Epigallocatechin Gallate Decreases the Micellar Solubility of

Article pubs.acs.org/JAFC

Epigallocatechin Gallate Decreases the Micellar Solubility of Cholesterol via Specific Interaction with Phosphatidylcholine Makoto Kobayashi,*,†,‡ Masato Nishizawa,§ Nao Inoue,‡ Takahiro Hosoya,§ Masahito Yoshida,∥ Yuichi Ukawa,† Yuko M Sagesaka,† Takayuki Doi,∥ Tsutomu Nakayama,⊥ Shigenori Kumazawa,§ and Ikuo Ikeda‡ †

Central Research Institute, ITO EN, Ltd., Shizuoka 421-0516, Japan Laboratory of Food and Biomolecular Science, Department of Food Function and Health, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi 981-8555, Japan § Department of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan ∥ Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 981-8578, Miyagi, Japan ⊥ Department of Food Science and Technology, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan ‡

ABSTRACT: The mechanisms underlying the effect of epigallocatechin gallate (EGCG) on the micellar solubility of cholesterol were examined. EGCG eliminated both cholesterol and phosphatidylcholine (PC) from bile salt micelles in a dose-dependent manner in vitro. When the bile salt micelles contained a phospholipid other than PC, neither cholesterol nor the phospholipid was eliminated following the addition of EGCG. When vesicles comprised of various phospholipids were prepared and, EGCG was added to the vesicles, EGCG effectively and exclusively eliminated only PC. An intermolecular nuclear Overhauser effect (NOE) was observed between PC and EGCG in bile salt micelles with EGCG added, but not between cholesterol and EGCG, by using a NOE-correlated spectroscopy nuclear magnetic resonance method. The results of binding analyses using surface plasmon resonance (SPR) showed that EGCG did not bind to cholesterol. These observations strongly suggest that EGCG decreases the micellar solubility of cholesterol via specific interaction with PC. KEYWORDS: epigallocatechin gallate, phosphatidylcholine, micellar solubility of cholesterol, nuclear Overhauser effect, surface plasmon resonance

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that were cannulated in the thoracic duct.16,17 Raederstorff et al. also showed that purified EGCG decreased the micellar solubility of cholesterol in vitro.25 They also showed, via light scattering, that the addition of EGCG to micelles altered the size of the micelles.25 Vermeer et al. showed in an in vitro study that green tea extract reduced the incorporation of cholesterol into mixed micelles.26 These micellar cholesterol-lowering activities mediated by green tea catechins with a galloyl moiety may be the cause of the increased fecal excretion of cholesterol observed in experimental animals and hypocholesterolemic activity in experimental animals and humans.14,18−22 However, how green tea catechins associate with cholesterol is not well understood. When green tea catechins with a galloyl moiety were added to a bile salt micellar solution, the solution immediately turned turbid and precipitates were observed.16,17 We previously reported the existence of a hydrophobic domain in catechins with a galloyl moiety that was not present in free catechins.27 We also showed that green tea catechins with a galloyl moiety have a higher affinity for hydrophobic lipid bilayers than free catechins.27 Therefore, it is possible that green tea catechins with a galloyl moiety can interact directly with cholesterol through their hydrophobicity since cholesterol

INTRODUCTION Tea, which is produced from the leaves of Camellia sinensis,, is the most popular beverage, and it is consumed worldwide. It is categorized as green, oolong, or black, depending on whether the tea leaves are nonfermented, partially fermented, or completely fermented/oxidized, respectively.1 Green tea contains catechins as its major polyphenols. The catechins extracted from green tea leaves mainly include (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), and (−)-epigallocatechin gallate (EGCG) (Figure 1). They are categorized into two classes: free catechins, such as EC and EGC, and catechins with a galloyl moiety, such as ECG and EGCG. During the pasteurization of tea drinks, approximately 50% of tea catechins are epimerized at the C2 position, and (−)-catechin (C), (−)-gallocatechin (GC), (−)-catechin gallate (CG), and (−)-gallocatechin gallate (GCG) are formed.2,3 Therefore, canned and bottled tea drinks mainly contain eight catechins. Green tea catechins have been shown to have various physiological functions, including antiatherogenic,4 antiobesity,5−8 antioxidative,9,10 anticarcinogenic,11,12 and hypotriacylglycerolemic13,14 activities. The hypocholesterolemic activity of green tea catechins has also been reported in experimental animals,14−20 humans,21,22 and m-analyses.23,24 We previously reported that green tea catechins with a galloyl moiety effectively eliminated cholesterol from bile salt micelles in vitro and reduced the lymphatic recovery of cholesterol in rats © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2881

