopherol in human low-density lipoprotein by green tea catechin. J Agric Food Chem 1999;47:2020–5. [33] Ishii T, Ichikawa T, Minoda K, Kusaka K, Ito S, Suzuki Y, et al. Human serum albumin as an antioxidant in the oxidation of (−)-epigallocatechin gallate: participation of reversible covalent binding for interaction and stabilization. Biosci Biotechnol Biochem 2011;75:100–6. [34] Chow HH, Cai Y, Alberts DS, Hakim I, Dorr R, Shahi F, et al. Phase I pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatechin gallate and polyphenon E. Cancer Epidemiol Biomarkers Prev 2001;10: 53–8. [35] Shimoi K, Okada H, Furugori M, Goda T, Takase S, Suzuki M, et al. Intestinal absorption of luteolin and luteolin 7-O-beta-glucoside in rats and humans. FEBS Lett 1998;438:220–4. [36] Piskula MK, Terao J. Accumulation of (−)-epicatechin metabolites in rat plasma after oral administration and distribution of conjugation enzymes in rat tissues. J Nutr 1998;128:1172–8. [37] Manach C, Morand C, Texier O, Favier ML, Agullo G, Demigne C, et al. Quercetin metabolites in plasma of rats fed diets containing rutin or quercetin. J Nutr 1995;125:1911–22. [38] Natsume M, Osakabe N, Yasuda A, Baba S, Tokunaga T, Kondo K, et al. in vitro antioxidative activity of (−)-epicatechin glucuronide metabolites present in human and rat plasma. Free Radic Res 2004;38:1341–8. [39] Oritani Y, Setoguchi Y, Ito R, Maruki-Uchida H, Ichiyanagi T, Ito T. Comparison of (−)-epigallocatechin-3-O-gallate (EGCG) and O-methyl EGCG bioavailability in rats. Biol Pharm Bull 2013;36:1577–82. [40] van het Hof KH, Wiseman SA, Yang CS, Tijburg LB. Plasma and lipoprotein levels of tea catechins following repeated tea consumption. Proc Soc Exp Biol Med 1999;220:203–9. [41] Kobayashi M, Nishizawa M, Inoue N, Hosoya T, Yoshida M, Ukawa Y, et al. Epigallocatechin gallate decreases the micellar solubility of cholesterol via specific interaction with phosphatidylcholine. J Agric Food Chem 2014;62:2881–90. [42] Muramatsu K, Fukuyo M, Hara Y. Effect of green tea catechins on plasma cholesterol level in cholesterol-fed rats. J Nutr Sci Vitaminol (Tokyo) 1986;32:613–22. [43] Ikeda I, Imasato Y, Sasaki E, Nakayama M, Nagao H, Takeo T, et al. Tea catechins decrease micellar solubility and intestinal absorption of cholesterol in rats. Biochim Biophys Acta 1992; 1127:141–6. [44] Ikeda I, Kobayashi M, Hamada T, Tsuda K, Goto H, Imaizumi K, et al. Heat-epimerized tea catechins rich in gallocatechin gallate and catechin gallate are more effective to inhibit cholesterol absorption than tea catechins rich in epigallocatechin gallate and epicatechin gallate. J Agric Food Chem 2003;51:7303–7. [45] Kajimoto O, Kajimoto Y, Yabune M, Nozawa A, Nagata K, Kakuda T. Tea catechins reduce serum cholesterol levels in mild and borderline hypercholesterolemia patients. J Clin Biochem Nutr 2003;33:101–11. [46] Rose G, Shipley M. Plasma cholesterol concentration and death from coronary heart disease: 10 year results of the Whitehall study. Br Med J (Clin Res Ed) 1986;293:306–7.
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Original Research
Green tea catechins prevent low-density lipoprotein oxidation via their accumulation in low-density lipoprotein particles in humans Norie Suzuki-Sugihara a , Yoshimi Kishimoto b,⁎, Emi Saita b , Chie Taguchi b , Makoto Kobayashi c , Masaki Ichitani c , Yuuichi Ukawa c , Yuko M. Sagesaka c , Emiko Suzuki a , Kazuo Kondo b, d a
Department of Nutrition and Food Science, Graduate School of Humanities and Sciences, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan b Endowed Research Department “Food for Health,” Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan c Central Reseach Institute, ITO EN, LTD., 21 Mekami, Makinohara, Shizuoka 421-0516, Japan d Institute of Life Innovation Studies, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma 374-0193, Japan
ARTI CLE I NFO
A BS TRACT
Article history:
Green tea is rich in polyphenols, including catechins which have antioxidant activities and are
Received 3 August 2015
considered to have beneficial effects on cardiovascular health. In the present study, we
Revised 29 October 2015
investigated the effects of green tea catechins on low-density lipoprotein (LDL) oxidation in vitro
Accepted 30 October 2015
and in human studies to test the hypothesis that catechins are incorporated into LDL particles and exert antioxidant properties. In a randomized, placebo-controlled, double-blind, crossover trial, 19 healthy men ingested green tea extract (GTE) in the form of capsules at a dose of 1 g total
Keywords:
catechin, of which most (>99%) was the gallated type. At 1 hour after ingestion, marked
Tea
increases of the plasma concentrations of (−)-epigallocatechin gallate and (−)-epicatechin
Epigallocatechin gallate
gallate were observed. Accordingly, the plasma total antioxidant capacity was increased, and
Lipoproteins
the LDL oxidizability was significantly reduced by the ingestion of GTE. We found that gallated
Low-density lipoprotein
catechins were incorporated into LDL particles in nonconjugated forms after the incubation of
Lipid peroxidation
GTE with plasma in vitro. Moreover, the catechin-incorporated LDL was highly resistant to
Humans
radical-induced oxidation in vitro. An additional human study with 5 healthy women confirmed that GTE intake sufficiently increased the concentration of gallated catechins, mainly in nonconjugated forms in LDL particles, and reduced the oxidizability of LDL. In conclusion, green tea catechins are rapidly incorporated into LDL particles and play a role in reducing LDL oxidation in humans, which suggests that taking green tea catechins is effective in reducing atherosclerosis risk associated with oxidative stress. © 2015 Elsevier Inc. All rights reserved.
