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Compounds contained in fruits and leaves of blackcurrant (Ribes nigrum L.) are known as agents acting preventively and therapeutically on the organism. The HPLC analysis showed they are rich in polyphenol anthocyanins in fruits and flavonoids in leaves, that have antioxidant activity and are beneficial for health. The aim of the research was to determine the effect of blackcurrant fruit and leaf extracts on the physical properties of the erythrocyte membranes and assess their antioxidant properties. The effect of the extracts on osmotic resistance, shape of erythrocytes and hemolytic and antioxidant activity of the extracts were examined with spectrophotometric methods. The FTIR investigation showed that extracts modify the erythrocyte membrane and protect it against free radicals induced by UV radiation. The results show that the extracts do not induce hemolysis and even protect erythrocytes against the harmful action of UVC radiation, while slightly strengthening the membrane and inducing echinocytes. The compounds contained in the extracts do not penetrate into the hydrophobic region, but bind to the membrane surface inducing small changes in the packing arrangement of the polar head groups of membrane lipids. The extracts have a high antioxidant activity. Their presence on the surface of the erythrocyte membrane entails protection against free radicals.
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Research Article
Biological Activity of Blackcurrant Extracts (Ribes nigrum L.) in
Relation to Erythrocyte Membranes
Dorota Bonarska-Kujawa,1Sylwia Cyboran,1Romuald gyBka,1
Jan OszmiaNski,2and Halina KleszczyNska1
1Department of Physics and Biophysics, Wrocław University of Environmental and Life Sciences,
Norwida 25, 50-375 Wrocław, Poland
2Department of Fruits, Vegetable and Cereals Technology, Wrocław University of Environmental and Life Sciences,
Chełmo´
nskiego 37/41, 51-630 Wrocław, Poland
Correspondence should be addressed to Dorota Bonarska-Kujawa; dorota.bonarska-kujawa@up.wroc.pl
Received  August ; Accepted  November ; Published  January 
Academic Editor: Pranee Winichagoon
Copyright ©  Dorota Bonarska-Kujawa et al. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Compounds contained in fruits and leaves of blackcurrant (Ribes nigrum L.)areknownasagentsactingpreventivelyand
therapeutically on the organism. e HPLC analysis showed they are rich in polyphenol anthocyanins in fruits and avonoids in
leaves, that have antioxidant activity and are benecial for health. e aim of the research was to determine the eect of blackcurrant
fruit and leaf extracts on the physical properties of the erythrocyte membranes and assess their antioxidant properties. e eect of
the extracts on osmotic resistance, shape of erythrocytes and hemolytic and antioxidant activity of the extracts were examined with
spectrophotometric methods. e FTIR investigation showed that extracts modify the erythrocyte membrane and protect it against
free radicals induced by UV radiation. e results show that the extracts do not induce hemolysis and even protect erythrocytes
against the harmful action of UVC radiation, while slightly strengtheningt he membrane and inducing echinocytes. e compounds
contained in the extracts do not penetrate into the hydrophobic region, but bind to the membrane surface inducing small changes in
the packing arrangement of the polar head groups of membrane lipids. e extracts have a high antioxidant activity. eir presence
on the surface of the erythrocyte membrane entails protection against free radicals.
1. Introduction
Blackcurrant (Ribes nigrum L.)isashrubcommonlygrown
in various parts of the world of temperate climate. Its tasteful
fruits are a rich source of vitamin C and other health
benecial substances such as: routine, organic acids, pectins,
micro- and macronutrients and essential oils [].
Blackcurrant fruits contain polyphenolic substances with
antioxidant, antimicrobial, antiviral, and antibacterial prop-
erties []. Owing to these properties, polyphenols protect
and support many functions of organs and systems and in
particular the digestive [,], nervous [], and circulatory
[,] systems. In cells cultured in vitro, polyphenols exhibit
anticancer activity, inhibiting the multiplication and growth
of cancer cells by inducing apoptosis in them [,]. Antho-
cyanins, in particular derivatives of cyanidin and delphinidin,
which are the main polyphenols in fruit extract, are used in
thetreatmentofeyedefectsanddiseasesoftheeye[,].
Contained in the leaves of blackcurrant, quercetin deriva-
tives, as indicated in many studies, have a range of activities,
including antimicrobial, anti-inammatory, antiviral, anti-
toxic, antiseptic, and antioxidant eects, and are supposed
to support the treatment of cancers []. Very good
antioxidant properties of quercetin--O-glucoside in relation
to biological membranes were showed in previous studies by
the authors [,].
For oxidation and destruction of biological systems are
responsible high concentrations of free radicals that are
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BioMed Research International
Volume 2014, Article ID 783059, 13 pages
http://dx.doi.org/10.1155/2014/783059
BioMed Research International
formed either during metabolic processes or as a result of
exposure to UV radiation. A conspicuous and important
place of attack of free radicals is the cell membrane. Oxi-
dation of its components and, in particular, the membrane
lipids by free radicals causes disorder in the structure and
function of the cell membrane, which leads to pathological
changes in the organism. Development of many dangerous
diseases linked directly with peroxidation of membrane lipids
can be prevented by providing the organism with natural
antioxidants, which are polyphenolic substances contained
in dierent parts of the plant. eir protective eects, as
scavengers of free radicals, depend on both the number of
hydroxyl groups in the polyphenolic molecule as well as the
number of molecules associated with the membrane. ey
protect the red blood cell membrane against oxidation and
hemolysis induced by free radicals []. As indicated in
our previous studies, the eectiveness of certain avonoids,
anthocyanins in particular, is much greater than the activity
of vitamin E and its synthetic counterpart Trolox [,].