December March 12, March 15, March 16,

19, 2013 2014 2014 2014

dx.doi.org/10.1021/jf405591g | J. Agric. Food Chem. 2014, 62, 2881−2890

Journal of Agricultural and Food Chemistry

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Figure 1. Chemical structures of various green tea catechins and heat-epimerized tea catechins: (left) green tea catechins; (right) heat-epimerized tea catechins. Louis, MO, USA). Sodium 2,2-silapentane-5-sulfonate (DSS), 2-(9anthroyloxy)stearic acid (2-AS), and 12-(9-anthroyloxy)stearic acid (12-AS) were purchased from Wako. Deuterium oxide (D2O; deuterium purity, 99.8%) was purchased from Kanto Chemicals Co. (Tokyo, Japan). Effects of EGCG, EGC, and GCG on the Micellar Solubility of Cholesterol and PC in Vitro. The effects of purified catechins on the micellar solubility of cholesterol and PC were examined as described in our previous study.17 A bile salt micellar solution containing 6.6 mmol/L sodium taurocholate, 0.6 mmol/L PC, 0.5 mmol/L cholesterol, 132 mmol/L NaCl, and 15 mmol/L sodium phosphate (pH 6.8) was prepared by sonication and stored at 37 °C for at least 24 h. Various amounts of EGCG, EGC, or GCG in deionized water (100 μL) stored at 37 °C were added to the 3 mL micellar solutions. The mixture was incubated for 1 h at 37 °C. The supernatant was passed through a 0.2 μm syringe filter (25 mm; GDD/X; Whatman Inc., Piscataway, NJ, USA), and the cholesterol and fatty acid contents originating from the phospholipids were analyzed by gas chromatography using an SPB-1 column (Supelco, Bellefonte, PA, USA) and a DB-WAX column (Agilent Technologies, Santa Clara, CA, USA), respectively. Effect of EGCG on the Micellar Solubility of Cholesterol and Different Types of Phospholipids in Vitro. Five types of bile salt micellar solutions containing 0.6 mmol/L PC, PA, PE, PI, or PS were prepared as previously described. The effects of EGCG on the micellar

is also a hydrophobic molecule. However, we provided direct experimental evidence that the EGCG molecule interacts with a lipid bilayer containing dimyristoylphosphatidylcholine by solid-state 31P and 2H nuclear magnetic resonance (NMR).28 We also showed that ECG and EGCG interact with the surface of lipid membranes through the trimethylammonium group in phosphatidylcholine (PC) by using solution NMR techniques.29 Therefore, it is possible that the interaction between green tea catechins with a galloyl moiety and phosphatidylcholine leads to the precipitation of micellar cholesterol and, as a result, the micellar solubility of cholesterol is decreased. In this study, the mechanisms underlying the EGCG-mediated decrease in the micellar solubility of cholesterol were examined.

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MATERIALS AND METHODS

The purified catechins, EGCG, GCG, ECG, CG, EGC, GC, EC, and C, were purchased from Wako Pure Chemicals (Osaka, Japan). The purity of all catechins was confirmed to be >98% by NMR analysis. PC from egg yolk, phosphatidic acid (PA) from egg yolk, phosphatidylethanolamine (PE) from egg yolk, phosphatidylinositol (PI) from soybean, phosphatidylserine (PS) from soybean, 1-palmitoyl-2oleoylphosphatidylcholine (POPC), and 1,2-dioleoylphosphatidic acid (DOPA) were purchased from Sigma-Aldrich Co. LLC (St. 2882



