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Protective effect of epigallocatechin gallate on human erythrocytes
Accepted Manuscript Title: Protective effect of epigallocatechin gallate on human erythrocytes Authors: Jos´e R. Colina, Mario Suwalsky, Marcela Manrique-Moreno, Karla Petit, Luis F. Aguilar, Malgorzata Jemiola-Rzeminska, Kazimierz Strzalka PII: DOI: Reference:
S0927-7765(18)30734-3 https://doi.org/10.1016/j.colsurfb.2018.10.038 COLSUB 9722
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
10-5-2018 12-10-2018 15-10-2018
Please cite this article as: Colina JR, Suwalsky M, Manrique-Moreno M, Petit K, Aguilar LF, Jemiola-Rzeminska M, Strzalka K, Protective effect of epigallocatechin gallate on human erythrocytes, Colloids and Surfaces B: Biointerfaces (2018), https://doi.org/10.1016/j.colsurfb.2018.10.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Protective effect of epigallocatechin gallate on human erythrocytes
José R. Colinaa, Mario Suwalskya*, Marcela Manrique-Morenob, Karla Petita, Luis F. Aguilarc,
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Facultad de Ciencias Químicas, Universidad de Concepción, Concepción, Chile
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Malgorzata Jemiola-Rzeminskad,e, Kazimierz Strzalkad,e
Faculty of Exact and Natural Sciences, University of Antioquia, Medellín, Colombia
Instituto de Química, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile
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Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
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Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
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* Corresponding author at: Faculty of Chemical Sciences, University of Concepción, Concepción,
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Chile.
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E-mail address: [email protected] (M. Suwalsky)
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Total number of words: 5223, without references and figure legends.
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Number of figures: 8 (1 as supplementary material)
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Number of tables: 2 (as supplementary material)
Graphical abstract
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Highlights
Structural and thermotropic perturbations in DMPC bilayers were induced by EGCG
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EGCG produced morphological alterations in RBC inducing the formation of echinocytes
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Deleterious effects of HClO on DMPE bilayers were neutralized by EGCG
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-EGCG protected RBC morphological alterations and lysis caused by HClO
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ABSTRACT
The interactions and the protective effect of epigallocatechin gallate (EGCG) on human erythrocytes
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(RBC) and molecular models of its membrane were investigated. The latter consisted of bilayers builtup of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE),
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representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. X-ray diffraction and differential scanning calorimetry experiments showed that EGCG induced significant structural and thermotropic perturbations in multilayers and vesicles of DMPC; however, these effects were not observed in DMPE. Fluorescence spectroscopy results revealed that EGCG produced alterations of the molecular dynamics at the level of
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the hydrophobic-hydrophilic interface in DMPC vesicles, and in isolated unsealed human erythrocyte membranes (IUM). EGCG also induced morphological alterations in RBC from their normal discoid form to echinocytes. These outcomes indicate that EGCG molecules were located in the outer monolayer of the erythrocyte membrane. The assessment of EGCG protective effect demonstrated that
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it inhibits the morphological alterations and lysis induced by HClO to human erythrocytes. The results obtained from this study suggest that the insertion of EGCG into the outer monolayer of the erythrocyte membrane might prevent the access and deleterious effects of oxidant molecules such as HClO and free radicals into the red cells, protecting them from oxidative damage.
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Keywords:
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Epigallocatechin gallate
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Erythrocyte membrane
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Phospholipid bilayer
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1. Introduction
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Antioxidant
Green tea (Camellia sinensis) is one of the most widely consumed beverages throughout the
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world, and its consumption has long been associated with health benefits [1]. Green tea is rich in a wide variety of polyphenols, particularly of catechins [2]. Epigallocatechin gallate (EGCG, (−)-cis3,3′,4′,5,5′,7-hexahydroxyflavane-3-gallate, Fig. 1) is the most abundant and biologically active catechin isolated from green tea, containing between 25-30 mg per bag [3]. It has been suggested that EGCG is responsible for many of the beneficial effects of green tea both in cell culture as well as in animal and clinical studies [4]. It has been reported to have a therapeutic effect in pathologies such as
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cancer, diabetes, stroke, obesity, Parkinson's and Alzheimer's disease [5]. The beneficial effects of EGCG are mainly attributed to its role as an antioxidant and free radical scavenger, which have been attributed to the presence of phenolic groups; they are sensitive to oxidation which is increased by the existence of the trihydroxyl group in the D-ring of this catechin [6] (Fig. 1). However, a role as pro-
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oxidant and cytotoxic agent in some cellular contexts has also been suggested [7]. A specific molecular mechanism explaining EGCG antioxidant properties has not been reported. However, it has been suggested that its biological activity can be related to its ability to interact with plasma membrane components, including lipids and proteins, affecting the stability of the lipid bilayers [8,9]. The particular structure of catechins significantly influences their interaction with biological membranes.
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The galloyl group present in some catechins plays a key role in the relative affinity of these compounds
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to membranes, which has been related to their bioactivity [2]. Thus, EGCG and epigallocatechin
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(EGC), both containing the galloyl moiety show the most potent biological effects among the catechins
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present in green tea [10]. The presence of the galloyl group also determines the location of the
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catechins in the lipid bilayer. Despite extensive studies on the interaction of EGCG with lipid bilayers,
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few have been carried out on real membranes, particularly that of the human erythrocyte. The erythrocyte membrane is an appropriate model for studying the interaction of natural or
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synthetic compounds with cell membranes. Erythrocytes, although less specialized than other cells
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retain sufficient functions in common to be considered a representative model of the plasma membrane in general [11]. In order to understand the molecular mechanisms involved in the interaction of EGCG
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with the cell membrane, human erythrocytes and molecular models of its membrane were used. The molecular models consisted of bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), which represent classes of phospholipids located in the outer and inner monolayer of the human erythrocyte membrane, respectively as well as in those of many other cells. This research involved the structural, dynamic and thermotropic study of EGCG interactions with the human erythrocyte membrane, and its protective capacity against damage caused
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by hypochlorous acid (HClO). HClO is a powerful natural oxidant and a potent antimicrobial agent [12], which is involved in a large number of pathological conditions related to inflammatory processes including atherosclerosis, neurodegenerative diseases and some cancers [13]. In human erythrocytes, HClO is capable of causing morphological alterations and lysis of the plasma membrane [14].
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Understanding the mechanisms by which EGCG protects cells from oxidation can contribute to the development of new therapies aimed at preventing the development of pathological conditions.
2. Materials and methods
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2.1. Chemicals and reagents
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Epigallocatechin gallate and sodium hypochlorite solution (4–5%) were from Sigma-Aldrich
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(St. Louis, MO, USA). Hypochlorous acid (HClO) samples were prepared by diluting sodium
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hypochlorite solution in the phosphate buffer PBS (150 mM NaCl, 1.9 mM NaH2PO4, 8.1 mM
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Na2HPO4, pH 7.4). The hypochlorite concentration was determined spectrophotometrically at 292 nm using = 350 M−1 cm−1. Under such conditions both forms HClO and ClO− are present in an
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approximately equimolar ratio [15]. Synthetic DMPC (lot 850345-02-270, MW 677.9) and DMPE (lot
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850745-01-069, MW 635.9) were from Avanti Polar Lipids Inc. (AL, USA); 1,6-diphenyl-1,3,5hexatriene (DPH) and 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan) fluorescent probes were
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from Molecular Probe (Eugene, OR, USA). All other chemicals were from Sigma-Aldrich. Distilled water was purified using a Milli-Q system (Millipore, Bedford, MA, USA).
2.2. X-ray diffraction studies of DMPC and DMPE multilayers
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The capacity of EGCG to interact with DMPC and DMPE multilayers was evaluated using Xray diffraction. 2 mg of each phospholipid were placed in Eppendorf tubes and then filled with 150 L of (a) distilled water (control), and (b) aqueous solutions of EGCG (0.1-2.0 mM). The protective capacity of EGCG was evaluated by pre-incubating each phospholipid with EGCG for 10 min and
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thereafter with 10 mM HClO. All samples were incubated for 30 min in a shaking bath at 30 ºC for DMPC and 60 ºC for DMPE, then transferred into 1.5 mm diameter special glass capillaries (WJMGlas, Berlin, Germany) and centrifuged at 2500 rpm for 15 min (MRC, Israel). A Bruker Kristalloflex 760 (Karlsruhe, Germany) generator with Ni-filtered CuK radiation was used for the X-ray diffraction
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experiments. The relative reflection intensities were collected in an MBraun PSD-50M linear position
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sensitive detector system (Garching, Germany). The experiments were carried out at 18 ± 1 °C, which
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is lower than the main phase transition temperature of DMPC (24.3 °C) and DMPE (50.2 °C) [16].
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Higher temperatures would have induced transitions to more fluid phases hampering the detection of
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structural changes. Each experiment was repeated at least twice.