High antioxidant activity of plant extracts and their ingre-
dients was shown in numerous studies conducted around the
world [,,,]. e authors of many works consider
the eective protection of biological membranes against
oxidation dependent on the capabilities of the polypheno-
licsubstancesbindingtomembranes[]. Due to the
amphiphilic nature of polyphenols, it can be expected that
they incorporate, mainly due to their structural similarity, at
dierent depths into the lipid phase of biological membranes,
changing their properties to varying degrees. However, there
is little work on the impact of plant extracts and polyphe-
nolic compounds on the properties of biological systems,
and in particular of biological membranes. An important
issue, therefore, seems to be the relationship between the
high antioxidant activity of plant polyphenols in relation to
biological membranes and the extent of physical changes that
these substances induce in membranes. Such studies have
been carried out to a minor extent for selected plant extracts
in relation to membrane of erythrocytes and lipid membrane
models [,,,].
e present study aimed to determine the antioxidative
activity of extracts from the fruit (BCF) and leaves (BCL)
of blackcurrant, with respect to the biological membrane,
andinparticularinrelationtomembranelipids.Giventhe
rich polyphenolic composition of blackcurrant extracts, one
could expect an eective protection of the membranes against
oxidation-inducing agents. e research was conducted with
respect to the membrane of erythrocytes, which was treated
as an example and model of the biological membrane.
Antioxidative activity of the extracts was determined on the
basis of uorimetric studies, with oxidation of erythrocyte
membranes induced by UVC radiation and the AAPH com-
pound. Also, the inuence of extracts from fruits and leaves of
blackcurrant on the properties of the erythrocyte membrane
was examined, taking into account their possible negative
impact. In particular, the hemolytic activity of the extracts
and their impact on osmotic resistance of erythrocytes were
assayed spectrophotometrically. Er ythrocyte shapes were also
examined in the presence of extracts, using the optical and
electron microscopes. Using the Fourier transform infrared
spectroscopy (FTIR) method, it was specied the location in
the membrane of red blood cells of the phenolic substances
contained in the extracts and their impact on its degree of
hydration. Also, the protective eect of the extracts towards
changes in the membrane of erythrocytes due to its exposure
to UVC radiation was determined.
2. Materials and Methods
2.1. Materials. Plant extracts of fruits and leaves of blackcur-
rant (Ribes nigrum L.) were obtained from the Department of
Fruit, Vegetable, and Cereal Technology, Wroclaw University
of Environmental and Life Sciences. e percent content
of polyphenols in the extracts was determined with liquid
chromatography (HPLC).
e studies were conducted on pig erythrocytes and iso-
lated erythrocyte membranes (ghosts), which were obtained
from fresh blood using the Dodge method []. e content
of erythrocyte membranes in the samples was determined on
the basis of protein concentration, which was assayed using
the Bradford method []. e choice of pig erythrocytes was
dictatedbythefactthatthiscellspercentageshareoflipidsis
closesttothatofthehumanerythrocyte,andthebloodwas
easily available.
e uorescent probe -(p-(-phenyl)-..-hexatriene)
propionicacid(DPH-PA)waspurchasedfromMolecular
Probes, Eugene, Oregon, USA. e oxidation inductor ,2󸀠-
diazobis (-amidinopropane) dihydrochloride (AAPH) and
Trolox was purchased from Sigma-Aldrich, Steinheim, Ger-
many.
Erythrocyte membranes were irradiated with UVC radi-
ationfrombactericidallamp(,mW/cm
2).
2.2. Methods
2.2.1. HPLC-DAD and UPLC-ESI/MS. Polyphenols were iso-
lated from leaves and fruits by extraction with water contain-
ing  ppm of SO2, the ratio of solvent to leaves or fruits
being  : . e extract was absorbed on Purolite AP  (UK)
for further purication. e polyphenols were then eluted out
with % ethanol, concentrated, and freeze-dried. e per-
cent content of polyphenols in individual preparations was
determined by means of liquid chromatography HPLC and
UPLC method. Phenolic compounds were identied with
the HPLC/DAD method and the method of UPLC/ESI/MS
analysis described in papers [].
2.2.2. Antioxidant Activity of Extract. e DPH-PA probe
was used in the uorimetric experiments. Erythrocyte mem-
branes with and without (control) addition of extracts were
suspended in a phosphate buer of pH . and UVC irradi-
ated or treated with the chemical oxidation compound AAPH
for  min. Free radicals, released in the process of membrane
lipids irradiation, cause quenching of DPH-PA uorescence,
decreasing the uorescence intensity. As a measure of the
extent of lipid oxidation was assumed relative uorescence,
that is, the ratio of an UVC-irradiated or AAPH oxidized
probe uorescence to the initial uorescence of the probe.
BioMed Research International
Here, as a control was assumed the relative uorescence of an
erythrocyte membranes suspension that contained the DPH-
PA probe, oxidized with UVC or AAPH compound, while
the blank was the relative uorescence of a suspension of the
same concentration but not oxidized.
A spectrouorimeter (Carry Eclipsce, Varian) was used
to measure free radicals concentration in the samples. Exci-
tationandemissionwavelengthswereex = 364 nm and
em = 430nm.emeasureoflipidoxidationwastherelative
change of uorescence intensity F/F0,whereF0is the initial
uorescence and Fthe one measured during an oxidation
procedure described in a paper []. e percentage of
lipid oxidation inhibition was calculated from the following
formula:
inhibition% =𝑥−
𝑢
𝑘−
𝑢⋅ 100%,()
where 𝑥is relative uorescence of an UVC irradiated sample,
or oxidized by AAPH, for min in the presence of the
compounds, 𝑢is relative uorescence of the control sample,
oxidized by AAPH or UVC irradiated, measured aer  min,
and 𝑘is relative uorescence of the blank sample, not
subjected to oxidation procedure, measured aer  min.
e results of the assay were expressed in reference to
Trolox, standard antioxidant.
