research article biological activity of blackcurrant...

Research ArticleBiological Activity of Blackcurrant Extracts (Ribes nigrum L) inRelation to Erythrocyte Membranes

Dorota Bonarska-Kujawa1 Sylwia Cyboran1 Romuald gyBka1

Jan OszmiaNski2 and Halina KleszczyNska1

1 Department of Physics and Biophysics Wrocław University of Environmental and Life SciencesNorwida 25 50-375 Wrocław Poland

2Department of Fruits Vegetable and Cereals Technology Wrocław University of Environmental and Life SciencesChełmonskiego 3741 51-630 Wrocław Poland

Correspondence should be addressed to Dorota Bonarska-Kujawa dorotabonarska-kujawaupwrocpl

Received 29 August 2013 Accepted 28 November 2013 Published 16 January 2014

Academic Editor Pranee Winichagoon

Copyright copy 2014 Dorota Bonarska-Kujawa et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Compounds contained in fruits and leaves of blackcurrant (Ribes nigrum L) are known as agents acting preventively andtherapeutically on the organism The HPLC analysis showed they are rich in polyphenol anthocyanins in fruits and flavonoids inleaves that have antioxidant activity and are beneficial for healthThe aim of the researchwas to determine the effect of blackcurrantfruit and leaf extracts on the physical properties of the erythrocyte membranes and assess their antioxidant propertiesThe effect ofthe extracts on osmotic resistance shape of erythrocytes and hemolytic and antioxidant activity of the extracts were examined withspectrophotometric methodsThe FTIR investigation showed that extracts modify the erythrocytemembrane and protect it againstfree radicals induced by UV radiation The results show that the extracts do not induce hemolysis and even protect erythrocytesagainst the harmful action ofUVC radiation while slightly strengthening themembrane and inducing echinocytesThe compoundscontained in the extracts do not penetrate into the hydrophobic region but bind to themembrane surface inducing small changes inthe packing arrangement of the polar head groups of membrane lipidsThe extracts have a high antioxidant activityTheir presenceon the surface of the erythrocyte membrane entails protection against free radicals

1 Introduction

Blackcurrant (Ribes nigrum L) is a shrub commonly grownin various parts of the world of temperate climate Its tastefulfruits are a rich source of vitamin C and other healthbeneficial substances such as routine organic acids pectinsmicro- and macronutrients and essential oils [1]

Blackcurrant fruits contain polyphenolic substances withantioxidant antimicrobial antiviral and antibacterial prop-erties [2ndash6] Owing to these properties polyphenols protectand support many functions of organs and systems and inparticular the digestive [3 7] nervous [8] and circulatory[9 10] systems In cells cultured in vitro polyphenols exhibitanticancer activity inhibiting the multiplication and growth

of cancer cells by inducing apoptosis in them [11 12] Antho-cyanins in particular derivatives of cyanidin and delphinidinwhich are the main polyphenols in fruit extract are used inthe treatment of eye defects and diseases of the eye [13 14]

Contained in the leaves of blackcurrant quercetin deriva-tives as indicated in many studies have a range of activitiesincluding antimicrobial anti-inflammatory antiviral anti-toxic antiseptic and antioxidant effects and are supposedto support the treatment of cancers [15ndash19] Very goodantioxidant properties of quercetin-3-O-glucoside in relationto biological membranes were showed in previous studies bythe authors [20 21]

For oxidation and destruction of biological systems areresponsible high concentrations of free radicals that are

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 783059 13 pageshttpdxdoiorg1011552014783059

2 BioMed Research International

formed either during metabolic processes or as a result ofexposure to UV radiation A conspicuous and importantplace of attack of free radicals is the cell membrane Oxi-dation of its components and in particular the membranelipids by free radicals causes disorder in the structure andfunction of the cell membrane which leads to pathologicalchanges in the organism Development of many dangerousdiseases linked directly with peroxidation ofmembrane lipidscan be prevented by providing the organism with naturalantioxidants which are polyphenolic substances containedin different parts of the plant Their protective effects asscavengers of free radicals depend on both the number ofhydroxyl groups in the polyphenolic molecule as well as thenumber of molecules associated with the membrane Theyprotect the red blood cell membrane against oxidation andhemolysis induced by free radicals [22ndash26] As indicated inour previous studies the effectiveness of certain flavonoidsanthocyanins in particular is much greater than the activityof vitamin E and its synthetic counterpart Trolox [20 27]

High antioxidant activity of plant extracts and their ingre-dients was shown in numerous studies conducted around theworld [4 16 17 24] The authors of many works considerthe effective protection of biological membranes againstoxidation dependent on the capabilities of the polypheno-lic substances binding to membranes [22ndash26] Due to theamphiphilic nature of polyphenols it can be expected thatthey incorporate mainly due to their structural similarity atdifferent depths into the lipid phase of biological membraneschanging their properties to varying degrees However thereis little work on the impact of plant extracts and polyphe-nolic compounds on the properties of biological systemsand in particular of biological membranes An importantissue therefore seems to be the relationship between thehigh antioxidant activity of plant polyphenols in relation tobiological membranes and the extent of physical changes thatthese substances induce in membranes Such studies havebeen carried out to a minor extent for selected plant extractsin relation to membrane of erythrocytes and lipid membranemodels [23 24 26 28ndash31]

The present study aimed to determine the antioxidativeactivity of extracts from the fruit (BCF) and leaves (BCL)of blackcurrant with respect to the biological membraneand in particular in relation to membrane lipids Given therich polyphenolic composition of blackcurrant extracts onecould expect an effective protection of themembranes againstoxidation-inducing agents The research was conducted withrespect to the membrane of erythrocytes which was treatedas an example and model of the biological membraneAntioxidative activity of the extracts was determined on thebasis of fluorimetric studies with oxidation of erythrocytemembranes induced by UVC radiation and the AAPH com-poundAlso the influence of extracts from fruits and leaves ofblackcurrant on the properties of the erythrocyte membranewas examined taking into account their possible negativeimpact In particular the hemolytic activity of the extractsand their impact on osmotic resistance of erythrocytes wereassayed spectrophotometrically Erythrocyte shapeswere alsoexamined in the presence of extracts using the optical andelectron microscopes Using the Fourier transform infrared

spectroscopy (FTIR) method it was specified the location inthe membrane of red blood cells of the phenolic substancescontained in the extracts and their impact on its degree ofhydration Also the protective effect of the extracts towardschanges in the membrane of erythrocytes due to its exposureto UVC radiation was determined

2 Materials and Methods

21 Materials Plant extracts of fruits and leaves of blackcur-rant (Ribes nigrum L) were obtained from the Department ofFruit Vegetable and Cereal Technology Wroclaw Universityof Environmental and Life Sciences The percent contentof polyphenols in the extracts was determined with liquidchromatography (HPLC)

The studies were conducted on pig erythrocytes and iso-lated erythrocyte membranes (ghosts) which were obtainedfrom fresh blood using the Dodge method [32] The contentof erythrocyte membranes in the samples was determined onthe basis of protein concentration which was assayed usingthe Bradfordmethod [33]The choice of pig erythrocytes wasdictated by the fact that this cellrsquos percentage share of lipids isclosest to that of the human erythrocyte and the blood waseasily available

The fluorescent probe 3-(p-(6-phenyl)-135-hexatriene)propionic acid (DPH-PA) was purchased from MolecularProbes Eugene Oregon USA The oxidation inductor 221015840-diazobis (2-amidinopropane) dihydrochloride (AAPH) andTrolox was purchased from Sigma-Aldrich Steinheim Ger-many

Erythrocyte membranes were irradiated with UVC radi-ation from bactericidal lamp (35mWcm2)

22 Methods

221 HPLC-DAD and UPLC-ESIMS Polyphenols were iso-lated from leaves and fruits by extraction with water contain-ing 200 ppm of SO

2 the ratio of solvent to leaves or fruits

being 3 1The extract was absorbed on Purolite AP 400 (UK)for further purificationThe polyphenols were then eluted outwith 80 ethanol concentrated and freeze-dried The per-cent content of polyphenols in individual preparations wasdetermined by means of liquid chromatography HPLC andUPLC method Phenolic compounds were identified withthe HPLCDAD method and the method of UPLCESIMSanalysis described in papers [34]

222 Antioxidant Activity of Extract The DPH-PA probewas used in the fluorimetric experiments Erythrocyte mem-branes with and without (control) addition of extracts weresuspended in a phosphate buffer of pH 74 and UVC irradi-ated or treatedwith the chemical oxidation compoundAAPHfor 30min Free radicals released in the process ofmembranelipids irradiation cause quenching of DPH-PA fluorescencedecreasing the fluorescence intensity As a measure of theextent of lipid oxidation was assumed relative fluorescencethat is the ratio of an UVC-irradiated or AAPH oxidizedprobe fluorescence to the initial fluorescence of the probe

BioMed Research International 3

Here as a control was assumed the relative fluorescence of anerythrocyte membranes suspension that contained the DPH-PA probe oxidized with UVC or AAPH compound whilethe blank was the relative fluorescence of a suspension of thesame concentration but not oxidized