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2.3. Differential scanning calorimetry (DSC) studies of DMPC and DMPE vesicles
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Multilamellar vesicles (MLV) were used to study the thermotropic behavior of DMPC and
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DMPE in the presence of EGCG. To prepare MLV, first the appropriate amount of each phospholipid was dissolved in chloroform. The solvent was evaporated by applying a gentle stream of nitrogen until
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a thin film was formed on the walls of the glass tubes. The samples were then exposed to vacuum for 1 h in order to remove traces of the solvent. MLV with a final lipid concentration of 1 mM were prepared by vortexing the samples at a temperature above the gel-to-liquid crystalline phase transition of the pure lipids (30 °C for DMPC and 70 °C for DMPE). EGCG dissolved in water was added in the concentration range of 0.01-0.5 mM. The samples were degassed prior to being loaded in the
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calorimeter cell by pulling a vacuum of 30.4–50.7 kPa on the solution at 25 °C for 10 min to avoid bubble formation during heating. The DSC experiments were carried out using a NANO DSC Series III System with Platinum Capillary Cell (TA Instruments, USA). A sample cell was filled with about 400 μL of MLV suspension, and an equal volume of water was used as a reference; the cells were sealed
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and thermally equilibrated for 10 min at the starting temperature of the run. All measurements were made on samples under 0.3 MPa pressure. The data were collected within a range of 5-40 °C (DMPC) and 30-70 °C (DMPE) at a scan rate of 1 °C min−1 for both heating and cooling. Water as a sample and a reference was also scanned to collect the apparatus baseline. In order to check the reproducibility, each sample was prepared and recorded at least three times. Each data set was analyzed for
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thermodynamic parameters with a software package supplied by TA Instruments.
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human erythrocyte membranes (IUM)
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2.4. Fluorescence spectroscopy studies of large unilamellar vesicles (LUV) and isolated unsealed
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The influence of EGCG on the dynamics of DMPC LUV and IUM was examined using fluorescence spectroscopy. The measurements of anisotropy (r) and generalized polarization (GP) were
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achieved using DPH and laurdan fluorescent probes, respectively according to the previously described
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method [17]. Briefly, DMPC LUV were prepared by extrusion through two polycarbonate filters of 400 nm pore size (Nucleopore, Corning Costar Corp., MA, USA) from a suspension of 0.4 mM MLV
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previously subjected to freeze-thaw cycles. IUM were prepared using the method proposed by Dodge et al. [18]. DPH and laurdan probes were incorporated into the LUV and IUM suspensions by adding 1 L/mL aliquots of 0.5 mM stock solutions of the probe in dimethyl sulfoxide and ethanol, respectively and incubated at 37 °C for 1 h. EGCG was incorporated into LUV and IUM suspensions by addition of adequate aliquots in order to obtain the different concentrations used in this study. Fluorescence spectra
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and anisotropy measurements were taken in a K-2 spectrofluorometer (ISS Inc., Champaign, IL, USA) using the software ISS in order to collect and analyze the data. For DPH and laurdan, the exciting light was from a modulable ISS 375 nm LED laser. Fluorescence anisotropy (r) was calculated according to the definition: r = (I∥ − I⊥)/(I∥ + 2I⊥), where I∥ and I⊥ are the corresponding parallel and perpendicular
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emission fluorescence intensities with respect to the vertically polarized excitation light [19]. Laurdan fluorescence spectral shifts were quantitatively evaluated using the GP concept, which is defined by the expression GP = (Ib − Ir)/(Ib + Ir), where Ib and Ir are the emission intensities at the blue and red edges of the emission spectrum, respectively. These intensities were measured at the emission wavelengths of 440 and 490 nm, which correspond to the emission maxima of laurdan in both gel and liquid crystalline
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phases, respectively [20]. The measurements were taken at 18 ºC and 37 ºC for LUV and 37 ºC for
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IUM suspensions. The data presented in the figures represent the mean values and a standard error of
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2.5. Scanning electron microscopy (SEM) studies on human erythrocytes
The morphological alterations induced by HClO and the protective role of EGCG in human
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erythrocytes were analyzed using SEM. 100 L of blood was obtained by finger puncture from a
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healthy donor and received in an Eppendorf tube containing 900 L of phosphate buffer saline (PBS) pH 7.4. The red blood cell suspension (RBCs) was homogenized and centrifuged at 1000 rpm for 10
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min (MRC, Israel), the supernatant was removed via aspiration and replaced with fresh PBS; this procedure was repeated three times. 50 L of RBCs were placed in Eppendorf tubes and then added 150 L of (a) PBS (Control), (b) EGCG (1-100 M), and (c) 50 M HClO dissolved in PBS. In order to determine the protective effect RBCs were pre-incubated with EGCG (1-10 M) for 20 min and then with 50 M HClO. All samples were incubated in a shaking bath for 1 h at 37 °C. The samples were
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centrifuged (1000 rpm x 10 min) and the supernatant discarded. Afterwards, the cells were fixed for 24 h at 4 °C with 500 L of 2.5% glutaraldehyde in PBS. The RBCs were washed three times with 500 L of distilled water, and one drop of each sample was placed on siliconized Al glass-covered stubs of 20x20 mm, air-dried at room temperature, and gold coated for 3 min at 13.3 Pa in a sputter device
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(Edwards S 150, Sussex, England). The observations were made at a resolution of 2500X in a scanning electron microscope (JEOL JSM-6380LV, Japan).
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2.6. Hemolysis assays in human erythrocytes
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The ability of EGCG to prevent membrane rupture of human erythrocytes by HClO was
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determined using hemolysis assays. RBC were obtained directly via venipuncture from a healthy donor
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who was not receiving pharmacological treatment. The blood (10 mL) was centrifuged at 2500 rpm for 10 min at 4 °C; after the plasma and buffy coat had been removed, the cells were washed three times
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with PBS. Next, the RBCs (10% v/v) were incubated with different concentrations of EGCG (5-100
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for 5 min. Hemolysis was determined spectrophotometrically (Shimadzu UV-mini, Japan) by the
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hemoglobin released in the supernatant at 540 nm. The protective effect was evidenced by a decrease in
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the percentage of hemolysis in a relation to a control consisting of 0.5 mM HClO.
2.7. Statistical Analyses
Statistical analyses were performed using Graph Pad Prism 6 (Graph Pad Software Inc.). Oneway ANOVA followed by Dunnett’s post hoc test at significance level α = 0.05 was used to estimate
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the differences between the samples treated with EGCG and the control.
3.1. X-ray diffraction studies of DMPC and DMPE multilayers
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3. Results
Fig. 2A shows the diffraction patterns of DMPC incubated with water and increasing concentrations of EGCG. As previously reported, water affected the structure of DMPC producing an increase in the bilayer repeat (bilayer width plus the width of the water layer between bilayers) from
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about 55 Å in its dry crystalline form to 64 Å when fully hydrated [21]. This interaction also caused a
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reduction in the small-angle reflections (SA in the figure), which were reduced to only two orders of
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the bilayer repeat. On the other hand, only one reflection of 4.2 Å was observed in the wide-angle
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region (WA in the figure), which corresponds to the average distance between the fully extended acyl
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chains packed in a hexagonal arrangement. The incubation of DMPC with increasing concentrations of
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EGCG produced a regular weakening of the low, and particularly of the wide angle 4.2 Å reflection intensities, which was practically absent with 2 mM EGCG. As shown in Fig. 2B, EGCG did not affect
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the DMPE structure in the assayed concentration range (0.1-2.0 mM). Figures 3A and 3B show the
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effects of hypochlorous acid (HClO) on DMPC and DMPE, respectively. As it can be appreciated, HClO had no significant effect on DMPC even at a 10 mM concentration, although 2.5 mM HClO
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produced a profound effect on the DMPE bilayer structure as its small angle reflection intensities suffered a considerably decrease, which almost disappeared at a concentration of 10 mM. Fig. 3C presents the protective capacity of EGCG against the structural effects of HClO on DMPE. In fact, preincubating DMPE with EGCG in the 0.1-2.0 mM range increasingly neutralized the deleterious effect of 10 mM HClO until completely eradicated by 2 mM EGCG.
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3.2. Differential scanning calorimetry (DSC) studies in DMPC and DMPE vesicles
Representative high-sensitivity DSC heating thermograms obtained for pure DMPC and DMPE multilayer vesicles and binary mixtures of each phospholipid with EGCG within a concentration range of 0.01 mM to 0.5 mM are presented in Fig. 4. Fig. 4A shows the representative thermograms obtained
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for pure DMPC and DMPC/EGCG mixtures. Pure DMPC bilayers within a thermal range of 5-30 ºC showed a strong and sharp main phase transition at 24.01 °C, with an enthalpy change (ΔH) of 17.72 kJ mol−1 which corresponds to the conversion of the rippled gel phase (Pβ′) to the lamellar liquid-crystal (Lα) phase. At 14.71 °C was observed a pre-transition arising from the conversion of a lamellar gel phase (Lβ′) to a rippled gel phase with ΔH of 2.93 kJ mol−1. The presence of EGCG caused a marked alteration in the thermotropic behavior of the DMPC bilayer, which was evidenced by a gradual
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decrease and broadening of the main phase transition peak, together with a continuous shift towards
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lower temperatures. This effect can also be demonstrated from the transition width at half-peak height
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(ΔT1/2), which is a measure of destabilization of the phospholipid assembly and the size of the cooperative unit [22]. Pure phospholipid melts in a cooperative domain showing a sharp transition peak
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(ΔT1/2 = 0.61 and 0.62 for heating and cooling, respectively). However, the presence of EGCG continuously increased the ΔT1/2 reaching at 0.25 mM values of 3.64 and 1.91 for heating and cooling,
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respectively (Table 1, supplementary material). This acute increase indicates that EGCG caused a less
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cooperative melting process to DMPC due to the coexistence of gel and liquid-crystalline phase domains, which produces the widening in the melting temperature range [23,24]. EGCG also strongly
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affected the pre-transition of DMPC liposomes, which was hardly observed at the very low concentration of 0.01 mM, which disappeared at a concentration of 0.025 mM EGCG. From the DSC profiles shown in Fig. 4B, it can be seen that within a temperature range of 41-55 ºC DMPE showed a
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single sharp transition at 50.71 °C, with an enthalpy change of 20.95 kJ mol−1. This transition is related to the change of the gel phase (Lβ) to the Lα phase. The heating scans recorded for DMPE showed no
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evidence of changes in the thermotropic behavior of this lipid, indicating that there was no interaction with the catechin. The thermodynamic data of pure DMPC and DMPE were consistent with previous reports [25,26]. The insets in Fig. 4 show the dependence of values of the main phase transition temperature of DMPC (Fig. 4C) and of DMPE (Fig. 4D) on EGCG concentration. With an increase in the catechin content in DMPC, Tm decreased reaching a maximum variation (ΔTm) of 4.76 ºC at 0.5 mM EGCG, although the dependence is nonlinear. From Fig. 4D, it can be seen that EGCG had a negligible effect on the DMPE phase transition temperature. At a concentration of 0.5 mM a very slight
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decrease in Tm was only observed in this system upon cooling (data not shown); for details on thermodynamic data see supplementary material.
