Molecular Mechanism by Which Tea Catechins Decrease the Micellar

May 31, 2019 - Tea polyphenols lower the levels of cholesterol in the blood by decreasing the cholesterol micellar solubility. To clarify this mechani...
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Article Cite This: J. Agric. Food Chem. 2019, 67, 7128−7135

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Molecular Mechanism by Which Tea Catechins Decrease the Micellar Solubility of Cholesterol Takumi Sakakibara,† Yoshiharu Sawada,‡ Jilite Wang,† Satoshi Nagaoka,† and Emiko Yanase*,† †

Graduate School of Natural Science and Technology, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Division of Instrumental Analysis Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

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ABSTRACT: Tea polyphenols lower the levels of cholesterol in the blood by decreasing the cholesterol micellar solubility. To clarify this mechanism, the interactions between taurocholic acid and (−)-epigallocatechin gallate (EGCg) and its derivatives were investigated. 13C NMR studies revealed remarkable chemical-shift changes for the carbonyl carbon atom and the 1″- and 4″-positions in the galloyl moiety. Furthermore, 1H NMR studies using (−)-EGCg derivatives showed that the number of hydroxyl groups on the B ring did not affect these interactions, whereas the carbonyl carbon atom and the aromatic ring of the galloyl moiety had remarkable effects. The configuration at the 2- and 3-positions of the catechin also influenced these interactions, with the trans-configuration resulting in stronger inhibition activity than the cis-configuration. Additionally, a 1:1 component ratio for the catechin−taurocholic acid complex was determined by electrospray ionization−mass spectrometry. These molecular mechanisms contribute to the development of cholesterol-absorption inhibitors. KEYWORDS: tea (Camellia sinensis), catechin, cholesterol, micellar solubility, taurocholic acid

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hydrolysis using tannase. Deuterium oxide (D2O, 99.8% D) was purchased from Kanto Chemical Company (Tokyo, Japan). Phosphatidylcholine (PC) from egg yolk, cholesterol, oleic acid, monoolein, sodium taurocholate, and all other regents and reactants used for the synthesis of the (−)-EGCg derivatives were purchased from Sigma-Aldrich Company (St. Louis, MO). (−)-EGCg Epimerization. Compound 3 was synthesized according to our previous report.16 A solution of 1 (100 mg) in 2-propanol (20 mL) containing 1% H2SO4 was stirred at 80 °C for 3 h. After this time, 2-propanol was removed under reduced pressure, and ethyl acetate (EtOAc) was added. The EtOAc layer was then washed with water to remove the H2SO4 and dried over anhydrous Na2SO4 prior to concentration. The residue was purified by preparative high-performance liquid chromatography (HPLC) using an Inertsil ODS-3 column (30% MeOH in H2O) to give 3 (50.5 mg, 51% yield). The 1H NMR data were in good agreement with the literature data.17 Compound 3: 1 H NMR (500 MHz, CD3OD) δ (ppm): 7.00 (2H, s, H-2″ and H-6″), 6.43 (2H, s, H-2′ and H-6′), 5.98 (2H, s, H-6 and H-8), 5.40 (1H, q, J = 5.2 Hz, H-3), 5.08 (1H, d, J = 5.8 Hz, H-2), 2.80 (1H, dd, J = 5.2 and 11.5 Hz, H-4a), 2.75 (1H, dd, J = 5.7 and 10.5 Hz, H-4b). Synthesis of the (−)-EGCg Derivatives. A synthetic scheme outlining the preparation of the (−)-EGCg derivatives is shown in Figure S1. Synthesis of 3′,4′,5,7-Tetra-O-benzyl-(+)-catechin (10). Potassium carbonate (574 mg) was added to a solution of (+)-catechin (9, 200 mg) in anhydrous N,N-dimethylformamide (DMF, 2 mL) under an Ar atmosphere. After the solution was stirred at 25 °C for 1 h, benzyl bromide (0.37 mL) was added. The reaction mixture was stirred at 25 °C overnight and then poured into toluene/water. After extraction, the organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified by recrystallization from MeOH to give 10 (298.3 mg, 67% yield).

INTRODUCTION Tea (Camellia sinensis) is a popular beverage globally. As the major polyphenols present in tea, catechins account for ∼30% of the total dry weight of tea leaves. Four main types of catechins exist (Figure 1), namely, (−)-epigallocatechin gallate (EGCg, 1) and (−)-epicatechin gallate (ECg, 2), which have gallate groups at their 3-positions, and (−)-epicatechin (EC) and (−)-epigallocatechin (EGC), which bear no gallate groups. Depending on the processing method employed, tea is mainly classified into three types, namely, green tea, oolong tea, and black tea, and the catechin contents in the tea leaves change during processing. Catechins have attracted growing attention in recent years because of their various health benefits, including their antioxidant1,2 and antitumor activities.2,3 Furthermore, catechins are known to lower blood cholesterol levels, and it has been reported that 1, which bears a gallate group, is particularly potent in this respect.4−13 It is assumed that the mechanism by which catechins decrease cholesterol absorption involves inhibition of the solubility of cholesterol in bile acid micelles in the small intestine.14 Our previous study, in which we investigated the molecular mechanism of this phenomenon using a model bile acid micelle system, indicated that the interaction of 1 with taurocholic acid was regiospecific.15 On the basis of this result, we suggest that the inhibition of cholesterol solubility in bile acid micelles is due to physical changes in the micelle caused by the interaction between catechin and taurocholic acid. Thus, to clarify the details of the inhibition mechanism, we herein report the synthesis of derivatives of 1 and subsequent investigations into the effects of these modifications on the regiospecific interactions.