A spectrofluorimeter (Carry Eclipsce Varian) was usedto measure free radicals concentration in the samples Exci-tation and emission wavelengths were 120582ex = 364 nm and120582em = 430 nmThemeasure of lipid oxidationwas the relativechange of fluorescence intensity FF

0 where F

0is the initial

fluorescence and F the one measured during an oxidationprocedure described in a paper [35] The percentage oflipid oxidation inhibition was calculated from the followingformula

inhibition =(119865119909minus 119865119906)

(119865119896minus 119865119906)sdot 100 (1)

where119865119909is relative fluorescence of anUVC irradiated sample

or oxidized by AAPH for 30min in the presence of thecompounds 119865

119906is relative fluorescence of the control sample

oxidized byAAPHorUVC irradiatedmeasured after 30minand 119865

119896is relative fluorescence of the blank sample not

subjected to oxidation procedure measured after 30minThe results of the assay were expressed in reference to

Trolox standard antioxidant

223 Hemolytic Activity of Extracts and Osmotic Resistanceof Erythrocytes The hemolytic experiments were conductedon fresh heparinized blood For washing the erythrocytesand in the experiments an isotonic phosphate solutionof pH 74 (131mMNaCl 179mMKCl 086mMMgCl

2

1179mMNa2HPO4times 2H

2O and 180mMNa

2H2PO4times

H2O) was used Full blood was centrifuged for 3min 1251 g

at 4∘C to remove the plasma and leucocytes Upon removingfrom plasma the erythrocytes were washed four times inphosphate solution and then incubated in the same solutionbut containing proper amounts of the compounds studiedThe modification was conducted at 37∘C for 1 h each samplecontaining 10mL of erythrocyte suspension of 2 hematocritwas stirred continuously After modification 1mL sampleswere taken centrifuged and the supernatant assayed forhemoglobin content using a UV-Vis spectrophotometer(Cary 300 Bio Varian) at 540 nm wavelength Hemoglobinconcentration in the supernatant expressed as percentageof hemoglobin concentration of totally hemolyzed cells wasassumed as the measure of the extent of hemolysis

For osmotic resistance upon removing from plasma theerythrocytes were washed three times with a cool (ca 4∘C)310 mosm PBS isotonic solution Next a 2 red cells suspen-sion containing plant extracts of 001mgmL concentrationwas prepared and left for 1 h at 37∘Cwith continuous stirringAfter this modification the suspension of erythrocytes wascentrifuged for 15min at room temperature in order toremove the cells from the extract solution From the cellsediment were taken 100120583L samples of the extract-modifiedcells and suspended in test tubes containing NaCl solutionsof 05ndash086 concentration and to an isotonic (09)NaClsolution In solutions of the same concentrations were also

suspended unmodified red blood cells that constituted thecontrol for osmotic resistance determinations Then thesuspension was stirred and centrifuged under the abovestated conditions After that the percentage of hemolysis wasmeasured with a spectrophotometer at 120582 = 540 nm On thebasis of the results obtained the relation was determinedbetween the percentage of hemolysis andNaCl concentrationin the solution Next using the obtained plots the NaClpercent concentrations (C

50) that caused 50 hemolysis were

found The C50

values were taken as a measure of osmoticresistance If a determined sodium chloride concentration ishigher than that of control cells the osmotic resistance of theerythrocytes is regarded to be lower and vice versa

224 Erythrocyte Shapes For investigation with the opticalmicroscope the red cells separated from plasma were washedfour times in saline solution and suspended in the samesolution but containing 001 and 01mgmL of BCL andBCF extracts respectively Hematocrit of the erythrocytes inthe modification solution was 2 the modification lasting1 h at 37∘C After modification the erythrocytes were fixedwith a 02 solution of glutaraldehyde After that the redcells were observed under a biological optical microscope(Nikon Eclipse E200) equipped with a digital camera Thephotographs obtained made it possible to count erythrocytesof various shapes and then percent share of the two basicforms (echinocytes and stomatocytes) in a population of ca800 cellswas determinedThe individual forms of erythrocytecells were assigned morphological indices according to theBessis scale [36] which for stomatocytes assume negativevalues from minus1 to minus4 and for echinocytes-positive from1 to 4

For investigation with the electron microscope the redcells separated from plasma were washed four times in salinesolution and suspended in the same solution but containing01mgmL of the blackcurrant leaf (BCL) and fruit (BCF)extracts Hematocrit of the erythrocytes in the modificationsolution was 2 the modification lasting 1 h at 37∘C Aftermodification the erythrocytes were fixed for 48 h in 25solution of glutaraldehyde After that the preparations werewashed in phosphate buffer for 20min and then the materialwas dehydrated in a rising series of acetone concentrations(30 50 60 70 80 90 and 100) Each sample was washedfor 15min in an appropriate concentration and the materialremain in pure acetone for 30min Next the erythrocyteswere dried for 12 h at room temperature Erythrocytes thusprepared were deposited on object stages and subjected toX-ray microanalysis by means of an X-ray analyzer BruckerAXS Quantax collaborating with the ESPRIT ver 182program Next the samples were coated with gold usingthe Scancoat 6 (Edwards London) sprinkler The materialultrastructure was analyzed using a scanning microscope(EVO LS15 ZEISS) with SE1 detector under high vacuum andaccelerating voltage EHT = 20 kV

225 FTIR Investigation of Erythrocyte Membrane Erythro-cyte ghosts were washed three times in 09 NaCl solutionNext the ghost suspension was incubated as 1mL samples


(150 120583L ghosts + 850 120583L physiological salt) for 24 h at 37∘CIn that way the control sample with ghosts suspensionwas prepared as well as samples with blackcurrant fruitand leaf extracts obtaining 005mgmL concentration Afterincubation the samples were centrifuged for 15min at16000 g and IR spectra were recorded from a condensedmembrane suspension Tests were also conducted with sam-ples containing erythrocyte membranes irradiated with UVprepared in a similar way After 24 h incubationwith extractsthe ghost suspension was exposed to UV lamp for 1 hand then centrifuged and spectra taken in the IR rangeThe measurements were performed with a Thermo Nicolet6700MCT spectrometer with ZnSe crystal applied at roomtemperature Each single spectrum was obtained from 128records at 2 cmminus1 resolution in the range 700ndash4000 cmminus1Preliminary elaboration of a spectrum was done using theEZ OMNIC v 80 program also of the Thermo Nicolet firmAfter filtering the noise out from the spectrum of the objectstudied the spectrum of the NaCl solution was subtracted inorder to remove the OH band of water and the base line wascorrected

3 Results

31 HPLC-DAD and UPLC-ESIMS Detailed quantitativeand qualitative contents of phenolic compounds in theextracts from leaves and fruits of blackcurrant are given inTable 1

The phenolic contents of blackcurrant leaves indicate thatthe dominant components in the leaves are quercetin deriva-tives which constitute 80 of polyphenolic compounds indrymass of extract Very interesting though is the polyphenolcomposition of blackcurrant fruits constituting over a half(5614) of total dry mass of extract

The blackcurrant fruit extract was analyzed by UPLC-ESI-MS and HPLC-DAD systems and is summarized inTable 1 and Figures 1 and 2 A total of 13 kinds of polyphenoliccompounds found in blackcurrant fruit extract were identi-fied and quantified Two hydroxycinnamates were detectedchlorogenic and p-coumaroylquinic acids The compoundsthat had a [M minusH]minus atmz 353 and 341 with 120582max 320 nm and311 nm after a fragmentation yielded a caffeic and p-coumaricacid respectively

Anthocyanins delphinidin-3-O-glucoside (peak tr1529min) delphinidin-3-O-rutinoside (peak tr 1621min)cyanidin-3-O-glucoside (peak tr 1703min) cyanidin-3-O-rutinoside (peak tr 1803min) petunidin-3-O-rutinoside(peak tr 1917min) (Figure 2) were identified on the basisof reference compounds UV-Vis spectra mass spectra andthe literature Delphinidin and cyanidin were the two majoranthocyanidins commonly known in blackcurrant fruit [37]The minor peak was identified as petunidin aglycone

Furthermore a total of 6 flavonol glycosides were detect-ed (Table 1) Three quercetin derivatives were detected quer-cetin-3-O-rutinoside quercetin-3-O-galactoside and quer-cetin-3-O-glucoside Peaks with (119877

119905) 2317min 2377min

and 2446min had 120582max of 355 nm respectively All com-pounds had a fragmentation yielded a quercetin ion at mz

303 Galactosides and glucosides have the samemolar massesand mass spectra and they differ according to their retentiontimes only Analogously myricetin galactoside (119877

119905) 2071min

was assumed to eluate before myricetin glucoside (119877119905)

2129min Myricetin-3-O-rutinoside (119877119905) 1904min was also

identified in our samples as defined in earlier investigations[38ndash40] The anthocyanidin derivatives were the most pre-dominant phenolic group found in blackcurrant fruit extractand constituted 5449 weight of powder Among identifiedanthocyanidin derivatives the most important compoundswere delphinidin and cyanidin rutinoside (4367)The othercompounds such as hydroxycinnamic acids and flavonolderivatives accounted total for 166 of blackcurrant fruitextract

