gi

, Hes, Nl and

kenrane's aition, the extract's effect on the physical properties of the erythrocyte membraneucted a detailed analysis of supplement ingredients using high-yield liquid chroma-

ted green tea extractand cell membranes,on biological systems.n tea supplement was

Life Sciences 126 (2015) 19

Contents lists available at ScienceDirect

Life Scie

e lsthat the biological activity of phenolics contained in green tea is signif-icantly different from the isolated single components [61,76,78]. Greentea ingredients such as epigallocatechin gallate (EGCG) and gallic acid

dened using different methods. Biological activity of the supplementwas dened in biophysical studies, and to indicate which substancesare mainly responsible for the activity, the research was conducted onfore they may be sold without detailed studies determining their effecton biological systems. Both bioprotective and negative effects of con-sumed plant supplements probably depend on a number of factors,not just the size of the dose intake. In addition, research has shown

determine the biological activity of concentracontained in a supplement with a focus on cellsin order to determine themechanism of its action

Composition of phenolic compounds in greeopment of cancer and diabetes [21,25,39,54,59,65,77].In recent years, however, there have been reports that green tea

polyphenols, when consumed in large quantities, cause unwanted sideeffects in the body [18,22,26,51]. Commonly available supplementsand plant extracts are not subject to stringent requirements, and there-

compounds contained therein, the mechanism of molecular interactionof these substances in the body is not yet fully explained. In particular,there is no denite explanation of the effects of concentrated extractsof green tea. Information regarding the impact of such extracts on cellmembrane properties is also lacking. Therefore it seems benecial to(GA) have both protective and therapeutic pr

Corresponding author. Tel.: +48 713205275; fax: +4E-mail address: sylwia.cyboran@up.wroc.pl (S. Cybora

http://dx.doi.org/10.1016/j.lfs.2014.12.0250024-3205/ 2015 Elsevier Inc. All rights reserved.nd its extracts are widelydiseases. They have anti-s, and prevent the devel-

effects and negative ones induced by extracts of green tea.Despite the large amount of information in the worldwide scientic

articles on biological activity of extracts of green tea and polyphenolic

used in the prevention and treatment of manyinammatory, antibacterial and antiviral effectGreen tea is a rich source of avan-Gallic acid (PubChem CID: 370)

Keywords:Green tea supplementAntioxidant activityAnti-inammatory activityUPLCPolyphenolsMembrane uidityErythrocytesHemolytic activity

with no deleterious effect on red blood cells. The extract modies the physical properties of the erythrocytemembrane, apparently by binding to its hydrophilic region, with consequent rigidity of the hydrophobic region,increased hydration and a moderate increase in its resistance to changes in tonicity of the medium. Because theextract's components anchor in the polar region of membrane lipids, they are able to effectively scavenge freeradicals in the immediate vicinity of the membrane and hinder their diffusion into its interior.Signicance: Green tea supplement at concentrations markedly exceeding the blood plasma physiological poly-phenol concentrations has no destructive effect on the erythrocyte membrane. Due to the high content ofavan-3-ols, the supplement exhibits high biological activity, which makes it an alternative source of thosesubstances to the commonly used infusion of green tea leaves.

2015 Elsevier Inc. All rights reserved.

Introduction

3-ols, a

damaging to cells and their membranes [7,11,24,34,35,38,41]. Currentknowledge does not, however, allow a full explanation of the healthy() Epigallocatechin gallate (PubChem CID: 65064)Key ndings: The study showed that green tea extract has a high antioxidant and anti-inammatory capacityChemical compounds studied in this article: tography, supplemented with standard tests of total content of polyphenols and avonoids in the supplement.inammatory actions. In addwas examined.We also condConcentrated green tea supplement: Biolomolecular mechanisms

S. Cyboran a,, P. Strugaa a, A. Woch a, J. Oszmiaski ba Department of Physics and Biophysics, Wrocaw University of Environmental and Life Sciencb Department of Fruit, Vegetable and Cereal Technology, Wrocaw University of Environmenta

a b s t r a c ta r t i c l e i n f o

Article history:Received 14 July 2014Accepted 17 December 2014Available online 20 February 2015

Aim: This study was undertacells and erythrocyte membMain methods: The extract

j ourna l homepage: www.operties, as well as being

8 713205167.n).cal activity and

. Kleszczyska a

orwida 25, 50-375 Wrocaw, PolandLife Sciences, Norwida 25, 50-375 Wrocaw, Poland

to determine the biological activity of a green tea supplement with respect tos and the molecular mechanism of that activity.ctivity was evaluated on the basis of its hemolytic, antioxidant and anti-

nces

ev ie r .com/ locate / l i fesc ietwo selected components of the supplement: () epigallocatechin-3-gallate (EGCG) and gallic acid (GA). These compounds were chosen be-cause they represent two different groups of polyphenolic compoundsfound in green tea; EGCG belongs to avan-3-ols and GA to phenolicacids. Biological activity of the selected substances was established on

2 S. Cyboran et al. / Life Sciences 126 (2015) 19the basis of hemolytic, antioxidant and anti-inammatory activities andtheir impact on the physical properties of the erythrocyte membrane.