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Received: Revised: Accepted: Published:

MATERIALS AND METHODS

Chemicals. (−)-EGCg was a kind gift from Nagara Science Company (Gifu, Japan). (−)-EGC was prepared by enzymatic © 2019 American Chemical Society

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April 11, 2019 May 20, 2019 May 31, 2019 May 31, 2019 DOI: 10.1021/acs.jafc.9b02265 J. Agric. Food Chem. 2019, 67, 7128−7135

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Journal of Agricultural and Food Chemistry

chromatography (15% EtOAc in hexane) to give 13 (9.3 mg, 83% yield). Synthesis of (−)-EGCg Analogue A (6). The debenzylation of compound 13 (8 mg) was performed using the same procedure described for the synthesis of 4. The crude product was purified by preparative HPLC using a COSMOSIL 5C18-MS-II column (4.6 mm i.d. × 150 mm, 20% MeCN and 0.5% HCOOH in H2O) to give 6 (1.9 mg, 51% yield). Compound 6: 1H NMR (500 MHz, CD3OD) δ (ppm): 7.88 (2H, d, J = 7.5 Hz, H-2″ and H-6″), 7.52 (1H, t, J = 7.5 Hz, H-4″), 7.39 (2H, t, J = 8.0 Hz, H-3″ and H-5″), 6.52 (2H, s, H-2′ and H-6′), 5.99 (1H, d, J = 2.3 Hz, H-8), 5.96 (1H, d, J = 2.3 Hz, H-6), 5.57 (1H, s, H-3), 5.01 (1H, s, H-2), 3.01 (1H, dd, J = 4.1 and 13.2 Hz, H-4a), 2.93 (1H, dd, J = 2.3 and 14.9 Hz); 13C NMR (150 MHz, CD3OD) δ (ppm): 167.4 (C-9), 157.7 (C-7), 157.6 (C-5), 157.0 (C-8a), 146.5 (C-3′, 5′), 134.0 (C-4′), 133.6 (C-4″), 131.1 (C-1′), 130.6 (C-1″), 130.5 (C-2″, 6″), 129.3 (C-3″, 5″), 106.6 (C-2′, 6′), 99.2 (C-4a), 96.5 (C-8), 95.8 (C-6), 78.3 (C-2), 70.6 (C-3), 26.6 (C-4); ESI-HRMS: m/z calcd for C22H18O8 [M − H]− 409.0923, found 409.0341. Synthesis of 3-O-3,4,5-Tris(phenylmethoxy)-benzoyl3′,4′,5,5′,7-penta-O-benzyl-(−)-epigallocatechin (14). Sodium hydride (20.4 mg) was added to a solution of 12 (340 mg) in anhydrous DMF (6.8 mL) and stirred at 0 °C for 5 min. Subsequently, 5(bromomethyl)-1,2,3-tris(phenylmethoxy)benzene (265.2 mg) was added, and the mixture was stirred at 0 °C for 2 h. After this, aqueous HCl was added to give a neutral pH, and the mixture was extracted with toluene. The resulting organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified by silica-gel chromatography (60% CH2Cl2 in hexane) to give 14 (396.1 mg, 76% yield). Synthesis of (−)-EGCg Analogue B (7). The debenzylation of compound 14 (390 mg) was performed using the same procedure as described for the synthesis of 4. The crude product was purified by preparative HPLC using an Inertsil Amide column (10 mm i.d. × 250 mm, 90% MeCN in H2O) to give 7 (55.8 mg, 38% yield). Compound 7: 1 H NMR (500 MHz, CD3OD) δ (ppm): 6.53 (2H, s, H-2″ and H-6″), 6.21 (2H, s, H-2′ and H-6′), 5.94 (1H, d, J = 2.3 Hz, H-8), 5.92 (1H, d, J = 2.3 Hz, H-6), 4.83 (1H, s, H-2), 4.17 (1H, d, J = 11.5 Hz, H-9a), 4.12 (1H, d, J = 12.0 Hz, H-9b), 3.96 (1H, s, H-3), 2.76 (1H, dd, J = 4.0 and 12.6 Hz, H-4a), 2.72 (1H, dd, J = 4.6 and 12.6 Hz, H-4b); 13C NMR (150 MHz, CD3OD) δ (ppm): 157.5 (C-7), 157.4 (C-5), 157.0 (C-8a), 146.4 (C-3′, 5′), 146.3 (C-3″, 5″), 134.0 (C-4′), 133.5 (C-4″), 131.3 (C-1′), 130.1 (C-1″), 108.4 (C-2″, 6″), 107.0 (C-2′, 6′), 100.1 (C-4a), 96.2 (C-8), 95.8 (C-6), 78.8 (C-2), 73.8 (C-9), 72.9 (C-3), 25.9 (C-4); ESI-HRMS: m/z calcd for C22H20O10 [M − H]− 443.0978, found 443.0373. Synthesis of 3-O-Acetyl-3′,4′,5,5′,7-penta-O-benzyl-(−)-epigallocatechin (15). Acetic anhydride (4.12 mL) was added to a solution of 12 (412 mg) in pyridine (4.12 mL) and stirred at 25 °C for 2 h. After the addition of MeOH (4.12 mL), the mixture was concentrated under reduced pressure. The crude product was purified by silica-gel chromatography (20% EtOAc in hexane) to give 15 (400 mg, 93% yield). Synthesis of (−)-EGCg Analogue C (8). The debenzylation of compound 15 (400 mg) was performed using the same procedure as described for the synthesis of 4. The crude product was purified by preparative HPLC using an Inertsil ODS-3 column (10 mm i.d. × 250 mm, 25% MeOH in H2O) to give 8 (98.8 mg, 56% yield). The 1H NMR data were in good agreement with the literature data.18 Compound 8: 1 H NMR (500 MHz, CD3OD) δ (ppm): 6.47 (2H, s, H-2′ and H-6′), 5.96 (1H, d, J = 2.1 Hz, H-8), 5.92 (1H, d, J = 2.8 Hz, H-6), 5.35 (1H, s, H-3), 4.86 (1H, s, H-2), 2.90 (1H, dd, J = 4.8 and 12.4 Hz, H-4a), 2.79 (1H, dd, J = 2.0 and 15.1 Hz, H-4b), 1.92 (3H, s, Ac); ESI-HRMS: m/z calcd for C17H16O8 [M − H]− 347.0767, found 347.0370. 13 C NMR Spectroscopy. Compound 1 (4.4 mmol/L) was added to a solution of the taurocholic acid sodium salt (7.4 mmol/L) in D2O. 13C NMR spectra were recorded on a Bruker Biospin AVANCE III 600 spectrometer (Billerica, MA) at 600 MHz. 1 H NMR Spectroscopy. Various concentrations (2−16 mM) of the catechin samples were added to a solution of the taurocholic acid sodium salt (7.4 mmol/L) in H2O containing 5% D2O. 1H NMR

