Synthesis of novel (?)-epicatechin derivatives as potential endothelial GPER agonists: Evaluation of biological effects

Article abstract of DOI:10.1016/j.bmcl.2018.01.025

To potentially identify proteins that interact (i.e. bind) and may contribute to mediate (?)-epicatechin (Epi) responses in endothelial cells we implemented the following strategy: 1) synthesis of novel Epi derivatives amenable to affinity column use, 2)

Full text of DOI:10.1016/j.bmcl.2018.01.025

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel (ꢀ)-epicatechin derivatives as potential endothelial
GPER agonists: Evaluation of biological effects
a
b,f
c
a
Viviana Sarmiento , Israel Ramirez-Sanchez , Aldo Moreno-Ulloa , Diego Romero-Perez ,
d
b
b
b
e
Daniel Chávez , Miguel Ortiz , Nayelli Najera , Jose Correa-Basurto , Francisco Villarreal ,
b,f,
⇑
Guillermo Ceballos
a
Universidad Autónoma de Baja California, Tijuana, BC, Mexico
b
c
Escuela Superior de Medicina del Instituto Politécnico Nacional, Sección de Posgrado, Mexico
Departamento de Innovación Biomédica, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), BC, Mexico
Centro de Graduados e Investigación en Química del Instituto Tecnológico de Tijuana, Apartado Postal 1166, Tijuana, BC 22510, Mexico
University of California, San Diego, School of Medicine, USA
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
To potentially identify proteins that interact (i.e. bind) and may contribute to mediate (ꢀ)-epicatechin
Received 10 November 2017
Revised 11 January 2018
Accepted 16 January 2018
Available online xxxx
(
Epi) responses in endothelial cells we implemented the following strategy: 1) synthesis of novel Epi
derivatives amenable to affinity column use, 2) in silico molecular docking studies of the novel derivatives
on G protein-coupled estrogen receptor (GPER), 3) biological assessment of the derivatives on NO produc-
tion, 4) implementation of an immobilized Epi derivative affinity column and, 5) affinity column based
isolation of Epi interacting proteins from endothelial cell protein extracts. For these purposes, the Epi
phenol and C3 hydroxyl groups were chemically modified with propargyl or mesyl groups. Docking stud-
ies of the novel Epi derivatives on GPER conformers at 14 ns and 70 ns demostrated favorable thermody-
namic interactions reaching the binding site. Cultures of bovine coronary artery endothelial cells (BCAEC)
treated with Epi derivatives stimulated NO production via Ser1179 phosphorylation of eNOS, effects that
were attenuated by the use of the GPER blocker, G15. Epi derivative affinity columns yielded multiple
proteins from BCAEC. Proteins were electrophoretically separated and inmmunoblotting analysis
revealed GPER as an Epi derivative binding protein. Altogether, these results validate the proposed strat-
egy to potentially isolate and identify novel Epi receptors that may account for its biological activity.
Ó 2018 Elsevier Ltd. All rights reserved.
Keywords:
(
ꢀ)-Epicatechin
Docking
GPER
Affinity chromatography
eNOS
Flavonoids are an important class of widely distributed natural
products that posses a diverse range of biological activities.
mimicks the beneficial vascular effects observed after the con-
1
,2
6,7
sumption of cocoa products.
A proposed mechanism through
Accumulating evidence indicates that the consumption of
which Epi mediates its vascular effects include the stimulation of
flavanol-rich foods such as those found in cacao-based products,
nitric oxide (NO) production via endothelial NO synthase (eNOS)
activation. Evidence indicates that eNOS activation can occur sec-
protects against cardiometabolic diseases.3
–5
(ꢀ)-Epicatechin
8
(
Epi) is the main flavanol present in cacao seeds and its oral intake
ondary to the stimulation of cell surface receptors including those
from the tyrosine kinase and G-protein-coupled receptor (GPCRs)
9
,10
families.