Materials and methods

Green tea supplement, polyphenols

A dietary supplement widely available on the world market, whichcontains green tea and high contents of polyphenolic compounds, wasused in the study. One supplement capsule contains 300mg of polyphe-nol extract of green tea. The raw material used to manufacture thisextract comes from the leaves and stems of green tea growing in themountainous areas of the province of Fujian (South China). The domi-nant species of green tea in the material from which the polyphenolswere extracted is Tieguanyin. EGCG, (+) catechin, () epicatechin(EC), quercetin-3-rutinoside (rutin) and gallic acid (GA) were pur-chased from Extrasynthese, Paris Cedex, France.

Cells and biological membranes

The investigation was conducted on erythrocytes and their mem-branes obtained from fresh heparinized pig blood according to themethod of Dodge et al. [15]. The content of erythrocyte membranes inthe samples was determined on the basis of protein concentration, whichwas assayed using the Bradford method [8], and it was 100 g/ml.The choice of pig erythrocytes was dictated by the fact that this cell'spercentage content of lipids is closest to that of the human erythrocyte,and the blood readily available. Each time, fresh blood was added to aphysiological solution of sodium chloride containing heparin.

Fluorescence probes, enzymes and reagents

The uorescent probes Laurdan (6-dodecanoyl-2-dimethylamino-naphthalene), DPH (1,6-diphenyl-1,3,5-hexatriene), and TMA-DPH(1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate) were purchased from Molecular Probes, Eugene,Oregon, USA. The FolinCiocalteu phenol reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-diazobis (2-amidinopropane) dihydro-chloride (AAPH), butylated hydroxyanisole (BHA), L(+) ascorbicacid (AA), and enzymes COX-1, COX-2 and 1-LOX were purchasedfrom Sigma-Aldrich, Inc., Steinheim, Germany. All other chemicalswere of analytical grade, obtained from Sigma-Aldrich, Inc., Steinheim,Germany.

Phenolic content

Total phenolic contentTotal phenolic content was determined using the FolinCiocalteu

(FC) reagent, adapted from Blainsky et al. [5]. The standard curvewas made for gallic acid. The results were expressed as mg gallic acidequivalents (GAE) per 1 g of dry sample.

Total avonoid contentGT extract was analyzed for total avonoid content according

to the colorimetric method described previously by Lamaison andCarnat [33].The standard curve was prepared for rutin (quercetin-3-0-rutinoside) under the same conditions. The results were expressedas mg rutin equivalents (Q3R) per 1 g of dry sample.

UPLCDAD and UPLCESI-MS analysesThe percentage content of polyphenols in the extract of green tea

was determined by means of UPLC/DAD and the UPLC/ESI/MS method

analyses described by Oszmiaski et al. [49].Hemolytic activity

The ability of polyphenolic compounds contained in the GT extract,EGCG and GA to induce hemolysis of red blood cells was studied usingspectrophotometric methods described earlier in Kleszczyska et al.[30] with minor modications.

Levels of hemolysis of blood cells modied with the substances atconcentrations of 0.005 to 0.1 mg/ml and of the control were deter-mined after different times of incubation. Briey, the modication wasconducted at 37 C thrice (1 h, 2 h and 3 h), each sample containing1ml of erythrocyte suspension of 1.2% hematocrit, stirred continuously.After modication, 2 ml of isotonic phosphate buffer of pH = 7.4 wasadded to each sample. Next, the samples were centrifuged and thesupernatant assayed for hemoglobin content using a spectrophotome-ter (Specord 40, Analytik Jena) at a 540 nm wavelength. Hemoglobinconcentration in the supernatant, expressed as percentage of hemoglo-bin concentration in the supernatant of totally hemolyzed cells, wasassumed as the measure of the extent of hemolysis.

Antioxidant activity

DPPH radical scavenging activity assayThe effect of studied substances on reduction of DPPH radical

concentration was measured spectrophotometrically, as previouslydescribed by Vidal et al. [71].

In that study, the measure of antioxidant activity of the studied sub-stances was assumed to be EC50 the concentration at which the freeradicals DPPH are reduced by 50%.