Abbreviations: ANOVA, analysis of variance; EC, (−)-epicatechin; ECG, (−)-epicatechin gallate; EDTA, ethylenediamine-tetraacetic acid; EGC, (−)-epigallocatechin; EGCG, (−)-epigallocatechin gallate; EGCG3″Me, (−)-epigallocatechin 3-O-(3-O-methyl) gallate; GTE, green tea extract; LDL, low-density lipoprotein; TAC, total antioxidant capacity. ⁎ Corresponding author. Tel.: +81 3 5978 5810; fax: +81 3 5978 2694. E-mail address: [email protected] (Y. Kishimoto). http://dx.doi.org/10.1016/j.nutres.2015.10.012 0271-5317/© 2015 Elsevier Inc. All rights reserved.
17
N U TR IT ION RE S EA RCH 3 6 ( 2 01 6 ) 1 6 –2 3
1.
Introduction
Various lines of research indicate that oxidized low-density lipoprotein (LDL) within the arterial wall promotes the development of atherosclerosis [1]. Protection against LDL oxidation is an effective strategy to prevent atherosclerosis [2], and growing evidence from epidemiologic studies has shown that dietary antioxidants contribute to the prevention of coronary heart disease [3,4]. Polyphenols are found in most foods and beverages of plant origin and are known to have antioxidant properties. Green tea is a large source of polyphenols among the Japanese population [5–7], and epidemiologic studies by the Japan Public Health Center–based Prospective Study group have revealed that the consumption of green tea can reduce the risk of all-cause mortality and the risk of mortality due to the 3 leading causes of death including heart disease, cerebrovascular disease, and respiratory disease [8]. Green tea extract (GTE) contains a number of catechins, including (−)-epigallocatechin gallate (EGCG), (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC), and (−)-epicatechin (EC) and has been known to have many beneficial properties that can help prevent atherosclerotic diseases via the regulation of obesity [9], hypertension [10], diabetes [11], and oxidative stress [12,13]. The molecular mechanism of the antiatherogenic activity of green tea catechins, particularly EGCG, which is the most abundant catechin, has also been determined in several studies [14,15]. Among the various antiatherogenic activities, the antioxidant effects are presumed to play a major role in mediating the cardioprotective role of green tea catechins. Our previous study reported that green tea catechins inhibited LDL oxidation when they were directly added to isolated LDL [16]. Moreover, we reported that the acute intake of green tea (5 g of green tea powder) successfully increased the resistance against LDL oxidation as well as the plasma catechin levels in healthy volunteers [17]. Nakagawa et al [18] reported that EGCG was incorporated into human plasma and could decrease the levels of plasma phosphatidylcholine hydroperoxide, a marker of oxidized lipoproteins, after a single oral intake of green tea catechins (254 mg of catechins containing 82 mg of EGCG). These findings prompted us to hypothesize that catechins, especially gallated catechins, may play a preventive role in LDL oxidation by being incorporated into LDL particles in plasma. It was recognized that catechins undergo glucuronidation and sulfation in the intestinal mucosa, liver, and kidney, and only 0.2% to 5.0% of the ingested doses were present in the circulating plasma after the ingestion of tea catechins [19–22]. Compared with other flavonoids like quercetin, catechins can be present in a free (nonconjugated) “active” form in human plasma [19]; however, no information regarding the incorporation of catechins into LDL is available. Thus, the aim of this study was to test our hypothesis that green tea catechins are incorporated into LDL particles and exert antioxidant properties. The protective mechanism of green tea catechins against LDL oxidation was investigated in vitro and in human studies by measuring the LDL oxidizability and catechin concentration both in plasma and in LDL.
2.
Methods and materials
2.1.
Reagents
Decaffeinated GTE powder (THEA-FLAN 90S), mainly composed of gallated catechins (as shown in Table 1), was obtained from ITO EN, LTD (Shizuoka, Japan). Purified catechins (EGCG, ECG, EGC, and EC), β-glucuronidase from Escherichia coli, and sulfatase type VIII from abalone entrails were purchased from Sigma-Aldrich (St Louis, MO, USA). The (−)-epigallocatechin-3O-(3-O-methyl) gallate (EGCG3″Me) was purchased from Nagara Science (Gifu, Japan). A Micro BCA Protein Assay Kit was obtained from Thermo Fisher Scientific (Rockford, IL, USA). Ethyl gallate was purchased from Tokyo Chemical Industry (Tokyo, Japan). Ethyl acetate, acetonitrile, and formic acid were purchased from Wako Pure Chemicals (Osaka, Japan). Green tea extract capsules containing 1 g of catechins and matching placebo capsules containing starch were formulated by the Sunsho Pharmaceutical (Shizuoka, Japan).
2.2.
