Molecular Mechanism by Which Tea Catechins Decrease the Micellar Solubility of Cholesterol

Article abstract of DOI:10.1021/acs.jafc.9b02265

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.

Full text of DOI:10.1021/acs.jafc.9b02265

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Food and Beverage Chemistry/Biochemistry
Molecular mechanism by which tea catechins
decrease the micellar solubility of cholesterol
Takumi SAKAKIBARA, Yoshiharu Sawada, Jilite Wang, Satoshi Nagaoka, and Emiko Yanase
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02265 • Publication Date (Web): 31 May 2019
Downloaded from http://pubs.acs.org on June 3, 2019
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Molecular mechanism by which tea catechins decrease the micellar solubility of cholesterol
Takumi Sakakibara,† Yoshiharu Sawada,‡ Jilite Wang,† Satoshi Nagaoka,† Emiko Yanase†*
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†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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This paper is dedicated to Prof. Koji Nakanishi (Columbia University).
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w4521040@edu.gifu-u.ac.jp (T.S.); y_sawada@gifu-u.ac.jp (Y.S.), wangjilite@126.com (J.W.),
nagaoka@gifu-u.ac.jp (S.N.)
*Corresponding author: e-yanase@gifu-u.ac.jp; TEL/FAX +81-58-293-2914
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ABSTRACT
Tea polyphenols lower the levels of cholesterol in blood by decreasing the cholesterol micellar
solubility. To clarify this mechanism, the interactions between taurocholic acid and (−)-
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epigallocatechin gallate (EGCg) and its derivatives were investigated. C 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, while 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
a 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 will contribute to the development of cholesterol absorption inhibitors.
KEYWORDS: Tea (Camellia sinensis), Catechin, Cholesterol, Micellar solubility, Taurocholic acid
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INTRODUCTION
Tea (Camellia sinensis) is a globally popular beverage. As the major polyphenols present in
tea, the catechins account for ~30% of the total dry weight of tea leaves. Four main types of catechins
exist (Fig. 1), namely (−)-epigallocatechin gallate (EGCg; 1) and (−)-epicatechin gallate (ECg; 2),
which have a gallate group at the 3-position, and (−)-epicatechin (EC) and (−)-epigallocatechin
(EGC), which bear no gallate group. Depending on the processing method employed, tea is mainly
classified into three types, namely green tea, oolong tea, and black tea, where the catechin contents
in the tea leaves change during processing.
Catechins have attracted growing attention in recent years due to their various health benefits,
including their antioxidant1,2 and antitumor activities.2,3 Furthermore, catechins are known to lower
blood cholesterol level, 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 Based on 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 syntheses of derivatives of 1 and subsequent investigations into the effect of these
modifications on the regiospecific interactions.
MATERIALS AND METHODS
Chemicals. (−)-EGCg was a kind gift from Nagara Science Co. (Gifu, Japan). (−)-EGC was
prepared by enzymatic hydrolysis using tannase. Deuterium oxide (D2O, 99.8% D) was purchased
from Kanto Chemical Co. (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
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(−)-EGCg derivatives were purchased from Sigma Aldrich Co. (St. Louis, MO, USA).
(−)-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). 1H NMR data was in good agreement with the literature data.17 Compound 3: 1H 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 Fig. S1.
Synthesis of 3ʹ,4ʹ,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 stirring 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).
Synthesis of 3ʹ,3ʹʹ,4ʹ,4ʹʹ,5,5ʹʹ,7-per-O-benzyl-(+)-catechin gallate (11). 3,4,5-
Tris(benzyloxy)benzoic acid (304 mg), N,N-dimethyl-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 stirring at 0 °C for 5 min, water-soluble carbodiimide (WSCD)·HCl (177 mg) was added and
the mixture was stirred at 25 °C overnight. After this time, 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
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= 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 stirring 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 ODS-3 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, H-3′), 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); ESI-HRMS: m/z calcd for C22H18O10 [M+H]+ 443.0978, found
443.0419.