Inhibition of membrane lipid peroxidationAntioxidant activities of GT extract, GA, and EGCGwere determined

using the uorimetric method described in our previous work [6] withminor modications. The studies were carried out on membranes oferythrocytes (RBC). The TMA-DPH probe was used in these experi-ments. Suspensions of erythrocyte membranes (ghosts) were treatedwith the chemical oxidation inductor AAPH for 30min at 37 C after ad-dition of appropriate amounts of antioxidants (GT extract, GA, EGCGdissolved in ethanol). Free radicals generated by thermal decompositionof AAPH at 37 C attacked the erythrocytemembranes and induced lipidperoxidation. They also caused quenching of TMA-DPH uorescenceand decreased uorescence intensity. A Cary Eclipse (Varian) spectro-uorimeter was used to measure free radical concentrations in thesamples. Excitation and emission wavelengths were ex = 362 nmand em = 428 nm. The measure of lipid oxidation was the relativechange of uorescence intensity, F/F0, where F0 is the initial uores-cence and F the one measured during an oxidation procedure [2]. Theconcentration of the compounds (IC50) at which 50% inhibition ofperoxidation occurred (fall in uorescence intensity) was assumed asa measure of their antioxidant activity. The results of the assays wereexpressed relative to Trolox and AA.

Inhibition of enzyme activity

Cyclooxygenase inhibitory activityThe anti-inammatory activity of the GT supplement and EGCG,

established on the basis of a modied method given in the work byKumar et al. [31], was assayedwith a spectrophotometric measurementof inhibition of activity of the cyclooxygenase COX-1 and COX-2.In short, the experiment was conducted as follows: into a cuvettecontaining TrisHCl buffer (pH 8.0) the following were successivelyadded: the studied enzyme inhibitor (GT, EGCG at 2.5 mg/ml initialconcentration), hematin (0.1026 mM) and cyclooxygenase (COX-1or COX-2) at 1 mg/ml. After mixing and incubation (approx. 3 min),TMPD was added at 24.35 mM. To initiate the inhibition reaction,arachidonic acid was added at a concentration of 35 mM. The nal vol-

ume of the sample was 1ml. Changes in absorbance of the sample were

Statistical analysis

Statistical analysis of the results was performed using theSTATISTICA 10.0 (StatSoft PL) software. Dunnett's test (post-hocANOVA) at signicance level=0.01 or=0.05was used to estimatethe differences between themean values ofmeasured parameters of thecontrol samples and the mean values of the parameters of the samplesmodied with the extracts. All the experiments were done in at leastve replicates, the results being presented as mean standarddeviation.

Results

Table 1Total phenolic content (TPC) and total avonoid content (TFC) in green tea supplement;EC50 valueswere determined for DPPH radical scavenging activity and IC50 values calculatedfor lipid peroxidation inhibitory activity of green tea extract (GT), gallic acid (GA), ()epigallocatechin-3-gallate (EGCG) and L(+) ascorbic acid (AA).

CompoundTPC[mgGAE/g]

TFC[mgQ3R/g]

EC50[g/ml]

IC50[g/ml]

GT extract 708.60 8.0 175.42 4.5 2.93 0.007 5.93 0.18EGCG 2.14 0.002 3.53 0.16GA 2.43 0.002 5.67 0.10AA 3.26 0.001 20.1 1.15Trolox 4.87 0.002 3.90 0.30

() Gallocatechin gallate 18.32 5.06 275 457 169/305Myricetin 3-galactoside 1.31 5.95 354 479 317Myricetin 3-glycoside 2.23 6.13 354 479 317() Epigallocatechin 3-methylgallate

10.89 6.27 276 471 183/287/305

Quercetin 3-rutinoside (rutin) 0.28 6.74 360 609 301() Epicatechin gallate 94.68 6.82 276 441 289Quercetin 3-galactoside 0.96 7.23 350 463 301Quercetin 3-rhamnoglucoside 0.41 7.32 360 609 301Quercetin 3-glucoside 2.94 7.49 350 463 301Kaempferol 3-galactoside 0.50 8.16 350 447 285Kaempferol 3-rutinoside 0.42 8.43 350 593 285Kaempferol 3-glycoside 1.50 8.63 350 447 285Myricetin 0.76 8.77 365 317 151Quercetin 1.23 11.21 365 301 146Total 829.76

3S. Cyboran et al. / Life Sciences 126 (2015) 19followed for 3min bymeasuring it at 1min intervals, using the spectro-photometer at awavelength of 611 nm (Cary 100 Bio Varian) in relationto the reference sample. The measurements were carried out at roomtemperature. The control sample, instead of the extract, contained theright amount of solvent. In this investigation, the concentration of thestudied substances at which 50% inhibition of enzyme activity occurredwas assumed as a measure of the compound's anti-inammatoryactivity.

Lipoxygenase inhibitory activityThe GT extract, GA and EGCG were also tested for inhibition of

soybean lipoxygenase (1-LOX) activity on the basis of a proceduredescribed by Mahesha et al. [37] with modication. For comparativepurposes the non-steroidal, anti-inammatory agent ibuprofen wasalso tested. Reactions were carried out in 10 mm path-length quartzcuvettes containing, in a nal volume of 2.6 ml: borate buffer pH 9and 0.1 mg/ml 1-LOX, the extracts tested, and 50 M linoleic acid(previously prepared in borate buffer pH 9.0 plus 1% Tween 20 and0.1 M NaOH). The incubation time was 3 min at room temperature.The ability of the substances tested to inhibit 1-LOX enzyme activitywas expressed by the agent's concentration (IC50-LOX) at which 50%inhibition of enzyme activity occurred.