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solubility of both cholesterol and the phospholipids were examined as previously described. Effect of EGCG on the Vesicular Solubility of Different Phospholipids in Vitro. For the purpose of conveniently examining the interaction between EGCG and phospholipids, five vesicular solutions containing 0.6 mmol/L PC, PA, PE, PI, or PS in ultrapure water were prepared by sonication and stored at 37 °C. Various amounts of EGCG in ultrapure water (in a volume of 200 μL) were added to the 6 mL vesicular solutions. The mixture was incubated for 1 h at 37 °C and then centrifuged at 25000g for 1 h. In a preliminary study, we confirmed that centrifugation did not precipitate the phospholipids from vesicular solution. The supernatant was passed through a 0.2 μm syringe filter (25 mm; GDD/X; Whatman), and the phospholipids in the filtrate were chemically analyzed as previously described.30 Effects of EGCG, GCG, and EGC on the Vesicular Solubility of PC in Vitro. A vesicular solution containing 0.6 mmol/L PC in ultrapure water was prepared by sonication and stored at 37 °C. The effects of EGCG, GCG, and EGC on the vesicular solubility of PC were examined as previously described. Determination of the Apparent Equilibrium Dissociation Constant (KD) for the Binding of Eight Green Tea Catechins to POPC Using an SPR-Based Biosensor Assay. Experiments were performed using L1 sensor chips (GE Healthcare U.K. Ltd., Buckinghamshire, England) on a Biacore X100 Plus package analytical system (GE Healthcare) at the Biomedical Research Core of the Tohoku University Graduate School of Medicine. The running buffer used for all experiments was a 0.01 mmol/L phosphate buffer containing 2.7 mmol/L KCl and 137 mmol/L NaCl (pH 6.8). The washing solution was 40 mmol/L N-octyl-β-D-glucopyranoside. All solutions were freshly prepared, degassed, and filtered through a 0.2 μm filter. A 50 μmol amount of POPC (the ligand; phase transition temperature, −2 °C) or DOPA (the control ligand; phase transition temperature, −8 °C) in CHCl3 was placed in a round-bottomed glass flask, and the solvent was removed under nitrogen. POPC was then resuspended in 0.5 mL of the running buffer, and DOPA was resuspended in 0.5 mL of the running buffer containing 500 mmol/L NaCl to suppress the electrostatic repulsion between DOPA and the alkyl surface of the L1 sensor chip for effective immobilization because DOPA is negatively charged. The resulting phospholipid suspension was mixed vigorously and then recapitulated by five freeze−thaw cycles. To obtain homogeneous liposomal solutions, the liposomal solutions were then passed through 50 nm pore polycarbonate filters 20 times using a mini-extruder (Avanti Polar Lipids Inc., Alabaster, AL, USA). The liposomal solutions were diluted 20-fold with the running buffer with or without 500 mmol/L NaCl. The final concentration of POPC or DOPA in the liposomal solution was 0.5 mmol/L. The liposome was immobilized on the L1 sensor chip surface using the method of Subasinghe et al.31 with some modifications. The alkyl surface of the L1 sensor chip was cleaned by a 50 μL injection of 40 mmol/L N-octyl-β-D- glucopyranoside at a flow rate of 10 μL/min. The POPC or DOPA liposomal solution was then immediately applied to the L1 sensor chip surface at a flow rate of 5 μL/min. The immobilization levels of POPC and DOPA were equalized as accurately as possible using an “aim for immobilized level” command. To remove any multilamellar structures from the synthetic phospholipid bilayer surface, 2 μL of 10 mmol/L sodium hydroxide was injected at a flow rate of 5 μL/min. Generally, the injection of bovine serum albumin solution is recommended for sealing the remainder of the alkyl surface of the L1 sensor chip, which is not bound to the phospholipid bilayer. However, in preliminary studies, we observed that EGCG (one of the analytes) nonspecifically and tightly bound to bovine serum albumin and the surface of the L1 sensor chip. Therefore, 5 μL of a 1 mmol/L EGCG solution prepared in the running buffer was repeatedly injected until the baseline elevation stopped to suppress nonspecific binding to the gap. Then, the running buffer was injected until a stable baseline was achieved. The aim of this injection was to dissociate the EGCG from the phospholipid bilayer. The operating temperature was 25 °C.

An SPR-based biosensor assay was performed using the single-cycle kinetics method.32 This method involves the sequential injection of various concentrations of an analyte without any regeneration steps. Eight green tea catechin solutions were prepared at 5−80 μmol/L (green tea catechins with a galloyl moiety) and 50−800 μmol/L (green tea catechins without a galloyl moiety) in running buffer. To associate the green tea catechins with POPC or DOPA, the green tea catechin solutions were injected over the lipid surface at a flow rate of 5 μL/min for 1,080 s. To dissociate the POPC− or DOPA−green tea catechin complexes, the green tea catechin solutions were replaced with the running buffer at a flow rate of 5 μL/min for 180 s. The sensorgrams obtained from the injection of the running buffer were subtracted from the sensorgrams obtained from the injection of green tea catechin solutions. Then, the sensorgram obtained from the reference flow cell, in which DOPA was immobilized, was subtracted from the sensorgram obtained from the measurement flow cell, in which POPC was immobilized, to yield apparent binding responses. The sensorgrams did not have appropriate curvature for curve fitting to determine association and dissociation rates. Furthermore, an unexpected finding was that EGCG weakly bound to DOPA in a dosedependent manner. Therefore, the apparent KD for the eight green tea catechins to POPC were calculated from the equilibrium resonance signals as a function of analyte concentration according to the manufacturer’s instructions (Biacore X100 evaluation software). 33 1 H NMR Measurement of EGCG in the Absence and Presence of Bile Salt Micelles. A bile salt micellar solution (in D2O) containing 6.6 mmol/L sodium taurocholate, 0.6 mmol/L PC, 0.5 mmol/L cholesterol, 132 mmol/L NaCl, and 15 mmol/L NaD2PO4 (pD 6.8) was prepared by sonication and stored at room temperature overnight. EGCG was then added to 1 mL of the micellar solution. The amount of EGCG added was adjusted to 0.5 mmol/L of micelles. Then, 20 μL of a 1 mg/mL DSS solution was added to the micellar solution as an internal standard, and the mixture was incubated for 1 h at room temperature. 1H NMR spectra were recorded on an AVANCE III 400 (Bruker BioSpin, Billerica, MA, USA) operating at a resonance frequency of 399.7 MHz with 20 Hz spinning at 300 K. Typically, an excitation pulse length of 14.0 μs and a pulse delay time of 1.0 μs were used. Signal assignments were performed as described in our previous study.29 NOESY Experiments on Bile Salt Micelle/EGCG Samples. Preparation of a bile salt micellar solution (in D2O) and addition of EGCG and DSS solution were performed as described above. NOESY spectra were acquired on an AVANCE III 400 (Bruker BioSpin) operating at a resonance frequency of 399.7 MHz with 0 Hz spinning at 300 K. The typical acquisition parameters used were as follows: a pulse delay of 2.0 s, 0.03 and 0.3 s acquisition times in the f1 and f 2 axes, respectively, a mixing time of 800 ms, 64 scans, and 1,024 data points acquired in both the f1 and f 2 axes. The sine-bell-squared function was adopted as a window function. Measuring the Fluorescence of 2-AS or 12-AS in Bile Salt Micelles in the Presence of EGCG. A bile salt micellar solution containing 6.6 mmol/L sodium taurocholate, 0.6 mmol/L PC from egg yolk, 0.5 mmol/L cholesterol, 132 mmol/L NaCl, and 15 mmol/L NaH2PO4 (pH 6.8) containing 1 mmol/L 2-AS or 12-AS was prepared by sonication and stored at room temperature overnight. The micellar solutions (100 μL) were incubated with 100 μL of phosphate-buffered saline (PBS) containing EGCG for 20 min at 20 °C. The amount of EGCG added was adjusted to 0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 mmol/L of micelles. The fluorescence of 2-AS (excitation, 362 nm; emission, 446 nm) or 12-AS (excitation, 381 nm; emission, 446 nm) in the bile salt micellar solution was measured.27 The fluorescence intensity was calculated as the percentage of the fluorescence intensity of 2-AS or 12-AS in the absence of EGCG. Binding Analyses of EGCG to Cholesterol by an SPR-Based Biosensor Assay. Binding analyses were performed using L1 sensor chips on the Biacore X100 Plus package analytical system. Preparations of 0.5 mmol/L POPC or DOPA liposomes with or without 0.05 mmol/L or 0.15 mmol/L cholesterol and immobilizations to the phospholipid bilayer with or without cholesterol on the surface of the L1 sensor chip were performed as described above with some 2883