Experimental design
To evaluate the effect of green tea catechins on LDL oxidizability and their ability to bind to LDL both in vitro and in vivo, we conducted human studies with healthy volunteers. These studies were approved by the Ethics Committee of Ochanomizu University in accordance with the principles of the Declaration of Helsinki. In addition, all participants were fully informed regarding the content and methods of this trial. Written informed consent was obtained prior to enrollment from each participant. Blood was drawn from an antecubital vein after fasting and was collected in tubes containing ethylenediamine-tetraacetic acid (EDTA) or clot accelerant for the separation of plasma and serum. Plasma samples were immediately prepared by centrifugation, and LDL was separated by single-spin density gradient ultracentrifugation at 417 000g for 40 minutes at 4°C [23]. The LDL protein concentration was determined using a Micro BCA Protein Assay Kit.
2.2.1.
Plasma loading with catechins (in vitro study)
Human plasma from healthy volunteers was incubated with purified catechins (EGCG, ECG, EGC, or EC, at 50 μmol/L) or GTE (3 μg/mL) for 20 minutes at 37°C, and LDL was immediately isolated [24,25]. The LDL oxidizability and catechins concentration in plasma or LDL were measured. Table 1 – Catechin composition of GTE (g/100 g) EGCG ECG Gallocatechin gallate (GCG) EGC Catechin gallate (CG) Gallocatechin (GC) EC Catechin (C) Abbreviation: ND, not detected.
53.6 12.5 2.8 0.4 0.4 0.1 ND ND
18 2.2.2.
N U TR IT ION RE S EAR CH 3 6 ( 2 01 6 ) 1 6 –2 3
Human study 1
We conducted a randomized, placebo-controlled, doubleblind, 2-way crossover trial (Fig. 1A). Participants in the study were recruited by public offering on our Web site. Nineteen healthy male volunteers ranging from 25 to 53 years of age were enrolled in the study. The sample size was estimated from previous studies with a similar design. Based on the power calculations made using G*power 3 software [26], the number of participants (n = 19) was sufficient for detecting a difference in LDL lag time with at least 80% power with P < .05 using a 2-tailed test. The participants were asked to complete 2 sessions in which GTE capsules containing 1 g of catechins or matching placebo capsules were ingested in a randomized order. Test days were separated by at least 2week washout periods. In the 12 hours before the tests, participants were not permitted to eat or drink except for water. Blood samples were collected from the participants before and 1 hour after consumption. The LDL oxidizability, serum total antioxidant capacity (TAC) value, and catechins concentration in plasma were measured.
2.2.3.
Human study 2
In order to confirm the amount of catechins in the LDL fraction after the intake of GTE, we recruited 5 healthy female volunteers (ranging from 22 to 30 years of age) and conducted
an additional human study with GTE capsules containing 1 g of catechin (Fig. 1B). The LDL oxidizability and catechins concentration in LDL were measured.
2.3.
Measurement of LDL oxidizability
Low-density lipoprotein oxidizability was determined by lag-time assay as described previously [16,27]. The LDL samples (final concentration of protein, 70 μg/mL) were oxidized by 2,2-azobis-4-methoxy-2,4-dimethylvaleronitrile. The kinetics of LDL oxidation were determined by monitoring the absorbance of conjugated dienes at 234 nm using a DU800 spectrophotometer (Beckman Coulter, Brea, CA, USA) at 4-minute intervals at 37°C. The lag time for the start of LDL oxidation was defined as the time interval between the initiation and intercept of the 2 tangents drawn to the lag and propagation phases of the absorbance curve at 234 nm.
2.4.
Determination of catechin levels in plasma and LDL
Sample preparation and extraction was carried out by the method of Unno et al [28] and Nakagawa et al [29] with some modifications. Each plasma (200 μL) or LDL fraction (80 μL) was mixed with 10 μL of 10% (wt/vol) ascorbic acid solution, and the mixture was stored at −80°C until analysis. To determine the
A. Randomized, placebo-controlled, double-blind, crossover trial (Human study-1)
B. Additional study (Human study-2)
Enrollment
Enrollment
Assessment for eligibility (n=19)
Randomization (n=19)
Study period-1
Placebo (n=13)
GTE (n=6)
Assessment for eligibility (n=5)
GTE (n=5)
Analyzed (n=5) LDL lag-time LDL catechin concentration
Washout (2 weeks)
Study period-2
Placebo (n=6)
GTE (n=13)
Analyzed (n=19) LDL lag-time TAC Plasma catechin concentration
Fig. 1 – Flowchart of the study design. Human study 1 was conducted with 19 healthy men in a randomized, placebo-controlled, double-blind, crossover method (A), and human study 2 was additionally conducted with 5 healthy women to confirm the amount of catechins in the LDL fraction (B).
total amounts of catechins, including free (nonconjugated) and conjugated forms as well as their metabolites, β-glucuronidase (250 units) and sulfatase (20 units) were added to the plasma samples. The mixture was incubated at 37°C for 45 minutes. To determine the nonconjugated form of catechins, the samples were analyzed without these enzymes. For the extraction of plasma catechins, the mixture was mixed with 300 μL of 0.1 mol/L phosphate buffer containing 0.5 mM EDTA-2Na (pH 3.5) and 40 μL of ethyl gallate (2.5 mg/L) solution containing 0.5% (wt/vol) ascorbic acid and 0.01 mg/mL EDTA-2Na as an internal standard. Then, 500 μL of ethyl acetate was added to the mixture, and this was vortexed. After centrifugation at 21 000g for 10 minutes at 4°C, the supernatants obtained were collected. This extraction was repeated 3 times, and the supernatants obtained were combined, evaporated, and dissolved in 200 μL of 6% (vol/vol) acetonitrile containing 0.094% (wt/vol) formic acid. The solution was centrifuged at 21 000g for 10 minutes at 4°C, and the supernatants were analyzed by ultraperformance liquid chromatography (UPLC)-/MS/MS using the method of Spáčila et al [30]. The UPLC–MS/MS system used included the Acquity UPLC (Waters, Milford, MA, USA) and the Acquity TQD Mass Spectrometer (Waters). The data were acquired and processed using MassLynx software version 4.1 (Waters). The analytical column was C18 BEH (100 mm × 2.1 mm i.d.; 1.7 μm; Waters). The concentration of catechins in the LDL was normalized to the protein content in the fraction.