Properties of the erythrocyte membrane

Osmotic resistance of the membraneThe osmotic resistance assay was performed according to the

method described in our earlier work [13]. The C50 values (the NaClconcentrations that caused 50% hemolysis) were taken as a measureof osmotic resistance. If a determined sodium chloride concentrationis higher than that of control cells, the osmotic resistance of the erythro-cytes is regarded to be lower, and vice versa.

Fluidity of the membraneThe effect of GT extract, GA, and EGCG on uidity of lipids in the

erythrocytemembrane (ghosts) was investigated using theuorimetricmethod described in our previous work [6], with minor modications.Control samples contained only erythrocytemembranes with a uores-cent probe at 1 g/23.2 g lipid:uorescent probemass ratio, an appropri-ate compound at a concentration in the range of 0.001 to 0.1 mg/mlbeing added to the remaining samples. Fluorescence intensitywasmea-suredwith the DPH probes at 37 C. Themeasurementswere conductedwith a uorimeter (CARRY Eclipse VARIAN) equippedwith a DBS Peltiertemperature controller (temp. accuracy 0.1 C). On the basis of themeasured uorescence intensity of the DPH probe, the valuesof uorescence anisotropy (A) were calculated, using the formuladescribed by Lakowicz [32]. An increase in DPH anisotropy indicates adecrease in membrane uidity and vice versa.

Degree of hydration of lipid polar headsThe effect of GT supplement, and EGCG and GA compounds on the

degree of hydration of the area occupied by the polar heads of lipidswas assessed using the method described by [13,72]. The study wascarried out on isolated, unsealed erythrocyte membranes. The amountof erythrocyte ghosts in the samples was about 100 g/ml, while the con-centration of the Laurdan probewas ca. 1 M.Appropriate amounts of GT,EGCG and GA were added to samples containing erythrocyte ghosts andthe probe in a buffer solution of pH 7.4 to obtain concentrations from 1to 100 g/ml. To evaluate the extent of alterations in the bilayer hydrationand lipid packing density upon addition of GT, EGCG, and GA, we tracedchanges in Laurdan-induced generalized polarization (GP). The GP wascalculated with the formula described by Lakowicz [32].

The rise in GP value was interpreted in terms of bilayer dehydration

and/or increase in lipid packing density and vice versa.Phenolic content

Using spectrophotometric methods, we examined the total contentof polyphenolic compounds (TPC) and the total content of avonoids(TFC) in green tea supplement; the results are presented in Table 1. Inaddition, using chromatographic methods, a detailed quantitative andqualitative analyses of the compounds contained in the supplementwas performed (Table 2).

The investigation showed that the green tea supplement is rich inpolyphenolic substances, which account for more than 80% of all itscomponents (Table 2). Compounds contained in the supplement belongto 3 groups of avonoids: avanols (catechins and their derivativesca. 88%), avonols (quercetin glycosides and kaempferol ca. 1%), and

Table 2Content and characterization of phenolic compounds of the green tea preparation, usingtheir spectral characteristic in UPLCDAD (retention time, max) and negative ions inUPLCESI-MS.

Compound Content[mg/g]

RT max[nm]

[MH]

(m/z)MS2(m/z)

Gallic acid 63.19 1.02 274 169 125() Gallocatechin 4.58 1.65 270 305 179() Epigallocatechin 83.84 3.04 269 305 179(+) Catechin 8.34 3.27 278 289 245Unknown 28.08 4.34 272 745 407/289() Epigallocatechin gallate 422.29 4.49 272 457 169/305() Epicatechin 83.01 4.82 278 289 179/205/245

phenolic acids (gallic acid ca. 7.5%). The dominant components arecatechins and their gallate esters epigallocatechin gallate (EGCG) andepicatechin gallate (ECG), epicatechin (EC), epigallocatechin (EGC)and gallic acid (GA).

Hemolytic activity

The results of hemolytic studies for the two selected concentrationsof the test substances, i.e. 0.01 and 0.1 mg/ml, are presented in Fig. 1.

The results indicate that test substances in a wide range of concen-trations do not induce hemolysis of blood cells, because virtually no sta-tistically signicant differences were found between levels of hemolysisin the modied samples and controls (Fig. 1A and B). With the highestconcentration of 0.1 mg/ml, only in the presence of EGCG was thelevel of hemolysis of modied cells signicantly higher compared tothat of control cells after 3 h of incubation. The observed increase inthe amount of hemolyzed cells in the presence of EGCG was negligibleand did not exceed 2%.