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Figure 2. Effect of green tea catechins on micellar solubility of cholesterol and phosphatidylcholine in vitro. Comparison of effect of epigallocatechin gallate (EGCG) and epigallocatechin (EGC) on the micellar solubility of cholesterol (A) and phosphatidylcholine (B) in vitro. Comparison of the effect of EGCG and gallocatechin gallate (GCG) on micellar solubility of cholesterol (C) and phosphatidylcholine (D) in vitro. Various amounts of EGCG, EGC, or GCG in deionized water (100 μL) stored at 37 °C were added to 3 mL micellar solutions. The micellar cholesterol and phosphatidylcholine concentration at 0 mmol/L tea catechins was adjusted to 100%. Data are means ± SE of triplicate experiments. Means not sharing a common letter differ significantly (P < 0.05). Two-way ANOVA: (A−D) effect of catechins type, P < 0.0001; effect of catechin concentration, P < 0.0001; interaction between catechins type and catechin concentration, P < 0.0001. modification. The 10 mmol/L sodium hydroxide injection was avoided because this decreased immobilization levels, particularly in the measurement flow cell, in which cholesterol was immobilized. EGCG solutions were prepared at concentrations from 0.05 to 1.6 mmol/L using the running buffer. The solutions were injected over the lipid surface at a flow rate of 5 μL/min for 60 s. The solutions were then replaced by the running buffer at a flow rate of 5 μL/min for 60 s. The operating temperature was routinely 25 °C. Statistical Analysis. Data are expressed as the means ± standard error (SE). Statistical analysis of the data was performed by two-way ANOVA followed by a Bonferroni/Dunn test to evaluate the significance of differences between pairs of means. Differences were considered significant at P < 0.05.

into the bile micelles in Figure 2A,C were 94.2% and 91.0%, respectively. These values were adjusted to 100% in Figure 2. EGCG and GCG effectively decreased the micellar solubilities of cholesterol and PC in a dose-dependent manner (Figure 2C,D). EGC slightly decreased the micellar solubilities of cholesterol and PC (Figure 2A,B). The concentrations of micellar cholesterol and PC following the addition of GCG were significantly lower than those following the addition of EGCG at concentrations of 2 and 3 mmol/L of micelles (Figure 2C,D). Effects of EGCG on the Micellar Solubility of Cholesterol and Different Phospholipids in Vitro. In the absence of EGCG, the percentages of cholesterol incorporated into the bile micelles containing PC, PA, PE, PI, and PS were 85.5%, 91.0%, 95.4%, 84.0%, and 94.8%, respectively. These values were adjusted to 100% in Figure 3A. When the bile salt

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RESULTS Effects of EGCG, EGC, and GCG on the Micellar Solubility of Cholesterol and PC in Vitro. In the absence of green tea catechins, the amounts of cholesterol incorporated 2884



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Figure 3. Effect of epigallocatechin gallate on the micellar solubility of cholesterol (A) and different phospholipids (B) in vitro. Various amounts of epigallocatechin gallate (EGCG) in deionized water (100 μL) stored at 37 °C were added to 3 mL micellar solution containing 0.6 mmol/L phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), or phosphatidylserine (PS). The micellar cholesterol and phospholipid concentrations at 0 mmol/L of EGCG were adjusted to 100%. EGCG was added at 1 and 2 mmol/L. Data are means ± SE of triplicate experiments. Means not sharing a common letter differ significantly (P < 0.05). Two-way ANOVA: (A, B) effect of phospholipid type, P < 0.0001; effect of EGCG concentration, P < 0.0001; interaction between phospholipid type and EGCG concentration, P < 0.0001.