3.3. Fluorescence spectroscopy studies of large unilamellar vesicles (LUV) and isolated unsealed
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human erythrocytes membranes (IUM)
Changes in the physical properties of DMPC LUV and IUM induced by EGCG were investigated at two different depths of the lipid bilayers. DPH is a non-polar fluorophore used as a probe to monitor changes in the hydrophobic regions of the phospholipid bilayers, while laurdan
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provides information about the polarity and/or molecular dynamics at the level of the phospholipid
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glycerol backbone. Fig. 5 shows the influence of EGCG on the values of the fluorescence spectral shift
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GP parameter and the steady-state fluorescence anisotropy in both DMPC LUV and IUM. A very sharp
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increase in GP, concentration-dependent of EGCG was observed in DMPC LUV at 37 °C (Fig. 5A), causing a variation (ΔGP) of 0.33 at 40 μM EGCG. At 18 °C EGCG caused only a mild increase in GP
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up to a concentration of 20 μM, and no significant changes were observed in the remaining
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concentrations. The results of the interaction of EGCG with erythrocyte membranes (solid triangles
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line, Fig. 5A) at 37 °C showed a moderate increase in GP values within a range of 40-100 μM, reaching a maximum ΔGP of 0.12 at 100 μM EGCG. These results indicate that EGCG caused a structural
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ordering of the hydrophilic region of DMPC LUV and RBC membrane. As it can be observed in Fig. 5B, changes induced by EGCG on DPH anisotropy in DMPC LUV and IUM were less marked than in
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the case of GP indicating a scanty interaction of EGCG with the hydrophobic environment of the lipid and the erythrocyte membrane.
3.4. Scanning electron microscopy (SEM) studies on human erythrocytes
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SEM was used to visualize the morphological changes of human erythrocytes (RBC) subjected to in vitro assays. Fig. 6 presents the protective effect of EGCG on the deleterious effects of HClO in human erythrocytes. RBC incubated with PBS pH 7.4 (Fig. 6A, control) shows the characteristic discoid-biconcave morphology of normal erythrocytes (discocytes) [27]. As it can be observed in Fig.
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6B, erythrocytes incubated with 10 μM EGCG did not show morphological changes; however, 50 μM EGCG changed their normal morphology to echinocytes (cells characterized by the presence of spicules that are regularly spaced and uniformly distributed) (Fig. 6C). Fig. 6D shows that 50 μM HClO induced morphological alterations in the RBC, causing the formation of stomatocytes (cells with deep concave invaginations) and of a few knizocytes (cells with two or three concavities). However, as
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shown in Figs. 6E and 6F the pre-incubation of RBC with 2.5 μM and 5 μM EGCG, respectively
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erythrocytes their normal biconcave shapes.
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neutralized in a dose-dependent manner the morphological effects caused by HClO, exhibiting the
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3.5. Hemolysis assays in human erythrocytes
In order to determine the protective role of EGCG in HClO-induced lysis in human
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erythrocytes, hemolysis assays were carried out. Fig. 7 shows that EGCG within a concentration range
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of 5-100 μM did not induced significant hemolysis in the RBC (black bars). However, the same EGCG concentrations progressively inhibited the hemolysis caused to red cells by 0.5 mM HClO (white bar),
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reaching a minimum of 4.5% hemolysis with 100 μM EGCG (gray bars). These results demonstrate the capacity of EGCG to protect human erythrocytes from the lysis caused by HClO.
4. Discussion
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In the present study, the interaction between EGCG and human erythrocytes and molecular models of its membrane was investigated, and the protective effect of this catechin against oxidantinduced damage was evaluated. The results of X-ray diffraction in DMPC and DMPE multilayers in the gel phase showed that 2 mM EGCG produced significant structural perturbations to DMPC but not to
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DMPE bilayers. Both phospholipids differ only in their terminal amino groups, these being +NH3 for DMPE and +N(CH3)3 for DMPC. DMPE molecules pack tighter than DMPC molecules given the smaller size of its head group and consequent higher effective charge. This results in a very stable system with strong electrostatic and hydrogen bond interactions which are not easily affected by water or by EGCG. On the other hand, DMPC inter bilayer interactions are much weaker due to its bulky
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head group. This allows water to fill the polar inter bilayer spaces resulting in an increase of their
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separation [21], condition that makes possible the entry of EGCG molecules resulting in the observed
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structural perturbations.
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DSC is a very sensitive method for investigating the changes in the enthalpy and the
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temperature of the phase transition of lipids. The incorporation of EGCG into DMPC bilayers gave rise
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to a pronounced distortion of its thermotropic behavior and as a result both the pre-transition and the main phase transition were affected. This result coincides with that reported by Caturla et al. [28].
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Interestingly, the pre-transition seemed to be much more effectively distorted than the conversion of
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the rippled gel phase to the lamellar liquid-crystal phase, known as the main phase transition. This finding is consistent with the alterations observed in the conformational rearrangement in the head
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group region of the lipid molecules [29,30]. The lowering of Tm and the increase in the main phase transition peak width indicates that EGCG molecules cause a significant perturbation in the DMPC chain melting process. The widening of the melting peak is consistent with a reduction of the melting cooperativity or the presence of intermediate melting states [31]. On the other hand, it was observed a small effect of EGCG in the case of DMPE, which can be explained in terms of its tighter molecular packing. The conclusion that EGCG molecules alter the DMPC array both at the head group and
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hydrocarbon chain regions is supported by the X-ray diffraction results. The ability of EGCG to affect the structure of systems composed of phosphatidylcholines is mainly due to its ability to form hydrogen bonds with the polar moiety of these phospholipids. The EGCG molecule can form up to 19 H-bonds, with 11 as an acceptor and 8 as a donor, the highest number among green tea catechins [32].
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Comparative studies have shown that the affinity of galloyl-type catechins EGCG and ECG to DMPC bilayers was 1000 times stronger than epicatechin gallate (EC) or epigallocatechin (EGC) (both without the galloyl group), which shows that the occurrence of galloyl moiety and the number of OH groups play a key role in these interactions [2]. Given its relatively low octanol-water partition coefficient (log P3=1.2, PubChem, 2011), EGCG does not show a marked lipophilicity. However, taking into account
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the amphiphilic character of the EGCG molecule it is very likely that its hydrophobic part is inserted
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into the inner lipophilic acyl chain region of DMPC, conclusion supported by Abram et al. [32] and
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Kajiya et al. [33], and the hydrophilic part preferentially interacts with the lipid polar head groups.
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The fluorescence measurements in DMPC vesicles showed that EGCG induced a strong dose-
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dependent increase in the GP value within a concentration range of 0-40 μM at 37 °C, and a more
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moderate one at 18 °C. At this last temperature, DMPC is in a gel state, which corresponds to a much more ordered phase with respect to the fluid liquid crystalline state in which the phospholipid is at 37
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°C. On the other hand, the DPH anisotropy measurements made at both temperatures do not present
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significant variations. These results indicate that the EGCG molecules were mostly located in the polar head group region of DMPC inducing a hydration and/or molecular dynamic decrease. These results
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contrast with those obtained by Ionescu et al. [8], in which they report a very slight decrease in GP values induced by EGCG in pure DMPC and DMPC/cholesterol liposomes. However, other studies have concluded that EGCG caused membrane-rigidifying effects on a tumor cell model membrane consisting of 20:80 mol% cholesterol-phosphatidylcholines [34], and a pronounced decrease in the membrane fluidity in PC:PS multilamellar liposomes 2.4:1 molar ratio [32]. In isolated unsealed human erythrocyte membranes (IUM), EGCG induced a moderate increase in laurdan GP, a result that implies
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a decrease in the molecular dynamics at the hydrophobic–hydrophilic interphase, which confirms an increase in the rigidity of this region of the membrane. SEM observations showed that EGCG induced morphological alterations in human erythrocytes from their normal discoid form to crenated echinocytes. According to the bilayer couple hypothesis
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[35] the shape changes induced in human erythrocytes by foreign molecules are due to a differential expansion of the two monolayers of the red blood cell membrane. Thus, when the exogenous molecules locate into the outer moiety echinocytes are produced whereas stomatocytes are formed when they insert into the inner monolayer of the membrane. The finding that EGCG caused echinocytes to form indicates that this catechin was preferentially sited into the outer monolayer of the erythrocyte
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membrane. Results obtained from X-ray diffraction and DSC studies that showed that EGCG interacts
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only with DMPC, a class of phospholipid that is preferentially located in the outer monolayer of human
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erythrocytes supports this conclusion. On the other hand, NMR studies showed that EGCG molecules
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are preferably located in regions near to the surface of the lipid bilayer with their B-ring and the galloyl
D
moiety at the level of the trimethylammonium groups of phosphatidylcholines [36]. This compound is
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also capable of binding to receptors located on the surface of the membrane [37]. The galloyl moiety together with the cis configuration of EGCG also significantly increase the ability of this catechin to
EP
form hydrogen bonds with the lipid head group as demonstrated in HepG2 liver cancer cells [38].