Fig. 1. Percentage of hemolyzed cells in modied samples of the control and test sub-stances at concentrations of: A) 0.01mg/ml and B) 0.1mg/ml after 1, 2 and 3 h incubationat 37 C (GT green tea supplement, GA gallic acid, EGCG () epigallocatechingallate). Statistically signicant differences betweenhemolysis of the control andmodiedcells are denoted as *= 0.05, **= 0.01.

Fig. 2. Relative uorescence of the TMA-DPH probe vs. time of oxidation by AAPH in

4 S. Cyboran et al. / Life Sciences 126 (2015) 19Antioxidant activity

The results on antioxidant activity of GT supplement, EGCG, and GAcompounds in the test with DPPH are shown in Table 1. Such studieswere additionally carried out for two other compounds found in theextract, () epicatechin (EC) and (+) catechin (C), for which the EC50values were respectively 3.48 0.012 and 4.24 0.007 g/ml. The an-tioxidant activities of the GT supplement and its avanols, on the basisof the results obtained, follow the sequence: EGCG N GA N GT N EC N C.

The results obtained by the uorimetric method are shown in theform of kinetic curves of relative uorescence intensity for the TMA-DPHprobe in thepresence of test substances. Examples of kinetic curvesfor the samples containing dietary supplement GT and EGCG are givenin Fig. 2.

The kinetics of oxidation inhibition by the supplement and EGCGcompound are similar. In therst 15min of the response, the two agentsalmost completely protect membrane lipids against oxidation inducedby AAPH. Then, as the antioxidant becomes depleted, a virtually lineardecline continues in the intensity of uorescence, which becomessmaller the higher the concentration of antioxidant present in the sam-ple. Similar oxidation kinetics were obtained for GA. The IC50 valuesfound for the test substances are presented in Table 1.

Anti-inammatory and lipoxygenase inhibitory activity

The investigation showed that all test substances, to varying de-grees, inhibit the enzyme cyclooxygenases COX-1 and COX-2 andthe presence of A) green tea supplement and B) () epigallocatechin gallate.

lipoxygenase 1-LOX. The results for the anti-inammatory activityof the test substances and certied standard medicines with anti-inammatory effect (indomethacin and ibuprofen) are presented inTable 3.

Assuming that among the supplement's components, the ability toinhibit enzymes COX-1, COX-2 and 1-LOX is shown only by EGCG andit is not inhibited by its other components, the supplement concentra-

than those for lower concentrations (0.50.65%). This means that athigher values of osmotic pressure the presence of the compounds testedin themembrane of erythrocytes practically does not affect the sensitiv-ity of the erythrocytes to osmotic pressure (Fig. 3). On the basis of C50values, determined from hemolytic curves, the osmotic resistance ofthe erythrocytes modied by various substances decreases as follows:C50GT N C50EGCG N C50GA.

Fluidity of the membraneUsing the uorimetric method, we examined the impact of the sup-

plement and polyphenols on uidity of the membrane. Fig. 4 showschanges in uorescence anisotropy of the DPH probe as a function ofsubstance concentration.

The observed increase in the value of A is largest for EGCG, at a con-centration of 4.22 g/ml, and is 32%, while the supplement at a concen-tration of 10 g/ml (containing the same amount of EGCG at 4.22 g/ml)increases anisotropy values by 37%. This means that the observedstiffening of supplement modied membrane is mainly caused by theepigallocatechin gallate (EGCG).

Table 3Values of IC50 concentrations for GT supplement, epigallocatechin gallate (EGCG),indomethacin (IND), and ibuprofen (IBUP), at which 50% inhibition of cyclooxygenase(COX-1, COX-2) and lipoxygenase (1-LOX) activity occurs.

Compound

IC50-COX [g/ml] IC50-LOX [g/ml]

COX-1 COX-2 1-LOX

GT 57.33 1.15 53.72 0.21 17.83 0.043EGCG 14.92 0.10 10.71 0.10 5.87 0.491IND 9.15 0.23 7.60 0.68 IBUP 1.28 0.093

5S. Cyboran et al. / Life Sciences 126 (2015) 19tion responsible for 50% inhibition of the enzymes' activity would be:24 (COX-1), 22 (COX-2) and 7.5 g/ml (LOX-1). Such an assumptionwould lead to the conclusion that the supplement concentration re-sponsible for 50% inhibition would be much lower than those obtainedin the tests, so the ability of EGCG to inhibit the activity of theseenzymes is likely to be diminished by other substances contained inthe supplement.

Properties of the erythrocyte membrane

Osmotic resistance of membraneThe effect of the supplement, EGCG, GT andGAon osmotic resistance

of erythrocytes was examined using spectrophotometric methods. Thiseffect was determined by comparing the percentage of modied bloodcell hemolysis induced by the compounds at a 0.01 mg/ml concentra-tionwith hemolysis of control cells suspended in isotonic and hypotonicsolutions of NaCl. Fig. 3 shows the obtained hemolytic curves, i.e. hemo-lysis of control and modied cells vs. sodium chloride concentration. Ashift of modied cell kinetics toward lower concentrations testies tothe increased osmotic resistance of red blood cells, while in the direc-tion of physiological concentration there is a drop in resistance.