Due to the healthy effects triggered by Epi, there is
Abbreviations: Epi, (ꢀ)-epicatechin; eNOS, endothelial nitric oxide synthase; NO,
nitric oxide; GPER, G-protein coupled estrogen receptor; BCAEC, bovine coronary
artery endothelial cells; GPCRs, G-protein coupled receptors; EPI-COLUMN, affinity
an increasing interest in elucidating the mechanisms by which this
flavanol mediates its cardiometabolic protective effects.1
1,12
0
0
column with Epi covalently bound; Epi-4-prop, 3,5,7,3 ,4 -penta-O-propargyl-(ꢀ)-
We recently demonstrated that Epi stimulates NO production
through the involvement of the G-protein coupled estrogen (GPER)
0
0
epicatechin; Epi-Ms, 3-O-mesyl-(ꢀ)-epicatechin; Epi-5-prop, 5,7,3 ,4 -tetra-O-
propargyl-(ꢀ)-epicatechin; Epi-prop, 3-O-propargyl-(ꢀ)-epicatechin; G15, GPER
antagonist; BPY, 5,5-difluoro-1,3,7,9-tetramethyl-N-(prop-2-yn-1-yl)-5H-4k4,5k4-
1
3
and epidermal growth factor receptors (EGFR). However, the use
of selective blockers or receptor gene silencing approaches resulted
in a partial blockade of Epi stimulated NO production. Thus, other
cell membrane receptors are likely involved in mediating the
effects of Epi and there is little knowledge about the identity of
0
0
dipyrrolo[1,2-c:2 ,1 -f][1,3,2]diazaborinin-10-amine;
SAR,
structure–activity
relationship.
⇑
Corresponding author.
E-mail address: gceballosr@ipn.mx (G. Ceballos).
Co-senior this work.
f
https://doi.org/10.1016/j.bmcl.2018.01.025
960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
0
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2
V. Sarmiento et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 1. Structures of (A) (ꢀ)-epicatechin (Epi) and its synthetized derivatives; Epi-5-prop (E), Epi-4-prop (D), Epi-Ms. (B) and Epi-prop (C).
such structures. Interestingly, several studies have suggested that
the biological properties of flavonoids are largely dependent on
the availability of ‘‘free” phenol groups on their structure
studies were implemented as previously described (see Supple-
mentary data).
Docking results (Fig. 2) suggest that the interactions between
Epi derivatives and GPER are energetically favorable and that the
2
0
1
4,15
(Fig. 1).
We thus, implemented a rational strategy comprising the fol-
type of interactions generated are via hydrogen and p-p bonding.
lowing steps: 1) synthesis of novel Epi derivatives (which relied
on the introduction of mesyl or propargyl groups) that may be
optimal for the generation of affinity columns and purification of
Epi binding proteins, 2) in silico molecular docking of the novel
Epi derivatives on a previously validated GPER platform, 3)
in vitro analysis of the novel Epi derivatives on NO production
and, 4) implementation of an inmobilized Epi derivative affinity
column to isolate binding Epi proteins from endothelial cell protein
extracts. Flavonoid effects appear to be structure-dependent and a
In general, the interactions of Epi derivatives on the GPER con-
former at 14 ns are similar to those of Epi. In contrast, Epi deriva-
tives docking results on GPER conformer at 70 ns evidenced
different binding modes between Epi derivatives and Epi. In the
1
3
14 ns GPER conformer, Epi (
G = ꢀ7.74 kcal/mol) reach some common aminoacid residues
L137, M141 by hydrophobic interaction whereas under with
F208, F223, W272 there are interactions and with S317 and
D
G = ꢀ7.9 kcal/mol) and Epi-Ms.
(D
p-p
A313 there are hydrogend bonds, Epi-Ms. also interacts with
major determinant factor is the presence of hydroxyl (i.e. phenols
S112 residue via hydrogen bonds.
and alcohol groups) moieties.1
4,15
The esterification and alkylation
Epi-5-prop (
D
G = ꢀ9.2 kcal/mol) reaches the aminoacid resi-
of the hydroxyl groups are commonly used methods used to gen-
erate flavonoid derivatives. Using this strategy, others and we have
synthesized novel flavonoid derivatives by targeting their phenol
dues L108 and L137 by hydrophobic interactions and W272,
F208 and Y142 by interactions and with S112 and Q138 under
hydrogen bonds (Fig. 2 upper panel). Epi-prop shows a
ꢀ8.05 kcal/mol and makes interactions with F278 and hydro-
gen bonds with N310. Epi-4-prop (
G = ꢀ8.68 kcal/mol) reaches
the aminoacid residues Q138, E218, Q215, E275 by hydrogen
bonds; Y142, F208 and F206 by interactions and; R286 by a
-cation interaction.
In contrast, using GPER conformer at 70 ns, docking analyses
DG =
1
6–19
groups.
. In this study, we modified the structure of Epi by tar-
p-p
0
0
geting its phenol (3 ,4 , 5 and 7 position) and alcohol (C-3 position)
groups (Fig. 1A). For the synthesis of the derivatives, native Epi was
used as a starting material. The detailed synthetic procedures used
to obtain each Epi derivative are presented in Supplementary data.