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Synthesis of 3ʹ,4ʹ,5,5ʹ,7-penta-O-benzyl-(-)-epigallocatechin (12). The phenolic groups of
(−)-epigallocatechin (5) (100 mg) were benzylated by 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).
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Synthesis of 3-O-benzoyl-3ʹ,4ʹ,5,5ʹ,7-penta-O-benzyl-(-)-epigallocatechin (13). The
benzoylation of 12 (10 mg) at 3-position was performed according to the procedure described for the
synthesis of 11. The crude product was purified by silica gel chromatography (15% EtOAc in hexane)
to give 13 (9.3 mg, 83% yield).
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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,
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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-
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5), 157.0 (C-8a), 146.5 (C-3ʹ, 5ʹ), 134.0 (C-4ʹ), 133.6 (C-4ʹʹ), 131.1 (C-1ʹ), 130.6 (C-1ʹʹ), 130.5 (C-2ʹʹ,
6ʹʹ), 129.3 (C-3ʹʹ, 5ʹʹ), 106.6 (C-2ʹ, 6ʹ), 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)-benzoyl-3ʹ,4ʹ,5,5ʹ,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 time, 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 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: 1H 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-3ʹ, 5ʹ), 146.3 (C-3ʹʹ,
5ʹʹ), 134.0 (C-4ʹ), 133.5 (C-4ʹʹ), 131.3 (C-1ʹ), 130.1 (C-1ʹʹ), 108.4 (C-2ʹʹ, 6ʹʹ), 107.0 (C-2ʹ, 6ʹ), 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-3ʹ,4ʹ,5,5ʹ,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).
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Synthesis of (−)-EGCg analogue C (8). The debenzylation of compound 15 (400 mg) was
performed using the same procedure 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)
1
to give 8 (98.8 mg, 56% yield). H NMR data was in good agreement with the literature data.18
Compound 8: 1H 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.
13C 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, USA) at 600 MHz.
1H NMR spectroscopy. Various concentrations (2 to 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 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/min) 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 100000 × g for 1 h at 37 °C. The supernatant was collected and analyzed by
DLS at 25 °C (Zetasizer Nano ZS, Malvern Panalytical Co., Malvern, UK). 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 Fig. S2.
In vitro analysis of the effect of catechins on the micellar solubility of cholesterol. The
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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/min) 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 100000 × g for 1 h at 37 °C. The amount of cholesterol in the
supernatant was measured using a PerkinElmer Tri-Carb-2900TR (Waltham, MA, USA).
Electrospray ionization–mass spectrometry (ESI-MS). ESI-MS was performed in flow
injection mode using a Waters Xevo G2 QTof mass spectrometer (Milford, MA, USA). 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,
2.5 kV; sampling cone, 30 V; desolvation temperature, 500 °C; source temperature, 150 °C;
desolvation gas flow, 1000 L N2/h; and cone gas flow, 50 L N2/h. 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
Figs. 5 and S3.
Nuclear Overhauser effect (NOE) measurements
Rotating-frame 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, 0.11 and 0.028 s for the f1 and
f2 axes, respectively; mixing time, 0.25 s; and relaxation delay, 2 s. The corresponding data are shown
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in Fig. 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, 5.5 s; and
relaxation delay, 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 using array measurements based on the T1 values obtained by the inversion recovery
method. The corresponding data are shown in shown in Figs. 6 and S5.
RESULTS
13C 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
1
number of protons for its molecular size. Although its sensitivity is lower than that of H NMR
spectroscopy, 13C NMR measurements 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 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-
1
position, in agreement with the previous H NMR observations. Significant changes were also
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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 contain hydroxyl groups. This result correlates well with the fact that catechins without
a galloyl moiety have little effect on the micellar solubility of cholesterol.
1H 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 similar to those induced by
the addition of 1 (Fig. 3A). This result indicated that the B ring has a small effect on the interaction
with taurocholic acid.