32 Antioxidant Activity of Extract The investigation ofantioxidative activity of BCF and BCL extracts were con-ducted on erythrocyte membranes based on the extent ofmembrane lipids oxidation The oxidation was induced withUVC and the AAPH compoundThe extent of lipid oxidationwas assayed fluorimetrically based on the kinetics of DPH-PA probe quenching caused by free radicals that arose duringoxidation

The relative fluorescence for the studied concentrationsof BCF and BCL extracts decreases with time of oxidationwith UVC radiation which shows that the degree of thelipid oxidation inhibition increases Quenching of DPH-PA fluorescence in the presence of BCF and BCL extractsat different concentrations was also observed when lipidoxidation was induced in erythrocyte membranes with thechemical compound AAPH at 60 120583M

Based on the kinetics of the oxidation curves obtainedfor various concentrations of both the extracts and comparedwith Trolox the concentration responsible for 50 inhibitionof membrane lipids oxidation (IC

50) was found The results

from the fluorimetric method are given in Table 2 andFigure 3

The results indicate that the extracts protect erythrocytemembrane lipids against free radicals induced with UVC andthe AAPH compound Extract concentration responsible for50 protection of the lipids is comparable with that of Troloxin the case of AAPH and smaller when oxidation was UVCinduced

As indicated by the IC50

values BCL and BCF extractsprotect membrane lipids against oxidation The resultsobtained have shown that BCL extract protect the erythrocytemembrane against UVC-induced oxidation better than BCFextract but worse than Trolox For protecting membranesagainst AAPH-induced oxidation the BCL and BCF extractswere equal in antioxidant activity to Trolox Such resultssuggest that the reaction with free radicals depends onthe oxidation inducer In the case of photo-oxidation thepercentage of inhibition and the kinetics of the process areslower whereas in the case of the AAPH compound theprocess is much faster and efficient in inhibiting oxidationThe results indicate that polyphenols mainly of the flavonoidgroup contained in blackcurrant extract have good antiox-idative properties being more effective towards free radicals


Table 1The percent content and characterization of phenolic compounds of the extract of blackcurrant fruits (BCF) and leaves (BCL) usingtheir spectral characteristic in HPLC-DAD (retention time 120582max) and positive and negative ions in UPLC-ESI-MS [MndashH]minus

Compounds BCF BCLlowast BCFBCL BCFBCL BCFBCLContent () 119877

119905(min) 120582max (nm) (MS) (mminus)

Chlorogenic acid 012 115 1071 320 353p-Coumaroylquinic acid 011 0 718 311 341Neochlorogenic acid 0 01 718 320 353Cryptochlorogenic acid 0 011 1087 320 353Delphinidin-3-O-glucoside 458 0 1529 526 465Delphinidin-3-O-rutinoside 2249 0 1621 525 611Cyanidin-3-O-glucoside 600 0 1703 516 449Cyanidin-3-O-rutinoside 2118 0 1803 516 595Petunidin-3-O-rutinoside 024 0 1917 527 625Quercetin-3-O-rutinoside 011 033 23171904 355 611609Quercetin-3-O-galactoside 017 252 23772071 355 465463Quercetin-3-O-glucoside 015 0 2446 355 465Quercetin-3-(610158401015840-malonyl)-glucoside 0 191 2129 355 549Quercetin-3-O-glucosyl-610158401015840-acetate 0 553 2317 354 505Myricetin-3-O-rutinoside 004 0 1904 355 627Myricetin-3-O-galactoside 088 0 2071 358 481Myricetin-3-O-glucoside 008 0 2129 356 481Kaempferol-3-O-rutinoside 0 023 2377 342 593Kaempferol-3-O-galactoside 0 041 2446 342 447Kaempferol-3-O-glucosyl-610158401015840-acetate 096 2696 346 489Total 5614 1323lowastPublished previously in Oszmianski et al 2011 [34]

()

0

100

mz

59530

4652830323

27521

24518

231192051817715 21928

28925

3473033332 44924

3773339131

4213443532 46622

49337 5074152333 56740

61128

61228

61329

175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850

Figure 1 Direct injection ESI-MS of polyphenols in the blackcurrant fruit extract Positively charged molecular ion are 44924 cyanidin-3-O-glucoside 46528 delphinidin-3-O-glucoside quercetin-3-O-glucoside and quercetin-3-O-galactoside 59530 cyanidin-3-O-rutinoside61128 delphinidin-3-O-rutinoside and quercetin-3-O-rutinoside and 625 petunidin-3-O-rutinoside


Sens 20N-Meth 1

025

020

015

010

005

000

10 12 14 16 18 20 26 28 3022 24

1529

1803

1621

1703

1917

2096

2853

Inte

nsity

(AU

)

Retention time (min)

Figure 2 HPLC profile (520 nm) of blackcurrant extracts tr1529min delphinidin-3-O-glucoside 1621min delphinidin-3-O-rutinoside 1703min cyanidin-3-O-glucoside 1803min cyanidin-3-O-rutinoside and 1917min petunidin-3-O-rutinoside

Table 2 Values of IC50 for BCL and BCF extracts and Trolox thatinhibit erythrocyte membrane lipids oxidation by 50 determinedwith the fluorimetric methodThe oxidation was induced with UVCradiation and the AAPH compound

Extractinducer Concentration IC50 (120583gmL)plusmn SDUVC AAPH

Blackcurrant leaves (BCL) 245 plusmn 17 72 plusmn 005

Blackcurrant fruits (BCF) 256 plusmn 15 48 plusmn 016

Trolox 146 plusmn 13 39 plusmn 030

that arise in the process of AAPH-induced oxidation oferythrocyte membranes than UVC-induced one

33 Hemolysis and Osmotic Resistance of Erythrocytes Theresults on the effect of blackcurrant extracts on hemolysis oferythrocytes compiled in Table 3 testify that in the presenceof BCF and BCL extracts red blood cells do not undergoincreased hemolysis comparedwith unmodified cells hemol-ysis with extract at 01mgmL not exceeding 5 in respect tothe control probeThe control probe contained suspension ofunmodified erythrocyte cells in phosphate buffer (pH 74)

In the studies of the effect of the extracts on erythrocyteosmotic resistance no significant differences between thedegree of hemolysis in the control cells and those modifiedwith BCF extract was found in aqueous solutions of variousconcentrations of sodium chloride Concentrations (C

50)

of sodium chloride expressed as a percentage determinedfor control blood cells and those modified with BCL andBCF extracts of 001mgmL concentration are C

50controlmdash

0725 BCLmdash0644 BCFmdash0714 These results indicatean increase in osmotic resistance of blackcurrant leaf extractmodified cells suggesting that the red blood cell membranetreatedwithBCL extract is less sensitive to changes in osmoticpressure (Figure 4)

Table 3 Percent content of hemolyzed erythrocytes in the presenceof BCL and BCF extracts of specific concentrations

Extracts Percentage of hemolysis plusmn SDBlackcurrant

Concentration (mgmL) BCF BCLControl 510 plusmn 056 510 plusmn 056

001 097 plusmn 011 098 plusmn 011

002 218 plusmn 026 160 plusmn 019

003 282 plusmn 034 201 plusmn 024

004 439 plusmn 053 250 plusmn 030

005 522 plusmn 063 338 plusmn 040

006 523 plusmn 063 422 plusmn 051

007 618 plusmn 074 514 plusmn 062

008 689 plusmn 083 595 plusmn 071

009 741 plusmn 089 769 plusmn 092

010 870 plusmn 105 941 plusmn 113

34 Erythrocyte Shapes Figure 5 shows the percent share ofthe various forms of cells in a population of erythrocytesmodified with BCL and BCF extracts of 01 and 001mgmLconcentration As seen in Figures 5(a) 5(b) and 6 theseextracts induce mostly various forms of echinocytes mainlydiscoechinocytes It can thus be assumed that the blackcur-rant extracts concentrate mainly in the outer monolayer ofthe erythrocyte membrane when inducing echinocytes andpractically do not permeate into the inner monolayer of themembrane[41ndash44]

The work [43 44] showed that the formation ofechinocytes occurred when amphiphilic molecules wereincorporated in the outer monolayer of the erythrocytemembrane Compounds penetrating to the inner monolayerof the membrane induced formation of stomatocytes We cantherefore assume that BCF and BCL extract components con-centrated mainly in the outer monolayer of the erythrocytemembrane

35 FTIR Investigation of Erythrocyte Membrane Spectro-scopic studies in the near infrared allowed to take absorp-tion spectra for unmodified and BCL and BCF modifiederythrocytes Spectra of unmodified and extract modifiedmembranes irradiated with UVC were also takenThe resultsobtained allowed us to observe changes induced in theerythrocyte membrane by UVC radiation and phenolic com-ponents of the extracts In the spectra the characteristicfrequency bands of the protein and lipid components wereanalysed We identified the characteristic for erythrocytesfrequency bands for individual membrane componentsmethyl groups and methylene hydrocarbon chains (2980ndash2830 cmminus1) carbonyl groups of lipids (1770ndash1710 cmminus1)the amide band I (1690ndash1590 cmminus1) and amide band II(1580ndash1500 cmminus1) and phosphate band (1280ndash1200 cmminus1)and choline band (1000ndash940 cmminus1) [45ndash48] Analysis ofthe spectra of membranes treated with aqueous extracts ofblackcurrant leaves and fruit showed no significant changesin the amide I and amide II bands (vibration bands of groups