Figure 1. Structures of the catechin derivatives and of taurocholic acid.

Synthesis of 3′,3″,4′,4″,5,5″,7-Per-O-benzyl-(+)-catechin Gallate (11). 3,4,5-Tris(benzyloxy)benzoic acid (304 mg), N,Ndimethyl-4-aminopyridine (DMAP, 11 mg), and trimethylamine (0.256 mL) were added to a solution of 10 (298 mg) in anhydrous CH2Cl2 (4.5 mL). After the solution was stirred at 0 °C for 5 min, watersoluble carbodiimide (WSCD)·HCl (177 mg) was added, and the mixture was stirred at 25 °C overnight. After this, the reaction mixture was poured into water/CH2Cl2 and extracted. The resulting organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified by silica-gel chromatography (hexane/toluene/ EtOAc = 3:3:1) to give 11 (302.9 mg, 61% yield). Synthesis of (+)-Catechin Gallate (Cg, 4). Compound 11 (303 mg) and 5% Pd/C (300 mg) were combined in tetrahydrofuran (THF, 30 mL). After the solution was stirred for 32 h at 25 °C under a hydrogen atmosphere, the mixture was filtered and concentrated. The crude product was purified by preparative HPLC using an Inertsil ODS3 column (10 mm i.d. × 250 mm, 35% MeOH and 0.5% HCOOH in H2O) to give 4 (58.4 mg, 46% yield). Compound 4: 1H NMR (500 MHz, CD3OD) δ (ppm): 6.70 (2H, s, H-2″ and H-6″), 6.85 (1H, s, H3′), 6.73 (2H, s, H-2′ and H-6′), 5.97 (2H, m, H-6 and H-8), 5.39 (1H, q, J = 5.5 Hz, H-3), 5.07 (1H, d, J = 6.2 Hz, H-2), 2.84 (1H, dd, J = 4.8 and 9.5 Hz, H-4a), 2.73 (1H, dd, J = 6.2 and 10.3 Hz, H-4b); ESIHRMS: m/z calcd for C22H18O10 [M + H]+ 443.0978, found 443.0419. Synthesis of 3′,4′,5,5′,7-Penta-O-benzyl-(−)-epigallocatechin (12). The phenolic groups of (−)-epigallocatechin (5, 100 mg) were benzylated in the same manner as described for the synthesis of 11. The crude product was purified by silica-gel chromatography (20% EtOAc in hexane) to give 12 (167.4 mg, 67% yield). Synthesis of 3-O-Benzoyl-3′,4′,5,5′,7-penta-O-benzyl(−)-epigallocatechin (13). The benzoylation of 12 (10 mg) at the 3-position was performed according to the procedure described for the synthesis of 11. The crude product was purified by silica-gel 7129