2.5.
Measurement of serum TAC
Serum TAC was determined by a 2,2′-azinobis-(3-ethylbenzothiazoline6-sulfonic acid) radical scavenging assay (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions [31]. The values were quantified as millimolar Trolox equivalents.
2.6.
Statistical analyses
All data are presented as means ± SE. Statistical analyses were performed using analysis of variance (ANOVA), followed by a post hoc test. When appropriate, repeated-measures ANOVA was used. Results were considered significant when P < .05. The statistical analyses were performed using the GraphPad Prism 5 software package (GraphPad Software, La Jolla, CA, USA).
3.
Results
3.1. Comparison of the effect of 4 major catechins on LDL oxidation lag time in vitro After the incubation of EGCG, EGC, ECG, or EC with plasma in vitro, the oxidizability was determined in each isolated LDL by lag-time assay. As shown in Fig. 2, the LDL oxidation lag time was prolonged the most by ECG, second-most by EGCG, and third-most by EC, and was unchanged by EGC at 50 μmol/L. In our preliminary experiment, the concentration of each catechin remaining after preincubation was higher in plasma with EGCG and ECG than in plasma with EGC and EC (data not shown).
19
LDL lag time (min)
N U TR IT ION RE S EA RCH 3 6 ( 2 01 6 ) 1 6 –2 3
Fig. 2 – Comparison of the effect of 4 major catechins on LDL oxidation lag time in vitro. Plasma was incubated with 4 major catechins (EGCG, ECG, EGC, or EC), and then LDL was isolated. The LDL oxidizability was measured by a lag-time assay. Values are means ±SE (n = 5). ***P < .001 and **P < .01, indicating a significant change in lag time compared with before intake by Dunnett test after 1-way repeated-measures ANOVA.
3.2. Human study 1: LDL oxidizability and plasma catechins concentration after GTE intake Nineteen healthy participants consumed GTE capsules containing 1 g of catechins or placebo capsules in a randomized order. Before this study, we performed a pilot trial to determine the time to the peak concentration of plasma catechins and found that EGCG and ECG appeared transiently in the plasma and reached their highest level at 1 hour after the GTE intake. As a result, the LDL oxidation lag time was significantly prolonged after the ingestion of GTE compared with that before the GTE intake (31.5 ± 1.4 minutes vs 38.6 ± 1.5 minutes; P < .001; Fig. 3A). This finding indicated that GTE could reduce LDL oxidizability in human participants as well as in vitro. We also found that GTE significantly increased the serum TAC value 1 hour after intake (P < .001; Fig. 3B). In contrast, no significant changes in either LDL lag time or TAC were observed after the ingestion of the placebo. The plasma catechins and caffeine concentration are shown in Table 2. The baseline concentrations of each catechin were at low levels as they were measured in the morning after fasting. (−)-Epigallocatechin gallate and ECG, 2 major components of GTE, and EGCG3″Me, a metabolite of EGCG, were markedly increased in the plasma 1 hour after GTE intake. The total concentration of catechins reached 2752.2 ± 456.7 μg/L; these consisted mostly of gallate-type catechins. The increased catechins were largely found in nonconjugated forms (73.8%). There were no significant changes in the concentrations of catechins after the intake of the placebo.
3.3. Concentration of catechins in LDL isolated after incubation of GTE with plasma in vitro To examine the possibility that the increased plasma catechins accumulate in the LDL fraction after GTE intake, GTE was preincubated with plasma in vitro, and then LDL was isolated from the plasma by ultracentrifugation. (−)-Epigallocatechin gallate and ECG were detected both in the plasma (EGCG: 1033.5 ± 153.3 μg/L and ECG: 251.8 ± 31.0 μg/L) and in the LDL
20
A
B
LDL lag time (min)
TAC (mM Trolox equivalent)
N U TR IT ION RE S EAR CH 3 6 ( 2 01 6 ) 1 6 –2 3
Fig. 3 – Low-density lipoprotein oxidizability and TAC before and after GTE intake in placebo-controlled study. After overnight fasting, 19 healthy volunteers consumed GTE capsules containing 1 g catechins or placebo capsules. The blood samples were collected before and at 1 hour after intake. A, The LDL oxidizability was measured by a lag-time assay. B, The serum TAC was determined by an ABTS radical scavenging assay. Values are means ± SE (n = 19). ***P < .001, indicating a significant change in LDL lag time or TAC compared with before intake by Bonferroni/Dunn test after 2-way repeated-measures ANOVA. ABTS indicates 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid).
fraction (EGCG: 64.8 ± 10.6 ng/mg LDL protein and ECG: 16.6 ± 2.9 ng/mg LDL protein; Table 3). These abundance ratios in both plasma and LDL were similar to the abundance rations in GTE (EGCG/ECG ≈ 4:1).
consisted largely of gallate-type catechins. The percentage of nonconjugated forms was 56.6%.
