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SUPPLEMENTARY MATERIAL
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Effect of Tamarindus indica L. leaves fluid extract on human blood cells
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Authors: Escalona-Arranz JC*1, Garcia-Diaz J1, Perez-Rosés R2, De la Vega J3, Rodríguez-Amado J1
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and Morris-Quevedo HJ4.
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Pharmacy Department, Oriente University. Santiago de Cuba, Cuba
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Departament Farmacología i Química Terapéutica. Universitat de Barcelona, Spain.
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Medical Toxicology Centre (TOXIMED). Medical Sciences University, Santia-go de Cuba, Cuba
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for Studies in Industrial Biotechnology (CEBI). Oriente University, Santiago de Cuba, Cuba
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de Cuba; Cuba. E-mail: jcea@cnt.uo.edu.cu, Phone: 0053 (22) 641411 Fax: 0053 (22) 641411
Pharmacy Department, Oriente University. Address: Avenida Patricio Lumumba s/n, 90500 Santiago
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Abstract
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Tamarind leaves are edible, but their saponin content could be toxic to human blood cells. In this
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paper a tamarind leaf fluid extract (TFE) is evaluated in several tests developed on human blood cells.
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Results revealed that TFE did not cause significant haemolysis on human red blood cells even at the
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lowest evaluated concentration (20 mg/mL). Blood protein denaturalization ratio was consistently
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lower than in control if TFE concentration was greater than 40mg/mL. Erythrocytes membrane
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damage caused by the oxidative H2O2 action displayed a steady reduction with increasing TFE
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concentrations. In the reactive oxygen species (ROS) measurement by flow cytometry assay,
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leucocytes viability was over 95% at tested concentrations and a high ROS inhibition was also
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recorded. Protective behaviour found in TFE should be attributed to its polyphenols content. Thus
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tamarind leaves should be regarded as a potential source of interesting phytochemicals.
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1. Experimental section
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1.1. Plant material, chemicals and reagents
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Tamarind leaves were collected from a tamarind population in Santiago de Cuba, eastern Cuba (GPS
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20º 2´38.9´´N y 075º 45´25.8´´W.), and were previously identified by Dr. Jorge Sierra Calzado. A
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voucher specimen registered as 052216 was deposited in the Docent Section of The BSC Herbarium
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at the Biology Department of the Oriente University. Collected leaves were sun dried (residual
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humidity below 10% by the stove method), milled by a (MLK, Russia), and passed across a 5 mm of
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mesh light sieve. All chemicals were of analytical grade and were obtained from Sigma Chemicals, St
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Louis, USA.
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1.2. Tamarind extracts preparation
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Extracting conditions were: 4 days of percolation and a mixture of ethanol/water 72% v:v as solvent for
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the procedure. Previous experiences of our research group had proved those conditions as a sure
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way to extract high quantities of metabolites (Escalona-Arranz et al., 2011). The tamarind fluid extract
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(TFE) was prepared from 4 extractions that were collected, mixed and concentrated up to 1 milliliter of
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extract for each milligram of dried leaves. A vacuum evaporation system at 42 ºC (KIKA WERKE,
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Germany) was used for the concentration of collected extractions. TFE had already been
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characterized in its physico-chemical and chemical properties: total soluble substances, pH, relative
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density, refraction index and total polyphenol and flavonoid content (Escalona-Arranz et al., 2011).
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Saponins presence was confirmed by the foam assay.
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1.3. Red blood cells (RBC) lyses and protein denaturation tests
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The methodology performed was INVITTOX n° 37, an in vitro technique using human blood (Lewis,
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1994). Human blood collected from a vein puncture of a healthy volunteer was added into a
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polyethylene vial containing citrate buffer to achieve a final concentration of 1:10 citrate:blood. A
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centrifugation at 1500 x g for 15 minutes at room temperature followed and supernatant (plasma) was
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carefully removed. Red blood cells were washed four times with isotonic Phosphate Buffered Saline
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(PBS), pH 7.4. In those cases in which a spontaneous hemolysis appeared, the solution was
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discarded. Morphology of cells was examined before use.
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1.3.1 Haemolysis
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Five different TFE concentrations were prepared by dilution on phosphate buffered saline (PBS). They
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consisted in 100, 80, 60, 40, or 20 L of TFE with the necessary volume of PBS to prepare 975 L of
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the test solution. Each test solution was incubated with 25 L of a RBC suspension for 10 minutes with
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constant shaking at room temperature. Incubation was finished by a high-speed centrifugation and the
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removing of cells and debris from the medium. Supernatant´s absorbance was monitored at 560 nm in
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a spectrophotometer CECIL CE 7200 (U.K.). Spontaneous haemolysis was settled as cero value. It
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was determined by adding 25 L of the RBC suspension to 975 L of PBS. The 100% haemolysis
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value was measured when 25 L of RBC suspension were added to 975 L of distilled water. Half-
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maximal effective concentration (H50) was calculated from the resulting dose-response curve.