DOI: 10.1021/acs.jafc.9b02265 J. Agric. Food Chem. 2019, 67, 7128−7135

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spectra were recorded on a JEOL ECA 500 spectrometer (Tokyo, Japan) at 500 MHz. Dynamic Light Scattering (DLS). A bile acid micellar solution containing 6.6 mM taurocholic acid sodium salt, 0.6 mM PC, 0.1 mM cholesterol, 1 mM oleic acid, 0.5 mM monoolein, 132 mM NaCl, and 15 mM sodium phosphate (pH 7.4) was prepared by sonication and stored at 37 °C with random shaking (125 times per minute) for at least 24 h (micelle A solution). Micelle B solution was prepared according to the same procedure but in the absence of oleic acid. After the addition of 1 (5 mg/mL) to the micellar solution, the mixture was incubated for 1 h at 37 °C and then subjected to ultracentrifugation at 100 000g for 1 h at 37 °C. The supernatant was collected and analyzed by DLS at 25 °C (Zetasizer Nano ZS, Malvern Panalytical Company, Malvern, U.K.). Disposable folded capillary cells (DTS 1070) were used for the measurements. Both micellar solutions were also prepared without the addition of 1 and were subjected to DLS analysis under the same conditions. The obtained data are shown in Figure S2. In Vitro Analysis of the Effect of Catechins on the Micellar Solubility of Cholesterol. The effects of the purified catechins on the micellar solubility of cholesterol were examined as described previously.19 Micellar solutions (1 mL) containing 6.6 mM taurocholic acid sodium salt, 0.6 mM PC, 0.1 mM cholesterol, 2.1 Gbp/mM 0.74 kBq [4-14C]-cholesterol, 1 mM oleic acid, 0.5 mM monoolein, 132 mM NaCl, and 15 mM sodium phosphate (pH 7.4) were prepared by sonication and stored at 37 °C with random shaking (125 times per minute) for at least 24 h. After the addition of each catechin (5 mg, the concentrations of 1, 3, and 4 in the micelles were 10.9, 10.9, and 11.3 mM each) or cholestyramine (positive control) to the micellar solutions, the mixtures were incubated for 1 h at 37 °C and then subjected to ultracentrifugation at 100 000g for 1 h at 37 °C. The amount of cholesterol in the supernatant was measured using a PerkinElmer Tri-Carb-2900TR (Waltham, MA). Electrospray Ionization−Mass Spectrometry (ESI-MS). ESIMS was performed in flow-injection mode using a Waters Xevo G2 QToF mass spectrometer (Milford, MA). Compound 1 or 5 (2 or 16 mM) was added to a solution of the taurocholic acid sodium salt (7.4 mM) in water. The sample solution was injected through the injector using a sampling tube (100 μL). The mobile phase was ultrapure water and the flow rate was set at 0.4 mL/min. The ESI interface was operated in negative-ion mode. The MS parameters employed for analysis were as follows: capillary potential of 2.5 kV, sampling cone at 30 V, desolvation temperature of 500 °C, source temperature of 150 °C, desolvation-gas flow for 1000 L/h N2, and cone-gas flow of 50 L/h N2. For analysis of the samples containing 1 (16 mM) and the taurocholic acid sodium salt (7.4 mM), the mass spectrometer was operated in MS/ MS mode with a collision-cell energy of 22 V. The corresponding data are shown in Figures 5 and S3. Nuclear-Overhauser-Effect (NOE) Measurements. RotatingFrame Nuclear-Overhauser-Effect Spectroscopy (ROESY). A D2O solution containing 3 (21.9 mM) and the taurocholic acid sodium salt (26.4 mM) was prepared. ROESY spectra were recorded on a JEOL ECA 600 spectrometer (Tokyo, Japan) at 600 MHz. The typical acquisition parameters were as follows: 64 scans; acquisition times of 0.11 and 0.028 s for the f1 and f2 axes, respectively; mixing time of 0.25 s; and relaxation delay of 2 s. The corresponding data are shown in Figure S4. Difference-NOE Measurements. A D2O solution containing 1 or 3 (8.7 mM) and the taurocholic acid sodium salt (10.4 mM) was prepared. Difference-NOE spectra were recorded on a JEOL ECX 400 P spectrometer (Tokyo, Japan) at 400 MHz. The typical acquisition parameters for measurement of the mixture containing 1 and the taurocholic acid sodium salt were as follows: 32 scans, irradiation at 0.1436 ppm (18-position of taurocholic acid), presaturation time of 5.5 s, and relaxation delay of 7 s. The parameters for measurement of the mixture containing 3 and the taurocholic acid sodium salt were identical, with the exception that irradiation at 0.3115 ppm (18-position of taurocholic acid sodium salt) and a presaturation time of 6.9 s were used. The presaturation times were chosen by using array measurements based on the T1 values obtained by the inversion recovery method. The corresponding data are shown in Figures 6 and S5.

Article

RESULTS C NMR Analysis. To elucidate the interactions between 1 and taurocholic acid, we previously examined the 1H NMR chemical shifts of taurocholic acid in the absence and presence of 1.15 A large chemical-shift change at the 3-position of 1 was observed in the presence of taurocholic acid, but this experiment provided insufficient information because 1 has three aromatic rings and a relatively small number of protons for its molecular size. Although its sensitivity is lower than that of 1H NMR spectroscopy, 13C NMR spectroscopy can provide information regarding the electron density on the carbon skeleton. In particular, information related to the carbon atoms bonded to hydroxyl groups is important because 1 has a number of phenolic hydroxyl groups that can potentially form hydrogen bonds with other molecules. It is assumed that the formation of a hydrogen bond not only directly affects the electron density of the hydroxyl group but also indirectly affects the carbon atom to which it is bonded. Figure 2 shows the differences in the 13

Figure 2. Changes in the 13C NMR chemical shifts of (−)-EGCg (1) induced by the addition of taurocholic acid: Δppm = [1 + taurocholic acid] − [1], in D2O.

chemical shifts of the carbon atoms in 1 in the absence and presence of taurocholic acid. The largest chemical shift change was observed at the 3-position, in agreement with the previous 1 H NMR observations. Significant changes were also observed on the gallate residue, with the chemical-shift changes decreasing in the following order: 1″-position > 4″-position > carbonyl carbon. These results suggested that the galloyl moiety, especially the 1″- and 4″-positions and the carbonyl group, strongly affected the interactions with taurocholic acid. In contrast, almost no chemical-shift changes were observed on the A- and B-ring positions that contained hydroxyl groups. This result correlates well with the fact that catechins without galloyl moieties have little effect on the micellar solubility of cholesterol. 1 H NMR Analysis. Various types of intermolecular interactions are known to exist, including hydrophobic interactions, ionic interactions, and hydrogen bonding. As 1 has a large number of hydroxyl groups, hydrogen bonding between these hydroxyl groups and taurocholic acid is presumed to be the major intermolecular force that drives the interaction between these two compounds. Thus, several derivatives of 1 were prepared to investigate the effect of modifying the catechin structure on the interaction with taurocholic acid. The interaction of compound 2, which is found in tea and has a smaller number of hydroxyl groups in the B ring than 1, with taurocholic acid was analyzed by 1H NMR spectroscopy. The seven characteristic signals of taurocholic acid (at the 3-, 7-, 12-, 18-, 19-, 25-, and 26-positions) tended to shift as the concentration of 2 increased, and the observed changes were 7130