4. 3.4. Human study 2: Catechins concentration in LDL after GTE intake Finally, we determined the catechins concentration in the LDL fraction after GTE intake in an additional human study. The plasma catechins and caffeine concentrations are shown in Table 4. As a result of the present human study 1, the LDL oxidation lag time was significantly prolonged after the ingestion of GTE compared with that before intake (28.7 ± 1.8 minutes vs 33.0 ± 2.5 minutes; P < .05; Fig. 4). (−)-Epigallocatechin gallate and ECG, 2 major components of GTE, were markedly increased in the plasma (Table 4) and in the LDL fraction (Table 5) at 1 hour after GTE intake. The total concentration of catechins in the LDL fraction reached 202.9 ± 32.4 ng/mg LDL protein; these catechins
Discussion
It is well established that the increase in the susceptibility of LDL to oxidative processes accelerates atherosclerosis formation. Green tea catechins have been known to protect human LDL against oxidative attack [17,18], but the mechanisms by which the catechins inhibit LDL oxidation after ingestion have been poorly understood. In this study, we observed that oral acute intake of GTE resulted in increases in the LDL catechins level that effectively reduced the LDL oxidizability in healthy participants. The ability of catechins to prevent LDL oxidation is considered to be due to their radical-trapping effects and also their function as hydrogen donors to α-tocopherol radicals [32]. Moreover, the presence of the catechol structure of the B ring is known to be a prime determinant of the
Table 2 – Concentration of catechins and caffeine in plasma before and after GTE intake in human study 1 Placebo Before (μg/L) Total (free + conjugated) EGCG 27.2 ECG 27.5 EGCG3″Me 3.6 Caffeine 506.1 Total catechins 58.3 Free (nonconjugated) EGCG 7.4 ECG 2.0 EGCG3″Me 0.0 Caffeine 589.9 Total catechins 9.4
GTE After 1 h (μg/L)
After − before (μg/L)
Before (μg/L)
After 1 h (μg/L)
After − before (μg/L)
Free/total (%)
± ± ± ± ±
8.6 10.6 3.6 103.5 21.2
26.9 11.7 3.0 532.6 41.6
± ± ± ± ±
7.4 5.8 3.0 121.8 15.4
−0.3 −15.8 −0.6 26.5 −16.7
25.4 24.8 4.7 481.4 54.9
± ± ± ± ±
6.8 8.7 3.4 113.1 17.0
1913.1 681.2 157.9 432.7 2752.2
± ± ± ± ±
324.5 131.0 31.2 92.4 456.7
1887.7 656.4 153.2 −48.7 2697.3
– – – – –
± ± ± ± ±
2.1 1.2 0.0 149.1 2.5
11.1 2.6 2.1 535.9 15.9
± ± ± ± ±
2.8 1.3 2.1 154.9 4.5
3.7 0.6 2.1 −54.0 6.5
17.7 2.8 4.0 452.5 24.5
± ± ± ± ±
5.6 2.8 2.8 87.0 9.5
1486.2 424.9 105.2 413.9 2016.0
± ± ± ± ±
238.8 80.1 20.3 100.2 320.6
1468.5 422.1 101.2 −38.6 1991.5
77.8 64.3 66.1 – 73.8
Values are means ± SE (n = 19).
21
Table 3 – Concentration of catechins in LDL isolated after incubation of GTE in vitro
EGCG ECG
Concentration in plasma (μg/L)
Concentration in LDL (ng/mg LDL protein)
1033.5 ± 153.3 251.8 ± 31.0
64.8 ± 10.6 16.6 ± 2.9
Values are expressed as means ± SE (n = 5).
prevention of LDL oxidation in vitro [16]. Our previous study was in line with these theories; it demonstrated that the coincubation of green tea catechins with LDL led to the prolongation of the oxidation lag time, with the magnitude of the effect of the catechins ordered as follows: ECG > EC > EGCG > EGC [17]. With regard to the in vivo situation, however, it remains to be discussed whether the structural difference affects the stability of green tea catechins in circulating plasma and/or the affinity of these catechins to LDL. In the present study, we found that gallated catechins showed a greater ability to resist LDL oxidation than non–gallated catechins after plasma loading in vitro (ECG > EGCG > EC > EGC). Our preliminary data also indicate that the stability of each catechin after preincubation was higher in gallated types. In support of our data, Ishii et al [33] reported that the galloyl moiety participated in the interaction of EGCG with human serum albumin, which was of critical importance to the stability of EGCG. In order to clarify the relationship between orally ingested gallated catechins and the oxidative susceptibility of LDL, we conducted studies of humans who ingested GTE. Green tea extract is an aqueous extract from green tea leaves and is composed mostly of EGCG and ECG. In a randomized, placebocontrolled, double-blind, crossover design, GTE intake was found to reduce the susceptibility of LDL to oxidation and increase TAC in healthy Japanese men. After 1 hour, significant increases of plasma total catechins were observed, and approximately 74% of those catechins were detected in nonconjugated forms, a finding
Table 4 – Concentration of catechins and caffeine in plasma before and after GTE intake in human study 2 Before (μg/L)
Total (free + conjugated) EGCG 34.3 ± 14.0 ECG 78.5 ± 1.3 EGCG3″Me 0.0 ± 0.0 Caffeine 1098.4 ± 366.9 Total catechins 112.8 ± 14.1 Free (nonconjugated) EGCG 30.6 ± 19.7 ECG 31.9 ± 19.5 EGCG3″Me 0.0 ± 0.0 Caffeine 1115.6 ± 349.7 Total catechins 62.5 ± 32.4
After 1 h (μg/L)
1585.4 ± 393.2 472.0 ± 105.9 33.9 ± 14.8 1024.6 ± 316.8 2091.3 ± 512.1
After − before (μg/L) Free/total (%) 1551.1 393.5 33.9 −73.8 1978.4
– – – – –
998.6 ± 245.2 968.0 62.4 278.9 ± 50.1 247.0 62.8 6.5 ± 3.2 6.5 19.1 985.8 ± 300.3 −129.8 – 1283.9 ± 297.9 1221.5 61.7
Values are expressed as means ± SE (n = 5).