micelles contained a phospholipid other than PC, neither cholesterol nor the phospholipid was eliminated following the addition of EGCG. (Figures 3A,B). Effect of EGCG on the Vesicular Solubility of Different Phospholipids in Vitro. EGCG effectively decreased the vesicular solubility of PC and slightly decreased the vesicular solubility of PS (Figure 4). EGCG did not decrease the vesicular solubilities of PA, PE, or PI. Effects of EGCG, GCG, and EGC on the Vesicular Solubility of PC in Vitro. EGCG and GCG similarly and effectively decreased the vesicular solubility of PC in a dosedependent manner (Figure 5). EGC slightly decreased the vesicular solubility of PC (Figure 5). The concentrations of vesicular PC following the addition of EGCG or GCG were significantly lower than those following the addition of EGC at concentrations of 0.5, 1, and 2 mmol/L of vesicles (Figure 5). Determination of the Apparent KD for the Binding of Eight Green Tea Catechins to POPC by an SPR-Based Biosensor Assay. We immobilized DOPA in the reference flow cell as the control ligand because green tea catechins were not expected to interact with DOPA based on the result shown in Figure 4. However, the green tea catechins weakly bound to DOPA, and the resonances in the reference flow cell with immobilized DOPA were slightly elevated. Therefore, we calculated the apparent KD values for the eight green tea catechins to POPC. The immobilization levels of POPC in the measurement flow cell and DOPA in the reference flow cell were 5,705.6 and 5,090.3 resonance units (RU), respectively. Figure 6 shows the sensorgrams of sequentially injected EGCG and EGC obtained using a single-cycle kinetics method. The sensorgrams were obtained by subtracting of the sensorgram obtained for the reference flow cell from the sensorgram obtained for the measurment flow cell. The binding of EGCG

Figure 4. Effect of epigallocatechin gallate on the vesicular solubility of different phospholipids in vitro. Various amounts of epigallocatechin gallate (EGCG) in ultrapure water (200 μL) were added to the vesicular solutions (6 mL). The mixture was incubated for 1 h at 37 °C and then centrifuged at 25000g for 1 h. In a preliminary study, we confirmed that centrifugation did not precipitate the phospholipids from the vesicular solution. The vesicular phospholipid concentration at 0 mmol/L EGCG was adjusted to 100%. EGCG was added at 2 mmol/L. Data are means ± SE of triplicate experiments. Means not sharing a common letter differ significantly (P < 0.05). Two-way ANOVA: effect of phospholipid type, P < 0.0001; effect of EGCG concentration, P < 0.0001; interaction between phospholipid type and EGCG concentration, P < 0.0001.

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Table 1. Apparent Equilibrium Dissociation Constants for the Binding of Eight Green Tea Catechins to POPC Obtained from Surface Plasmon Resonance-Based Biosensor Assaysa apparent KD (mol/L) EGCG GCG ECG CG EGC GC EC C

1.535 2.485 2.888 3.662 6.685 9.488 1.036 4.316

× × × × × × × ×

10−5 10−5 10−5 10−5 10−4 10−4 10−3 10−4

a Eight green tea catechin solutions were prepared at 5−80 μmol/L (green tea catechins with a galloyl moiety) and 50−800 μmol/L (green tea catechins without a galloyl moiety) in the running buffer. To measure the association of green tea catechin with POPC or DOPA, the solutions were injected over the lipid surface at a flow rate of 5 μL/min for 1,080 s. To measure the dissociation of the POPC− or DOPA−green tea catechin complex, the green tea catechin solutions were then replaced with the running buffer at a flow rate of 5 μL/min for 180 s. The sensorgrams obtained from the injection of the running buffer were subtracted from the sensorgrams obtained from the injection of the green tea catechin solutions. Then, the sensorgram obtained from the reference flow cell, in which DOPA was immobilized, was subtracted from the sensorgram obtained from the measurement flow cell, in which POPC was immobilized, to yield the apparent binding responses. Because the sensorgrams did not have the appropriate curvature for curve fitting to determine the association and dissociation rates and because EGCG weakly bound to DOPA, the apparent equilibrium dissociation constants (KD) for eight green tea catechins to POPC were calculated from the equilibrium resonance signals as a function of analyte concentration according to the manufacturer’s instructions (Biacore X100 evaluation software).33

Figure 5. Effect of epigallocatechin gallate, gallocatechin gallate, and epigallocatechin on the vesicular solubility of phosphatidylcholine in vitro. Various amounts of epigallocatechin gallate (EGCG), gallocatechin gallate (GCG), and epigallocatechin (EGC) in ultrapure water (200 μL) were added to the PC vesicles (6 mL). The mixture was incubated for 1 h at 37 °C, and then centrifuged at 25000g for 1 h. The vesicular phosphatidylcholine concentration at 0 mmol/L of tea catechins was adjusted to 100%. Tea catechins were added at 0.5, 1, and 2 mmol/L. Data are means ± SE of triplicate experiments. Means not sharing a common letter differ significantly (P < 0.05). Two-way ANOVA: effect of phospholipid type, P < 0.0001; effect of EGCG concentration, P < 0.0001; interaction between phospholipid type and EGCG concentration, P < 0.0001.

and EGC to POPC was initially fast but then slowed, and concentration-dependent binding was observed (Figure 6A,B). Table 1 shows the apparent KD for eight green tea catechins to POPC. The apparent KD values of the green tea catechins with a galloy moiety to POPC were lower than those of the green tea catechins without a galloyl moiety. These results show that green tea catechins with a galloyl moiety have higher affinity for POPC than those without a galloyl moiety.