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chemical shift at the 18-position of taurocholic acid upon the addition of 8 or 7 (Figure 3A). Moreover, no further changes in the chemical shifts of taurocholic acid were observed when the concentration of 1 exceeded 16 mM, whereas a concentration of 48 mM was required with 6, and the magnitude of the chemical-shift change induced by the addition of 6 was larger at the saturation point (Figure 3B). This difference in the saturation points may be attributable to a shift in the equilibrium between free and complexed 6 owing to the interaction between 6 and taurocholic acid being weaker than that between 1 and taurocholic acid. As the chemical shift corresponds to an average of the free and complexed states, these results indicate that a higher concentration of 6 is required to reach the interaction-rich condition. DLS Analysis. To evaluate the interaction between 1 and bile acid micelles, we measured the particle sizes of bile acid micelles containing 1 by DLS. In the bile acid micelle without 1, only a single peak corresponding to a size of 147.9 ± 5 nm was observed (Figure S2a). In contrast, two peaks were observed in the distribution curve of the bile acid micelle containing 1: one peak at a larger size of 278.6 ± 30 nm and a second peak at 20.53 ± 1 nm (Figure S2b). Although the micelle system was different from that used in this study, Raederstorff et al. reported7 a similar phenomenon, with an increase in particle size caused by 1, which is in good agreement with our results. It is known that cholesterol is rendered soluble in bile salt mixed micelles and then absorbed from the small intestine, and so it is likely that bile acid micelles play an important role in cholesterol absorption.14 Indeed, various researchers have conducted in vitro assays to evaluate the effects of various compounds on the micellar solubility of cholesterol.7,13,15,19−23 For example, two types of model micellar systems were used to easily evaluate the inhibition activity in vitro: a micellar solution containing cholesterol, PC, and taurocholic acid7,13,22 and a micellar solution consisting of these three regents in addition to oleic acid or monoolein (micelle A solution).15,19−21,23 A precipitate formed upon the addition of 1 to the micelle solution and the removal of monoolein from micelle A solution did not affect the degree of precipitation. In contrast, larger amounts of precipitate were observed when 1 was added to the micelle A solution in the absence of oleic acid (i.e., micelle B solution). To further investigate the effect of oleic acid on the interaction between 1 and taurocholic acid, 1H NMR and DLS measurements were performed for micelle A and B solutions. Following the addition of 1, 1H NMR analyses of the two supernatants revealed no difference between the two micelle solutions in terms of the chemical-shift changes (data not shown). In contrast, remarkable differences were observed by DLS. Although a single peak at 147.9 ± 5 nm was observed for the micelle A solution (Figure S2a), two peaks were observed for the micelle B solution (189.0 ± 10 and 7.901 ± 0.2 nm, Figure S2c). Furthermore, when 1 was added to the micelle B solution, the diameter of the larger particle increased to 351.6 ± 8 nm, whereas the smaller particles disappeared (Figure S2d). Effect of Catechins on the Micellar Solubility of Cholesterol in Vitro. The inhibition activities of 1, 3, and 4 on cholesterol micellar solubility were evaluated using a model micelle system. Cholestyramine was used as a positive control. In the presence of 1, 3, or 4, the percentages of cholesterol incorporated into the bile acid micelles were 33.5 ± 1.7, 27.4 ± 2.1, and 18.0 ± 1.1%, respectively (Figure 4). Thus, all catechins effectively decreased the micellar solubility of cholesterol.

similar to those induced by the addition of 1 (Figure 3A). This result indicates that the B ring has a small effect on the interaction with taurocholic acid.

Figure 3. (A) Effect of catechin-derivative concentration on the change in the chemical shift at the 18-position of taurocholic acid: ●, (−)-EGCg (1); ■, (−)-ECg (2); ▲, (−)-GCg (3); ◆, (+)-Cg (4); ○, (−)-EGCg analogue A (6); □, (−)-EGCg analogue B (7); △, (−)-EGCg analogue C (8). (B) Saturation point of (−)-EGCg analogue A, which occurred at a higher concentration than those of the other derivatives (●, 1; ○, 6).

To investigate the effect of stereochemistry, the interactions of taurocholic acid with 3 and 4, which have different stereochemistries from 1 in the 2- and 3-positions, respectively, were analyzed by 1H NMR spectroscopy. An upfield shift at the 18-position of taurocholic acid was observed upon the addition of 3 or 4, which suggested that these compounds interact with taurocholic acid regiospecifically, as in the case of compound 1. However, the magnitudes of the changes induced by 3 and 4 were approximately two-thirds of that observed upon the addition of 1 (Figure 3A). To investigate which part of the galloyl moiety is involved in the interactions with taurocholic acid, we synthesized three analogues of 1 bearing modified galloyl moieties (Figure S1): one without a hydroxyl group (analogue A, 6), one without a carbonyl group (analogue B, 7), and one without an aromatic ring (analogue C, 8). The 1H NMR chemical shifts of taurocholic acid were altered upon the addition of 6, but the magnitude of the chemical-shift change at the 18-position was smaller than that observed when the same amount of 1 was added. In contrast, little or no changes were observed in the 7131