LDL lag time (min)
N U TR IT ION RE S EA RCH 3 6 ( 2 01 6 ) 1 6 –2 3
Fig. 4 – LDL oxidizability before and after GTE intake in human participants. After overnight fasting, 5 healthy volunteers consumed GTE capsules containing 1 g catechins. Blood samples were collected before and at 1 hour after intake. The LDL oxidizability was measured by a lag-time assay. Values are means ± SE (n = 5). *P < .05, indicating a significant change in lag-time compared with before intake by paired t test.
that was in line with a previous report suggesting that EGCG appeared mostly in nonconjugated forms [34]. On the other hand, other flavonols such as quercetin, rutin, and epicatechin have been reported to appear predominantly in the conjugated form [35–37]. In plasma, the nonconjugated form of catechins has been believed to be an important factor in antioxidant activity, because the chemical structure of catechins determines their antioxidant activities, and catechin metabolites have fewer antioxidant activities than nonconjugated catechins [38]. We therefore speculated that nonconjugated EGCG could accumulate in LDL after GTE intake and could exert a protective effect against oxidation. In addition, methylated EGCG was also detected in plasma after the intake of GTE. Although some reports demonstrated that O-methyl EGCG showed higher bioactivity than EGCG [39], O-methyl EGCG might not be greatly involved in the antioxidant effect of GTE intake due to the small amount of it present in plasma.
Table 5 – Concentration of catechins and caffeine in LDL before and after GTE intake in human study 2 Before After 1 h After − before (ng/mg LDL (ng/mg LDL (ng/mg LDL Free/total protein) protein) protein) (%) Total (free + conjugated) EGCG 10.7 ± 7.9 ECG 10.8 ± 6.8 EGCG3″Me 0.0 ± 0.0 Caffeine 41.6 ± 10.0 Total 21.5 ± 8.9 catechins Free (nonconjugated) EGCG 21.0 ± 13.8 ECG 31.9 ± 5.4 EGCG3″Me 0.0 ± 0.0 Caffeine 37.9 ± 9.5 Total 53.0 ± 18.5 catechins
145.9 ± 27.5 56.9 ± 5.1 0.0 ± 0.0 43.6 ± 11.2 202.9 ± 32.4
135.3 46.2 0.0 2.1 181.5
– – – – –
108.8 ± 24.9 46.8 ± 6.3 0.0 ± 0.0 43.6 ± 12.3 155.6 ± 30.4
87.8 14.9 0.0 5.8 102.7
64.9 32.2 – – 56.6
Values are expressed as means ± SE (n = 5).
22
N U TR IT ION RE S EAR CH 3 6 ( 2 01 6 ) 1 6 –2 3
To identify whether gallated catechins could bind to the LDL fraction, the concentration of catechins in the LDL fraction was measured after plasma loading with GTE. The EGCG and ECG concentrations in the LDL fraction were significantly increased, suggesting that gallated catechins were incorporated into the LDL in vitro. To support these data, the catechin levels in the LDL fraction after the intake of GTE were determined in an additional study involving 5 volunteers. As expected, the ingestion of GTE containing 1 g catechins caused a significant increase of catechins in the LDL fraction as well as a reduction of the susceptibility of LDL to oxidation. To our knowledge, only one published study evaluated the plasma and lipoprotein levels of catechins after repeated tea consumption and found that the accumulation of catechins in LDL particles was not sufficient to improve the resistance of LDL to oxidation [40]. Generally, catechins and proteins have strong interactions through hydrogen bonding and/or hydrophobic attractions. Catechins were reported to be localized mainly in the proteinrich fraction of plasma after repeated green tea consumption, and they were distributed in lipoprotein, with the high-density lipoprotein amount being the highest, the LDL amount the second highest, and the very low-density lipoprotein amount the lowest [40]. Low-density lipoprotein particles contain not only proteins but also phospholipids. Catechins, particular gallated catechins, also interact with the trimethylammonium group of phosphatidylcholine, which is the predominant phospholipid in humans [41]. Because the trimethylammonium group of phosphatidylcholine is a hydrophilic group, it might be located on the surface of LDL particles. In the present study, the amount of total catechins ingested was higher than in the previous study. High levels of EGCG in the nonconjugated form were detected in the LDL fraction, a finding that supports our hypothesis that EGCG directly contributed to the protection against LDL oxidation via its interaction with proteins and phosphatidylcholine. Importantly, the hypocholesterolemic activity of green tea catechins has also been reported in experimental animals [42–44] and humans [45]. The increase of plasma cholesterol concentration is an independent risk factor for atherosclerosis [46]. These previous reports, combined with our present results, suggest that green tea may prevent the progression of oxidized LDL–triggered atherosclerosis by means of the synergistic action between the cholesterol-lowering effect and the antioxidative property in humans. There are several potential limitations to our study. The LDL lag time was the only marker of LDL oxidation that we determined. Human study 2 included a limited number of participants (n = 5), but a marked accumulation of gallated catechins was observed after GTE intake in the LDL in all participants. The effect of a lower GTE intake must be evaluated. However, the determination of catechin levels not only in plasma but also in LDL enabled us to precisely evaluate the stability and distribution of each catechin in human blood and the contribution of each to the reduction of LDL oxidizability. In conclusion, after the ingestion of GTE, sufficient amounts of gallated catechins were accumulated in the LDL fraction and led to reduced oxidation of LDL in human participants. These results are consistent with our hypothesis that green tea catechins prevent LDL oxidation through a direct mechanism, at least in part. Taking green tea and/or gallated catechins could be effective for reducing the atherosclerosis risk associated with oxidative stress.