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 up-field 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 magnitude of the change induced by 3
or 4 was approximately two-thirds of that observed upon the addition of 1 (Fig. 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 (Fig. S1), i.e., without a
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hydroxyl group (analogue A; 6), without a carbonyl group (analogue B; 7), and 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 chemical
shift at the 18-position of taurocholic acid upon the addition of 8 or 7, respectively (Fig. 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 for 6 and
the magnitude of the chemical shift change induced by the addition of 6 was larger at the saturation
point (Fig. 3B). This difference in the saturation points may be attributed 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 shifts correspond 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 (Fig. S2 [a]). 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 (Fig. S2 [b]). 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, 20–23 For example, two types of
model micellar system were used to easily evaluate the inhibition activity in vitro, i.e., a micellar
solution containing cholesterol, PC, and taurocholic acid,7,13, 22 and a micellar solution consisting
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of these three regents in addition to oleic acid, monoolein, etc. (micelle A solution).15, 19, 20, 21, 23
A
precipitate formed upon the addition of 1 to the micelle solution, and the removal of monoolein from
the 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 (Fig. S2 [a]), two peaks were observed for the micelle B solution (189.0 ±
10 nm and 7.901 ± 0.2 nm, Fig. S2 [c]). Furthermore, when 1 was added to the micelle B solution,
the diameter of the larger particle increased to 351.6 ± 8 nm, while the smaller particles disappeared
(Fig. S2 [d]).
Effect of catechins on the micellar solubility of cholesterol in vitro. The inhibition activities
of 1, 3, and 4 on the 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 (Fig. 4). Thus, all catechins effectively decreased the micellar solubility of cholesterol.
Furthermore, the concentration of micellar cholesterol following the addition of 3 and 4 tended to be
lower than that following the addition of 1.
ESI-MS analysis. Although the NMR experiments indicated that regiospecific interactions
take place between the catechins and taurocholic acid, the molar ratio of these complexes remains
unclear, since the NMR spectra show the equilibrium state between the bound and free catechins.
Thus, we applied MS as a valuable tool to determine the ratio of the components in this complex.
Among the various ionization methods available, we applied ESI since the interactions between the
catechins and taurocholic acid are only observed in aqueous solution.
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The MS spectrum of the mixture containing 1 (16 mM) and taurocholic acid (7.4 mM) exhibited
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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 (Figs. 5A, 5B and S3A). Thus, the observed molecular ion peaks at m/z 972 and m/z 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 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 the 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 m/z 457 (Fig. 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 2-position; 1 has a 2,3-cis-configuration, whereas 3 has a 2,3-trans-configuration. This
difference, which affects the conformation between the B ring and the gallate moiety, may contribute
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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 (Fig. 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 mixed solution of 1 or 3 with
taurocholic acid. The obtained spectra correspond to the difference between spectra recorded with
and without NOEs caused by the presaturation of individual resonances. Thus, only signals that have
a 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 owing to 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 a 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 NOE difference experiments using a solution 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 (Fig. 6A), whereas the
NOE between taurocholic acid and 3 was observed at a presaturation time of 6.9 s (Fig. 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 using presaturation times of 5.3 and 7.1 s,
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respectively (Fig. S5). These results suggested that the distance between 3 and taurocholic acid is
greater than that between taurocholic acid and 1.
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, 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 (Fig. 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 1ʹʹ- and 4ʹʹ-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
analogs 6, 7, and 8 by 1H NMR spectroscopy, chemical shift changes were only observed when
analog 6 was added (Fig. 3). In our previous study, we suggested that hydrogen bonding induced by
the hydroxyl group was important because the magnitude of the chemical shift 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, while 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
hydrogen bond forming ability.
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In terms of the effect of 2, chemical shift changes comparable to 1 were observed. This result
is 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. Based on this result, we consider that
the contribution of the B ring to the interaction is small.