2.2.3. Hemolytic Activity of Extracts and Osmotic Resistance
of Erythrocytes. e hemolytic experiments were conducted
on fresh, heparinized blood. For washing the erythrocytes,
and in the experiments, an isotonic phosphate solution
of pH . ( mM NaCl, . mM KCl, . mM MgCl2,
. mM Na2HPO4×H2O, and . mM Na2H2PO4×
H2O) was used. Full blood was centrifuged for  min,  g
at Ctoremovetheplasmaandleucocytes.Uponremoving
from plasma, the erythrocytes were washed four times in
phosphate solution and then incubated in the same solution
but containing proper amounts of the compounds studied.
e modication was conducted at C for  h, each sample
containing  mL of erythrocyte suspension of % hematocrit
was stirred continuously. Aer modication  mL samples
were taken, centrifuged and the supernatant assayed for
hemoglobin content using a UV-Vis spectrophotometer
(Cary  Bio, Varian) at  nm wavelength. Hemoglobin
concentration in the supernatant, expressed as percentage
of hemoglobin concentration of totally hemolyzed cells, was
assumed as the measure of the extent of hemolysis.
For osmotic resistance, upon removing from plasma the
erythrocytes were washed three times with a cool (ca. C),
 mosm PBS isotonic solution. Next, a % red cells suspen-
sion containing plant extracts of . mg/mL concentration
was prepared and le for  h at Cwithcontinuousstirring.
Aer this modication the suspension of erythrocytes was
centrifuged for  min at room temperature in order to
removethecellsfromtheextractsolution.Fromthecell
sediment were taken  L samples of the extract-modied
cells and suspended in test tubes containing NaCl solutions
of .–.% concentration and to an isotonic (.%) NaCl
solution. In solutions of the same concentrations were also
suspended unmodied red blood cells that constituted the
control for osmotic resistance determinations. en, the
suspension was stirred and centrifuged under the above
stated conditions. Aer that the percentage of hemolysis was
measured with a spectrophotometer at  = 540nm. On the
basis of the results obtained, the relation was determined
between the percentage of hemolysis and NaCl concentration
in the solution. Next, using the obtained plots, the NaCl
percent concentrations (C50)thatcaused%hemolysiswere
found. e C50 values were taken as a measure of osmotic
resistance. If a determined sodium chloride concentration is
higher than that of control cells, the osmotic resistance of the
erythrocytes is regarded to be lower, and vice versa.
2.2.4. Erythrocyte Shapes. For investigation with the optical
microscope,theredcellsseparatedfromplasmawerewashed
four times in saline solution and suspended in the same
solution but containing . and . mg/mL of BCL and
BCF extracts, respectively. Hematocrit of the erythrocytes in
the modication solution was %, the modication lasting
h at C. Aer modication the erythrocytes were xed
with a .% solution of glutaraldehyde. Aer that the red
cells were observed under a biological optical microscope
(Nikon Eclipse E) equipped with a digital camera. e
photographs obtained made it possible to count erythrocytes
of various shapes and then percent share of the two basic
forms (echinocytes and stomatocytes) in a population of ca.
 cells was determined. e individual forms of erythrocyte
cells were assigned morphological indices according to the
Bessis scale [], which for stomatocytes assume negative
values from to and for echinocytes-positive from
to.
For investigation with the electron microscope, the red
cells separated from plasma were washed four times in saline
solution and suspended in the same solution but containing
. mg/mL of the blackcurrant leaf (BCL) and fruit (BCF)
extracts. Hematocrit of the erythrocytes in the modication
solution was %, the modication lasting h at C. Aer
modication the erythrocytes were xed for  h in .%
solution of glutaraldehyde. Aer that the preparations were
washed in phosphate buer for  min, and then the material
was dehydrated in a rising series of acetone concentrations
(,,,,,and%).Eachsamplewaswashed
for  min in an appropriate concentration and the material
remain in pure acetone for  min. Next, the erythrocytes
were dried for  h at room temperature. Erythrocytes thus
prepared were deposited on object stages and subjected to
X-ray microanalysis by means of an X-ray analyzer, Brucker
AXS Quantax, collaborating with the ESPRIT ver. ...
program. Next, the samples were coated with gold using
the Scancoat  (Edwards, London) sprinkler. e material
ultrastructure was analyzed using a scanning microscope
(EVO LS ZEISS) with SE detector, under high vacuum and
accelerating voltage EHT =  kV.
2.2.5. FTIR Investigation of Erythrocyte Membrane. Erythro-
cyte ghosts were washed three times in .% NaCl solution.
Next, the ghost suspension was incubated as mL samples
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( Lghosts+Lphysiologicalsalt)forhat
C.
In that way the control sample with ghosts suspension
was prepared, as well as samples with blackcurrant fruit
and leaf extracts, obtaining . mg/mL concentration. Aer
incubation, the samples were centrifuged for  min at
 g and IR spectra were recorded from a condensed
membrane suspension. Tests were also conducted with sam-
ples containing erythrocyte membranes irradiated with UV,
prepared in a similar way. Aer  h incubation with extracts,
the ghost suspension was exposed to UV lamp for  h
andthencentrifugedandspectratakenintheIRrange.
e measurements were performed with a ermo Nicolet
 MCT spectrometer, with ZnSe crystal, applied at room
temperature. Each single spectrum was obtained from 
records at  cm−1 resolution in the range –cm−1.
Preliminary elaboration of a spectrum was done using the
EZ OMNIC v . program, also of the ermo Nicolet rm.
Aer ltering the noise out from the spectrum of the object
studied, the spectrum of the NaCl solution was subtracted in
ordertoremovetheOHbandofwaterandthebaselinewas
corrected.