DOI: 10.1021/acs.jafc.9b02265 J. Agric. Food Chem. 2019, 67, 7128−7135

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ESI-MS Analysis. Although the NMR experiments indicated that regiospecific interactions take place between the catechins and taurocholic acid, the molar ratios of these complexes remain unclear, because the NMR spectra show the equilibrium states between the bound and free catechins. Thus, we applied MS as a valuable tool for determining the ratio of the components in this complex. Among the various ionization methods available, we applied ESI because the interactions between the catechins and taurocholic acid are only observed in aqueous solution. The MS spectrum of the mixture containing 1 (16 mM) and taurocholic acid (7.4 mM) exhibited peaks at m/z 457 and 514, which corresponded to the molecular-ion peaks of 1 and taurocholic acid, respectively; pseudo-ion peaks ([2M − 1]− and [3M − 1]−) were also observed. Additionally, an ion peak was observed at m/z 972, which corresponded to the complex formed between 1 and taurocholic acid. Although no regiospecific interaction was expected between 5 and taurocholic acid, the MS spectrum of the mixture of 5 (16 mM) and taurocholic acid (7.4 mM) exhibited a peak at m/z 820, which is consistent with the complex formed between 5 and taurocholic acid (data not shown). In contrast, no obvious molecular-ion peak was observed at m/z 820 when the concentration of 5 was reduced to 2 mM, although a peak at m/z 972 was clearly observed when the concentration of 1 was reduced to 2 mM (Figures 5A,B and S3A). Thus, the observed molecular-ion peaks at m/z 972 and 820 were considered to correspond to a cluster ion formed by nonspecific interactions between molecules at high catechin concentrations. In fact, cluster ions formed by two or more molecules of taurocholic acid

Figure 4. Cholesterol micellar solubility in the presence of various catechin derivatives. The values are expressed as means ± SEM (n = 4). Means with different superscript letters indicate significant differences (P < 0.05) as determined by Tukey’s test.

Furthermore, the concentrations of micellar cholesterol following the addition of 3 and 4 tended to be lower than that following the addition of 1.

Figure 5. MS spectra of (A) mixture containing 1 (2 mM) and taurocholic acid (7.4 mM) and (B) mixture containing 5 (2 mM) and taurocholic acid (7.4 mM). 7132

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Figure 6. Difference-NOE spectra of (A) mixture containing (−)-EGCg (8.7 mM) and taurocholic acid (10.4 mM) at a presaturation time of 5.5 s and (B) mixture containing (−)-GCg (8.7 mM) and taurocholic acid (10.4 mM) at a presaturation time of 6.9 s.

were also detected (e.g., [2M − 1]− and [3M − 1]−). In contrast, at low concentrations of 1, the molecular-ion peak at m/z 972 was considered to correspond to the (−)-EGCg−taurocholic acid complex formed by the regiospecific interaction of 1 with taurocholic acid. This assumption was confirmed by the disappearance of the molecular-ion peak at m/z 820 following dilution. Furthermore, no ion peaks were observed that corresponded to 1:2 or 2:1 ratios of 1 and taurocholic acid. This result indicated a 1:1 interaction between 1 and taurocholic acid. Furthermore, MS/MS analysis of the ion peak at m/z 972 revealed two fragment peaks at m/z 514 and 457 (Figure S3B), which were attributed to taurocholic acid and 1, respectively. These results indicated that the ion peak at m/z 972 is derived from the (−)-EGCg−taurocholic acid complex. NOE Experiments. In our previous study, intermolecular correlations between the gallate protons of 1 and the methyl protons at the 18- and 19-positions of taurocholic acid were observed by ROESY experiments in D2O.15 Compounds 1 and 3 are stereoisomers, differing in the configuration of the 2position; 1 has a 2,3-cis-configuration, whereas 3 has a 2,3-transconfiguration. This difference, which affects the conformation between the B ring and the gallate moiety, may contribute to the different interaction abilities of these compounds and their inhibition activities on the cholesterol micellar solubility. Hence, to clarify the effect of this conformational change on the interaction with taurocholic acid, ROESY experiments were performed for a mixture of 3 and taurocholic acid in D2O. Intermolecular correlations between the gallate protons of 3 and the methyl groups at the 18- and 19-positions of taurocholic acid were observed (Figure S4). However, the observed correlations were the same as those obtained for a mixture of 1 and taurocholic acid in our previous study. Subsequently, difference-NOE experiments were performed using a mixed solution of 1 or 3 with taurocholic acid. The obtained spectra correspond to the difference between the spectra recorded with and without NOEs caused by the presaturation of individual resonances. Thus, only signals that have an NOE with a saturated resonance are detected. Furthermore, the integral ratios of the detected signals provide

relative-distance information. However, correct measurement of the NOE values was expected to be difficult because of the similar chemical shifts of 1 and 3 in the presence of taurocholic acid. For this reason, to evaluate the differences between 1 and 3, the presaturation times were compared. The presaturation time is the period required for the development of an NOE. Selective saturation is achieved by irradiation, and this saturation is transferred to the correlation points. In general, the transfer ratio is inversely proportional to the atomic distance between the saturated resonance and the transferred resonance.24 Therefore, it was assumed that the effect of the conformational difference between 1 and 3 on the distance to taurocholic acid could be evaluated by comparing the presaturation times. In differenceNOE experiments with solutions of 1 or 3 and taurocholic acid in D2O, the NOE between the 18-position of taurocholic acid (saturated) and the gallate protons of 1 (transferred) was observed at a presaturation time of 5.5 s (Figure 6A), whereas the NOE between taurocholic acid and 3 was observed at a presaturation time of 6.9 s (Figure 6B). Thus, a longer presaturation time was required for 3−taurocholic acid than for 1−taurocholic acid to observe NOE signals with sufficient intensity. Additionally, the NOEs between taurocholic acid and 1 or 3 were observed by irradiation of the gallate protons with presaturation times of 5.3 and 7.1 s, respectively (Figure S5). These results suggested that the distance between 3 and taurocholic acid is greater than that between taurocholic acid and 1.