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H NMR Measurement of EGCG in the Absence and Presence of Bile Salt Micelles. Interaction of EGCG with the bile salt micelles resulted in a considerable shift of the A-

Figure 6. Sensorgrams showing the binding of various concentrations of (−)-epigallocatechin gallate (A) and (−)-epigallocatechin (B) obtained from single-cycle kinetics assays. EGCG in running buffer at 5, 10, 20, 40, and 80 μmol/L (A) and EGC in running buffer at 50, 100, 200, 400, and 800 μmol/L (B) were sequentially injected over the lipid surface at a flow rate of 5 μL/min for 1,080 s. To dissociate the POPC− or DOPA−green tea catechin complex, the green tea catechin solutions were then replaced with the running buffer at a flow rate of 5 μL/min for 180 s. The sensorgrams obtained from the injection of the running buffer were subtracted from the sensorgrams obtained from the injection of the green tea catechin solutions. Then, the sensorgram obtained from the reference flow cell, in which DOPA was immobilized, was subtracted from the sensorgram obtained from the measurement flow cell, in which POPC was immobilized, to yield the apparent binding responses. 2886



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ring (6- and 8-H), the B-ring (2′- and 6′-H), C-ring (2- and 3H), and the galloyl moiety (2″- and 6″-H) signals in EGCG (Figure 7). These changes of chemical shift following the

Figure 7. Change values of the chemical shift of (−)-epigallocatechin gallate following its addition to the bile salt micelles. The changes of (−)-epigallocatechin gallate (EGCG) signals in the presence of the bile salt micelles are compared to those in the absence of the bile salt micelles.

addition of EGCG to the bile salt micelles were similar to those to isotropic bicelles, which are composed of PC and frequently used as a lipid bilayer model, in our previous study.29 These observations suggest that EGCG interacts with PC in the bile salt micells as well as in lipid bilayers. 4α- and 4β-H signals were not observed because they overlapped large signals derived from several molecules that existed in the bile salt micelles. NOESY Experiment on Bile Salt Micelles/EGCG Samples. NOESY experiments are suitable for estimating both intermolecular and intramolecular proton distances up to 5 Å.34 NOESY spectra were first measured with various mixing times. By increasing the mixing time, an NOE was observed. We found that a mixing time of 800 ms was optimal for observing the cross-peak in the bile salt micelle/EGCG sample. Figure 8A shows the NOESY spectra of EGCG interacting with bile salt micelles containing cholesterol and PC prepared with D2O at 300 K. Intermolecular NOE was observed between the γ-H of the PC molecule and the 2″- and 6″-H on the galloyl moiety in EGCG (Figure 8B). An intermolecular NOE between cholesterol and EGCG was not observed. Several intramolecular cross-peaks in the EGCG molecule were observed between the protons on the B-ring and the protons on the galloyl moiety (data not shown). Measuring the Fluorescence of 2-AS or 12-AS in Bile Salt Micelles in the Presence of EGCG. The localization of EGCG in bile salt micelles containing cholesterol and PC was investigated by fluorescence quenching of 2-AS and 12-AS with EGCG. Fluorescence was measured using a Flex Station II system (Molecular Devices, Sunnyvale, CA, USA). The anthroyl group of 2-AS should be located on the surface of the bile salt micelles, whereas that of 12-AS should be located in the hydrophobic core of the bile salt micelles. EGCG effectively quenched the fluorescence of 2-AS in a dose-dependent manner (Figure 9). EGCG (at 0.1, 0.3, 0.5, 0.7, 1, and 2 mmol/L) more strongly quenched the fluorescence of 2-AS than that of 12-AS (Figure 9).

Figure 8. NOESY spectra of epigallocatechin gallate and bile salt micelles containing cholesterol and phosphatidylcholine, with a 800 ms mixing time at 300 K. (A) Overall view of the NOESY spectra. (B) Enlarged view of the NOESY spectra.

Binding Analyses of EGCG to Cholesterol by an SPRBased Biosensor Assay. Four immobilization experiments were performed (reference flow cell/measurement flow cell: 0.5 mmol/L POPC/0.5 mmol/L POPC and 0.05 mmol/L cholesterol, 0.5 mmol/L POPC/0.5 mmol/L POPC and 0.15 mmol/L cholesterol, 0.5 mmol/L DOPA/0.5 mmol/L DOPA and 0.05 mmol/L cholesterol, and 0.5 mmol/L DOPA/0.5 mmol/L DOPA and 0.15 mmol/L cholesterol). The immobilization levels of phospholipid on the measurement flow cell and the reference flow cell were equalized as accurately as possible. The EGCG binding to cholesterol was not observed in any immobilization experiments (data not shown).