3. Results
3.1. HPLC-DAD and UPLC-ESI/MS. Detailed quantitative
and qualitative contents of phenolic compounds in the
extracts from leaves and fruits of blackcurrant are given in
Table .
e phenolic contents of blackcurrant leaves indicate that
the dominant components in the leaves are quercetin deriva-
tives, which constitute % of polyphenolic compounds in
dry mass of extract. Very interesting though is the polyphenol
composition of blackcurrant fruits, constituting over a half
(.%) of total dry mass of extract.
e blackcurrant fruit extract was analyzed by UPLC-
ESI-MS and HPLC-DAD systems and is summarized in
Table  and Figures and . A total of  kinds of polyphenolic
compounds found in blackcurrant fruit extract were identi-
ed and quantied. Two hydroxycinnamates were detected:
chlorogenic and p-coumaroylquinic acids. e compounds
that had a [M H]at m/z  and  with max  nm and
 nm aer a fragmentation yielded a caeic and p-coumaric
acid, respectively.
Anthocyanins, delphinidin--O-glucoside (peak tr.
. min), delphinidin--O-rutinoside (peak tr. . min),
cyanidin--O-glucoside (peak tr. . min), cyanidin--O-
rutinoside (peak tr. . min), petunidin--O-rutinoside
(peak tr. . min) (Figure ),wereidentiedonthebasis
of reference compounds, UV-Vis spectra, mass spectra, and
the literature. Delphinidin and cyanidin were the two major
anthocyanidins, commonly known in blackcurrant fruit [].
e minor peak was identied as petunidin aglycone.
Furthermore, a total of  avonol glycosides were detect-
ed (Tabl e ). ree quercetin derivatives were detected: quer-
cetin--O-rutinoside, quercetin--O-galactoside and quer-
cetin--O-glucoside. Peaks with (𝑡). min, . min
and . min had max of  nm, respectively. All com-
pounds had a fragmentation yielded a quercetin ion at m/z
. Galactosides and glucosides have the same molar masses
and mass spectra, and they dier according to their retention
tim es only. A nalog ousl y, myr i cetin ga l actos ide (𝑡). min
was assumed to eluate before myricetin glucoside (𝑡)
. min. Myricetin--O-rutinoside (𝑡). min was also
identied in our samples, as dened in earlier investigations
[]. e anthocyanidin derivatives were the most pre-
dominant phenolic group found in blackcurrant fruit extract
and constituted .% weight of powder. Among identied
anthocyanidin derivatives the most important compounds
were delphinidin and cyanidin rutinoside (.%) e other
compounds such as hydroxycinnamic acids and avonol
derivatives accounted total for .% of blackcurrant fruit
extract.
3.2. Antioxidant Activity of Extract. e investigation of
antioxidative activity of BCF and BCL extracts were con-
ducted on erythrocyte membranes, based on the extent of
membrane lipids oxidation. e oxidation was induced with
UVC and the AAPH compound. e extent of lipid oxidation
was assayed uorimetrically, based on the kinetics of DPH-
PA probe quenching caused by free radicals that arose during
oxidation.
e relative uorescence for the studied concentrations
of BCF and BCL extracts decreases with time of oxidation
with UVC radiation, which shows that the degree of the
lipid oxidation inhibition increases. Quenching of DPH-
PA uorescence in the presence of BCF and BCL extracts
at dierent concentrations was also observed when lipid
oxidation was induced in erythrocyte membranes with the
chemical compound AAPH at  M.
Basedonthekineticsoftheoxidationcurvesobtained
for various concentrations of both the extracts and compared
with Trolox, the concentrationresponsible for % inhibition
of membrane lipids oxidation (IC50)wasfound.eresults
from the uorimetric method are given in Table  and
Figure .
e results indicate that the extracts protect erythrocyte
membrane lipids against free radicals induced with UVC and
the AAPH compound. Extract concentration responsible for
% protection of the lipids is comparable with that of Trolox
in the case of AAPH and smaller when oxidation was UVC
induced.
As indicated by the IC50 values, BCL and BCF extracts
protect membrane lipids against oxidation. e results
obtained have shown that BCL extract protect the erythrocyte
membrane against UVC-induced oxidation better than BCF
extract but worse than Trolox. For protecting membranes
against AAPH-induced oxidation, the BCL and BCF extracts
were equal in antioxidant activity to Trolox. Such results
suggest that the reaction with free radicals depends on
theoxidationinducer.Inthecaseofphoto-oxidationthe
percentage of inhibition and the kinetics of the process are
slower, whereas in the case of the AAPH compound the
process is much faster and ecient in inhibiting oxidation.
e results indicate that polyphenols, mainly of the avonoid
group, contained in blackcurrant extract have good antiox-
idative properties, being more eective towards free radicals
BioMed Research International
T : e percent content and characterization of phenolic compounds of the extract of blackcurrant fruits (BCF) and leaves (BCL) using
their spectral characteristic in HPLC-DAD (retention time, max) and positive and negative ions in UPLC-ESI-MS [M–H].
Compounds BCF BCLBCF/BCL BCF/BCL BCF/BCL
Content (%) 𝑡(min) max (nm) (MS) (m)
Chlorogenic acid . . .  
p-Coumaroylquinic acid . .  
Neochlorogenic acid . .  
Cryptochlorogenic acid . .  
Delphinidin--O-glucoside .  .  
Delphinidin--O-rutinoside .  .  
Cyanidin--O-glucoside .  .  
Cyanidin--O-rutinoside .  .  
Petunidin--O-rutinoside .  .  
Quercetin--O-rutinoside . . ./.  /
Quercetin--O-galactoside . . ./.  /
Quercetin--O-glucoside .  .  
Quercetin--(󸀠󸀠-malonyl)-glucoside  . .  
Quercetin--O-glucosyl-󸀠󸀠-acetate  . .  
Myricetin--O-rutinoside .  .  
Myricetin--O-galactoside .  .  
Myricetin--O-glucoside .  .  
Kaempferol--O-rutinoside  . .  
Kaempferol--O-galactoside  . .  
Kaempferol--O-glucosyl-󸀠󸀠-acetate . .  
Total . .
Published previously in Oszmia´
nski et al.,  [].