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DISCUSSION In our previous study, we proposed that the mechanism by which the addition of 1 lowers the solubility of cholesterol in micelles involves the interaction between 1 and taurocholic acid. In particular, the 1H NMR experiments suggested that the steroid skeleton of 5 and the gallate group of 1 are key to this interaction.15 The magnitudes of the chemical shifts in the 13C NMR experiments were observed surrounding the gallate group (Figure 2), and these were found to be in good agreement with previous 1H NMR results. This suggested the 13C NMR analysis is also useful for interaction analysis. In particular, the 1″- and 7133

DOI: 10.1021/acs.jafc.9b02265 J. Agric. Food Chem. 2019, 67, 7128−7135

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magnitude of the chemical-shift change at the 18-position of taurocholic acid induced by the addition of 3 was smaller than that induced by 1, even though 3 has higher inhibition activity. In addition, the ROESY spectrum did not show any significant difference between 1 and 3 (Figure S4), although the presaturation times in the NOE experiments (Figures 6 and S5) showed that the distance between the gallate moiety and taurocholic acid was affected by the conformation of the catechin, with 3 being located further from taurocholic acid than 1. This suggestion supports the results of the 1H NMR experiments that indicated the lesser effect of 3 on the chemical shifts of taurocholic acid compared with that of 1. Because of the difference in the 2−3 configuration, placement of the A and B rings relative to the gallate group differs between 1 and 3. It is presumed that this induces steric effects, although the A and B rings do not directly contribute to interactions between the gallate group and taurocholic acid. NMR and ESI-MS data suggested that 1 and taurocholic acid form a 1:1 complex that could be attributed to hydrophobic interactions between the galloyl group and steroid skeleton. In the DLS experiment of the bile acid micelle containing 1, it appeared that the particle size expanded as a result of the incorporation of 1 into the bile acid micelle. Although the DLS experiment indicated an increase in the particle size for the micelle containing 3, there was no significant difference in particle size between the micelles containing 1 and 3 (data not shown). However, under equal molar conditions, the influence of 3 on the micelles was suspected to be greater than that of 1 because of the larger distance to taurocholic acid upon interaction. We therefore consider that this accounts for the conflicting results in terms of the chemical-shift change induced by 3 being smaller than that induced by 1 despite the higher capacity of 1 to inhibit cholesterol solubility. From these results, we propose the molecular mechanism by which 1 decreases the micellar solubility of cholesterol, as follows: the structural change of the micelle is caused by the insertion of 1 in the micelle via hydrophobic interactions with taurocholic acid. Meanwhile, the presence or absence of oleic acid in the micelles affects micelle size (Figure S2) and the solubility of cholesterol. We assumed that oleic acid does not contribute to the interaction with 1, instead affecting cholesterol micellar solubility by changing the shape and aggregation state, for example. As bile acid micelles are known to contain many other components in addition to taurocholic acid, our results suggest that other components may also affect the inhibition activity. Further investigations into the effects of other micelle components is therefore necessary to clarify the inhibition mechanism of cholesterol micellar solubility by catechins.

4″-positions and the carbonyl carbon in the galloyl moiety play an important role in the interaction of 1 with taurocholic acid. It is assumed that possible intermolecular forces between 1 and taurocholic acid include hydrogen bonds through the carbonyl group and hydrophobic bonds through the aromatic ring. As a result of examining the effects of analogues 6, 7, and 8 by 1H NMR spectroscopy, chemical-shift changes were only observed when analogue 6 was added (Figure 3). In our previous study, we suggested that hydrogen bonding induced by the hydroxyl group was important because the magnitude of the chemicalshift change depended on the number of hydroxyl groups in taurocholic acid.15 However, this result indicated that the carbonyl group and the aromatic ring of the galloyl moiety are necessary for catechin−taurocholic acid interactions, whereas the hydroxyl group of the galloyl moiety only has a secondary effect on the interaction with taurocholic acid. In addition, we note that the structures of 1 and 7 differ at the 9-position, with 1 having a carbonyl group bearing an sp2 orbital and 7 having a methylene group bearing an sp3 orbital. Although the carbonyl group of 1 was able to form a hydrogen bond with taurocholic acid, no chemical-shift changes were observed upon the addition of 7. We therefore believe that the interaction was affected by the planarity of the carbonyl carbon atom rather than its hydrogenbond-forming ability. In terms of the effect of 2, chemical-shift changes comparable to those with 1 were observed. This result is in good agreement with the 13C NMR study, which revealed that the effects of the A and B rings on the chemical shift were smaller than that of the galloyl moiety. On the basis of this result, we consider that the contribution of the B ring to the interaction is small. In the 1H NMR experiments of 3 and 4, which are stereoisomers of 1, regiospecific interactions with taurocholic acid were observed; however, the magnitude of the chemicalshift change induced was smaller than that of 1. There are two possible explanations for this difference: (1) the position that interacts with taurocholic acid changes, or (2) the interaction between 3 or 4 and taurocholic acid is weaker than that between 1 and taurocholic acid. However, no significant differences exist between the functional groups, and the changes in the chemical shifts at the other positions of taurocholic acid induced by the addition of 3 or 4 were similar to, though smaller than (data not shown), those induced by the addition of 1. Furthermore, the magnitudes of the chemical-shift changes induced by the addition of 3 or 4 were comparable. It is therefore likely that the interactions of 3 and 4 with taurocholic acid are weaker than that of 1. The difference between these compounds is the relative conformations of the 2- and 3-positions; 1 has a cisconfiguration, whereas 3 and 4 have trans-configurations. Hence, it is suggested that the chemical-shift change is not affected by the stereochemistry at the 2- and 3-positions but by the relative position between the galloyl moiety and the A or B ring caused by the trans-configuration. Ikeda et al. previously reported6 that the inhibition activity of a heated catechin mixture is higher than that without heat treatment. It is known that catechin epimerization is induced by heat treatment, and the C2-position epimer is produced (i.e., compound 1 is converted to 3).16,17 The observation of stronger activity for compound 3 than for compound 1 is therefore consistent with their report (Figure 4). In our previous study, the magnitude of the chemical-shift change induced by the addition of catechins and related compounds to this evaluation system was correlated with the capacity to inhibit cholesterol solubility.15 However, the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b02265. Synthesis of catechin derivatives, DLS spectra of each micellar solution with and without 1, negative-mode-ESIMS analysis of a mixtures containing catechin and taurocholic acid, ROESY spectra of the mixture containing taurocholic acid and 3, and difference-NOE spectra with gallate proton irradiation of the mixture containing 1 and taurocholic acid and the mixture containing 3 and taurocholic acid (PDF) 7134