0 5 1510 20 3025

Time (min)

BlankControl001mgmL002mgmL

003mgmL004mgmL005mgmL

Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

0

(a)

Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

00 5 1510 20 3025

Time (min)



(b)

Figure 3 Relation between relative fluorescence intensity and time of UVC induced oxidation of erythrocyte membranes for control and testsample at different concentrations of blackcurrant extracts of leaves (a) and fruits (b)

0

10

20

30

40

50

60

70

80

90

100

05 055 06 065 07 075 08 085 09 095NaCl concentration ()

ControlBCFBCL

Hem

olys

is (

)

Figure 4 Percent of hemolysis of cells modified with blackcurrantleaf (BCL) and fruit (BCF) extract of 001mgmL concentrationversus sodium chloride concentration

C=O and NndashH proteins) There were also no changes inthe vibrational bands of methyl and methylene groups alkylchains of lipids and C=O vibrations of carbonyl groups oflipids This result suggests that the polyphenolic compoundscontained in the extracts do not significantly affect the struc-ture of the proteins contained in the membrane of red bloodcells and they do not modify the hydrocarbon chains of lipid

molecules UVC radiation exposure of control unmodifiedmembranes and membranes modifies with the extracts doesnot cause visible changes in the above mentioned bands Thepresence of the extracts in erythrocytes membranes causes aslight shift of the phosphate band in the direction of smallerwave numbers which points to a slight increase in the degreeof hydration of PO

2group A slightly larger displacement

of this band is caused by the extract from BCL Exposureof the membranes to UVC radiation results in a significantshift of the phosphate band towards higher values of wavenumbers suggesting a clear reduction in the number of OHgroups associated with lipid phosphate groups via hydrogenbonds The presence of the extracts in a suspension reducedthe transfer effect of phosphate bands distinctly Phosphatebands of irradiatedmembranes in the presence of extracts areshifted toward lower wave numbers relative to the irradiatedghosts which is particularly visible with BCL extract Sowe can assume that the presence of extracts in membranesexposed to UVC protects them against changes induced bythe radiation causing the membrane properties becomingcloser to those of membrane not exposed to radiation Aparticularly high level of membrane protection was observedin the presence of blackcurrant leaf extract (Figure 7)

The presence of extract and exposure toUVC light causedvisible changes also in the choline N+ (CH

3) band of lipids In

the case of ghosts not exposed toUVC the presence of extractscaused distinct decrease in the half-width of the choline bandwhich suggests a change in the packing order in that part ofmembrane and can be linked with presence of sugar groupsin the extract components UV radiation caused shift of theband towards lower wave numbers For ghosts irradiated in


ControlBCL 001mgmLBCL 01mgmL

Morphological index

SfSt(minus4)

StII(

minus3)

StI(minus2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

(a)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

SfSt(minus4)

StII(

minus3)

StI(minus

2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)

ControlBCF 001mgmLBCF 01mgmL

Morphological index

(b)

Figure 5 Percentage share of different shapes of erythrocytes induced by BCL and BCF extracts at 01 (black bar) and 001 (white bar) mgmLconcentration control (grey bar) On the abscissa there aremorphological indices for the respective shapes of cells spherostomatocytes (minus4)stomatocytes II (minus3) stomatocytes I (minus2) discostomatocytes (minus1) discocytes (0) discoechinocytes (1) echinocytes (2) spheroechinocytes(3) and spherocytes (4)

(a) (b) (c)

Figure 6 Shapes of unmodified erythrocytes (a) andmodified with BCF (b) and BCL (c) at 01mgmL concentration observed with electronmicroscope

the presence of fruit extract the effect of light was markedlysmaller while in the presence of leaf extract no changes wereobserved compared with nonirradiated ghosts (Figure 8)

36 Statistical Analysis Statistical analysis was carried outusing Statistica 90 (StatSoft Inc) All the experiments wereperformed at least in triplicate unless otherwise specifiedAnalysis of variance was carried out and significance betweenmeans was determined using Dunnettrsquos post hoc test Resultsare presented as mean plusmn SD Significant levels were defined at119875 lt 005

4 Discussion

The results of the presented research have shown that black-currant leaves (BCL) and blackcurrant fruits (BCF) extractsinduce small changes in erythrocyte membranes Their

polyphenolic compositionmakes possible an interactionwiththe lipids of biological membranes The main polyphenolicconstituent of BCF extract are anthocyanins which constituteover 98 of the total polyphenol content of the extract Itwas shown that the beneficial properties of anthocyaninscyanidins and their glycosides are connected with theirability to scavenge free radicals including the reactive formsof oxygen [49ndash51] Cyanidins were found to bind with thebiological membrane protecting it against oxidation [21 52ndash54] It can thus be assumed that mainly anthocyanins areresponsible for the protective action of blackcurrant fruitextracts with respect to the biological membrane

BCL extract differs in polyphenolic composition fromBCF extract where quercetin derivatives dominate whichconstitute ca 80 of the total polyphenols content of theextract An antioxidation activity of quercetin glycosides inrelation to the erythrocyte membrane was shown in our


1260 1240 1220 1200 11800

001

002

003Ab

sorb

ance

ControlBCLBCF

Wavenumber (cmminus1)

(a)

ControlBCLBCF

1260 1240 1220 1200 11800

001

002

003

Abso

rban

ce


(b)

Figure 7 Phosphate band of erythrocyte membrane modified with BCF and BCL extract (a) and of erythrocyte membrane modified withBCF andBCL extract irradiatedwithUVC (b) Solid line controlmembrane dashed linemembranewith fruit extract dotted linemembranewith leaf extract

1000 980 960 9400

001

002

003

004

Abso

rban

ce

ControlBCLBCF


(a)

01020 1000 980 960 940

001

002

003

Abso

rban

ce

004

ControlBCLBCF


(b)

Figure 8 Choline band of erythrocyte membrane modified with BCF and BCL extract (a) and of erythrocyte membranemodified with BCFand BCL extract irradiated with UVC (b) Solid line control membrane dashed line membrane with fruit extract dotted line membranewith leaf extract

previous studies [20] However it is not yet fully elucidatedthe molecular mechanism of the antioxidant action of thesecompounds in relation to the biological membrane

The present study aimed to determine the antioxida-tive activity of blackcurrant extracts with respect to themembrane of red blood cells treated as a model of thebiological membrane The impact of the extracts on ery-throcyte membrane properties was also examined Selectedresearch methods helped to determine the location of thecompounds contained in the extracts based on erythrocyteshape changes and hemolytic and FTIR studies The results

of the investigations undertaken enabled us to determine therelationship between the antioxidative activity of the extractsand their affinity to the membrane

Hemolytic tests showed that polyphenolic compoundscontained in the extracts do not induce hemolysis thereforedo not cause lytic effect on red blood cells in the usedrange of concentrations The lack of hemolysis and cyto-toxicity for chosen flavonoids was also confirmed in thework [55] One can therefore assume on the basis of thestudies [56ndash60] that the compounds do not embed deepinto the hydrophobic membrane area Hemolysis caused


by various lytic compounds occurs when such substancespenetrate deeply into the membrane weakening the inter-action between its components Disturbed is the structureof the membrane and facilitated transport of water swellsthe cell and the membrane is permanently damaged Studiesof osmotic resistance confirmed the hemolytic results theyshowed that as a result of incorporation of the extractcontained compounds the osmotic resistance does not changeor is even greater in the case of BCL extract

The extract from the fruit does not alter the osmoticresistance that is does not change the properties of theerythrocyte membrane whereas the leaf extract causes anincrease in the resistance which means that substancespresent in the extract bind to the membrane and makeit stronger so that it becomes less sensitive to changes inosmotic pressure

Both the extracts induced an alternation in the ery-throcyte morphology from normal discoid shape to anechinocytic form BCF and BCL extracts are responsiblefor creation of varied forms of echinocytes BCL mainlyechinocytes and BCF echinocytes and spheroechinocytesIt can thus be assumed according to the bilayer cou-ple hypothesis [42] that the extracts concentrate mainlyin the outer monolayer of the erythrocyte membranewhen inducing various forms of echinocytes and practi-cally do not permeate into the inner monolayer of themembrane

The changes observed in the IR spectrum suggest asuperficial character of the interaction of BCF and BCLcomponents with the hydrophilic part of the erythrocytemembrane FTIR spectra for extract modified membranesshowed no significant changes in the hydrophobic partof the hydrocarbon chains but showed a change in thedegree of hydration in the phosphate and choline bandof membrane phospholipids As the authors suggest [4861] as a result of the effect of polyphenols on lipid mem-brane hydrogen bonds are formed between the hydroxylgroups of polyphenols and the polar groups of lipidsIn addition numerous studies showed that plant extractsparticularly those rich in polyphenol compounds withsugar residues reduce the packing order of the hydrophilicarea of membrane causing increased hydration of thisarea