(%)
0
100
m/z
595.30
465.28
303.23
275.21
245.18
231.19
205.18
177.15 219.28
289.25
347.30
333.32 449.24
377.33
391.31
421.34
435.32 466.22
493.37 507.41
523.33 567.40
611.28
612.28
613.29
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
F : Direct injection ESI-MS of polyphenols in the blackcurrant fruit extract. Positively charged molecular ion are . cyanidin-
-O-glucoside, . delphinidin--O-glucoside, quercetin--O-glucoside and quercetin--O-galactoside, . cyanidin--O-rutinoside,
. delphinidin--O-rutinoside and quercetin--O-rutinoside, and  petunidin--O-rutinoside.
BioMed Research International
Sens 20
N-Meth 1
0.25
0.20
0.15
0.10
0.05
0.00
10 12 14 16 18 20 26 28 3022 24
15.29
18.03
16.21
17.03
19.17
20.96
28.53
Intensity (AU)
Retention time (min)
F : HPLC prole ( nm) of blackcurrant extracts tr.
. min delphinidin--O-glucoside, . min delphinidin--O-
rutinoside, . min cyanidin--O-glucoside, . min cyanidin--
O-rutinoside, and . min petunidin--O-rutinoside.
T : Values of IC for BCL and BCF extracts, and Trolox that
inhibit erythrocyte membrane lipids oxidation by %, determined
with the uorimetric method. e oxidation was induced with UVC
radiation and the AAPH compound.
Extract/inducer Concentration IC (g/mL) ±SD
UVC AAPH
Blackcurrant leaves (BCL) 24.5 ± 1.7 7.2 ± 0.05
Blackcurrant fruits (BCF) 25.6 ± 1.5 4.8 ± 0.16
Tro l ox 14.6 ± 1.3 3.9 ± 0.30
that arise in the process of AAPH-induced oxidation of
erythrocyte membranes than UVC-induced one.
3.3. Hemolysis and Osmotic Resistance of Erythrocytes. e
results on the eect of blackcurrant extracts on hemolysis of
erythrocytes compiled in Table  testify that in the presence
of BCF and BCL extracts red blood cells do not undergo
increased hemolysis compared with unmodied cells, hemol-
ysis with extract at . mg/mL not exceeding % in respect to
the control probe. e control probe contained suspension of
unmodied erythrocyte cells in phosphate buer (pH .).
In the studies of the eect of the extracts on erythrocyte
osmotic resistance no signicant dierences between the
degree of hemolysis in the control cells and those modied
with BCF extract was found, in aqueous solutions of various
concentrations of sodium chloride. Concentrations (C50)
of sodium chloride, expressed as a percentage, determined
for control blood cells and those modied with BCL and
BCF extracts of . mg/mL concentration are C50 control—
.%, BCL—.%, BCF—.%. ese results indicate
an increase in osmotic resistance of blackcurrant leaf extract
modied cells, suggesting that the red blood cell membrane
treated with BCL extract is less sensitive to changes in osmotic
pressure (Figure ).
T : Percent content of hemolyzed erythrocytes in the presence
of BCL and BCF extracts of specic concentrations.
Extracts Percentage of hemolysis ±SD
Blackcurrant
Concentration (mg/mL) BCF BCL
Control 5.10 ± 0.56 5.10 ± 0.56
. 0.97 ± 0.11 0.98 ± 0.11
. 2.18 ± 0.26 1.60 ± 0.19
. 2.82 ± 0.34 2.01 ± 0.24
. 4.39 ± 0.53 2.50 ± 0.30
. 5.22 ± 0.63 3.38 ± 0.40
. 5.23 ± 0.63 4.22 ± 0.51
. 6.18 ± 0.74 5.14 ± 0.62
. 6.89 ± 0.83 5.95 ± 0.71
. 7.41 ± 0.89 7.69 ± 0.92
. 8.70 ± 1.05 9.41 ± 1.13
3.4. Erythrocyte Shapes. Figure  shows the percent share of
the various forms of cells in a population of erythrocytes
modied with BCL and BCF extracts of . and . mg/mL
concentration. As seen in Figures (a),(b),and,these
extracts induce mostly various forms of echinocytes, mainly
discoechinocytes. It can thus be assumed that the blackcur-
rant extracts concentrate mainly in the outer monolayer of
the erythrocyte membrane when inducing echinocytes, and
practically do not permeate into the inner monolayer of the
membrane[].
e work [,] showed that the formation of
echinocytes occurred when amphiphilic molecules were
incorporated in the outer monolayer of the erythrocyte
membrane. Compounds penetrating to the inner monolayer
of the membrane induced formation of stomatocytes. We can
therefore assume that BCF and BCL extract components con-
centratedmainlyintheoutermonolayeroftheerythrocyte
membrane.
3.5. FTIR Investigation of Erythrocyte Membrane. Spectro-
scopic studies in the near infrared allowed to take absorp-
tion spectra for unmodied and BCL and BCF modied
erythrocytes. Spectra of unmodied and extract modied
membranes irradiated with UVC were also taken. e results
obtained allowed us to observe changes induced in the
erythrocyte membrane by UVC radiation and phenolic com-
ponents of the extracts. In the spectra, the characteristic
frequency bands of the protein and lipid components were
analysed.Weidentiedthecharacteristicforerythrocytes
frequency bands for individual membrane components:
methyl groups and methylene hydrocarbon chains (–
 cm−1), carbonyl groups of lipids (– cm−1),
theamidebandI(cm
−1) and amide band II
(– cm−1), and phosphate band (– cm−1)
and choline band (– cm−1)[]. Analysis of
the spectra of membranes treated with aqueous extracts of
blackcurrant leaves and fruit showed no signicant changes
in the amide I and amide II bands (vibration bands of groups
BioMed Research International
05 1510 20 3025
Time (min)
Blank
Control
0.01 mg/mL
0.02 mg/mL
0.03 mg/mL
0.04 mg/mL
0.05 mg/mL
Relative fluorescence F/F0
0.2
0.4
0.6
0.8
1
1.2
0
(a)
Relative fluorescence F/F0
0.2
0.4
0.6
0.8
1
1.2
0
05 1510 20 3025
Time (min)
Blank
Control
0.001 mg/mL
0.005 mg/mL
0.01 mg/mL
0.05 mg/mL
0.1 mg/mL
(b)
F : Relation between relative uorescence intensity and time of UVC induced oxidation of erythrocyte membranes for control and test
sample at dierent concentrations of blackcurrant extracts of leaves (a) and fruits (b).