As a first step, we introduced mesyl or propargyl group substitu-
ents in the Epi molecule at the C-3 alcohol group in order to keep
the four phenolic groups available (Fig. 1B and C respectively). We
also alkylated the four phenol groups of Epi, and kept free the 3-
alcohol group (Fig. 1D). Finally, we alkylated the four phenolic
and the alcohol groups of Epi, which led to a molecule with no free
hydroxyl groups (Fig. 1E).
D
p-p
p
demonstrate that Epi derivatives interact with aminoacid residues
distinct than those observed with Epi derivatives at 14 ns (Fig. 1
bottom panel). Epi-Ms. establishes hydrogend bonds with N310,
S62, Q54, E115 and C205 and
p-p interactions with Y123. Epi-5-
prop makes hydrogen bonds with P226, T220 and W150;
interactions with F146, W150 and; hydrophobic interactions with
p-p
The resultant Epi derivatives were 3-O-mesyl-(ꢀ)-epicatechin
L221, V225, F146 and L176. Epi-prop, establishes hydrogen bonds
with S317, D111 and D105; p-p interactions with F268 and W272
and; hydrophobic interactions with L108. Epi-4-prop recognized
Q138 and C207 using hydrogen bonds. Additionally, this Epi
0
(
3
3
Epi-Ms), 5,7,3,4 -tetra-O-propargyl-(ꢀ)-epicatechin (Epi-4-prop),
0
0
,5,7,3 ,4 -penta-O-propargyl-(ꢀ)-epicatechin (Epi-5-prop), and
-O-propargyl-(ꢀ)-epicatechin (Epi-prop).
On the other hand, to ascertain for their possible bioactivity and
derivative established p-p interactions with F208 and Y123 as well
coupling to a known receptor, the novel Epi derivatives were eval-
uated in silico. For this purpose, molecular docking and dynamics
as hydrophobic interactions with L129, V196 and M133. Using
both GPER conformers, docking modeling estimates that Epi-4-
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V. Sarmiento et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
prop and Epi-5-prop interact with more aminoacid residues than
Epi-prop and Epi. We therefore propose, that the alkyne groups
enable additional interactions with aminoacid residues of the GPER
binding pocket, which contributes to a higher binding free energy
when compared to the natural flavanol.
Once the derivatives were synthesized and pre-validated in sil-
ico, we tested their in vitro efficacy using the GPER expressing
bovine coronary artery endothelial cells (BCAEC). Our primary end-
point was to evaluate the capacity of derivatives to stimulate
eNOS/NO pathway activation (Fig. 3). Cells were stimulated for
stimulated to a lesser extent the eNOS/NO pathway vs. Epi (Fig. 3
and Table 1). Interestingly, although Epi-prop also possesses a sub-
stituent at the C-3 alcohol group, displayed increased efficacy on
NO/eNOS activation pathway endpoints when compared to Epi-
Ms. and Epi. These results suggest that the modification of the C-
3 alcohol group in Epi molecule increase the efficacy of the flavanol
to stimulate the eNOS pathway. It may be possible that the intro-
duction of the hydrophilic mesylate group in the Epi molecule
affects its ability to interact with GPER present on the cell mem-
brane, where it has been localized. Interestingly, Epi-Ms. also
denoted the lowest free binding energy in docking studies, sug-
gesting a close relationship between our in silico and in vitro data.
In addition, all other derivatives showed higher free binding energy
values vs. Epi that was also accompanied by higher efficacy on
eNOS/NO activation endpoints by derivatives.
Our in vitro and docking results indicate that GPER is capable of
interacting and be activated by Epi and its derivatives. However,
other receptors are also likely involved as G15 only partially inhib-
ited eNOS activation and NO production. In order to explore this
possibility, we implemented affinity chromatography. This
approach is based on the immobilization of the molecule of inter-
est to a solid support as a means to isolate and eventually charac-
terize specific binding proteins including receptors. However, the
immobilization of ligands to a solid support may alter the biologi-
cal properties of the free ligand. Therefore, we implemented an
affinity column (Epi-based column) using Epi-prop. The assump-
1
3
1
0 min by either 1 lM Epi (used as a positive control) or 1 lM of
each Epi derivative. Nitrates/nitrites levels in the supernatant
and immunodetection of phosphorylated (at Ser1179 residue)
eNOS (p-eNOS by Western blots) were measured as a surrogate
of NO synthesis and eNOS activation, respectively (Fig. 3A). To
explore the participation of GPER the antagonist G15 was used.