As indicated by the results after one hour of exposureto UVC radiation changes occurred in the erythrocytemembrane in particular in its hydrophilic region This isshown by a clear shift of the phosphate band of polar heads oflipids whichmay be related to oxidation of OH groups whichare connected by hydrogen bonds with the phosphate groupsor lipids The presence of the extracts in ghosts suspensionmarkedly reduced the effect of phosphate band transfer thatwas caused by exposure to UV radiation In particular suchan effect was observed in the case of BCL extract whichcaused a complete abolition of UVC induced changes incontrol ghosts and even a visible widening of the bandThis may suggest that during exposure to UVC radiation inthe presence of the extracts (BCL and BCF) the membranephosphate groups were not oxidized but had undergonehydration (BCL) which also indicates the presence of extract

components in this part of the membrane In addition it isbelieved that the polyphenolic compounds especially thoseoccurring in the form of glycosides cause hydration of themembrane transporting numerous water molecules boundto them [31 40 48ndash51 53 54 56ndash61] Such protective actionof the extracts can be attributed to their phenolic componentsthat bind to themembrane surface and containing numerousOH groups in their structure they possibly as the firstundergo oxidation

The location of the compounds in the hydrophilic partof membrane seems to constitute a protective shield of thecell against other substances the reactive forms of oxygen inparticular which finds its reflection in their antioxidant prop-erties BCL and BCF extracts exhibited a high antioxidantactivity towards reactive forms of oxygen which developedas a result of membrane photo-oxidation induced by UVCradiation and by the AAPH compound

5 Conclusion

The results obtained indicate that BCL and BCF extractsexert beneficial effect on the organism protecting the cellmembrane against oxidation The polyphenolic compoundscontained in the extracts do not disturb the structure of thebiological membrane to which they bind only superficiallypenetrating its hydrophilic region and do not penetratethe membrane hydrophobic region as is evident from thehemolytic microscopic and FTIR investigations It can thusbe expected that they do not disturb the function of thebiological membrane

In this context a protection of the organisms against theharmful effects of free radicals and reactive oxygen speciesis made possible by providing the body with appropriatequantities of plant polyphenolic substances Thus the resultsof this research encourage the consumption of fruits andextracts from blackcurrant leaves to fight free radicals andthus increase our resistance to many diseases

Conflict of Interests

The authors confirm that the paper has been read andapproved by all of the authors They also confirm that thereare no other persons who participated in the preparation ofthis paper other than the authors listed In addition theyconfirm that the order with which the authors are listed isagreed and approved by all authorsThe authors confirm thatthere is no conflict of interests associated with this paperFurthermore this work has not been financially supported byother institutions which could have influenced its outcomeThe authors confirm that they have followed the regulationsof Wroclaw University of Environmental and Life Sciencesconcerning intellectual property They declare that there areno impediments to the publication of this work because theyhave taken into account the protection of the intellectualproperty associatedwith thiswork Furthermore they declarethat this paper is not under consideration for publicationelsewhere


Acknowledgment

This work was sponsored by the Ministry of Science andEducation scientific Project nos NN312 422340 andNN304173840

References

[1] P HMattila J Hellstrom GMcDougall et al ldquoPolyphenol andvitamin C contents in European commercial blackcurrant juiceproductsrdquo Food Chemistry vol 127 no 3 pp 1216ndash1223 2011

[2] J Krisch L Ordogh L Galgoczy T Papp and C VagvolgyildquoAnticandidal effect of berry juices and extracts from Ribesspeciesrdquo Central European Journal of Biology vol 4 no 1 pp86ndash89 2009

[3] A-L Molan Z Liu andM Kruger ldquoThe ability of blackcurrantextracts to positively modulate key markers of gastrointestinalfunction in ratsrdquoWorld Journal of Microbiology and Biotechnol-ogy vol 26 no 10 pp 1735ndash1743 2010

[4] J Tabart T Franck C Kevers et al ldquoAntioxidant and anti-inflammatory activities of Ribes nigrum extractsrdquo Food Chem-istry vol 131 no 4 pp 1116ndash1122 2012

[5] H L Brangoulo and P C Molan ldquoAssay of the antioxidantcapacity of foods using an iron(II)-catalysed lipid peroxidationmodel for greater nutritional relevancerdquo Food Chemistry vol125 no 3 pp 1126ndash1130 2011

[6] B Szachowicz-Petelska I Dobrzynska E Skrzydlewska andZ Figaszewski ldquoProtective effect of blackcurrant on livercell membrane of rats intoxicated with ethanolrdquo Journal ofMembrane Biology vol 245 pp 191ndash200 2012

[7] G J McDougall N N Kulkarni and D Stewart ldquoBerrypolyphenols inhibit pancreatic lipase activity in vitrordquo FoodChemistry vol 115 no 1 pp 193ndash199 2009

[8] B Shukitt-Hale R L Galli V Meterko et al ldquoDietary sup-plementation with fruit polyphenolics ameliorates age-relateddeficits in behavior and neuronal markers of inflammation andoxidative stressrdquo Age vol 27 no 1 pp 49ndash57 2005

[9] H Matsumoto E Takenami K Iwasaki-Kurashige T OsadaT Katsumura and T Hamaoka ldquoEffects of blackcurrant antho-cyanin intake on peripheral muscle circulation during typingwork in humansrdquo European Journal of Applied Physiology vol94 no 1-2 pp 36ndash45 2005

[10] S Dannapfel T Lowe and R Cummins ldquoThe acute effect ofprolonged intense cycling and blackcurrant extract on proteincarbonyls in well-trained male cyclistrdquo Journal of Science andMedicine in Sport vol 12S article S69 2009

[11] N Jia B Kong Q Liu X Diao and X Xia ldquoAntioxidant activityof black currant (Ribes nigrum L) extract and its inhibitoryeffect on lipid and protein oxidation of pork patties duringchilled storagerdquoMeat Science vol 91 no 4 pp 533ndash539 2012

[12] A Bishayee T Mbimba R J Thoppil et al ldquoAnthocyanin-rich black currant (Ribes nigrum L) extract affords chemo-prevention against diethylnitrosamine-induced hepatocellularcarcinogenesis in ratsrdquo Journal of Nutritional Biochemistry vol22 no 11 pp 1035ndash1046 2011

[13] H Matsumoto K E Kamm J T Stull and H AzumaldquoDelphinidin-3-rutinoside relaxes the bovine ciliary smoothmuscle through activation of ETB receptor and NOcGMPpathwayrdquo Experimental Eye Research vol 80 no 3 pp 313ndash3222005

[14] H Matsumoto Y Nakamura H Iida K Ito and H OhguroldquoComparative assessment of distribution of blackcurrant antho-cyanins in rabbit and rat ocular tissuesrdquo Experimental EyeResearch vol 83 no 2 pp 348ndash356 2006

[15] L Movileanu I Neagoe and M L Flonta ldquoInteraction ofthe antioxidant flavonoid quercetin with planar lipid bilayersrdquoInternational Journal of Pharmaceutics vol 205 no 1-2 pp 135ndash146 2000

[16] C A Rice-Evans N J Miller and G Paganga ldquoStructure-antioxidant activity relationships of flavonoids and phenolicacidsrdquo Free Radical Biology andMedicine vol 20 no 7 pp 933ndash956 1996

[17] C A Rice-Evans N J Miller and G Paganga ldquoAntioxidantproperties of phenolic compoundsrdquoTrends in Plant Science vol2 no 4 pp 152ndash159 1997

[18] I F F Benzie ldquoEvolution of dietary antioxidantsrdquo ComparativeBiochemistry and Physiology vol 136 no 1 pp 113ndash126 2003

[19] L Hou B Zhou L Yang and Z-L Liu ldquoInhibition of freeradical initiated peroxidation of human erythrocyte ghostsby flavonols and their glycosidesrdquo Organic and BiomolecularChemistry vol 2 no 9 pp 1419ndash1423 2004

[20] S Cyboran D Bonarska-Kujawa I Kapusta J Oszmianskiand H Kleszczynska ldquoAntioxidant potentials of polyphenolicextracts from leaves of trees and fruit bushesrdquo Current Topics inBiophysics vol 34 pp 15ndash21 2011

[21] D Bonarska-Kujawa S Cyboran J Oszmianski and HKleszczynska ldquoExtracts from apple leaves and fruits as effectiveantioxidantsrdquo Journal of Medicinal Plant Research vol 5 no 11pp 2339ndash2347 2011

[22] K A Arbos L M Claro L Borges C A M Santos and AM Weffort-Santos ldquoHuman erythrocytes as a system for eval-uating the antioxidant capacity of vegetable extractsrdquo NutritionResearch vol 28 no 7 pp 457ndash463 2008

[23] B Bukowska J Michałowicz A Krokosz and P SicinskaldquoComparison of the effect of phenol and its derivatives onprotein and free radical formation in human erythrocytes (invitro)rdquo Blood Cells Molecules and Diseases vol 39 no 3 pp238ndash244 2007