0
10
20
30
40
50
60
70
80
90
100
0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95
NaCl concentration (%)
Control
BCF
BCL
Hemolysis (%)
F : Percent of hemolysis of cells modied with blackcurrant
leaf (BCL) and fruit (BCF) extract of . mg/mL concentration
versus sodium chloride concentration.
C=O and N–H proteins). ere were also no changes in
the vibrational bands of methyl and methylene groups, alkyl
chains of lipids, and C=O vibrations of carbonyl groups of
lipids. is result suggests that the polyphenolic compounds
contained in the extracts do not signicantly aect the struc-
ture of the proteins contained in the membrane of red blood
cells, and they do not modify the hydrocarbon chains of lipid
molecules. UVC radiation exposure of control unmodied
membranes and membranes modies with the extracts does
not cause visible changes in the above mentioned bands. e
presence of the extracts in erythrocytes membranes causes a
slight shi of the phosphate band in the direction of smaller
wave numbers, which points to a slight increase in the degree
of hydration of PO2group. A slightly larger displacement
of this band is caused by the extract from BCL. Exposure
of the membranes to UVC radiation results in a signicant
shi of the phosphate band towards higher values of wave
numbers, suggesting a clear reduction in the number of OH
groups associated with lipid phosphate groups via hydrogen
bonds. e presence of the extracts in a suspension reduced
the transfer eect of phosphate bands distinctly. Phosphate
bands of irradiated membranes in the presence of extracts are
shied toward lower wave numbers relative to the irradiated
ghosts, which is particularly visible with BCL extract. So
we can assume that the presence of extracts in membranes
exposed to UVC protects them against changes induced by
the radiation, causing the membrane properties becoming
closer to those of membrane not exposed to radiation. A
particularly high level of membrane protection was observed
in the presence of blackcurrant leaf extract (Figure ).
e presence of extract and exposure to UVC light caused
visible changes also in the choline N+(CH3)bandoflipids.In
the case of ghosts not exposed to UVC the presence of extracts
caused distinct decrease in the half-width of the choline band,
which suggests a change in the packing order in that part of
membrane and can be linked with presence of sugar groups
in the extract components. UV radiation caused shi of the
band towards lower wave numbers. For ghosts irradiated in
BioMed Research International
Control
BCL 0.01 mg/mL
BCL 0.1 mg/mL
Morphological index
SfSt(−4)
StII(−3)
StI(−2)
DSt(−1)
D(0)
E(2)
Sf(4)
DE(1)
SfE(3)
70
60
50
40
30
20
10
0
Erythrocytes shapes (%)
(a)
70
60
50
40
30
20
10
0
Erythrocytes shapes (%)
SfSt(−4)
StII(−3)
StI(−2)
DSt(−1)
D(0)
E(2 )
Sf(4)
DE(1)
SfE(3)
Control
BCF 0.01 mg/mL
BCF 0.1 mg/mL
Morphological index
(b)
F : Percentage share of dierent shapes of erythrocytes induced by BCL and BCF extracts at . (black bar) and . (white bar) mg/mL
concentration, control (grey bar). On the abscissa, there are morphological indices for the respective shapes of cells: spherostomatocytes (),
stomatocytes II (), stomatocytes I (), discostomatocytes (), discocytes (), discoechinocytes (), echinocytes (), spheroechinocytes
(), and spherocytes ().
(a) (b) (c)
F : Shapes of unmodied erythrocytes (a) and modied with BCF (b) and BCL (c) at . mg/mL concentration, observed with electron
microscope.
thepresenceoffruitextracttheeectoflightwasmarkedly
smaller, while in the presence of leaf extract no changes were
observed compared with nonirradiated ghosts (Figure ).
3.6. Statistical Analysis. Statistical analysis was carried out
using Statistica . (StatSo Inc.). All the experiments were
performed at least in triplicate unless otherwise specied.
Analysis of variance was carried out and signicance between
means was determined using Dunnett’s post hoc test. Results
are presented as mean ±SD. Signicant levels were dened at
 < 0.05.
4. Discussion
e results of the presented research have shown that black-
currant leaves (BCL) and blackcurrant fruits (BCF) extracts
induce small changes in erythrocyte membranes. eir
polyphenolic composition makes possible an interaction with
the lipids of biological membranes. e main polyphenolic
constituent of BCF extract are anthocyanins, which constitute
over % of the total polyphenol content of the extract. It
was shown that the benecial properties of anthocyanins,
cyanidins and their glycosides, are connected with their
ability to scavenge free radicals, including the reactive forms
of oxygen []. Cyanidins were found to bind with the
biological membrane, protecting it against oxidation [,
].Itcanthusbeassumedthatmainlyanthocyaninsare
responsible for the protective action of blackcurrant fruit
extracts with respect to the biological membrane.
BCL extract diers in polyphenolic composition from
BCF extract, where quercetin derivatives dominate, which
constitute ca. % of the total polyphenols content of the
extract. An antioxidation activity of quercetin glycosides in
relation to the erythrocyte membrane was shown in our
BioMed Research International
1260 1240 1220 1200 1180
0
0.01
0.02
0.03
Absorbance
Control
BCL
BCF
Wavenumber (cm−1)
(a)
Control
BCL
BCF
1260 1240 1220 1200 1180
0
0.01
0.02
0.03
Absorbance
Wavenumber (cm−1)
(b)
F : Phosphate band of erythrocyte membrane modied with BCF and BCL extract (a), and of erythrocyte membrane modied with
BCF and BCL extract, irradiated with UVC (b). Solid line, control membrane; dashed line, membrane with fruit extract; dotted line, membrane
with leaf extract.