The starting point at which hemolysis occurred in the untreated andmodied cells (Fig. 3A, B, C) was approximately 0.85% NaCl. In the pres-ence of modifying substances, a slight shift of the curves in the directionof lower concentrations of sodium chloride was observed, the largestwas for GT modied cells (Fig. 3A). In addition, we observed greaterchanges for higher NaCl concentrations (in the range of 0.650.80%)Fig. 3. Extent of hemolysis as a function of sodium chloride concentration in the presence of: Asignicant results were determined at: = 0.05 (*) and = 0.01 (**).Degree of hydration of lipid polar headsThis investigation showed that for erythrocyte membranes the

dietary supplement GT and the compounds EGCG and GA, dependingon the concentration, in varying degrees affect the value of the general-ized polarization of the Laurdan probe (Fig. 5). A concentration-dependent decrease in the value of GP was observed for the GT supple-ment. The inuence of compound EGCG on GP, as the research showed,does not depend on the concentration. Initially, with an increase in theconcentration of EGCG a drop in GP value of approximately 50 g/mlwas observed, while a further increase in concentration practically didnot inuence the value of GP. Modication of erythrocyte membraneswith GA did not affect the value of GP up to 75 g/ml (Fig. 5).

Comparing the impact of pure EGCG and GA on the GP value ofthe Laurdan probe and the impact of the supplement that contains thesame amounts of these substances, we can conclude that among thesupplement components, in addition to EGCG andGA, other polypheno-lic compounds also share responsibility for decreased GP.

Discussion

The UPLC analysis showed that GT is rich in polyphenolic substances(Table 2). The dominant constituents are epigallocatechin gallate,epicatechin gallate, epigallocatechin, epicatechin, and gallic acid. Suchcompounds were identied previously in extracts of green tea [3,56,60]. In addition, the content of polyphenols and avonoids in 1 g ofthe supplement may exceed by several times the quantities of these

) GT supplement, B) epigallocatechin gallate (EGCG) and C) gallic acid (GA). Statistically

Fig. 4. DPH probe uorescence anisotropy as a function of concentration of the testcompounds: EGCG epigallocatechin gallate, GT green tea supplement and GA gallicacid. Statistically signicant differences between anisotropy values for the modied andcontrol membranes are denoted as: = 0.05 (*), = 0.01 (**).

6 S. Cyboran et al. / Life Sciences 126 (2015) 19compounds present in 1 g of commercially available varieties of greentea [28,77]. The content of catechins in a capsule of supplement(300 g) on average corresponds to their content in two cups of greentea [9].

In connection with the toxic actions reported for some polyphenols[17,23], in this work hemolytic activity of the supplement and its con-stituents, EGCGandGAwere specied on the basis of their hemolytic ef-fect. These studies have shown that in a wide range of concentrationsthey practically do not induce hemolysis (Fig. 1). The exception isEGCG, which when used in a high concentration and after a long timeof action on erythrocytes showed low hemolytic activity. The adverseeffects of EGCG in relation to different cells were noted by other authors.They demonstrated, among other things, that EGCG induced injury inmouse blastocysts [16]; and could be hepatotoxic, in particular, whenpredisposing conditions, such as inammation, exacerbate its harmfuleffect on liver cells [58]. It is believed that the damaging effects ofEGCG, in particular at high concentrations, are mainly due to its proox-idative properties related to its katecholic structure (two hydroxylgroups in the ortho position of the B ring). The katecholic group canFig. 5. Generalized polarization (GP) of the Laurdan probe as a function of green teasupplement (GT), epigallocatechin gallate (EGCG) and gallic acid (GA) concentration.Statistically signicant differences between GP values of the Laurdan probe for the controland modied membranes are denoted as: = 0.05 (*), = 0.01 (**).generate superoxide anion radical from molecular oxygen through for-mation of an electrophilic o-quinone counterpart [43]. In addition, theharmful activity of EGCG may be also connected with the presence ofa gallate group in its molecule, as suggested in the work by Ugartondoet al. [68]. This supposition is conrmed by our earlier studies [13,73],where we found that there is no hemolytic action of polyphenolic ex-tracts which do not contain phenols of the gallate group. It is believedthat the observed slight hemolytic activity of EGCG may by connectedwith its ability to make covalent bonds with thiolic groups (SH) ofproteins and their modication [42,45].

The concentrations of the GT, EGCG and GA compounds used in thehemolytic investigationwere signicantly higher than the physiologicalconcentrations of polyphenolic compounds in the blood (several toseveral dozen nM). Van Amelsvoort et al. [70] found that themaximumconcentration of catechins in plasma does not exceed 0.5 M in theamount of 12 mg/kg body weight. This means that the tested com-pounds used at a concentration that is ca. 100 times higher than physi-ological concentrations of polyphenols in blood (0.01 mg/ml) do notinterfere with the structure of erythrocyte membrane.