Cells were pre-incubated during 30 min with 1 lM of G15, a GPER
inhibitor. Results demonstrate that as predicted by in silico model-
ing, the derivatives were able to stimulate to different degrees NO
production (Fig. 3A left and rigth panels) via eNOS activation (Fig.
3B left and rigth panels) and that effects were notably attenuated
by the GPER antagonist G15 (Table 1).
Noteworthy, Epi-4-prop, Epi-5-prop induced an effect compa-
rable to Epi, suggesting that the presence of ‘‘free” phenolic groups
1
4
might not be crucial for the stimulation of eNOS/NO pathway. On
the other hand, Epi-Ms, which possesses a mesylate group at C-3,
Fig. 2. GPER 3D model docked with (ꢀ)-epicatechin (Epi) derivatives. GPER 3D model at 14 ns docked with (A) Epi-Ms, (B) Epi-5-prop, (C) Epi-prop and (D) Epi-4-prop
(
(
upper panel right). Ligands superimposed into the binding site of 14 ns GPER conformer (upper panel left). GPER 3D model at 70 ns docked with (A) Epi-Ms, (B) Epi-5-prop,
C) Epi-prop and (D) Epi-4-prop (bottom panel right). Ligands superimposed into the binding site of 70 ns GPER conformer (bottom panel left).
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4
V. Sarmiento et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 3. Effects of (ꢀ)-epicatechin (Epi) and Epi derivatives on eNOS/NO pathway activation. (A) Epi derivatives induced nitric oxide (NO) production and (B) endothelial nitric
oxide synthase (eNOS) activation (phosphorylation at Ser-1179 [p-eNOS]) in bovine coronary artery endothelial cells (BCAEC). All compounds were tested at 1
was arbritarily set to 1 and values are reported as mean ± SEM, p < 0.05 * vs control or ^ vs Epi (n = 5 per group).
lM. Control
Table 1
Quantification of the effects of (ꢀ)-epicatechin (Epi) and Epi derivatives on eNOS/NO pathway activation in BCAEC.
Compound
NO production (%)
p-eNOS activation (%)
+G15 NO production (%)b
+G15 p-eNOS activation (%)b
Epia
Epi-Ms
Epi-5-prop
Epi-4-prop
Epi-prop
100.0
54.5
101.8
107.3
141.8
100.0
64.8
92.6
111.1
137.0
60.0
41.8
60.0
63.6
70.9
55.6
53.0
54.8
57.4
59.3
a
Epi levels were arbritarily set to 100% and values of derivatives are adjusted to Epi values and expressed as percentages.
For GPER blocking, cells were pre-incubated with G15 (1 lM) for 30 min before the addition of 1 lM of ligands.
b
tion is that this molecule as per its enhanced in vitro bioactivity
and free in silico binding energy retains intact, the core bioactive
elements of Epi. For this purpose, Epi-prop was covalently immo-
bilized by the use of Click chemistry to commercially available
agarose azide beads, which were then packed into a mini-
These columns were used to identify possible nonspecific protein
binding to the matrix. The experimental column was obtained add-
ing all the reactants to the agarose beads. The binding of Epi-prop
to the agarose-azide beads was determined indirectly by fluores-
cence and quantification of Epi-prop recovered after column
washings. An external calibration curve using known concentra-
tion solutions of Epi-prop served to extrapolate the fluorescence
levels measured in the column washings. Results indicated that
86.6% of added Epi-prop to the agarose-azide beads was attached
(data not shown).
Moreover, to test the feasibility of the Click chemistry reaction,
agarose-azide beads were treated with an alkyne-fluorescein (exci-
tation/emission peak at 400/460 nm, [BPY]) reactive under the
same conditions employed in the reaction with Epi-prop. The
BPY was successfully attached to the agarose beads (ꢁ80%), as
indicated by the fluorescence levels noted under ultraviolet light
(Fig. 4) and the fluorescent quantification levels (using an external
2
1,22
column
(see Supplementary data). To achieve this goal, we
took advantage of the propargyl substituent at the C-3 alcohol
group in Epi-prop that reacts selectively with organic azides
groups such as those present on agarose azide beads, leaving free
the Epi phenol groups. The specific method used for the synthesis
of the affinity columns are described in detail in Supplementary
data. As controls, we generated three different agarose columns,
A) a column where all reactants except copper (II) sulfate pentahy-
drate were added, B) a column where all reactants except sodium
ascorbate were added and, C) a column where all reactants except
Epi-prop were omitted in the reaction (Fig. 4). Reagents on the col-
umn surfaces were removed by exhaustive washing using EDTA.