[24] S Chaudhuri A Banerjee K Basu B Sengupta and PK Sengupta ldquoInteraction of flavonoids with red blood cellmembrane lipids and proteins antioxidant and antihemolyticeffectsrdquo International Journal of Biological Macromolecules vol41 no 1 pp 42ndash48 2007

[25] M Suwalsky P Orellana M Avello F Villena and C PSotomayor ldquoHuman erythrocytes are affected in vitro byextracts of Ugni molinae leavesrdquo Food and Chemical Toxicologyvol 44 no 8 pp 1393ndash1398 2006

[26] M Suwalsky K Oyarce M Avello F Villena and C PSotomayor ldquoHuman erythrocytes and molecular models ofcell membranes are affected in vitro by Balbisia peduncularis(Amancay) extractsrdquo Chemico-Biological Interactions vol 179no 2-3 pp 413ndash418 2009

[27] D Bonarska-Kujawa J Sarapuk K Bielecki and J OszmianskildquoAntioxidant activity of extracts from apple chokeberry andstrawberryrdquo Polish Journal of Food and Nutrition Sciences vol62 no 4 pp 229ndash234 2012

[28] M Suwalsky P Orellana M Avello and F Villena ldquoProtectiveeffect ofUgni molinaeTurcz against oxidative damage of humanerythrocytesrdquo Food and Chemical Toxicology vol 45 no 1 pp130ndash135 2007


[29] M Suwalsky P Vargas M Avello F Villena and C PSotomayor ldquoHuman erythrocytes are affected in vitro byflavonoids of Aristotelia chilensis (Maqui) leavesrdquo InternationalJournal of Pharmaceutics vol 363 no 1-2 pp 85ndash90 2008

[30] S Cyboran J Oszmianski and H Kleszczynska ldquoInteractionbetween plant polyphenols and the erythrocyte membranerdquoCellular amp Molecular Biology Letters vol 17 pp 77ndash88 2012

[31] A Arora T M Byrem M G Nair and G M StrasburgldquoModulation of liposomal membrane fluidity by flavonoids andisoflavonoidsrdquo Archives of Biochemistry and Biophysics vol 373no 1 pp 102ndash109 2000

[32] J T Dodge C Mitchell and D J Hanahan ldquoThe preparationand chemical characteristics of hemoglobin-free ghosts ofhuman erythrocytesrdquo Archives of Biochemistry and Biophysicsvol 100 no 1 pp 119ndash130 1963

[33] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[34] J Oszmianski A Wojdyło J Gorzelany and I KapustaldquoIdentification and characterization of low molecular weightpolyphenols in berry leaf extracts by HPLC-DAD and LC-ESIMSrdquo Journal of Agricultural and Food Chemistry vol 59 no24 pp 12830ndash12835 2011

[35] A Arora and G M Strasburg ldquoDevelopment and validationof fluorescence spectroscopic assays to evaluate antioxidantefficacy Application tometal chelatorsrdquo Journal of the AmericanOil Chemistsrsquo Society vol 74 no 9 pp 1031ndash1040 1997

[36] M Bessis ldquoLa forme et la deformabilite des erythrocytesnormaux et dans certaines anemies hemolytiques congenitalesrdquoNouvelle Revue Francaise DrsquoHematologie vol 18 pp 75ndash94 1977

[37] R Slimestad and H Solheim ldquoAnthocyanins from black cur-rants (Ribes nigrum L)rdquo Journal of Agricultural and FoodChemistry vol 50 no 11 pp 3228ndash3231 2002

[38] M J Anttonen and R O Karjalainen ldquoHigh-performanceliquid chromatography analysis of black currant (Ribes nigrumL) Fruit phenolics grown either conventionally or organicallyrdquoJournal of Agricultural and Food Chemistry vol 54 no 20 pp7530ndash7538 2006

[39] M Sojka S Guyot K Kołodziejczyk B Krol and A BaronldquoComposition and properties of purified phenolics prepa-rations obtained from an extract of industrial blackcurrant(Ribes nigrum L) pomacerdquo Journal of Horticultural Science andBiotechnology vol 84 pp 100ndash106 2009

[40] N Jia Y L Xiong B Kong Q Liu and X Xia ldquoRadicalscavenging activity of black currant (Ribes nigrum L) extractand its inhibitory effect on gastric cancer cell proliferation viainduction of apoptosisrdquo Journal of Functional Foods vol 4 no1 pp 382ndash390 2012

[41] B Deuticke ldquoTransformation and restoration of biconcaveshape of human erythrocytes induced by amphiphilic agentsand changes of ionic environmentrdquo Biochimica et BiophysicaActa vol 163 no 4 pp 494ndash500 1968

[42] M P Sheetz and S J Singer ldquoBiological membranes as bilayercouples A molecular mechanism of drug erythrocyte interac-tionsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 71 no 11 pp 4457ndash4461 1974

[43] A Iglic V Kralj-Iglic and H Hagerstrand ldquoAmphiphileinduced echinocyte-spheroechinoeyte transformation of redblood cell shaperdquo European Biophysics Journal vol 27 no 4 pp335ndash339 1998

[44] B Isomaa H Hagerstrand and G Paatero ldquoShape transforma-tions induced by amphiphiles in erythrocytesrdquo Biochimica etBiophysica Acta vol 899 no 1 pp 93ndash103 1987

[45] R Zyłka H Kleszczynska J Kupiec D Bonarska-Kujawa JHladyszowski and S Przestalski ldquoModifications of erythrocytemembrane hydration induced by organic tin compoundsrdquo CellBiology International vol 33 no 7 pp 801ndash806 2009

[46] C Petibois and G Deleris ldquoEvidence that erythrocytes arehighly susceptible to exercise oxidative stress FT-IR spectro-metric studies at the molecular levelrdquo Cell Biology Internationalvol 29 no 8 pp 709ndash716 2005

[47] C Petibois and G Deleris ldquoErythrocyte adaptation to oxidativestress in endurance trainingrdquo Archives of Medical Research vol36 no 5 pp 524ndash531 2005

[48] B E Pawlikowska-Pawlęga L E Misiak B Zarzyka RPaduch A Gawron and W I Gruszecki ldquoLocalization andinteraction of genistein with model membranes formed withdipalmitoylphosphatidylcholine (DPPC)rdquo Biochimica et Bio-physica Acta vol 1818 no 7 pp 1785ndash1793 2012

[49] P-N Chen S-C Chu H-L Chiou W-H Kuo C-L ChiangandY-SHsieh ldquoMulberry anthocyanins cyanidin 3-rutinosideand cyanidin 3-glucoside exhibited an inhibitory effect on themigration and invasion of a human lung cancer cell linerdquoCancerLetters vol 235 no 2 pp 248ndash259 2006

[50] F Galvano L La Fauci G Lazzarino et al ldquoCyanidinsmetabolism and biological propertiesrdquo Journal of NutritionalBiochemistry vol 15 no 1 pp 2ndash11 2004

[51] R Feng H-M Ni S Y Wang et al ldquoCyanidin-3-rutinoside anatural polyphenol antioxidant selectively kills leukemic cellsby induction of oxidative stressrdquo Journal of Biological Chemistryvol 282 no 18 pp 13468ndash13476 2007

[52] J-M Kong L-S Chia N-K Goh T-F Chia and R BrouillardldquoAnalysis and biological activities of anthocyaninsrdquo Phytochem-istry vol 64 no 5 pp 923ndash933 2003

[53] D Bonarska-Kujawa H Pruchnik J Oszmianski J Sarapukand H Kleszczynska ldquoChanges caused by fruit extracts inthe lipid phase of biological and model membranesrdquo FoodBiophysics vol 6 no 1 pp 58ndash67 2011

[54] D Bonarska-KujawaH Pruchnik andH Kleszczynska ldquoInter-action of selected anthocyanins with erythrocytes and liposomemembranesrdquo Cellular amp Molecular Biology Letters vol 17 pp289ndash308 2012

[55] M Bobrowska-Hagerstrand A Wrobel L Mrowczynska et alldquoFlavonoids as inhibitors of MRP1-like efflux activity in humanerythrocytes A structure-activity relationship studyrdquo OncologyResearch vol 13 pp 463ndash469 2003

[56] E Ponder Hemolysis and Related Phenomena J A ChurchillLondon UK 1948

[57] Y Tanaka K Mashino K Inoue and S Nojima ldquoMecha-nism of human erythrocyte hemolysis induced by short-chainphosphatidylcholines and lysophosphatidylcholinerdquo Journal ofBiochemistry vol 94 no 3 pp 833ndash840 1983

[58] H Kleszczynska J Sarapuk S Przestalski and S Witek ldquoTherole of the alkyl chain in the interaction of glycine esters witherythrocyte and model erythrocyte lipid membranesrdquo StudiaBiophysica vol 116 pp 115ndash122 1986

[59] H Kleszczynska J Sarapuk S Przestalski and M KilianldquoMechanical properties of red cell and BLM in the presenceof some mono- and bis-quaternary ammonium saltsrdquo StudiaBiophysica vol 135 pp 191ndash199 1990