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The protective role of EGCG in human erythrocytes as well as in DMPC and DMPE membranes was evaluated using assays in which these systems were exposed to HClO. Hypochlorous
A
acid is a biological oxidant generated by neutrophils and monocytes that functions as a powerful antimicrobial agent. It is also considered one of the most important factors that cause tissue injuries during inflammation [12]. HClO reacts with a wide range of biological target molecules including lipids, proteins, and nucleic acids. It also compromises the function of important membrane proteins, causing alterations in the elasticity of the erythrocyte membrane [39]. In human erythrocytes the major consequence of their exposure to HClO is cell lysis. Prior to this process, HClO causes morphological
17
transformations, changes in membrane fluidity and surface area, membrane inhibition of Na+, K+-, and Mg2+-ATPase activities, oxidation of thiol groups and chloramine formation [14,40,41]. Interestingly, X-ray results showed that HClO caused structural perturbations to DMPE but not to DMPC (Fig. 3). Both assayed phospholipids have completely saturated acyl chains of 14 carbon atoms. This perturbing
SC RI PT
effect is therefore explained by the structural differences that exist in their head groups. In DMPE, a small polar molecule such as HClO (pKa = 7.53) can insert between DMPE neighboring phosphate and amino groups disrupting the narrow net of electrostatic and H-bond interactions that ensure that the molecules in bilayers remain tightly stuck together. On the other hand, DMPC with its bulky methyl groups and the presence of considerable amount of water between its bilayers presents relatively weak
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inter bilayer interactions. Thus, HClO molecules remain mainly in the water layer causing some
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structural perturbation to DMPC only at high concentrations. As shown in Fig. 3C, increasing EGCG
A
concentrations gradually neutralized the deleterious effect of HClO on DMPE bilayers. SEM images of
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human erythrocytes incubated with 50 μM HClO (Fig. 6D) show the presence of stomatocytes,
D
expected result since HClO only interacts with DMPE, a class of lipid mostly located in the inner
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monolayer of RBC. This result also agrees with the bilayer couple hypothesis which states that stomatocytes are produced when a foreign compound interacts with the inner moiety of the human
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erythrocytes membrane [35]. However, the previous incubation of erythrocytes with EGCG prevented
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the morphological alterations of red cells. Based on this evidence, it is possible to hypothesize that the location of EGCG molecules in the outer monolayer and the resulting rigidifying effect prevents HClO
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from causing structural damage and diffusion through the membrane. This would also explain the protective effect of EGCG against the lysis produced by HClO observed in the hemolysis assays (Fig. 7), an effect that has also recently been reported by Grzesik et al. [42]. A similar explanation for the antioxidant capacity of galloylated catechins has been proposed by Martínez et al. [43] and Caturla et al. [28]. The protective mechanism exhibited by EGCG in this study might be an important factor against the pathogenesis and the development of chronic disorders associated with inflammation and
18
oxidative stress such as cardiovascular diseases [44], neurodegenerative diseases [45], hemolytic pathologies and cancer [46]. In summary, the results of this investigation showed that EGCG, the most abundant and biologically active catechin present in green tea is capable of interacting strongly with the human
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erythrocyte membrane protecting red cells from the deleterious effects caused by hypochlorous acid. These findings provide an insight into the potent antioxidant and pharmacological activity of epigallocatechin gallate in biological systems.
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Acknowledgments
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To Fernando Neira for technical assistance, the Graduate Program in Chemistry for a
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scholarship to J.R.C., and FONDECYT (research project 1130043). Calorimetric measurements were
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carried out using the instrument purchased thanks to financial support of European Regional
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Development Fund (contract No. POIG.02.01.00-12-167/08, project Malopolska Centre of
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Biotechnology). The Jagiellonian University is a partner of the Leading National Research Center
M. Afzal, A.M. Safer, M. Menon, Green tea polyphenols and their potential role in health and
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[1]
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Figure legends Fig. 1. Structural formula of epigallocatechin gallate (EGCG).
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Fig. 2. X-ray diffraction patterns of (A) dimyristoylphosphatidylcholine (DMPC) and (B) dimyristoylphosphatidylethanolamine (DMPE) in water and incubated with epigallocatechin gallate (EGCG). (SA) small-angle and (WA) wide-angle reflections.
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Fig. 3. X-ray diffraction patterns of (A) dimyristoylphosphatidylcholine (DMPC) and (B) dimyristoylphosphatidylethanolamine (DMPE) in water and HClO; (C) DMPE in water, epigallocatechin gallate (EGCG) and HClO. (SA) small-angle and (WA) wide-angle reflections.
Fig. 4. Representative DSC heating curves obtained for multilamellar liposomes of (A)
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dimyristoylphosphatidylcholine (DMPC) and (B) dimyristoylphosphatidylethanolamine (DMPE)
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containing epigallocatechin gallate (EGCG) at various concentrations. (C) and (D) inserts: Plots of
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were obtained at a heating rate of 1 °C min−1.
A
phase transition temperatures versus EGCG concentration for DMPC and DMPE, respectively. Scans
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Fig. 5. Fluorescence measurement of (A) generalized polarization (GP) of laurdan and (B) anisotropy of DPH in large unilamellar vesicles (LUV) of DMPC at 18 ºC and 37 °C, and isolated unsealed human
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erythrocyte membranes (IUM) at 37 °C treated with different concentrations of epigallocatechin gallate
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(EGCG). Statistically significant differences (α = 0.05) between generalized polarization (GP) and
A
anisotropy values for the samples treated with EGCG respect to the control (0 μM) are denoted as (*).
Fig. 6. Protective effect of epigallocatechin gallate (EGCG) on the morphology of human erythrocytes. SEM images of (A) untreated erythrocytes; incubated with (B) 10 μM EGCG; (C) 50 μM EGCG; (D) 50 μM HClO; (E) 2.5 μM EGCG and 50 μM HClO; (F) 5 μM EGCG and 50 μM HClO. Full and empty arrows point to a selected echinocyte and stomatocyte, respectively.
26
Fig. 7. Percentage of hemolysis of red blood cells (RBC) incubated with 0.5 mM HClO and increasing
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concentrations of epigallocatechin gallate (EGCG); n = 3. Values are the mean ± SD.
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A
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A
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M
A
N
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Table 1. Thermodynamic parameters of the pretransition and main phase transition of pure fully hydrated DMPC multilamellar liposomes and DMPC/epigallocatechin gallate (EGCG) mixtures determined from heating and cooling scans collected at a heating (cooling) rate of 1 °C min -1.
A
Compound [μM]
Pretransition heating
Main transition heating
ΔH [kJ/mol]
ΔS [J/mol K]
Tp [ºC]
ΔH [kJ/mol]
ΔS [J/mol K]
Tm [ºC]
DMPC
2.53
8.78
15.18
17.72
59.63
24.01
+ EGCG 10
1.11
3.87
13.57
15.53
52.27
23.98
25
-
-
-
18.41
61.97
23.91
29 50
-
-
-
15.47
52.08
23.79
100
-
-
-
14.88
50.20
23.35
250
-
-
-
5.97
20.21
22.39
500
-
-
-
13.46
46.05
19.25
Main transition cooling
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Pretransition cooling ΔS [J/mol K]
Tp [ºC]
ΔH [kJ/mol]
ΔS [J/mol K]
Tm [ºC]
DMPC
1.73
6.11
9.30
17.00
57.34
23.23
+ EGCG 10
1.22
4.35
8.47
16.07
54.24
23.15
25
-
-
-
18.09
61.06
23.06
50
-
-
-
17.39
58.71
22.97
100
-
-
-
14.76
49.90
22.61
250
-
-
-
5.12
17.37
21.71
500
-
-
-
13.07
44.86
18.24
A
N
ΔH [kJ/mol]
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Compound [μM]
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The accuracy for the main phase transition temperature and enthalpy was ± 0.01ºC and ± 0.8 kJ/mol, respectively.
Compound [μM]
Main transition heating
Main transition cooling
ΔS [J/mol K]
Tm [ºC]
ΔH [kJ/mol]
ΔS [J/mol K]
Tm [ºC]
20.95
64.68
50.71
18.19
56.42
49.24
20.29
62.65
50.68
17.27
53.57
49.19
25
20.95
64.70
50.68
19.57
60.68
49.22
50
20.71
63.94
50.72
19.45
60.31
49.29
100
20.24
62.50
50.72
19.43
60.26
49.29
250
19.51
60.24
50.67
18.61
50.74
49.10
500
22.68
70.07
50.57
22.46
69.73
48.88
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+ EGCG 10
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ΔH [kJ/mol]
DMPE
A
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Table 2. Thermodynamic parameters of the phase transition of pure fully hydrated DMPE multilamellar liposomes and DMPE/epigallocatechin gallate (EGCG) mixtures determined from heating and cooling scans collected at a heating (cooling) rate of 1 °C min -1.