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1.3.2 Denaturation of blood proteins
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The same five samples of TFE prepared in the haemolysis assay were prepared in PBS and
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incubated with 25 L of a RBC suspension for 10 minutes, with constant shaking, at room
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temperature. Incubation finished by a high-speed centrifugation that stopped the denaturing of just
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released oxyhemoglobin. Supernatant’s absorbance was determined in a dual-beam UV/VIS
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spectrophotometer CECIL CE 7200 (U.K.) at 575 nm and 540 nm against a blank. The cero value of
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protein denaturation was determined by adding 25 L of RBC suspension to 975 L of PBS. The
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100% protein denaturation value was measured when 25 L of RBC suspension were added to 975
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L of sodium dodecyl sulfate (SDS) 10 g/L in PBS. Extinction measured at 575nm was divided by
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values of extinction measured at 540nm to obtain the so-called alfa/beta ratio (R1). This ratio is
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subsequently used to characterize the haemoglobin denaturation index (DI), which is expressed as the
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relative percentage inhibition of the total protein denaturation value.
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1.4. H2O2-induced oxidative damage in human erythrocytes membrane
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The same five samples of TFE prepared in the previous assays were added to 750 L of RBC and
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970 L of PBS. The traditional H2O2-induced oxidative damage in human erythrocytes test
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(Konyalioglu & Karamenderes, 2005) was carried out by the addition of 50L of hydrogen peroxide to
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the RBC treated solutions. Experimental groups were incubated in a water-bath at 37 ºC for an hour.
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After incubation and a high-speed centrifugation the supernatant extinction was determined in a dual-
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beam UV/VIS spectrophotometer CECIL CE 7200 (U.K.) at 575 nm and 540 nm against a blank,
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following the same procedure that was used in the blood protein denaturation method. Control groups
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without H2O2 nor TFE and a positive peroxide group (not treated with TFE) were also considered.
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Results were expressed as relative percentage inhibition of induced oxidative damage in human
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erythrocytes membrane.
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1.5. Reactive Oxygen Species (ROS) measurement by flow cytometry assay
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Human leukocytes were purified from "buffy coats" bought at the Blood & Tissue Bank of Catalunya. A
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leukocyte suspension was produced through a controlled hemolytic shock with an ammonium chloride
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solution (Bossuyt et al., 1997). The method for ROS measurement described by Perez-Garcia et al.
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(1996) was followed with minor modifications to adapt its use to microplates. A leukocyte suspension
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was incubated with 2′,7′-dichlorofluorescin diacetate (10 µM) and sodium azide (1 mM) for 10 min at
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37º. After centrifugation for 5 min at 2500 rpm, supernatant was removed and the cell pellet
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resuspended in 4 mL of Hanks’ balanced salt solution (HBSS). In a 96-well flat bottomed microtiter
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plate, 200 μL of the already sensitive suspension of leukocytes (approximately 10 6 cells) were added
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to all experimental wells. Afterwards, 20 μL of eight sample concentrations of TFE (350, 175, 87.5,
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43.8, 21.9, 10.9 and 5.5 µg/mL) and different controls: quercetin (positive control, 1 µg/mL), base line
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control and stimulated control were added. After incubation for 5 min at 37 ºC, 20 μL of a 10 μM
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dimethyl sulfoxide solution of phorbol 12-myristate 13-acetate (PMA), was placed in all experimental
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wells except those designated as base line control where 20 μL of HBSS were added. Microtiterplates
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were again incubated for 5 min and 50 μL of paraformaldehyde (1% w/v) were added in all wells to fix
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cells. Before result evaluation, 2 μL of propidium iodide (10 µg/mL) were added to all wells. A flow-
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cytometer, model Cytomics FC 500 MPL system was employed (Beckman coulter, Inc., Bea, CA,
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USA) for analysis. More than 50,000 cells were counted by flow-cytometry, using “live” discriminating
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gate in the red fluorescence histogram (607 nm) of propidium iodide. A second gate surrounding
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neutrophils was set in the forward scatter vs side scatter biparametric histogram, where 20,000 cells
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were counted. Measures were always made in the viable population. A green fluorescence histogram
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(515-540 nm) in which “live” and “neutrophils” gates were applied, allowed measuring ROS
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production. Relative inhibition of ROS production was calculated by the following formula:
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Inh ROS (%) =100 ─ [(Fts─Fbc) / (Fsc ─Fbc)] x 100.
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Where: Inh ROS (%): relative inhibition of ROS production (%), Fts: fluorescence value in treated
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samples, Fbc: fluorescence value in baseline control, Fsc: fluorescence value in stimulated control.
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1.6. Data processing and statistics
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Flow-cytometry results were stored as listmodes files for later processing with the specialized software
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Summit version 4.2 for Windows. Results of relative inhibition of ROS production were recorded as the
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mean ± standard deviation (SD) of four independent measurements while the Hemolysis, Blood
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protein denaturation and H2O2-induced oxidative damage in human erythrocytes membrane test were
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recorded as the mean ± standard deviation (SD) of five independent measurements. An analysis of
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variance followed by a Bonferroni´s test was used to compare means of untreated controls to each
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treatment and were considered significant at p<0.05. Inhibitory concentration 50% (IC50) was
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calculated by interpolation in a concentration/effect curve when possible. For data analysis, software
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STATISTICA Version 8.0 for Windows, Oklahoma, USA, was used.
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References
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Bossuyt, X., Marti, G.E., & Fleisher, T.A. (1997). Comparative analysis of whole blood lysis methods
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G., Morris-Quevedo, H., & Licea-Jiménez, I. (2011). Metabolites extraction optimization in
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Lewis, R.W. (1994). Red blood cell lyses and protein denaturation. The ERGATT/FRAME databank of
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