The healthful properties of polyphenols are believed to followmainly from their high antioxidant capacity, due to which they preventthe development of many serious diseases associated with oxidativestress [29,74]. Therefore, in the presented work we determined theantioxidant activity of green tea supplement. Both applied researchmethods showed that substances contained therein are very good anti-oxidants. The results obtained in the DPPH test indicate that antioxidantactivity of GT is close to the activity of EGCG and GA contained in it, butis higher than the activity of the standard antioxidants AA and Trolox(Table 1). These results correlate well with results of other authors ob-tained for individual avan-3-ols [46,55] and green tea extracts [19]. Itis believed that the antioxidant activity of avan-3-ols, abundant inthe supplement, depends largely on their structure. Higher antioxidantactivity, in the DPPH test, is shown by compounds with a galloyl moietysubstitute in position 3 of the C ring, for instance EGCG, comparedwiththosewith an unsubstituted OHgroup in that position, and havingmorehydroxyl groups in the B ring [48]. Moreover, the GT supplement ismore effective in reducing DPPH radicals than other green tea extractsthat show lower antiradical ability than AA, e.g. the extract of Camelliasinensis leaves [27], and brands of green tea commercially available inAsian markets [10].

A uorimetric study of antioxidant activity showed that the activityof GT in relation to membrane lipid is only slightly weaker than that ofTrolox, but it is four times higher than that of AA activity (Table 1). Inaddition, the results showed that using a dietary supplement will havea similar effect as in the application of a single polyphenol of the sameconcentration, as the activity of the supplement is close to the activityof a single EGCG or GA. In addition, the results obtained indicate thatthe GT supplement protection of red blood cell membranes againstAAPH induced oxidation (IC50 = 5.93 g/ml) is on a level similar tothe protection shown by extracts from the leaves of mini kiwi (IC50 =5.5 g/ml) [14]. Analysis of the values of IC50 and EC50 (Table 1) andthe content of individual components in the supplement (Table 2)leads to the conclusion that the antioxidant activity of the supplementis the result of the additive action of its components.

Polyphenols, aside from their high antioxidant efcacy, also havean anti-inammatory effect, which consists in inhibiting enzymesconnected with the inammatory process. In particular, their ability toinhibit the COX-2 enzyme is noteworthy, since COX-2-mediated prosta-glandin production can stimulate cell proliferation, invasion and angio-genesis in cancer development [40]. Therefore, this study examined thesupplement's ability to inhibit two types of cyclooxygenase, COX-1 andCOX-2, that are likely to have fundamentally different biological roles[62]. Research showed that GT inhibits the activity of both isomericforms of the enzyme. Its inhibitory activity on COX-1 and COX-2 is at asimilar level and is 4 times less than EGCG (Table 3). Other authors

also demonstrated anti-inammatory properties of EGCG and GT,

7S. Cyboran et al. / Life Sciences 126 (2015) 19which was evaluated on the basis of inhibition of cytokines, COX-2activity and other inammatory factors [12,36,53,75,79].

Some polyphenols are also potent inhibitors of LOX enzyme activity.It is believed that their inhibitory action in relation to this enzyme de-pends on their type, on the physico-chemical state of the substrate,and on the conditions of the experiment [47,63]. Literature data indicatethat green tea polyphenols, particularly EGCG, are effective inhibitors oflipoxygenases, e.g. mackerel-mmLOX [4], rabbit reticulocyte 15-LOXand soybean 15-LOX [69]. Our studies have conrmed that the sub-stances contained in the supplement inhibit the activity of 1-LOX. How-ever, the ability of GT to inhibit this enzyme is much weaker than thatof EGCG and some recognized substances with anti-inammatory(ibuprofen, indomethacin) action. In addition, the results suggest thatEGCG's ability to inhibit the activity of both enzymes is likely to beinhibited by other substances contained in the supplement.

GT polyphenols can modulate both the structure and propertiesof biological membranes [50,57], which are responsible for their protec-tive and therapeutic effects but are also closely linked to their cytotoxic-ity [64]. Therefore, determination of their impact on the propertiesof biological membranes is necessary to understand the mechanismresponsible for their biological activity. For this reason, the study wasperformed to determine the impact of the GT extract on erythrocyteosmotic resistance, uidity of the membrane and degree of hydrationof the polar heads of membrane lipids.