Acknowledgment This study was supported, in part, by JSPS KAKENHI (Grant No. 2510977) and by ITO EN, LTD. N.S. is supported as a research fellow of the Japan Society for the Promotion of Science. The authors are grateful to the volunteers for participating in the study. The authors declare that there is no conflict of interest associated with this manuscript.
REFERENCES
[1] Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989;320:915–24. [2] Steinberg D. The LDL, modification hypothesis of atherogenesis: an update. J Lipid Res 2009;50:S376–81 [Suppl.]. [3] Hertog MG, Feskens EJ, Kromhout D. Antioxidant flavonols and coronary heart disease risk. Lancet 1997;349:699. [4] Huxley RR, Neil HA. The relation between dietary flavonol intake and coronary heart disease mortality: a meta-analysis of prospective cohort studies. Eur J Clin Nutr 2003;57:904–8. [5] Fukushima Y, Ohie T, Yonekawa Y, Yonemoto K, Aizawa H, Mori Y, et al. Coffee and green tea as a large source of antioxidant polyphenols in the Japanese population. J Agric Food Chem 2009;57:1253–9. [6] Taguchi C, Fukushima Y, Kishimoto Y, Saita E, Suzuki-Sugihara N, Yoshida D, et al. Polyphenol intake from beverages in Japan over an 18-year period (1996-2013): trends by year, age, gender and season. J Nutr Sci Vitaminol (Tokyo) 2015;61:338–44. [7] Fukushima Y, Tashiro T, Kumagai A, Ohyanagi H, Horiuchi T, Takizawa K, et al. Coffee and beverages are the major contributors to polyphenol consumption from food and beverages in Japanese middle-aged women. J Nutr Sci 2014;3:e48. [8] Saito E, Inoue M, Sawada N, Shimazu T, Yamaji T, Iwasaki M, et al. Association of green tea consumption with mortality due to all causes and major causes of death in a Japanese population: the Japan Public Health Center-based Prospective Study (JPHC Study). Ann Epidemiol 2015;25:512–518 e3. [9] Nagao T, Hase T, Tokimitsu I. A green tea extract high in catechins reduces body fat and cardiovascular risks in humans. Obesity (Silver Spring) 2007;15:1473–83. [10] Khalesi S, Sun J, Buys N, Jamshidi A, Nikbakht-Nasrabadi E, Khosravi-Boroujeni H. Green tea catechins and blood pressure: a systematic review and meta-analysis of randomised controlled trials. Eur J Nutr 2014;53:1299–311. [11] Tsuneki H, Ishizuka M, Terasawa M, Wu JB, Sasaoka T, Kimura I. Effect of green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and on glucose metabolism in healthy humans. BMC Pharmacol 2004;4:18. [12] Hirano-Ohmori R, Takahashi R, Momiyama Y, Taniguchi H, Yonemura A, Tamai S, et al. Green tea consumption and serum malondialdehyde-modified LDL concentrations in healthy subjects. J Am Coll Nutr 2005;24:342–6. [13] Ohmori R, Kondo K, Momiyama Y. Antioxidant beverages: green tea intake and coronary artery disease. Clin Med Insight Cardiol 2014;8:7–11. [14] Zheng Y, Morris A, Sunkara M, Layne J, Toborek M, Hennig B. Epigallocatechin-gallate stimulates NF-E2–related factor and heme oxygenase-1 via caveolin-1 displacement. J Nutr Biochem 2012;23:163–8. [15] Naito Y, Yoshikawa T. Green tea and heart health. J Cardiovasc Pharmacol 2009;54:385–90. [16] Hirano R, Sasamoto W, Matsumoto A, Itakura H, Igarashi O, Kondo K. Antioxidant ability of various flavonoids against
N U TR IT ION RE S EA RCH 3 6 ( 2 01 6 ) 1 6 –2 3
[17]
[18]
[19] [20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
DPPH radicals and LDL oxidation. J Nutr Sci Vitaminol (Tokyo) 2001;47:357–62. Ohmori R, Iwamoto T, Tago M, Takeo T, Unno T, Itakura H, et al. Antioxidant activity of various teas against free radicals and LDL oxidation. Lipids 2005;40:849–53. Nakagawa K, Ninomiya M, Okubo T, Aoi N, Juneja LR, Kim M, et al. Tea catechin supplementation increases antioxidant capacity and prevents phospholipid hydroperoxidation in plasma of humans. J Agric Food Chem 1999;47:3967–73. Miyazawa T. Absorption, metabolism and antioxidative effects of tea catechin in humans. Biofactors 2000;13:55–9. Nakagawa K, Okuda S, Miyazawa T. Dose-dependent incorporation of tea catechins, (−)-epigallocatechin-3-gallate and (−)-epigallocatechin, into human plasma. Biosci Biotechnol Biochem 1997;61:1981–5. Lee MJ, Maliakal P, Chen L, Meng X, Bondoc FY, Prabhu S, et al. Pharmacokinetics of tea catechins after ingestion of green tea and (−)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability. Cancer Epidemiol Biomarkers Prev 2002;11:1025–32. Lee MJ, Wang ZY, Li H, Chen L, Sun Y, Gobbo S, et al. Analysis of plasma and urinary tea polyphenols in human subjects. Cancer Epidemiol Biomarkers Prev 1995;4:393–9. Chung BH, Segrest JP, Ray MJ, Brunzell JD, Hokanson JE, Krauss RM, et al. Single vertical spin density gradient ultracentrifugation. Methods Enzymol 