The accuracy for the main phase transition temperature and enthalpy was ± 0.01ºC and ± 0.8 kJ/mol, respectively.
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A
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S0927-7765(18)30734-3 https://doi.org/10.1016/j.colsurfb.2018.10.038 COLSUB 9722
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
10-5-2018 12-10-2018 15-10-2018
Please cite this article as: Colina JR, Suwalsky M, Manrique-Moreno M, Petit K, Aguilar LF, Jemiola-Rzeminska M, Strzalka K, Protective effect of epigallocatechin gallate on human erythrocytes, Colloids and Surfaces B: Biointerfaces (2018), https://doi.org/10.1016/j.colsurfb.2018.10.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Protective effect of epigallocatechin gallate on human erythrocytes
José R. Colinaa, Mario Suwalskya*, Marcela Manrique-Morenob, Karla Petita, Luis F. Aguilarc,
a
Facultad de Ciencias Químicas, Universidad de Concepción, Concepción, Chile
b
c
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Malgorzata Jemiola-Rzeminskad,e, Kazimierz Strzalkad,e
Faculty of Exact and Natural Sciences, University of Antioquia, Medellín, Colombia
Instituto de Química, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile
d
Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
e
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Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
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* Corresponding author at: Faculty of Chemical Sciences, University of Concepción, Concepción,
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Chile.
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E-mail address: [email protected] (M. Suwalsky)
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Total number of words: 5223, without references and figure legends.
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Number of figures: 8 (1 as supplementary material)
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Number of tables: 2 (as supplementary material)
Graphical abstract
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2
Highlights
Structural and thermotropic perturbations in DMPC bilayers were induced by EGCG
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EGCG produced morphological alterations in RBC inducing the formation of echinocytes
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Deleterious effects of HClO on DMPE bilayers were neutralized by EGCG
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-EGCG protected RBC morphological alterations and lysis caused by HClO
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ABSTRACT
The interactions and the protective effect of epigallocatechin gallate (EGCG) on human erythrocytes
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(RBC) and molecular models of its membrane were investigated. The latter consisted of bilayers builtup of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE),
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representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. X-ray diffraction and differential scanning calorimetry experiments showed that EGCG induced significant structural and thermotropic perturbations in multilayers and vesicles of DMPC; however, these effects were not observed in DMPE. Fluorescence spectroscopy results revealed that EGCG produced alterations of the molecular dynamics at the level of
3
the hydrophobic-hydrophilic interface in DMPC vesicles, and in isolated unsealed human erythrocyte membranes (IUM). EGCG also induced morphological alterations in RBC from their normal discoid form to echinocytes. These outcomes indicate that EGCG molecules were located in the outer monolayer of the erythrocyte membrane. The assessment of EGCG protective effect demonstrated that
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it inhibits the morphological alterations and lysis induced by HClO to human erythrocytes. The results obtained from this study suggest that the insertion of EGCG into the outer monolayer of the erythrocyte membrane might prevent the access and deleterious effects of oxidant molecules such as HClO and free radicals into the red cells, protecting them from oxidative damage.
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Keywords:
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Epigallocatechin gallate
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Erythrocyte membrane
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Phospholipid bilayer
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1. Introduction
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Antioxidant
Green tea (Camellia sinensis) is one of the most widely consumed beverages throughout the
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world, and its consumption has long been associated with health benefits [1]. Green tea is rich in a wide variety of polyphenols, particularly of catechins [2]. Epigallocatechin gallate (EGCG, (−)-cis3,3′,4′,5,5′,7-hexahydroxyflavane-3-gallate, Fig. 1) is the most abundant and biologically active catechin isolated from green tea, containing between 25-30 mg per bag [3]. It has been suggested that EGCG is responsible for many of the beneficial effects of green tea both in cell culture as well as in animal and clinical studies [4]. It has been reported to have a therapeutic effect in pathologies such as
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cancer, diabetes, stroke, obesity, Parkinson's and Alzheimer's disease [5]. The beneficial effects of EGCG are mainly attributed to its role as an antioxidant and free radical scavenger, which have been attributed to the presence of phenolic groups; they are sensitive to oxidation which is increased by the existence of the trihydroxyl group in the D-ring of this catechin [6] (Fig. 1). However, a role as pro-
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oxidant and cytotoxic agent in some cellular contexts has also been suggested [7]. A specific molecular mechanism explaining EGCG antioxidant properties has not been reported. However, it has been suggested that its biological activity can be related to its ability to interact with plasma membrane components, including lipids and proteins, affecting the stability of the lipid bilayers [8,9]. The particular structure of catechins significantly influences their interaction with biological membranes.
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The galloyl group present in some catechins plays a key role in the relative affinity of these compounds
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to membranes, which has been related to their bioactivity [2]. Thus, EGCG and epigallocatechin
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(EGC), both containing the galloyl moiety show the most potent biological effects among the catechins
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present in green tea [10]. The presence of the galloyl group also determines the location of the
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catechins in the lipid bilayer. Despite extensive studies on the interaction of EGCG with lipid bilayers,
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few have been carried out on real membranes, particularly that of the human erythrocyte. The erythrocyte membrane is an appropriate model for studying the interaction of natural or
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synthetic compounds with cell membranes. Erythrocytes, although less specialized than other cells
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retain sufficient functions in common to be considered a representative model of the plasma membrane in general [11]. In order to understand the molecular mechanisms involved in the interaction of EGCG
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with the cell membrane, human erythrocytes and molecular models of its membrane were used. The molecular models consisted of bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), which represent classes of phospholipids located in the outer and inner monolayer of the human erythrocyte membrane, respectively as well as in those of many other cells. This research involved the structural, dynamic and thermotropic study of EGCG interactions with the human erythrocyte membrane, and its protective capacity against damage caused
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by hypochlorous acid (HClO). HClO is a powerful natural oxidant and a potent antimicrobial agent [12], which is involved in a large number of pathological conditions related to inflammatory processes including atherosclerosis, neurodegenerative diseases and some cancers [13]. In human erythrocytes, HClO is capable of causing morphological alterations and lysis of the plasma membrane [14].
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Understanding the mechanisms by which EGCG protects cells from oxidation can contribute to the development of new therapies aimed at preventing the development of pathological conditions.
2. Materials and methods
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2.1. Chemicals and reagents
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Epigallocatechin gallate and sodium hypochlorite solution (4–5%) were from Sigma-Aldrich
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(St. Louis, MO, USA). Hypochlorous acid (HClO) samples were prepared by diluting sodium
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hypochlorite solution in the phosphate buffer PBS (150 mM NaCl, 1.9 mM NaH2PO4, 8.1 mM
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Na2HPO4, pH 7.4). The hypochlorite concentration was determined spectrophotometrically at 292 nm using = 350 M−1 cm−1. Under such conditions both forms HClO and ClO− are present in an
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approximately equimolar ratio [15]. Synthetic DMPC (lot 850345-02-270, MW 677.9) and DMPE (lot
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850745-01-069, MW 635.9) were from Avanti Polar Lipids Inc. (AL, USA); 1,6-diphenyl-1,3,5hexatriene (DPH) and 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan) fluorescent probes were
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from Molecular Probe (Eugene, OR, USA). All other chemicals were from Sigma-Aldrich. Distilled water was purified using a Milli-Q system (Millipore, Bedford, MA, USA).
2.2. X-ray diffraction studies of DMPC and DMPE multilayers
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The capacity of EGCG to interact with DMPC and DMPE multilayers was evaluated using Xray diffraction. 2 mg of each phospholipid were placed in Eppendorf tubes and then filled with 150 L of (a) distilled water (control), and (b) aqueous solutions of EGCG (0.1-2.0 mM). The protective capacity of EGCG was evaluated by pre-incubating each phospholipid with EGCG for 10 min and
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thereafter with 10 mM HClO. All samples were incubated for 30 min in a shaking bath at 30 ºC for DMPC and 60 ºC for DMPE, then transferred into 1.5 mm diameter special glass capillaries (WJMGlas, Berlin, Germany) and centrifuged at 2500 rpm for 15 min (MRC, Israel). A Bruker Kristalloflex 760 (Karlsruhe, Germany) generator with Ni-filtered CuK radiation was used for the X-ray diffraction
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experiments. The relative reflection intensities were collected in an MBraun PSD-50M linear position
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sensitive detector system (Garching, Germany). The experiments were carried out at 18 ± 1 °C, which
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is lower than the main phase transition temperature of DMPC (24.3 °C) and DMPE (50.2 °C) [16].
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Higher temperatures would have induced transitions to more fluid phases hampering the detection of
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structural changes. Each experiment was repeated at least twice.
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2.3. Differential scanning calorimetry (DSC) studies of DMPC and DMPE vesicles
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Multilamellar vesicles (MLV) were used to study the thermotropic behavior of DMPC and
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DMPE in the presence of EGCG. To prepare MLV, first the appropriate amount of each phospholipid was dissolved in chloroform. The solvent was evaporated by applying a gentle stream of nitrogen until
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a thin film was formed on the walls of the glass tubes. The samples were then exposed to vacuum for 1 h in order to remove traces of the solvent. MLV with a final lipid concentration of 1 mM were prepared by vortexing the samples at a temperature above the gel-to-liquid crystalline phase transition of the pure lipids (30 °C for DMPC and 70 °C for DMPE). EGCG dissolved in water was added in the concentration range of 0.01-0.5 mM. The samples were degassed prior to being loaded in the
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calorimeter cell by pulling a vacuum of 30.4–50.7 kPa on the solution at 25 °C for 10 min to avoid bubble formation during heating. The DSC experiments were carried out using a NANO DSC Series III System with Platinum Capillary Cell (TA Instruments, USA). A sample cell was filled with about 400 μL of MLV suspension, and an equal volume of water was used as a reference; the cells were sealed
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and thermally equilibrated for 10 min at the starting temperature of the run. All measurements were made on samples under 0.3 MPa pressure. The data were collected within a range of 5-40 °C (DMPC) and 30-70 °C (DMPE) at a scan rate of 1 °C min−1 for both heating and cooling. Water as a sample and a reference was also scanned to collect the apparatus baseline. In order to check the reproducibility, each sample was prepared and recorded at least three times. Each data set was analyzed for
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thermodynamic parameters with a software package supplied by TA Instruments.