The results obtained indicate that the GT extract not only does notwork destructively on the erythrocytemembrane but it also exerts a for-tifying effect,making themembrane less sensitive to changes in osmoticpressure. In addition, the research showed that the observed increase inosmotic resistance of GT-modied cellswasmuch larger than that in theobserved changes for individual compounds such as EGCG and GA(Fig. 3). This means that the mixture of polyphenols present in the sup-plement strengthens the membrane much more than the individualcompounds used at the same concentration. In addition, the literaturesuggests that EGCG not only increases the osmotic resistance of redblood cells, but also protects them against cigarette smoke-induced bio-chemical perturbations [20]. Changes in osmotic resistance of erythro-cytes induced by polyphenols were also documented by other authors[23,44].

The investigation of the effect of GT on uidity of the erythrocytemembrane showed that this compound increases the value of uores-cence anisotropy (A) of the DPH probe, which means that it causesconcentration-dependent stiffening of the lipid double layer (Fig. 4).In addition, the results indicate that the increase in rigidity of GT-modied membrane is mainly due to EGCG contained in the supple-ment. Increased rigidity of various membranes in the presence ofavanols has also been reported by other authors. Abram et al. [1]found that the avan-3-ols cause an increase in packing order and de-cline of the dynamics of lipid acyl chains, as in our results. The observedincrease in the stiffness of themembranes is the result of the binding ofGT compounds on the surface of the membrane. Such interaction maycause the formation of larger areas of increased order in the outer partof the membrane, which affects its hydrophobic area. Tsuchiya [66]suggests that the reduction in membrane uidity is responsible for theantiplaque and hepatoprotective effects of green tea catechins.

Localization of these compounds in the hydrophilic area of themem-branewas conrmed in the test using the Laurdan probe. It showed thatGTmodulatesmembrane properties in the polar region by changing thevalue of the Laurdan probe GP. With the increase in the concentrationof GT, declining GP values were seen, which can be interpreted as anincrease in hydration of the polar heads of lipids [32]. That increase isprobably due to the presence in this area of additional water moleculesbound to the hydroxyl groups of polyphenolic compounds attached tothe polar heads of lipids [50]. Due to the number of hydroxyl groupsin the structure of the compounds contained in GT (e.g. EGCG has 8OH groups), they do not penetrate the hydrophobic region membrane,

but bind to the polar heads of lipids [52], probably at the level of thecholine groups [67]. This localization makes the substances able to suc-cessfully scavenge free radicals from around themembrane and protectit against free radicals diffusing to its interior.

Conclusion

Green tea supplement (GT) has a high content of polyphenols ofhigh biological activity, which, in particular, are very good antioxidants.The GT supplement can protect the organism, because apart from thehigh antioxidant activity it also inhibits enzymes involved in inamma-tion. It does not work destructively on the red blood cells, because itdoes not induce hemolysis even at concentrations 100-fold higherthan physiological concentrations of polyphenols in the blood plasma.The supplement modies the physical properties of the membrane,resulting in increased resistance to changes in medium tonicity, de-creased uidity and increased hydration. The observed effects are prob-ably due to the presence of the compounds contained in the supplementin the hydrophilic region of the erythrocyte membrane. The resultssuggest that GT supplement can protect the organism against the devel-opment of many dangerous diseases, in particular those caused byoxidative stress. The properties of the supplement shown here in bio-physical studies, consisting of protecting the biological membraneagainst harmful physicochemical factors, require further specialized re-search, which could be aimed at a precise explanation of the molecularmechanism of the effects of supplement on biological membranes, par-ticularly, e.g. their impact on the electric potential of the membrane.Comprehensive knowledge concerning the supplement and its com-ponents can be the basis for the use of these substances to protectthe organisms in the form of not only supplements but also medicines,e.g. anticancer medication.

Conict of interest statement

The authors declare that there are no conicts of interest.

Acknowledgments

This work was nancially supported from funds of the statutoryactivities of theDepartment of Physics and Biophysics,WroclawUniver-sity of Environmental and Life Sciences (PT/249/2014/S).Wewould liketo thank the Gonoderma Company for providing the green teasupplement.

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9S. Cyboran et al. / Life Sciences 126 (2015) 19

Concentrated green tea supplement: Biological activity and molecular mechanismsIntroductionMaterials and methodsGreen tea supplement, polyphenolsCells and biological membranesFluorescence probes, enzymes and reagentsPhenolic contentTotal phenolic contentTotal flavonoid contentUPLCDAD and UPLCESI-MS analyses

Hemolytic activityAntioxidant activityDPPH radical scavenging activity assayInhibition of membrane lipid peroxidation

Inhibition of enzyme activityCyclooxygenase inhibitory activityLipoxygenase inhibitory activity

Properties of the erythrocyte membraneOsmotic resistance of the membraneFluidity of the membraneDegree of hydration of lipid polar heads

Statistical analysis

ResultsPhenolic contentHemolytic activityAntioxidant activityAnti-inflammatory and lipoxygenase inhibitory activityProperties of the erythrocyte membraneOsmotic resistance of membraneFluidity of the membraneDegree of hydration of lipid polar heads

DiscussionConclusionConflict of interest statementAcknowledgmentsReferences