1986;128:181–209. Lourenco CF, Gago B, Barbosa RM, de Freitas V, Laranjinha J. LDL isolated from plasma-loaded red wine procyanidins resist lipid oxidation and tocopherol depletion. J Agric Food Chem 2008;56:3798–804. Wu CH, Lin JA, Hsieh WC, Yen GC. Low-density-lipoprotein (LDL)–bound flavonoids increase the resistance of LDL to oxidation and glycation under pathophysiological concentrations of glucose in vitro. J Agric Food Chem 2009;57: 5058–64. Faul F, Erdfelder E, Lang AG, Buchner A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 2007;39:175–91. Nakayama S, Kishimoto Y, Saita E, Sugihara N, Toyozaki M, Taguchi C, et al. Pine bark extract prevents low-density lipoprotein oxidation and regulates monocytic expression of antioxidant enzymes. Nutr Res 2015;35:56–64. Unno T, Sagesaka YM, Kakuda T. Analysis of tea catechins in human plasma by high-performance liquid chromatography with solid-phase extraction. J Agric Food Chem 2005;53:9885–9. Nakagawa K, Nakayama K, Nakamura M, Sookwong P, Tsuduki T, Niino H, et al. Effects of co-administration of tea epigallocatechin-3-gallate (EGCG) and caffeine on absorption and metabolism of EGCG in humans. Biosci Biotechnol Biochem 2009;73:2014–7. Spáčila Zdeněk, Nováková Lucie, Solich Petr. Comparison of positive and negative ion detection of tea catechins using tandem mass spectrometry and ultra high performance liquid chromatography. Food Chem 2010;123:535–41. Miller NJ, Rice-Evans CA. Factors influencing the antioxidant activity determined by the ABTS.+ radical cation assay. Free Radic Res 1997;26:195–9.
23
[32] Zhu QY, Huang Y, Tsang D, Chen ZY. Regeneration of alpha-tocopherol in human low-density lipoprotein by green tea catechin. J Agric Food Chem 1999;47:2020–5. [33] Ishii T, Ichikawa T, Minoda K, Kusaka K, Ito S, Suzuki Y, et al. Human serum albumin as an antioxidant in the oxidation of (−)-epigallocatechin gallate: participation of reversible covalent binding for interaction and stabilization. Biosci Biotechnol Biochem 2011;75:100–6. [34] Chow HH, Cai Y, Alberts DS, Hakim I, Dorr R, Shahi F, et al. Phase I pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatechin gallate and polyphenon E. Cancer Epidemiol Biomarkers Prev 2001;10: 53–8. [35] Shimoi K, Okada H, Furugori M, Goda T, Takase S, Suzuki M, et al. Intestinal absorption of luteolin and luteolin 7-O-beta-glucoside in rats and humans. FEBS Lett 1998;438:220–4. [36] Piskula MK, Terao J. Accumulation of (−)-epicatechin metabolites in rat plasma after oral administration and distribution of conjugation enzymes in rat tissues. J Nutr 1998;128:1172–8. [37] Manach C, Morand C, Texier O, Favier ML, Agullo G, Demigne C, et al. Quercetin metabolites in plasma of rats fed diets containing rutin or quercetin. J Nutr 1995;125:1911–22. [38] Natsume M, Osakabe N, Yasuda A, Baba S, Tokunaga T, Kondo K, et al. in vitro antioxidative activity of (−)-epicatechin glucuronide metabolites present in human and rat plasma. Free Radic Res 2004;38:1341–8. [39] Oritani Y, Setoguchi Y, Ito R, Maruki-Uchida H, Ichiyanagi T, Ito T. Comparison of (−)-epigallocatechin-3-O-gallate (EGCG) and O-methyl EGCG bioavailability in rats. Biol Pharm Bull 2013;36:1577–82. [40] van het Hof KH, Wiseman SA, Yang CS, Tijburg LB. Plasma and lipoprotein levels of tea catechins following repeated tea consumption. Proc Soc Exp Biol Med 1999;220:203–9. [41] Kobayashi M, Nishizawa M, Inoue N, Hosoya T, Yoshida M, Ukawa Y, et al. Epigallocatechin gallate decreases the micellar solubility of cholesterol via specific interaction with phosphatidylcholine. J Agric Food Chem 2014;62:2881–90. [42] Muramatsu K, Fukuyo M, Hara Y. Effect of green tea catechins on plasma cholesterol level in cholesterol-fed rats. J Nutr Sci Vitaminol (Tokyo) 1986;32:613–22. [43] Ikeda I, Imasato Y, Sasaki E, Nakayama M, Nagao H, Takeo T, et al. Tea catechins decrease micellar solubility and intestinal absorption of cholesterol in rats. Biochim Biophys Acta 1992; 1127:141–6. [44] Ikeda I, Kobayashi M, Hamada T, Tsuda K, Goto H, Imaizumi K, et al. Heat-epimerized tea catechins rich in gallocatechin gallate and catechin gallate are more effective to inhibit cholesterol absorption than tea catechins rich in epigallocatechin gallate and epicatechin gallate. J Agric Food Chem 2003;51:7303–7. [45] Kajimoto O, Kajimoto Y, Yabune M, Nozawa A, Nagata K, Kakuda T. Tea catechins reduce serum cholesterol levels in mild and borderline hypercholesterolemia patients. J Clin Biochem Nutr 2003;33:101–11. [46] Rose G, Shipley M. Plasma cholesterol concentration and death from coronary heart disease: 10 year results of the Whitehall study. Br Med J (Clin Res Ed) 1986;293:306–7.