1
In the H NMR experiments of 3 and 4, which are stereoisomers of 1, regiospecific
interactions with taurocholic acid were observed, however, the magnitude of the chemical shift
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 of 1. 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, although 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 cis-configuration, 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 without heat treatment. It is known that catechin epimerization is induced by heat
treatment and the C2-position epimer is produced, that is, compound 1 is converted to 3.16,17 The
observation of a stronger activity for compound 3 than for compound 1 is therefore consistent with
their report (Fig. 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
the cholesterol solubility.15 However, the magnitude of the chemical shift change at the 18-position
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of taurocholic acid induced by the addition of 3 was smaller than that induced by 1, even though 3
has a higher inhibition activity. In addition, the ROESY spectrum did not show any significant
difference between 1 and 3 (Fig. S4), although the presaturation times in the NOE experiments (Figs.
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
1
suggestion supports the results of the H NMR experiments that indicated the lesser effect of 3 on
the chemical shifts of taurocholic acid compared to 1. Due to the difference of 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 owing to
the incorporation of 1 into the bile acid micelle. Although DLS experiments 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 due to 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 micelles is caused by the insertion of
1 in a micelle by hydrophobic interactions with taurocholic acid.
Meanwhile, the presence or absence of oleic acid in the micelles affects the micelle size (Fig.
S2) and the solubility of cholesterol. We assumed that oleic acid does not contribute to the interaction
with 1, instead affecting the cholesterol micellar solubility by changing the shape and aggregation
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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.
Acknowledgments
This work was partially supported by Nagara Science Co., Ltd. and by a JSPS Kakenhi Grant
(15K07427).
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FIGURE CAPTIONS
Figure 1 Structures of the catechin derivatives and of taurocholic acid.
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.
Figure 3 (A) Effect of the 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), and △: (−)-EGCg analogue C (8)]. (B) The
saturation point of (−)-EGCg analogue A occurred at a higher concentration than those of the other
derivatives (●: 1, ○: 6).
Figure 4 The cholesterol micellar solubility in the presence of various catechin derivatives. The
values are expressed as a mean ± SEM (n = 4). Means with different superscript letters indicate
significant differences (P < 0.05) as determined by Tukey’s test.
Figure 5 MS spectra of (A) the mixture containing 1 (2 mM) and taurocholic acid (7.4 mM), and (B)
the mixture containing 5 (2 mM) and taurocholic acid (7.4 mM).
Figure 6 Difference NOE spectra of (A) the mixture containing (−)-EGCg (8.7 mM) and taurocholic
acid (10.4 mM) at a presaturation time of 5.5 s, and (B) the mixture containing (−)-GCg (8.7 mM)
and taurocholic acid (10.4 mM) at a presaturation time of 6.9 s.
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R1
O
9
2"
OH
OH
OH
2'
B
G=
6"
8
HO
O
2
3
OH
OH
2"
6'
C
9
R3
A
R4
R4
6
R2
G'=
4
6"
OH
R4
R1: OH R2: OG
(-)-EGCg (1): 2:
3:
R1: H R2: OG
R1: OH R2: OG
R1: H R2: OG
(-)-ECg (2): 2:
(-)-GCg (3): 2:
(+)-Cg (4): 2:
3:
3:
3:
R1: OH R2: OH
(-)-EGC (5): 2:
3:
R1: OH R2: OG' R3: C=O R4: H
R1: OH R2: OG' R3: CH2 R4: OH
R1: OH R2: OAc
(-)-EGCg analogue A (6): 2:
(-)-EGCg analogue B (7): 2:
(-)-EGCg analogue C (8): 2:
3:
3:
3:
O
21
OH
18
NH
12
19
25
26
H
SO3H
H
H
7
3
HO
OH
H
Taurocholic acid
523
524
Figure 1
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526
527
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Figure 2
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532
(A)
533
534
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536
Figure 3
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Figure 4
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Figure 5
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(A)
18 (irradiated)
G
(B)
G
18 (irradiated)
547
548
Figure 6
549
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Table of Contents graphic
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