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DISCUSSION We previously showed that the addition of green tea catechins with a galloyl moiety to a bile salt micellar solution precipitated cholesterol and decreased the micellar solubility of cholesterol in a dose-dependent manner.16 In contrast, green tea catechins without a galloyl moiety did not precipitated cholesterol. When purified EGCG was added to the bile salt micellar solution, the amount of EGCG precipitated from the micellar solution was highly positively correlated with the amount of cholesterol precipitated (correlation coefficient = 0.99). These results strongly suggested that EGCG eliminated the cholesterol from 2887



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rated into the bile salt micelles was extremely low, and no more than 2.47% was incorporated, whereas the rest of the cholesterol was precipitated. These results suggest that because PC is eliminated from the bile salt micelles through its interaction with green tea catechins with a galloyl moiety, the micellar solubility of cholesterol is reduced; therefore, the cholesterol precipitated. The result from Figure 7 suggested that EGCG interacted with PC in the bile salt micelles. To clearly investigate how EGCG interacts with the PC in a bile salt micellar solution and whether the interaction between EGCG and PC indirectly leads to the precipitation of cholesterol from the bile salt micelles or the formation of a complex containing EGCG, PC, and cholesterol, we performed a NOESY experiment and estimated the location of EGCG in the bile salt micelles containing cholesterol and PC. Intermolecular NOE was observed between the γ-H of PC and 2″- and 6″-H on the galloyl moiety of EGCG when EGCG was added to the bile salt micelles containing cholesterol and PC (Figure 8B). The cation−π interaction between the trimethylammonium group of PC and the galloyl moiety of EGCG was inferred from this observation. Furthermore, several intramolecular cross-peaks in the EGCG molecule were observed between the protons on the B-ring and the protons on the galloyl moiety (data not shown). Therefore, there is a strong possibility that the cation−π interaction between the trimethylammonium group of PC and the galloyl moiety of EGCG may lead not only to a considerable shift of the galloyl moiety (2″- and 6″-H) signals in EGCG but also to a considerable shift of the A-ring (6- and 8-H), the B-ring (2′- and 6′-H), and the C-ring (2- and 3-H) signals in EGCG (Figure 7). In contrast, the intermolecular NOE between cholesterol and EGCG was not observed. These observations support our expectation that EGCG specifically interacts with only PC, but not cholesterol in the bile salt micelles, and agree with the results of our previous study in which we showed that EGCG specifically interacts with isotropic bicelles, which are composed of PC and frequently used as lipid bilayer models.29 When the localization of EGCG in the bile salt micelles containing cholesterol and PC was investigated, EGCG effectively quenched the fluorescence of 2-AS in a dosedependent manner (Figure 9). Because the trimethylammonium group of PC is a hydrophilic group, it should be located on the surface of the bile salt micelles. Therefore, this observation suggests that EGCG is located on the surface of the bile salt micelles via its interaction with micellar PC. The concentration of EGCG added to the micelles in this experiment was less than the amount that precipitated following the addition of EGCG to the bile salt micelles. It is thought that PC could be eliminated from the bile salt micelles by increasing the EGCG concentration added to the micelles. The results from our previous study suggested that cholesterol directly precipitated with EGCG.16 To investigate whether EGCG interacts with cholesterol, we performed binding analyses using an SPR-based biosensor assay. Binding of EGCG to cholesterol was not observed (data not shown). This observation strongly suggests that EGCG does not directly interact with cholesterol in a bile salt micelle. The results from the present study suggest that EGCG formed a complex with PC, and then this complex interacts with cholesterol. Hénin et al. showed that cholesterol and dimyristoylphosphatidylcholine formed a complex with a 1:1 stoichiometry in a fully hydrated cholesterol−dimyristoylphosphatidylcholine bilayer through

Figure 9. Effects of epigallocatechin gallate on the fluorescence intensity of 2-(9-anthroyloxy) stearic acid or 12-(9-anthroyloxy)stearic acid in bile salt micelles. A bile salt micellar solution containing 1 mmol/L 2-AS or 12-AS was prepared by sonication and stored at room temperature overnight. The micellar solutions (100 μL) were incubated with 100 μL of PBS containing epigallocatechin gallate (EGCG) for 20 min at 20 °C. The amounts of EGCG added were adjusted to 0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 mmol/L micelles. The fluorescence of 2-AS or 12-AS in the bile salt micellar solution were measured. The fluorescence intensity was calculated as the percentage of the fluorescence intensity of 2-AS or 12-AS in the absence of EGCG. Data are means ± SE of triplicate experiments. Means not sharing a common letter differ significantly (P < 0.05). Two-way ANOVA: effect of AS type, P < 0.0001; effect of EGCG concentration, P < 0.0001; interaction between AS type and EGCG concentration, P < 0.0001.