1000 980 960 940
0
0.01
0.02
0.03
0.04
Absorbance
Control
BCL
BCF
Wavenumber (cm−1)
(a)
0
1020 1000 980 960 940
0.01
0.02
0.03
Absorbance
0.04
Control
BCL
BCF
Wavenumber (cm−1)
(b)
F : Choline band of erythrocyte membrane modied with BCF and BCL extract (a), and of erythrocyte membrane modied with BCF
and BCL extract, irradiated with UVC (b). Solid line, control membrane; dashed line, membrane with fruit extract; dotted line, membrane
with leaf extract.
previous studies []. However, it is not yet fully elucidated
the molecular mechanism of the antioxidant action of these
compounds in relation to the biological membrane.
e present study aimed to determine the antioxida-
tive activity of blackcurrant extracts with respect to the
membrane of red blood cells, treated as a model of the
biological membrane. e impact of the extracts on ery-
throcyte membrane properties was also examined. Selected
research methods helped to determine the location of the
compounds contained in the extracts, based on erythrocyte
shape changes and hemolytic and FTIR studies. e results
of the investigations undertaken enabled us to determine the
relationship between the antioxidative activity of the extracts
and their anity to the membrane.
Hemolytic tests showed that polyphenolic compounds
contained in the extracts do not induce hemolysis, therefore
donotcauselyticeectonredbloodcellsintheused
range of concentrations. e lack of hemolysis and cyto-
toxicity for chosen avonoids was also conrmed in the
work []. One can therefore assume, on the basis of the
studies [], that the compounds do not embed deep
into the hydrophobic membrane area. Hemolysis caused
 BioMed Research International
by various lytic compounds occurs when such substances
penetrate deeply into the membrane, weakening the inter-
action between its components. Disturbed is the structure
of the membrane and facilitated transport of water swells
the cell, and the membrane is permanently damaged. Studies
of osmotic resistance conrmed the hemolytic results, they
showed that as a result of incorporation of the extract
contained compounds the osmotic resistance does not change
or is even greater in the case of BCL extract.
e extract from the fruit does not alter the osmotic
resistance, that is, does not change the properties of the
erythrocyte membrane, whereas the leaf extract causes an
increase in the resistance, which means that substances
present in the extract bind to the membrane and make
it stronger, so that it becomes less sensitive to changes in
osmotic pressure.
Both the extracts induced an alternation in the ery-
throcyte morphology, from normal discoid shape to an
echinocytic form. BCF and BCL extracts are responsible
for creation of varied forms of echinocytes, BCL mainly
echinocytes and BCF echinocytes and spheroechinocytes.
Itcanthusbeassumed,accordingtothebilayercou-
ple hypothesis [], that the extracts concentrate mainly
in the outer monolayer of the erythrocyte membrane
when inducing various forms of echinocytes, and practi-
cally do not permeate into the inner monolayer of the
membrane.
e changes observed in the IR spectrum suggest a
supercial character of the interaction of BCF and BCL
components with the hydrophilic part of the erythrocyte
membrane. FTIR spectra for extract modied membranes
showed no signicant changes in the hydrophobic part
of the hydrocarbon chains, but showed a change in the
degree of hydration in the phosphate and choline band
of membrane phospholipids. As the authors suggest [,
],asaresultoftheeectofpolyphenolsonlipidmem-
brane, hydrogen bonds are formed between the hydroxyl
groups of polyphenols and the polar groups of lipids.
In addition, numerous studies showed that plant extracts,
particularly those rich in polyphenol compounds with
sugar residues, reduce the packing order of the hydrophilic
area of membrane, causing increased hydration of this
area.
As indicated by the results, aer one hour of exposure
to UVC radiation, changes occurred in the erythrocyte
membrane, in particular in its hydrophilic region. is is
shown by a clear shi of the phosphate band of polar heads of
lipids, which may be related to oxidation of OH groups which
are connected by hydrogen bonds with the phosphate groups
or lipids. e presence of the extracts in ghosts suspension
markedly reduced the eect of phosphate band transfer that
wascausedbyexposuretoUVradiation.Inparticular,such
an eect was observed in the case of BCL extract, which
caused a complete abolition of UVC induced changes in
control ghosts, and even a visible widening of the band.
is may suggest that during exposure to UVC radiation in
the presence of the extracts (BCL and BCF) the membrane
phosphate groups were not oxidized but had undergone
hydration (BCL), which also indicates the presence of extract
components in this part of the membrane. In addition, it is
believed that the polyphenolic compounds, especially those
occurring in the form of glycosides, cause hydration of the
membrane, transporting numerous water molecules bound
to them [,,,,,]. Such protective action
of the extracts can be attributed to their phenolic components
that bind to the membrane surface, and containing numerous
OH groups in their structure they, possibly, as the rst
undergo oxidation.
e location of the compounds in the hydrophilic part
of membrane seems to constitute a protective shield of the
cell against other substances, the reactive forms of oxygen in
particular, which nds its reection in their antioxidant prop-
erties. BCL and BCF extracts exhibited a high antioxidant
activity towards reactive forms of oxygen, which developed
as a result of membrane photo-oxidation induced by UVC
radiation and by the AAPH compound.
5. Conclusion
e results obtained indicate that BCL and BCF extracts
exert benecial eect on the organism protecting the cell
membrane against oxidation. e polyphenolic compounds
contained in the extracts do not disturb the structure of the
biological membrane, to which they bind only supercially,
penetrating its hydrophilic region, and do not penetrate
the membrane hydrophobic region, as is evident from the
hemolytic, microscopic, and FTIR investigations. It can thus
be expected that they do not disturb the function of the
biological membrane.