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human erythrocyte membranes (IUM)
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2.4. Fluorescence spectroscopy studies of large unilamellar vesicles (LUV) and isolated unsealed
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The influence of EGCG on the dynamics of DMPC LUV and IUM was examined using fluorescence spectroscopy. The measurements of anisotropy (r) and generalized polarization (GP) were
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achieved using DPH and laurdan fluorescent probes, respectively according to the previously described
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method [17]. Briefly, DMPC LUV were prepared by extrusion through two polycarbonate filters of 400 nm pore size (Nucleopore, Corning Costar Corp., MA, USA) from a suspension of 0.4 mM MLV
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previously subjected to freeze-thaw cycles. IUM were prepared using the method proposed by Dodge et al. [18]. DPH and laurdan probes were incorporated into the LUV and IUM suspensions by adding 1 L/mL aliquots of 0.5 mM stock solutions of the probe in dimethyl sulfoxide and ethanol, respectively and incubated at 37 °C for 1 h. EGCG was incorporated into LUV and IUM suspensions by addition of adequate aliquots in order to obtain the different concentrations used in this study. Fluorescence spectra
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and anisotropy measurements were taken in a K-2 spectrofluorometer (ISS Inc., Champaign, IL, USA) using the software ISS in order to collect and analyze the data. For DPH and laurdan, the exciting light was from a modulable ISS 375 nm LED laser. Fluorescence anisotropy (r) was calculated according to the definition: r = (I∥ − I⊥)/(I∥ + 2I⊥), where I∥ and I⊥ are the corresponding parallel and perpendicular
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emission fluorescence intensities with respect to the vertically polarized excitation light [19]. Laurdan fluorescence spectral shifts were quantitatively evaluated using the GP concept, which is defined by the expression GP = (Ib − Ir)/(Ib + Ir), where Ib and Ir are the emission intensities at the blue and red edges of the emission spectrum, respectively. These intensities were measured at the emission wavelengths of 440 and 490 nm, which correspond to the emission maxima of laurdan in both gel and liquid crystalline
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phases, respectively [20]. The measurements were taken at 18 ºC and 37 ºC for LUV and 37 ºC for
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15 measurements in three independent experiments.
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IUM suspensions. The data presented in the figures represent the mean values and a standard error of
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2.5. Scanning electron microscopy (SEM) studies on human erythrocytes
The morphological alterations induced by HClO and the protective role of EGCG in human
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erythrocytes were analyzed using SEM. 100 L of blood was obtained by finger puncture from a
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healthy donor and received in an Eppendorf tube containing 900 L of phosphate buffer saline (PBS) pH 7.4. The red blood cell suspension (RBCs) was homogenized and centrifuged at 1000 rpm for 10
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min (MRC, Israel), the supernatant was removed via aspiration and replaced with fresh PBS; this procedure was repeated three times. 50 L of RBCs were placed in Eppendorf tubes and then added 150 L of (a) PBS (Control), (b) EGCG (1-100 M), and (c) 50 M HClO dissolved in PBS. In order to determine the protective effect RBCs were pre-incubated with EGCG (1-10 M) for 20 min and then with 50 M HClO. All samples were incubated in a shaking bath for 1 h at 37 °C. The samples were
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centrifuged (1000 rpm x 10 min) and the supernatant discarded. Afterwards, the cells were fixed for 24 h at 4 °C with 500 L of 2.5% glutaraldehyde in PBS. The RBCs were washed three times with 500 L of distilled water, and one drop of each sample was placed on siliconized Al glass-covered stubs of 20x20 mm, air-dried at room temperature, and gold coated for 3 min at 13.3 Pa in a sputter device
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(Edwards S 150, Sussex, England). The observations were made at a resolution of 2500X in a scanning electron microscope (JEOL JSM-6380LV, Japan).
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2.6. Hemolysis assays in human erythrocytes
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The ability of EGCG to prevent membrane rupture of human erythrocytes by HClO was
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determined using hemolysis assays. RBC were obtained directly via venipuncture from a healthy donor
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who was not receiving pharmacological treatment. The blood (10 mL) was centrifuged at 2500 rpm for 10 min at 4 °C; after the plasma and buffy coat had been removed, the cells were washed three times
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with PBS. Next, the RBCs (10% v/v) were incubated with different concentrations of EGCG (5-100
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M). HClO was added to the mixture in a single aliquot of a stock of 5 mM in PBS. The samples were incubated in a shaking bath for 15 min at 37 °C; after that, the samples were centrifuged at 2500 rpm
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for 5 min. Hemolysis was determined spectrophotometrically (Shimadzu UV-mini, Japan) by the
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hemoglobin released in the supernatant at 540 nm. The protective effect was evidenced by a decrease in
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the percentage of hemolysis in a relation to a control consisting of 0.5 mM HClO.
2.7. Statistical Analyses
Statistical analyses were performed using Graph Pad Prism 6 (Graph Pad Software Inc.). Oneway ANOVA followed by Dunnett’s post hoc test at significance level α = 0.05 was used to estimate
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the differences between the samples treated with EGCG and the control.
3.1. X-ray diffraction studies of DMPC and DMPE multilayers
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3. Results
Fig. 2A shows the diffraction patterns of DMPC incubated with water and increasing concentrations of EGCG. As previously reported, water affected the structure of DMPC producing an increase in the bilayer repeat (bilayer width plus the width of the water layer between bilayers) from
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about 55 Å in its dry crystalline form to 64 Å when fully hydrated [21]. This interaction also caused a
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reduction in the small-angle reflections (SA in the figure), which were reduced to only two orders of
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the bilayer repeat. On the other hand, only one reflection of 4.2 Å was observed in the wide-angle
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region (WA in the figure), which corresponds to the average distance between the fully extended acyl
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chains packed in a hexagonal arrangement. The incubation of DMPC with increasing concentrations of
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EGCG produced a regular weakening of the low, and particularly of the wide angle 4.2 Å reflection intensities, which was practically absent with 2 mM EGCG. As shown in Fig. 2B, EGCG did not affect
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the DMPE structure in the assayed concentration range (0.1-2.0 mM). Figures 3A and 3B show the
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effects of hypochlorous acid (HClO) on DMPC and DMPE, respectively. As it can be appreciated, HClO had no significant effect on DMPC even at a 10 mM concentration, although 2.5 mM HClO
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produced a profound effect on the DMPE bilayer structure as its small angle reflection intensities suffered a considerably decrease, which almost disappeared at a concentration of 10 mM. Fig. 3C presents the protective capacity of EGCG against the structural effects of HClO on DMPE. In fact, preincubating DMPE with EGCG in the 0.1-2.0 mM range increasingly neutralized the deleterious effect of 10 mM HClO until completely eradicated by 2 mM EGCG.
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3.2. Differential scanning calorimetry (DSC) studies in DMPC and DMPE vesicles
Representative high-sensitivity DSC heating thermograms obtained for pure DMPC and DMPE multilayer vesicles and binary mixtures of each phospholipid with EGCG within a concentration range of 0.01 mM to 0.5 mM are presented in Fig. 4. Fig. 4A shows the representative thermograms obtained
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for pure DMPC and DMPC/EGCG mixtures. Pure DMPC bilayers within a thermal range of 5-30 ºC showed a strong and sharp main phase transition at 24.01 °C, with an enthalpy change (ΔH) of 17.72 kJ mol−1 which corresponds to the conversion of the rippled gel phase (Pβ′) to the lamellar liquid-crystal (Lα) phase. At 14.71 °C was observed a pre-transition arising from the conversion of a lamellar gel phase (Lβ′) to a rippled gel phase with ΔH of 2.93 kJ mol−1. The presence of EGCG caused a marked alteration in the thermotropic behavior of the DMPC bilayer, which was evidenced by a gradual
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decrease and broadening of the main phase transition peak, together with a continuous shift towards
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lower temperatures. This effect can also be demonstrated from the transition width at half-peak height
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(ΔT1/2), which is a measure of destabilization of the phospholipid assembly and the size of the cooperative unit [22]. Pure phospholipid melts in a cooperative domain showing a sharp transition peak
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(ΔT1/2 = 0.61 and 0.62 for heating and cooling, respectively). However, the presence of EGCG continuously increased the ΔT1/2 reaching at 0.25 mM values of 3.64 and 1.91 for heating and cooling,
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respectively (Table 1, supplementary material). This acute increase indicates that EGCG caused a less
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cooperative melting process to DMPC due to the coexistence of gel and liquid-crystalline phase domains, which produces the widening in the melting temperature range [23,24]. EGCG also strongly
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affected the pre-transition of DMPC liposomes, which was hardly observed at the very low concentration of 0.01 mM, which disappeared at a concentration of 0.025 mM EGCG. From the DSC profiles shown in Fig. 4B, it can be seen that within a temperature range of 41-55 ºC DMPE showed a
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single sharp transition at 50.71 °C, with an enthalpy change of 20.95 kJ mol−1. This transition is related to the change of the gel phase (Lβ) to the Lα phase. The heating scans recorded for DMPE showed no
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evidence of changes in the thermotropic behavior of this lipid, indicating that there was no interaction with the catechin. The thermodynamic data of pure DMPC and DMPE were consistent with previous reports [25,26]. The insets in Fig. 4 show the dependence of values of the main phase transition temperature of DMPC (Fig. 4C) and of DMPE (Fig. 4D) on EGCG concentration. With an increase in the catechin content in DMPC, Tm decreased reaching a maximum variation (ΔTm) of 4.76 ºC at 0.5 mM EGCG, although the dependence is nonlinear. From Fig. 4D, it can be seen that EGCG had a negligible effect on the DMPE phase transition temperature. At a concentration of 0.5 mM a very slight
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decrease in Tm was only observed in this system upon cooling (data not shown); for details on thermodynamic data see supplementary material.