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V. Sarmiento et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
Fig. 4. Scheme depicting the generation of the affinity columns by the use of Click chemistry. Three control columns (CTRL(s)-COLUMN) were fabricated by the addition of all
components except (A) Epi-prop, (B) copper(II) sulfate pentahydrate and sodium ascorbate and, (C) Epi-prop, copper(II) sulfate pentahydrate and sodium ascorbate.
Fig. 5. Identification of GPER in isolated proteins from the affinity chromatography columns. Lysates from endothelial cells were loaded into the (ꢀ)-epicatechin (Epi) affinity
column (EPI-COLUMN) and control columns (CTRL(A), CTRL(B) and CTRL(C)). Western blot analysis using a monoclonal GPER antibody on proteins separated by affinity
columns. UP: Unbound proteins fraction, E: Elution fraction.
calibration curve) of BPY recovered in continuous washings (data
not shown).
appears that substitutions with hydrophilic groups (i.e. mesylate)
at the C-3 alcohol in the Epi molecule reduces its in vitro efficacy,
which may be secondary to an increase in polarity that could affect
its ability to interact with cell membrane targets. Also, we demon-
strate that the Epi-prop derivative possesses a favorable thermo-
dynamic interaction using a tridimensional model of GPER that is
validated in vitro by noting an improved efficacy on eNOS/NO
activation vs. Epi. The chemical blockade of GPER and affinity
chromatography columns with immobilized Epi-prop confirmed
the role of this receptor in mediating the effects of Epi.
Based on our in vitro results where the antagonist G15 only par-
tially blocks the responses and on the presence of several proteins
interacting with the Epi-column, the possibility of more receptors
or effectors participating on Epi-induced effects is highly likely.
More work is necessary to identify and characterize these
molecules.
Once implemented, the ability of the affinity Epi-column to iso-
late protein targets was evaluated using protein extracts from
BCAEC. Proteins (0.8–1.0 mg) were loaded into control columns
and Epi-Column (incubated overnight at 4 °C with mild tumbling)
and columns were then washed thoroughly with at least 5 volumes
of wash buffer (5 mM EDTA, 0.5% SDS, 250 mM NaCl in 1X PBS pH
7
.5), 1X PBS (20% in wash buffer) and 1X PBS, or until no detectable
protein was found in the eludates. Attached proteins were eluted
using PBS (1X) buffer containing 8 M urea. Eludates containing iso-
lated proteins were concentrated using centrifugal filters (Amicon
Ultra 10K), and samples were separated using SDS-PAGE gel elec-
trophoresis and proteins stained using Coomassie-Blue. Consistent
bands were evidenced in the elution fractions of Epi-column (data
not shown). The control columns eludates were analyzed under the
same conditions in parallel. However, no bands were observed
when stained with Coomassie-Blue (data not shown) suggesting
specific binding to the experimental Epi-column.
Disclosures
SDS-PAGE proteins derived from Epi-column were transferred
onto PVDF membranes. Western blot analysis revealed the
presence of a 42-kDa protein that was reactive to a GPER antibody
Dr. Villarreal is a co-founder and stockholder of Cardero
Therapeutics Inc. and Dr. Ceballos is a stockholder.
(Fig. 5). These findings are in agreement with our previous
Acknowledgements
published data using BCAEC to determine the role of GPER in medi-
ating the effects of Epi on eNOS activation.1 In such study, using
siRNA and chemical blockers approaches, we validated GPER as a
likely receptor candidate for Epi in endothelial cells.
3
V. Sarmiento acknowledges support from CONACYT in the form
of graduate scholarships. Dr. Villarreal was supported by NIH
DK98717 and Dr. Ceballos by a Conacyt # 253769 grant. We thank
CONACyT for ITT NMR and HRMS facilities (Grants INFR-2011-3-
In summary, four new Epi derivatives were synthesized by
modifying Epi at either its phenol or C-3 alcohol groups in order
to assess the potential contribution of such positions on Epi biolog-
ical effects. Our limited SAR data suggest that propargyl substitu-
1
73395 and INFR- 2012-01-187686).
We would like to thank Dr. IA Rivera for the generous donation
of the Alkyne-fluorescein (BPY) and Dr. A Ochoa for allowing to use
his lab during this research both from Centro de Graduados e
Investigación en Química del Instituto Tecnológico de Tijuana.