In this context, a protection of the organisms against the
harmful eects of free radicals and reactive oxygen species
is made possible by providing the body with appropriate
quantities of plant polyphenolic substances. us, the results
of this research encourage the consumption of fruits and
extracts from blackcurrant leaves to ght free radicals and
thus increase our resistance to many diseases.
Conflict of Interests
e authors conrm that the paper has been read and
approvedbyalloftheauthors.eyalsoconrmthatthere
are no other persons who participated in the preparation of
this paper other than the authors listed. In addition, they
conrm that the order with which the authors are listed is
agreed and approved by all authors. e authors conrm that
there is no conict of interests associated with this paper.
Furthermore, this work has not been nancially supported by
other institutions, which could have inuenced its outcome.
e authors conrm that they have followed the regulations
of Wroclaw University of Environmental and Life Sciences
concerning intellectual property. ey declare that there are
no impediments to the publication of this work, because they
havetakenintoaccounttheprotectionoftheintellectual
property associated with this work. Furthermore, they declare
that this paper is not under consideration for publication
elsewhere.
BioMed Research International 
Acknowledgment
is work was sponsored by the Ministry of Science and
Education, scientic Project nos. N N  and N N
.
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... Juicing is one of the most popular ways to process fruits and berries. Due to the high content of biologically active substances, the consumption of juices and extracts from black currant is recommended for athletes and people leading an active lifestyle [3,4]. Directly pressed blackcurrant juice, as a final product entering the market, must have a color, taste and smell characteristic of the berries from which it was produced. ...
... For sensory analysis, it is important to select a list of descriptors that adequately describe the product and its properties. Our analysis showed that most studies of blackcurrant juices are related to the study of the influence of various factors on aroma, astringency and stability of sensory properties [4][5][6][7][8][9]. At the same time, there are very few publications on the sensory profiles of mono-varietal blackcurrant juices. ...
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The results of a comparative study of the quality of blackcurrant juices of five varieties of ARSRIFCB breeding suitable for industrial commercial use are presented: Azhurnaya, Orlovskaya Serenada, Orlovsky Vals, Ocharovanye, Chudnoye Mgnovenye. The sensory profiles of juices and their relation to scoring and varietal characteristics were studied. An analysis of the sensory qualities of mono-varietal blackcurrant juices showed that they largely depend on varietal characteristics, such as transparency and thickness, the sensation of acidity and sweetness, and the softness of the taste (absence of harsh acid in the taste). The indicators of the point analysis confirm the results of the descriptor. Sensory analysis data (point and descriptor) showed that preference is given to products with a ruby-red color, typical for blackcurrant juices, bright, not cloudy. Among the studied varieties, of great interest for juice production are the varieties Ocharovanye and Azhurnaya.
... Phenolic fraction of R. nigrum extract was investigated for the identification of compounds and the results have revealed the presence of around 30 major constituents comprising mainly of flavan-3-ols, flavonols, hydroxycinnamates, lignans, naphthols and furanocoumarins. The literature data also suggest quercetin derivatives as the main components of R. nigrum leaf extracts, which constitute around 80% of polyphenolic compounds on a dry mass basis [41]. The presence of such a variety of active ingredients explains the excellent antioxidant as well as reducing capacity of R. nigrum extract. ...
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Silver nanoparticles (Ag NPs) represent one of the most widely employed metal-based engineered nanomaterials with a broad range of applications in different areas of science. Plant extracts (PEs) serve as green reducing and coating agents and can be exploited for the generation of Ag NPs. In this study, the phytochemical composition of ethanolic extract of black currant (Ribes nigrum) leaves was determined. The main components of extract include quercetin rutinoside, quer-cetin hexoside, quercetin glucuronide, quercetin malonylglucoside and quercitrin. The extract was subsequently employed for the green synthesis of Ag NPs. Consequently, R. nigrum leaf extract and Ag NPs were evaluated for potential antibacterial activities against Gram-negative bacteria (Esche-richia coli ATCC 25922 and kanamycin-resistant E. coli pARG-25 strains). Intriguingly, the plant extract did not show any antibacterial effect, whilst Ag NPs demonstrated significant activity against tested bacteria. Biogenic Ag NPs affect the ATPase activity and energy-dependent H +-fluxes in both strains of E. coli, even in the presence of N,N'-dicyclohexylcarbodiimide (DCCD). Thus, the antibac-terial activity of the investigated Ag NPs can be explained by their impact on the membrane-associated properties of bacteria.
... The most useful preparations on the basis of Ribes nigrum leaves were alcohol liquid extracts, which were then lyophilized in order to determine the exact concentrations of orally or intraperitoneally administered portions [11]. According to the recent year literature data, the extracts, obtained from the leaves of R. nigrum, possess remarkable antioxidant activity [12]. No data on acute toxicity, genotoxicity, reproductive and developmental toxicity or carcinogenicity [11]. ...
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Phytotherapy is the branch of alternative medicine that studies the treatment of various diseases with the help of plants and herbal preparations. One of the plants commonly used in phytotherapeutic treatments is blackcurrant. All its component parts are rich in active principles that are responsible for its phytotherapeutic properties. Numerous studies have approached the phytotherapeutic properties of blackcurrant. In this material we focused on researching data from the literature that include studies related to antioxidant action of blackcurrant.
... As already stated, the bioactivities of the hydroalcoholic extract obtained from a 2 h maceration of blackcurrant pomace in EtOH/H2O 50/50 (pomace/solvent ratio 1/5 w/w) with an extraction yield of 3.3 ± 0.2% (extraction performed in triplicates) were evaluated in vitro. It displayed promising, but not surprising antioxidant and antihyaluronidase bioactivities (Figure 1), given the rich polyphenolic composition and notably the high anthocyanins and their aglycones content of blackcurrant berries [34][35][36][37] and pomace [38]. It also displayed to a lesser extend some interesting whitening properties, anti-inflammatory, and anticollagenase activities, but no anti-elastase activity. ...
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