3.3. Fluorescence spectroscopy studies of large unilamellar vesicles (LUV) and isolated unsealed
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human erythrocytes membranes (IUM)
Changes in the physical properties of DMPC LUV and IUM induced by EGCG were investigated at two different depths of the lipid bilayers. DPH is a non-polar fluorophore used as a probe to monitor changes in the hydrophobic regions of the phospholipid bilayers, while laurdan
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provides information about the polarity and/or molecular dynamics at the level of the phospholipid
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glycerol backbone. Fig. 5 shows the influence of EGCG on the values of the fluorescence spectral shift
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GP parameter and the steady-state fluorescence anisotropy in both DMPC LUV and IUM. A very sharp
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increase in GP, concentration-dependent of EGCG was observed in DMPC LUV at 37 °C (Fig. 5A), causing a variation (ΔGP) of 0.33 at 40 μM EGCG. At 18 °C EGCG caused only a mild increase in GP
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up to a concentration of 20 μM, and no significant changes were observed in the remaining
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concentrations. The results of the interaction of EGCG with erythrocyte membranes (solid triangles
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line, Fig. 5A) at 37 °C showed a moderate increase in GP values within a range of 40-100 μM, reaching a maximum ΔGP of 0.12 at 100 μM EGCG. These results indicate that EGCG caused a structural
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ordering of the hydrophilic region of DMPC LUV and RBC membrane. As it can be observed in Fig. 5B, changes induced by EGCG on DPH anisotropy in DMPC LUV and IUM were less marked than in
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the case of GP indicating a scanty interaction of EGCG with the hydrophobic environment of the lipid and the erythrocyte membrane.
3.4. Scanning electron microscopy (SEM) studies on human erythrocytes
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SEM was used to visualize the morphological changes of human erythrocytes (RBC) subjected to in vitro assays. Fig. 6 presents the protective effect of EGCG on the deleterious effects of HClO in human erythrocytes. RBC incubated with PBS pH 7.4 (Fig. 6A, control) shows the characteristic discoid-biconcave morphology of normal erythrocytes (discocytes) [27]. As it can be observed in Fig.
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6B, erythrocytes incubated with 10 μM EGCG did not show morphological changes; however, 50 μM EGCG changed their normal morphology to echinocytes (cells characterized by the presence of spicules that are regularly spaced and uniformly distributed) (Fig. 6C). Fig. 6D shows that 50 μM HClO induced morphological alterations in the RBC, causing the formation of stomatocytes (cells with deep concave invaginations) and of a few knizocytes (cells with two or three concavities). However, as
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shown in Figs. 6E and 6F the pre-incubation of RBC with 2.5 μM and 5 μM EGCG, respectively
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erythrocytes their normal biconcave shapes.
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neutralized in a dose-dependent manner the morphological effects caused by HClO, exhibiting the
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3.5. Hemolysis assays in human erythrocytes
In order to determine the protective role of EGCG in HClO-induced lysis in human
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erythrocytes, hemolysis assays were carried out. Fig. 7 shows that EGCG within a concentration range
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of 5-100 μM did not induced significant hemolysis in the RBC (black bars). However, the same EGCG concentrations progressively inhibited the hemolysis caused to red cells by 0.5 mM HClO (white bar),
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reaching a minimum of 4.5% hemolysis with 100 μM EGCG (gray bars). These results demonstrate the capacity of EGCG to protect human erythrocytes from the lysis caused by HClO.
4. Discussion
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In the present study, the interaction between EGCG and human erythrocytes and molecular models of its membrane was investigated, and the protective effect of this catechin against oxidantinduced damage was evaluated. The results of X-ray diffraction in DMPC and DMPE multilayers in the gel phase showed that 2 mM EGCG produced significant structural perturbations to DMPC but not to
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DMPE bilayers. Both phospholipids differ only in their terminal amino groups, these being +NH3 for DMPE and +N(CH3)3 for DMPC. DMPE molecules pack tighter than DMPC molecules given the smaller size of its head group and consequent higher effective charge. This results in a very stable system with strong electrostatic and hydrogen bond interactions which are not easily affected by water or by EGCG. On the other hand, DMPC inter bilayer interactions are much weaker due to its bulky
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head group. This allows water to fill the polar inter bilayer spaces resulting in an increase of their
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separation [21], condition that makes possible the entry of EGCG molecules resulting in the observed
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structural perturbations.
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DSC is a very sensitive method for investigating the changes in the enthalpy and the
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temperature of the phase transition of lipids. The incorporation of EGCG into DMPC bilayers gave rise
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to a pronounced distortion of its thermotropic behavior and as a result both the pre-transition and the main phase transition were affected. This result coincides with that reported by Caturla et al. [28].
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Interestingly, the pre-transition seemed to be much more effectively distorted than the conversion of
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the rippled gel phase to the lamellar liquid-crystal phase, known as the main phase transition. This finding is consistent with the alterations observed in the conformational rearrangement in the head
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group region of the lipid molecules [29,30]. The lowering of Tm and the increase in the main phase transition peak width indicates that EGCG molecules cause a significant perturbation in the DMPC chain melting process. The widening of the melting peak is consistent with a reduction of the melting cooperativity or the presence of intermediate melting states [31]. On the other hand, it was observed a small effect of EGCG in the case of DMPE, which can be explained in terms of its tighter molecular packing. The conclusion that EGCG molecules alter the DMPC array both at the head group and
15
hydrocarbon chain regions is supported by the X-ray diffraction results. The ability of EGCG to affect the structure of systems composed of phosphatidylcholines is mainly due to its ability to form hydrogen bonds with the polar moiety of these phospholipids. The EGCG molecule can form up to 19 H-bonds, with 11 as an acceptor and 8 as a donor, the highest number among green tea catechins [32].
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Comparative studies have shown that the affinity of galloyl-type catechins EGCG and ECG to DMPC bilayers was 1000 times stronger than epicatechin gallate (EC) or epigallocatechin (EGC) (both without the galloyl group), which shows that the occurrence of galloyl moiety and the number of OH groups play a key role in these interactions [2]. Given its relatively low octanol-water partition coefficient (log P3=1.2, PubChem, 2011), EGCG does not show a marked lipophilicity. However, taking into account
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the amphiphilic character of the EGCG molecule it is very likely that its hydrophobic part is inserted
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into the inner lipophilic acyl chain region of DMPC, conclusion supported by Abram et al. [32] and
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Kajiya et al. [33], and the hydrophilic part preferentially interacts with the lipid polar head groups.
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The fluorescence measurements in DMPC vesicles showed that EGCG induced a strong dose-
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dependent increase in the GP value within a concentration range of 0-40 μM at 37 °C, and a more
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moderate one at 18 °C. At this last temperature, DMPC is in a gel state, which corresponds to a much more ordered phase with respect to the fluid liquid crystalline state in which the phospholipid is at 37
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°C. On the other hand, the DPH anisotropy measurements made at both temperatures do not present
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significant variations. These results indicate that the EGCG molecules were mostly located in the polar head group region of DMPC inducing a hydration and/or molecular dynamic decrease. These results
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contrast with those obtained by Ionescu et al. [8], in which they report a very slight decrease in GP values induced by EGCG in pure DMPC and DMPC/cholesterol liposomes. However, other studies have concluded that EGCG caused membrane-rigidifying effects on a tumor cell model membrane consisting of 20:80 mol% cholesterol-phosphatidylcholines [34], and a pronounced decrease in the membrane fluidity in PC:PS multilamellar liposomes 2.4:1 molar ratio [32]. In isolated unsealed human erythrocyte membranes (IUM), EGCG induced a moderate increase in laurdan GP, a result that implies
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a decrease in the molecular dynamics at the hydrophobic–hydrophilic interphase, which confirms an increase in the rigidity of this region of the membrane. SEM observations showed that EGCG induced morphological alterations in human erythrocytes from their normal discoid form to crenated echinocytes. According to the bilayer couple hypothesis
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[35] the shape changes induced in human erythrocytes by foreign molecules are due to a differential expansion of the two monolayers of the red blood cell membrane. Thus, when the exogenous molecules locate into the outer moiety echinocytes are produced whereas stomatocytes are formed when they insert into the inner monolayer of the membrane. The finding that EGCG caused echinocytes to form indicates that this catechin was preferentially sited into the outer monolayer of the erythrocyte
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membrane. Results obtained from X-ray diffraction and DSC studies that showed that EGCG interacts
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only with DMPC, a class of phospholipid that is preferentially located in the outer monolayer of human
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erythrocytes supports this conclusion. On the other hand, NMR studies showed that EGCG molecules
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are preferably located in regions near to the surface of the lipid bilayer with their B-ring and the galloyl
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moiety at the level of the trimethylammonium groups of phosphatidylcholines [36]. This compound is
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also capable of binding to receptors located on the surface of the membrane [37]. The galloyl moiety together with the cis configuration of EGCG also significantly increase the ability of this catechin to
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form hydrogen bonds with the lipid head group as demonstrated in HepG2 liver cancer cells [38].