[60] H Kleszczynska J Hładyszowski H Pruchnik and S Przestal-ski ldquoErythrocyte hemolysis by organic tin and lead com-poundsrdquo Zeitschrift fur Naturforschung vol 52 pp 65ndash69 1997

[61] B Pawlikowska-Pawlęga L E Misiak B Zarzycka R PaduchA Gawron and W I Gruszecki ldquoFTIR 1H NMR and EPRspectroscopy studies on the interaction of flavone apigeninwith dipalmitoylphosphatidylcholine liposomesrdquo Biochimica etBiophysica Acta vol 1828 pp 518ndash527 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


formed either during metabolic processes or as a result ofexposure to UV radiation A conspicuous and importantplace of attack of free radicals is the cell membrane Oxi-dation of its components and in particular the membranelipids by free radicals causes disorder in the structure andfunction of the cell membrane which leads to pathologicalchanges in the organism Development of many dangerousdiseases linked directly with peroxidation ofmembrane lipidscan be prevented by providing the organism with naturalantioxidants which are polyphenolic substances containedin different parts of the plant Their protective effects asscavengers of free radicals depend on both the number ofhydroxyl groups in the polyphenolic molecule as well as thenumber of molecules associated with the membrane Theyprotect the red blood cell membrane against oxidation andhemolysis induced by free radicals [22ndash26] As indicated inour previous studies the effectiveness of certain flavonoidsanthocyanins in particular is much greater than the activityof vitamin E and its synthetic counterpart Trolox [20 27]

High antioxidant activity of plant extracts and their ingre-dients was shown in numerous studies conducted around theworld [4 16 17 24] The authors of many works considerthe effective protection of biological membranes againstoxidation dependent on the capabilities of the polypheno-lic substances binding to membranes [22ndash26] Due to theamphiphilic nature of polyphenols it can be expected thatthey incorporate mainly due to their structural similarity atdifferent depths into the lipid phase of biological membraneschanging their properties to varying degrees However thereis little work on the impact of plant extracts and polyphe-nolic compounds on the properties of biological systemsand in particular of biological membranes An importantissue therefore seems to be the relationship between thehigh antioxidant activity of plant polyphenols in relation tobiological membranes and the extent of physical changes thatthese substances induce in membranes Such studies havebeen carried out to a minor extent for selected plant extractsin relation to membrane of erythrocytes and lipid membranemodels [23 24 26 28ndash31]

The present study aimed to determine the antioxidativeactivity of extracts from the fruit (BCF) and leaves (BCL)of blackcurrant with respect to the biological membraneand in particular in relation to membrane lipids Given therich polyphenolic composition of blackcurrant extracts onecould expect an effective protection of themembranes againstoxidation-inducing agents The research was conducted withrespect to the membrane of erythrocytes which was treatedas an example and model of the biological membraneAntioxidative activity of the extracts was determined on thebasis of fluorimetric studies with oxidation of erythrocytemembranes induced by UVC radiation and the AAPH com-poundAlso the influence of extracts from fruits and leaves ofblackcurrant on the properties of the erythrocyte membranewas examined taking into account their possible negativeimpact In particular the hemolytic activity of the extractsand their impact on osmotic resistance of erythrocytes wereassayed spectrophotometrically Erythrocyte shapeswere alsoexamined in the presence of extracts using the optical andelectron microscopes Using the Fourier transform infrared

spectroscopy (FTIR) method it was specified the location inthe membrane of red blood cells of the phenolic substancescontained in the extracts and their impact on its degree ofhydration Also the protective effect of the extracts towardschanges in the membrane of erythrocytes due to its exposureto UVC radiation was determined

2 Materials and Methods

21 Materials Plant extracts of fruits and leaves of blackcur-rant (Ribes nigrum L) were obtained from the Department ofFruit Vegetable and Cereal Technology Wroclaw Universityof Environmental and Life Sciences The percent contentof polyphenols in the extracts was determined with liquidchromatography (HPLC)

The studies were conducted on pig erythrocytes and iso-lated erythrocyte membranes (ghosts) which were obtainedfrom fresh blood using the Dodge method [32] The contentof erythrocyte membranes in the samples was determined onthe basis of protein concentration which was assayed usingthe Bradfordmethod [33]The choice of pig erythrocytes wasdictated by the fact that this cellrsquos percentage share of lipids isclosest to that of the human erythrocyte and the blood waseasily available

The fluorescent probe 3-(p-(6-phenyl)-135-hexatriene)propionic acid (DPH-PA) was purchased from MolecularProbes Eugene Oregon USA The oxidation inductor 221015840-diazobis (2-amidinopropane) dihydrochloride (AAPH) andTrolox was purchased from Sigma-Aldrich Steinheim Ger-many

Erythrocyte membranes were irradiated with UVC radi-ation from bactericidal lamp (35mWcm2)

22 Methods

221 HPLC-DAD and UPLC-ESIMS Polyphenols were iso-lated from leaves and fruits by extraction with water contain-ing 200 ppm of SO

2 the ratio of solvent to leaves or fruits

being 3 1The extract was absorbed on Purolite AP 400 (UK)for further purificationThe polyphenols were then eluted outwith 80 ethanol concentrated and freeze-dried The per-cent content of polyphenols in individual preparations wasdetermined by means of liquid chromatography HPLC andUPLC method Phenolic compounds were identified withthe HPLCDAD method and the method of UPLCESIMSanalysis described in papers [34]

222 Antioxidant Activity of Extract The DPH-PA probewas used in the fluorimetric experiments Erythrocyte mem-branes with and without (control) addition of extracts weresuspended in a phosphate buffer of pH 74 and UVC irradi-ated or treatedwith the chemical oxidation compoundAAPHfor 30min Free radicals released in the process ofmembranelipids irradiation cause quenching of DPH-PA fluorescencedecreasing the fluorescence intensity As a measure of theextent of lipid oxidation was assumed relative fluorescencethat is the ratio of an UVC-irradiated or AAPH oxidizedprobe fluorescence to the initial fluorescence of the probe




0 where F

0is the initial



(119865119896minus 119865119906)sdot 100 (1)









2


2O and 180mMNa

2H2PO4times






found The C50







3 Results






119905) 2317min 2377min



119905) 2071min

















()

0

100

mz

59530

4652830323

27521

24518

231192051817715 21928

28925

3473033332 44924

3773339131

4213443532 46622

49337 5074152333 56740

61128

61228

61329

175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850



Sens 20N-Meth 1

025

020

015

010

005

000

10 12 14 16 18 20 26 28 3022 24

1529

1803

1621

1703

1917

2096

2853

Inte

nsity

(AU

)











50)


50controlmdash





001 097 plusmn 011 098 plusmn 011

002 218 plusmn 026 160 plusmn 019

003 282 plusmn 034 201 plusmn 024

004 439 plusmn 053 250 plusmn 030

005 522 plusmn 063 338 plusmn 040

006 523 plusmn 063 422 plusmn 051

007 618 plusmn 074 514 plusmn 062

008 689 plusmn 083 595 plusmn 071

009 741 plusmn 089 769 plusmn 092

010 870 plusmn 105 941 plusmn 113





0 5 1510 20 3025

Time (min)



Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

0

(a)

Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

00 5 1510 20 3025

Time (min)



(b)


0

10

20

30

40

50

60

70

80

90

100


ControlBCFBCL

Hem

olys

is (

)











Morphological index

SfSt(minus4)

StII(

minus3)

StI(minus2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

(a)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

SfSt(minus4)

StII(

minus3)

StI(minus

2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)


Morphological index

(b)


(a) (b) (c)




4 Discussion





1260 1240 1220 1200 11800

001

002

003Ab

sorb

ance

ControlBCLBCF


(a)

ControlBCLBCF

1260 1240 1220 1200 11800

001

002

003

Abso

rban

ce


(b)


1000 980 960 9400

001

002

003

004

Abso

rban

ce

ControlBCLBCF


(a)

01020 1000 980 960 940

001

002

003

Abso

rban

ce

004

ControlBCLBCF


(b)














5 Conclusion






Acknowledgment


References







































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




0 where F

0is the initial



(119865119896minus 119865119906)sdot 100 (1)









2


2O and 180mMNa

2H2PO4times






found The C50







3 Results






119905) 2317min 2377min



119905) 2071min

















()

0

100

mz

59530

4652830323

27521

24518

231192051817715 21928

28925

3473033332 44924

3773339131

4213443532 46622

49337 5074152333 56740

61128

61228

61329

175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850



Sens 20N-Meth 1

025

020

015

010

005

000

10 12 14 16 18 20 26 28 3022 24

1529

1803

1621

1703

1917

2096

2853

Inte

nsity

(AU

)











50)


50controlmdash





001 097 plusmn 011 098 plusmn 011

002 218 plusmn 026 160 plusmn 019

003 282 plusmn 034 201 plusmn 024

004 439 plusmn 053 250 plusmn 030

005 522 plusmn 063 338 plusmn 040

006 523 plusmn 063 422 plusmn 051

007 618 plusmn 074 514 plusmn 062

008 689 plusmn 083 595 plusmn 071

009 741 plusmn 089 769 plusmn 092

010 870 plusmn 105 941 plusmn 113





0 5 1510 20 3025

Time (min)



Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

0

(a)

Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

00 5 1510 20 3025

Time (min)



(b)


0

10

20

30

40

50

60

70

80

90

100


ControlBCFBCL

Hem

olys

is (

)











Morphological index

SfSt(minus4)

StII(

minus3)

StI(minus2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

(a)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

SfSt(minus4)

StII(

minus3)

StI(minus

2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)


Morphological index

(b)


(a) (b) (c)




4 Discussion





1260 1240 1220 1200 11800

001

002

003Ab

sorb

ance

ControlBCLBCF


(a)

ControlBCLBCF

1260 1240 1220 1200 11800

001

002

003

Abso

rban

ce


(b)


1000 980 960 9400

001

002

003

004

Abso

rban

ce

ControlBCLBCF


(a)

01020 1000 980 960 940

001

002

003

Abso

rban

ce

004

ControlBCLBCF


(b)














5 Conclusion






Acknowledgment


References







































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3 Results






119905) 2317min 2377min



119905) 2071min

















()

0

100

mz

59530

4652830323

27521

24518

231192051817715 21928

28925

3473033332 44924

3773339131

4213443532 46622

49337 5074152333 56740

61128

61228

61329

175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850



Sens 20N-Meth 1

025

020

015

010

005

000

10 12 14 16 18 20 26 28 3022 24

1529

1803

1621

1703

1917

2096

2853

Inte

nsity

(AU

)











50)


50controlmdash





001 097 plusmn 011 098 plusmn 011

002 218 plusmn 026 160 plusmn 019

003 282 plusmn 034 201 plusmn 024

004 439 plusmn 053 250 plusmn 030

005 522 plusmn 063 338 plusmn 040

006 523 plusmn 063 422 plusmn 051

007 618 plusmn 074 514 plusmn 062

008 689 plusmn 083 595 plusmn 071

009 741 plusmn 089 769 plusmn 092

010 870 plusmn 105 941 plusmn 113





0 5 1510 20 3025

Time (min)



Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

0

(a)

Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

00 5 1510 20 3025

Time (min)



(b)


0

10

20

30

40

50

60

70

80

90

100


ControlBCFBCL

Hem

olys

is (

)











Morphological index

SfSt(minus4)

StII(

minus3)

StI(minus2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

(a)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

SfSt(minus4)

StII(

minus3)

StI(minus

2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)


Morphological index

(b)


(a) (b) (c)




4 Discussion





1260 1240 1220 1200 11800

001

002

003Ab

sorb

ance

ControlBCLBCF


(a)

ControlBCLBCF

1260 1240 1220 1200 11800

001

002

003

Abso

rban

ce


(b)


1000 980 960 9400

001

002

003

004

Abso

rban

ce

ControlBCLBCF


(a)

01020 1000 980 960 940

001

002

003

Abso

rban

ce

004

ControlBCLBCF


(b)














5 Conclusion






Acknowledgment


References







































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






()

0

100

mz

59530

4652830323

27521

24518

231192051817715 21928

28925

3473033332 44924

3773339131

4213443532 46622

49337 5074152333 56740

61128

61228

61329

175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850



Sens 20N-Meth 1

025

020

015

010

005

000

10 12 14 16 18 20 26 28 3022 24

1529

1803

1621

1703

1917

2096

2853

Inte

nsity

(AU

)











50)


50controlmdash





001 097 plusmn 011 098 plusmn 011

002 218 plusmn 026 160 plusmn 019

003 282 plusmn 034 201 plusmn 024

004 439 plusmn 053 250 plusmn 030

005 522 plusmn 063 338 plusmn 040

006 523 plusmn 063 422 plusmn 051

007 618 plusmn 074 514 plusmn 062

008 689 plusmn 083 595 plusmn 071

009 741 plusmn 089 769 plusmn 092

010 870 plusmn 105 941 plusmn 113





0 5 1510 20 3025

Time (min)



Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

0

(a)

Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

00 5 1510 20 3025

Time (min)



(b)


0

10

20

30

40

50

60

70

80

90

100


ControlBCFBCL

Hem

olys

is (

)











Morphological index

SfSt(minus4)

StII(

minus3)

StI(minus2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

(a)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

SfSt(minus4)

StII(

minus3)

StI(minus

2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)


Morphological index

(b)


(a) (b) (c)




4 Discussion





1260 1240 1220 1200 11800

001

002

003Ab

sorb

ance

ControlBCLBCF


(a)

ControlBCLBCF

1260 1240 1220 1200 11800

001

002

003

Abso

rban

ce


(b)


1000 980 960 9400

001

002

003

004

Abso

rban

ce

ControlBCLBCF


(a)

01020 1000 980 960 940

001

002

003

Abso

rban

ce

004

ControlBCLBCF


(b)














5 Conclusion






Acknowledgment


References







































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Sens 20N-Meth 1

025

020

015

010

005

000

10 12 14 16 18 20 26 28 3022 24

1529

1803

1621

1703

1917

2096

2853

Inte

nsity

(AU

)











50)


50controlmdash





001 097 plusmn 011 098 plusmn 011

002 218 plusmn 026 160 plusmn 019

003 282 plusmn 034 201 plusmn 024

004 439 plusmn 053 250 plusmn 030

005 522 plusmn 063 338 plusmn 040

006 523 plusmn 063 422 plusmn 051

007 618 plusmn 074 514 plusmn 062

008 689 plusmn 083 595 plusmn 071

009 741 plusmn 089 769 plusmn 092

010 870 plusmn 105 941 plusmn 113





0 5 1510 20 3025

Time (min)



Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

0

(a)

Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

00 5 1510 20 3025

Time (min)



(b)


0

10

20

30

40

50

60

70

80

90

100


ControlBCFBCL

Hem

olys

is (

)











Morphological index

SfSt(minus4)

StII(

minus3)

StI(minus2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

(a)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

SfSt(minus4)

StII(

minus3)

StI(minus

2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)


Morphological index

(b)


(a) (b) (c)




4 Discussion





1260 1240 1220 1200 11800

001

002

003Ab

sorb

ance

ControlBCLBCF


(a)

ControlBCLBCF

1260 1240 1220 1200 11800

001

002

003

Abso

rban

ce


(b)


1000 980 960 9400

001

002

003

004

Abso

rban

ce

ControlBCLBCF


(a)

01020 1000 980 960 940

001

002

003

Abso

rban

ce

004

ControlBCLBCF


(b)














5 Conclusion






Acknowledgment


References







































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 5 1510 20 3025

Time (min)



Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

0

(a)

Relat

ive fl

uore

scen

ceFF

0

02

04

06

08

1

12

00 5 1510 20 3025

Time (min)



(b)


0

10

20

30

40

50

60

70

80

90

100


ControlBCFBCL

Hem

olys

is (

)











Morphological index

SfSt(minus4)

StII(

minus3)

StI(minus2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

(a)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

SfSt(minus4)

StII(

minus3)

StI(minus

2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)


Morphological index

(b)


(a) (b) (c)




4 Discussion





1260 1240 1220 1200 11800

001

002

003Ab

sorb

ance

ControlBCLBCF


(a)

ControlBCLBCF

1260 1240 1220 1200 11800

001

002

003

Abso

rban

ce


(b)


1000 980 960 9400

001

002

003

004

Abso

rban

ce

ControlBCLBCF


(a)

01020 1000 980 960 940

001

002

003

Abso

rban

ce

004

ControlBCLBCF


(b)














5 Conclusion






Acknowledgment


References







































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Morphological index

SfSt(minus4)

StII(

minus3)

StI(minus2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

(a)

70

60

50

40

30

20

10

0

Eryt

hroc

ytes

shap

es (

)

SfSt(minus4)

StII(

minus3)

StI(minus

2)

DSt(minus1)

D(0)

E(2)

Sf(4)

DE(

1)

SfE(3)


Morphological index

(b)


(a) (b) (c)




4 Discussion





1260 1240 1220 1200 11800

001

002

003Ab

sorb

ance

ControlBCLBCF


(a)

ControlBCLBCF

1260 1240 1220 1200 11800

001

002

003

Abso

rban

ce


(b)


1000 980 960 9400

001

002

003

004

Abso

rban

ce

ControlBCLBCF


(a)

01020 1000 980 960 940

001

002

003

Abso

rban

ce

004

ControlBCLBCF


(b)














5 Conclusion






Acknowledgment


References







































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1260 1240 1220 1200 11800

001

002

003Ab

sorb

ance

ControlBCLBCF


(a)

ControlBCLBCF

1260 1240 1220 1200 11800

001

002

003

Abso

rban

ce


(b)


1000 980 960 9400

001

002

003

004

Abso

rban

ce

ControlBCLBCF


(a)

01020 1000 980 960 940

001

002

003

Abso

rban

ce

004

ControlBCLBCF


(b)














5 Conclusion






Acknowledgment


References







































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









5 Conclusion






Acknowledgment


References







































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgment


References







































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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