0
0
tions at the 3,5,7,3 ,4 phenols or C-3 alcohol positions on Epi
molecule do not impair its capacity to stimulate eNOS. In fact, it
Please cite this article in press as: Sarmiento V., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.01.025

6
V. Sarmiento et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
1. Panneerselvam M et al. Dark chocolate receptors: epicatechin-induced cardiac
1
A. Supplementary data
protection is dependent on d-opioid receptor stimulation. Am J Physiol-Heart
Circulatory Physiol. 2010;299:H1604–H1609.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.01.025.
12. Moreno-Ulloa A et al. Cell membrane mediated (ꢀ)-epicatechin effects on
upstream endothelial cell signaling: evidence for a surface receptor. Bioorg Med
Chem Lett. 2014;24:2749–2752.
1
3. Moreno-Ulloa A et al. The effects of (ꢀ)-epicatechin on endothelial cells involve
the protein-coupled estrogen receptor (GPER). Pharmacol Res.
015;100:309–320.
References
G
2
1
. Arts ICW, van de Putte B, Hollman PCH. Catechin contents of foods commonly
consumed in The Netherlands. 1. Fruits, vegetables, staple foods, and processed
foods. J Agric Food Chem. 2000;48:1746–1751.
14. Vaya J et al. Inhibition of LDL oxidation by flavonoids in relation to their
structure and calculated enthalpy. Phytochemistry. 2003;62:89–99.
15. Garg R, Kurup A, Hansch C. Comparative QSAR: on the toxicology of the
phenolic OH moiety. Crit Rev Toxicol. 2001;31:223–245.
2
3
4
5
. Pan M-H, Lai C-S, Ho C-T. Anti-inflammatory activity of natural dietary
0
flavonoids. Food Funct. 2010;1:15–31.
16. Spencer JP et al. Epicatechin and its in vivo metabolite, 3 -O-methyl
. Buijsse B et al. Cocoa intake, blood pressure, and cardiovascular mortality: the
zutphen elderly study. Arch Intern Med. 2006;166:411–417.
. Buijsse B et al. Chocolate consumption in relation to blood pressure and risk of
cardiovascular disease in German adults. Eur Heart J. 2010;31:1616–1623.
. Mostofsky E et al. Chocolate intake and incidence of heart failure: a population-
based prospective study of middle-aged and elderly women. Clin Perspect.
epicatechin, protect human fibroblasts from oxidative-stress-induced cell
death involving caspase-3 activation. Biochem J. 2001;354:493–500.
17. Uesato S et al. Inhibitory effects of 3-O-acyl-(+)-catechins on Epstein-Barr virus
activation. Chem Pharm Bull. 2003;51:1448–1450.
18. Park KD et al. Anticancer activity of 3-O-acyl and alkyl-(ꢀ)-epicatechin
derivatives. Bioorg Med Chem Lett. 2004;14:5189–5192.
2
010;3:612–616.
19. Moreno-Ulloa A et al. Effects of (ꢀ)-epicatechin and derivatives on nitric oxide
mediated induction of mitochondrial proteins. Bioorg Med Chem Lett.
2013;23:4441–4446.
20. Méndez-Luna D et al. Deciphering the GPER/GPR30-agonist and antagonists
interactions using molecular modeling studies, molecular dynamics, and
docking simulations. J Biomol Struct Dyn. 2015;33:2161–2172.
21. Nwe K, Brechbiel MW. Growing applications of ‘‘click chemistry” for
bioconjugation in contemporary biomedical research. Cancer Biother
Radiopharm. 2009;24:289–302.
6
. Schroeter H et al. (–)-Epicatechin mediates beneficial effects of flavanol-rich
cocoa on vascular function in humans. PNAS. 2006;103:1024–1029.
. Ottaviani JI et al. The stereochemical configuration of flavanols influences the
level and metabolism of flavanols in humans and their biological activity
in vivo. Free Radical Biol Med. 2011;50:237–244.
7
8
. Ramirez-Sanchez
I
et al. (ꢀ)-Epicatechin activation of endothelial cell
endothelial nitric oxide synthase, nitric oxide, and related signaling
pathways. Hypertension. 2010;55:1398–1405.
9
. Dudzinski DM, Michel T. Life history of eNOS: partners and pathways.
Cardiovasc Res. 2007;75:247–260.
22. Anseth KS, Klok H-A. Click chemistry in biomaterials, nanomedicine, and drug
delivery. Biomacromolecules. 2016;17:1–3.
1
0. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell.
007;129:1261–1274.
2
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