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The protective role of EGCG in human erythrocytes as well as in DMPC and DMPE membranes was evaluated using assays in which these systems were exposed to HClO. Hypochlorous
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acid is a biological oxidant generated by neutrophils and monocytes that functions as a powerful antimicrobial agent. It is also considered one of the most important factors that cause tissue injuries during inflammation [12]. HClO reacts with a wide range of biological target molecules including lipids, proteins, and nucleic acids. It also compromises the function of important membrane proteins, causing alterations in the elasticity of the erythrocyte membrane [39]. In human erythrocytes the major consequence of their exposure to HClO is cell lysis. Prior to this process, HClO causes morphological
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transformations, changes in membrane fluidity and surface area, membrane inhibition of Na+, K+-, and Mg2+-ATPase activities, oxidation of thiol groups and chloramine formation [14,40,41]. Interestingly, X-ray results showed that HClO caused structural perturbations to DMPE but not to DMPC (Fig. 3). Both assayed phospholipids have completely saturated acyl chains of 14 carbon atoms. This perturbing
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effect is therefore explained by the structural differences that exist in their head groups. In DMPE, a small polar molecule such as HClO (pKa = 7.53) can insert between DMPE neighboring phosphate and amino groups disrupting the narrow net of electrostatic and H-bond interactions that ensure that the molecules in bilayers remain tightly stuck together. On the other hand, DMPC with its bulky methyl groups and the presence of considerable amount of water between its bilayers presents relatively weak
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inter bilayer interactions. Thus, HClO molecules remain mainly in the water layer causing some
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structural perturbation to DMPC only at high concentrations. As shown in Fig. 3C, increasing EGCG
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concentrations gradually neutralized the deleterious effect of HClO on DMPE bilayers. SEM images of
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human erythrocytes incubated with 50 μM HClO (Fig. 6D) show the presence of stomatocytes,
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expected result since HClO only interacts with DMPE, a class of lipid mostly located in the inner
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monolayer of RBC. This result also agrees with the bilayer couple hypothesis which states that stomatocytes are produced when a foreign compound interacts with the inner moiety of the human
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erythrocytes membrane [35]. However, the previous incubation of erythrocytes with EGCG prevented
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the morphological alterations of red cells. Based on this evidence, it is possible to hypothesize that the location of EGCG molecules in the outer monolayer and the resulting rigidifying effect prevents HClO
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from causing structural damage and diffusion through the membrane. This would also explain the protective effect of EGCG against the lysis produced by HClO observed in the hemolysis assays (Fig. 7), an effect that has also recently been reported by Grzesik et al. [42]. A similar explanation for the antioxidant capacity of galloylated catechins has been proposed by Martínez et al. [43] and Caturla et al. [28]. The protective mechanism exhibited by EGCG in this study might be an important factor against the pathogenesis and the development of chronic disorders associated with inflammation and
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oxidative stress such as cardiovascular diseases [44], neurodegenerative diseases [45], hemolytic pathologies and cancer [46]. In summary, the results of this investigation showed that EGCG, the most abundant and biologically active catechin present in green tea is capable of interacting strongly with the human
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erythrocyte membrane protecting red cells from the deleterious effects caused by hypochlorous acid. These findings provide an insight into the potent antioxidant and pharmacological activity of epigallocatechin gallate in biological systems.
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Acknowledgments
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To Fernando Neira for technical assistance, the Graduate Program in Chemistry for a
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scholarship to J.R.C., and FONDECYT (research project 1130043). Calorimetric measurements were
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carried out using the instrument purchased thanks to financial support of European Regional
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Development Fund (contract No. POIG.02.01.00-12-167/08, project Malopolska Centre of
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Biotechnology). The Jagiellonian University is a partner of the Leading National Research Center
M. Afzal, A.M. Safer, M. Menon, Green tea polyphenols and their potential role in health and
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[1]
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Figure legends Fig. 1. Structural formula of epigallocatechin gallate (EGCG).
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Fig. 2. X-ray diffraction patterns of (A) dimyristoylphosphatidylcholine (DMPC) and (B) dimyristoylphosphatidylethanolamine (DMPE) in water and incubated with epigallocatechin gallate (EGCG). (SA) small-angle and (WA) wide-angle reflections.
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Fig. 3. X-ray diffraction patterns of (A) dimyristoylphosphatidylcholine (DMPC) and (B) dimyristoylphosphatidylethanolamine (DMPE) in water and HClO; (C) DMPE in water, epigallocatechin gallate (EGCG) and HClO. (SA) small-angle and (WA) wide-angle reflections.
Fig. 4. Representative DSC heating curves obtained for multilamellar liposomes of (A)
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dimyristoylphosphatidylcholine (DMPC) and (B) dimyristoylphosphatidylethanolamine (DMPE)
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containing epigallocatechin gallate (EGCG) at various concentrations. (C) and (D) inserts: Plots of
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were obtained at a heating rate of 1 °C min−1.
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phase transition temperatures versus EGCG concentration for DMPC and DMPE, respectively. Scans
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Fig. 5. Fluorescence measurement of (A) generalized polarization (GP) of laurdan and (B) anisotropy of DPH in large unilamellar vesicles (LUV) of DMPC at 18 ºC and 37 °C, and isolated unsealed human
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erythrocyte membranes (IUM) at 37 °C treated with different concentrations of epigallocatechin gallate
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(EGCG). Statistically significant differences (α = 0.05) between generalized polarization (GP) and
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anisotropy values for the samples treated with EGCG respect to the control (0 μM) are denoted as (*).
Fig. 6. Protective effect of epigallocatechin gallate (EGCG) on the morphology of human erythrocytes. SEM images of (A) untreated erythrocytes; incubated with (B) 10 μM EGCG; (C) 50 μM EGCG; (D) 50 μM HClO; (E) 2.5 μM EGCG and 50 μM HClO; (F) 5 μM EGCG and 50 μM HClO. Full and empty arrows point to a selected echinocyte and stomatocyte, respectively.
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Fig. 7. Percentage of hemolysis of red blood cells (RBC) incubated with 0.5 mM HClO and increasing
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concentrations of epigallocatechin gallate (EGCG); n = 3. Values are the mean ± SD.
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Table 1. Thermodynamic parameters of the pretransition and main phase transition of pure fully hydrated DMPC multilamellar liposomes and DMPC/epigallocatechin gallate (EGCG) mixtures determined from heating and cooling scans collected at a heating (cooling) rate of 1 °C min -1.
A
Compound [μM]
Pretransition heating
Main transition heating
ΔH [kJ/mol]
ΔS [J/mol K]
Tp [ºC]
ΔH [kJ/mol]
ΔS [J/mol K]
Tm [ºC]
DMPC
2.53
8.78
15.18
17.72
59.63
24.01
+ EGCG 10
1.11
3.87
13.57
15.53
52.27
23.98
25
-
-
-
18.41
61.97
23.91
29 50
-
-
-
15.47
52.08
23.79
100
-
-
-
14.88
50.20
23.35
250
-
-
-
5.97
20.21
22.39
500
-
-
-
13.46
46.05
19.25
Main transition cooling
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Pretransition cooling ΔS [J/mol K]
Tp [ºC]
ΔH [kJ/mol]
ΔS [J/mol K]
Tm [ºC]
DMPC
1.73
6.11
9.30
17.00
57.34
23.23
+ EGCG 10
1.22
4.35
8.47
16.07
54.24
23.15
25
-
-
-
18.09
61.06
23.06
50
-
-
-
17.39
58.71
22.97
100
-
-
-
14.76
49.90
22.61
250
-
-
-
5.12
17.37
21.71
500
-
-
-
13.07
44.86
18.24
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ΔH [kJ/mol]
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Compound [μM]
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The accuracy for the main phase transition temperature and enthalpy was ± 0.01ºC and ± 0.8 kJ/mol, respectively.
Compound [μM]
Main transition heating
Main transition cooling
ΔS [J/mol K]
Tm [ºC]
ΔH [kJ/mol]
ΔS [J/mol K]
Tm [ºC]
20.95
64.68
50.71
18.19
56.42
49.24
20.29
62.65
50.68
17.27
53.57
49.19
25
20.95
64.70
50.68
19.57
60.68
49.22
50
20.71
63.94
50.72
19.45
60.31
49.29
100
20.24
62.50
50.72
19.43
60.26
49.29
250
19.51
60.24
50.67
18.61
50.74
49.10
500
22.68
70.07
50.57
22.46
69.73
48.88
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+ EGCG 10
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ΔH [kJ/mol]
DMPE
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Table 2. Thermodynamic parameters of the phase transition of pure fully hydrated DMPE multilamellar liposomes and DMPE/epigallocatechin gallate (EGCG) mixtures determined from heating and cooling scans collected at a heating (cooling) rate of 1 °C min -1.
The accuracy for the main phase transition temperature and enthalpy was ± 0.01ºC and ± 0.8 kJ/mol, respectively.
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