DOI: 10.1021/acs.jafc.9b02265 J. Agric. Food Chem. 2019, 67, 7128−7135

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(14) Nagaoka, S.; Nakamura, A.; Shibata, H.; Kanamaru, Y. Soystatin (VAWWMY), a novel bile acid-binding peptide, decreased micellar solubility and inhibited cholesterol absorption in rats. Biosci., Biotechnol., Biochem. 2010, 74, 1738−1741. (15) Ogawa, K.; Hirose, S.; Nagaoka, S.; Yanase, E. Interaction between tea polyphenols and bile acid inhibits micellar cholesterol solubility. J. Agric. Food Chem. 2016, 64, 204−209. (16) Ishino, N.; Yanase, E.; Nakatsuka, S. Epimerization of tea catechins under weakly acidic and alkaline conditions. Biosci., Biotechnol., Biochem. 2010, 74, 875−877. (17) Sang, S.; Lee, M. J.; Hou, Z.; Ho, C. T.; Yang, C. S. Stability of tea polyphenol (−)-epigallocatechin-3-gallate and formation of dimers and epimers under common experimental conditions. J. Agric. Food Chem. 2005, 53, 9478−9484. (18) Lin, S. F.; Lin, Y. H.; Lin, M.; Kao, Y. F.; Wang, R. W.; Teng, L. W.; Chuang, S. H.; Chang, J. M.; Yuan, T. T.; Fu, K. C.; Huang, K. P.; Lee, Y. S.; Chiang, C. C.; Yang, S. C.; Lai, C. L.; Liao, C. B.; Chen, P.; Lin, Y. S.; Lai, K. T.; Huang, H. J.; Yang, J. Y.; Liu, C. W.; Wei, W. Y.; Chen, C. K.; Hiipakka, R. A.; Liao, S.; Huang, J. J. Synthesis and structure-activity relationship of 3-O-acylated (−)-epigallocatechins as 5α-reductase inhibitors. Eur. J. Med. Chem. 2010, 45, 6068−6076. (19) Ogawa, K.; Hirose, S.; Yamamoto, H.; Shimada, M.; Nagaoka, S.; Yanase, E. Synthesis of oolongtheanins and their inhibitory activity on micellar cholesterol solubility in vitro. Bioorg. Med. Chem. Lett. 2015, 25, 749−752. (20) Megías, C.; Pedroche, J.; Del Mar Yust, M.; Alaiz, M.; GirónCalle, J.; Millán, F.; Vioque, J. Sunflower Protein Hydrolysates Reduce Cholesterol Micellar Solubility. Plant Foods Hum. Nutr. 2009, 64, 86− 93. (21) Ngamukote, S.; Mäkynen, K.; Thilawech, T.; Adisakwattana, S. Cholesterol-lowering activity of the major polyphenols in grape seed. Molecules 2011, 16, 5054−5061. (22) Tamura, T.; Inoue, N.; Ozawa, M.; Shimizu-Ibuka, A.; Arai, S.; Abe, N.; Koshino, H.; Mura, K. Peanut-skin polyphenols, procyanidin A1 and epicatechin-(4 β→6)-epicatechin-(2β→O→7, 4 β→8)catechin, exert cholesterol micelle-degrading activity in vitro. Biosci., Biotechnol., Biochem. 2013, 77, 1306−1309. (23) Marques, M. R.; Soares Freitas, R. A. M.; Corrêa Carlos, A. C.; Siguemoto, É . S.; Fontanari, G. G.; Arêas, J. A. Peptides from cowpea present antioxidant activity, inhibit cholesterol synthesis and its solubilisation into micelles. Food Chem. 2015, 168, 288−293. (24) Wagner, G.; Wüthrich, K. (1979).Truncated driven nuclear Overhauser effect (TOE). A new technique for studies of selective 1 H-1H Overhauser effects in the presence of spin diffusion. J. Magn. Reson. 1979, 33, 675−680.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel. and Fax: +81-58-293-2914. ORCID

Satoshi Nagaoka: 0000-0002-5348-3187 Emiko Yanase: 0000-0002-6652-4259 Funding

This work was partially supported by Nagara Science Company, Ltd., and by a JSPS Kakenhi Grant (15K07427). Notes

The authors declare no competing financial interest.

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DEDICATION This paper is dedicated to Professor Koji Nakanishi, Columbia University. REFERENCES

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DOI: 10.1021/acs.jafc.9b02265 J. Agric. Food Chem. 2019, 67, 7128−7135