the bile salt micelles and co-precipitated with cholesterol. Therefore, we hypothesized that the micellar cholesterollowering activities of green tea catechins with a galloyl moiety could be a major cause for the inhibition of cholesterol absorption in rats.16,17 In the present study, we showed for the first time that green tea catechins with a galloyl moiety, but not those without a galloyl moiety, eliminated not only cholesterol but also PC from bile salt micelles (Figure 2). When bile salt micelles contained a phospholipid other than PC, neither cholesterol nor the phospholipid was eliminated by the addition of EGCG (Figure 3A,B). The same phenomenon was also observed in vesicles containing various phospholipids. EGCG effectively eliminated only the PC in the vesicles (Figure 4). We also showed that EGCG and GCG, but not EGC, effectively decreased the vesicular solubility of PC in a dose-dependent manner (Figure 5). Furthermore, we showed that green tea catechins with a galloyl moiety had higher affinity for POPC than those without a galloyl moiety in an SPR-based biosensor assay (Figure 6A,B and Table 1). The different affinity to PC between green tea catechins with a galloyl moiety and those without a galloyl moiety may be a reason why the concentrations of vesicular PC following the addition of EGCG or GCG were significantly lower than those following the addition of EGC (Figure 5). These observations suggest that green tea catechins with a galloyl moiety interact with PC and the binding of EGCG to PC decreases PC solubility. In our preliminary study, when a bile salt micellar solution containing 0.5 mmol/L cholesterol was prepared by sonication in the absence of phospholipids, the amount of cholesterol incorpo2888



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molecular dynamics simulations.35 Furthermore, in preliminary studies, we observed that cholesterol and PC formed a complex with a 1:1 stoichiometry in CDCl3 (data not shown). These observations suggest that cholesterol and PC form a complex in bile salt micelles and that the interaction between EGCG and PC leads to the formation of a complex containing EGCG, PC, and cholesterol. However, we could not provide direct evidence that the interaction between EGCG and PC leads to the formation of a complex containing EGCG, PC, and cholesterol in the present study. We could not assign all spectra in the bile salt micelles in the NOESY study, because too many protons derived from several molecules existed in the bile salt micelles. Furthermore, NOESY experiments are suitable for estimating specific interactions, but not nonspecific interactions. If cholesterol nonspecifically interacts with the EGCG/PC complex, it would be difficult to observe the intermolecular NOE between the protons in the cholesterol molecule and the EGCG/PC complex. Further research is required to confirm these results. We previously suggested the possibility that the B-ring and the plane of the ring in the galloyl moiety in EGCG or ECG were stacked via the π−π interaction in lipid bilayers and that the cation−π interaction between the trimethylammonium group of PC and the galloyl moiety in EGCG or ECG was enhanced by the π−π interaction between the B-ring and the plane of the ring in their galloyl moiety of EGCG or ECG.36 Several intramolecular cross-peaks were observed between the protons on the B-ring and the protons on the galloyl moiety in the EGCG molecule in the NOESY experiment in this study (data not shown). The intramolecular NOE between the protons on the B-ring and the protons on the galloyl moiety was also observed in our previous NOESY experiment using bicelle/EGCG and ECG samples.29 Therefore, there is a strong possibility that the intramolecular interactions in the EGCG molecule enhanced the cation−π interaction between the trimethylammonium group of PC and the galloyl moiety of EGCG in bile salt micelles as well as in lipid bilayers. Both exogenous and endogenous phospholipids exist in the intestinal lumen. The dietary intake of phospholipids is typically estimated to be 2−8 g/day in humans, and the biliary pathway delivers 10−20 g/day to the intestinal lumen.37 The predominant phospholipid in the intestinal lumen is PC. Therefore, the intestinal lumen can be a suitable environment for the inhibitory effect of EGCG on cholesterol absorption because the interaction between green tea catechins with a galloyl moiety and PC is important in eliminating cholesterol from bile salt micelles. In conclusion, our study showed that EGCG decreased the micellar solubility of cholesterol via specific interaction with PC, but not through a direct interaction with cholesterol. At any rate, the limited solubility of cholesterol by green tea catechins with a galloyl moiety can be a major cause of the inhibition of cholesterol absorption.16,17 In this study, we could not sufficiently describe the mechanism underlying the EGCGmediated elimination of cholesterol from bile salt micelles. Therefore, detailed studies are necessary to understand how green tea catechins with a galloyl moiety cause the precipitation of cholesterol from bile salt micelles.16,17

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Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED EGCG, epigallocatechin gallate; ECG, epicatechin gallate; EGC, epigallocatechin; EC, epicatechin; GCG, gallocatechin gallate; CG, catechin gallate; GC, gallocatechin; C, catechin; PC, phosphatidylcholine; PA, phosphatidic acid; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; DOPA, 1,2-dioleoylphosphatidic acid; SPR, surface plasmon resonance; NOE, nuclear Overhauser effect; NOESY, NOEcorrelated spectroscopy; DSS, sodium 2,2-silapentane-5-sulfonate; 2-AS, 2-(9-anthroyloxy)stearic acid; 12-AS, 12-(9anthroyloxy)stearic acid

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Epigallocatechin Gallate Decreases the Micellar Solubility of

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