Phenolic content and biological activity of extracts of blackcurrant fruit and leaves

    Phenolic Content And Biological Activity Of Extracts Of Blackcurrant Fruit And Leaves Sylwia Cyboran, Dorota Bonarska-Kujawa, Hanna Pruchnik, Romuald ˙ Zyłka, Jan Oszmia´nski, Halina Kleszczy´nska PII: DOI: Reference:

S0963-9969(14)00352-4 doi: 10.1016/j.foodres.2014.05.037 FRIN 5279

To appear in:

Food Research International

Received date: Revised date: Accepted date:

18 December 2013 7 May 2014 29 May 2014

˙ lka, Please cite this article as: Cyboran, S., Bonarska-Kujawa, D., Pruchnik, H., Zy R., Oszmia´ nski, J. & Kleszczy´ nska, H., Phenolic Content And Biological Activity Of Extracts Of Blackcurrant Fruit And Leaves, Food Research International (2014), doi: 10.1016/j.foodres.2014.05.037

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ACCEPTED MANUSCRIPT PHENOLIC CONTENT AND BIOLOGICAL ACTIVITY OF EXTRACTS OF BLACKCURRANT FRUIT AND LEAVES

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Cyboran Sylwiaa*, Bonarska-Kujawa Dorotaa, Pruchnik Hannaa, Żyłka Romualda, Oszmiański Janb, a

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Kleszczyńska Halinaa

Department of Physics and Biophysics, Wrocław University of Environmental and Life Sciences,

Norwida 25, 50-375 Wrocław, Poland,

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e-mail: [email protected], [email protected], [email protected], [email protected]

Department of Fruit, Vegetable and Cereals Technology, Wrocław University of Environmental and

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b

e-mail: [email protected] *

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Life Sciences, Norwida 25, 50-375 Wrocław, Poland,

Corresponding author tel. +48713205275, fax. +48713205167, e-mail: [email protected]

Address: Department of Physics and Biophysics, Wroclaw University of Environmental and Life

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Sciences, Norwida 25, 50-375 Wrocław, Poland

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ACCEPTED MANUSCRIPT Abstract The studies were designed to determine the polyphenolic composition and biological activity of extracts from fruit and leaves of blackcurrant in relation to biological and lipid membranes. A detailed

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quantitative and qualitative analysis of extracts was conducted, using the UPLC-DAD and UPLC-

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PDA−Q/TOF-MS methods. Furthermore, stability of the phenolic content in the extracts was determined using the Folin-Ciocalteu method. The antioxidant activity of the extracts in relation to the membrane of

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erythrocytes and lipids extracted from red blood cell membranes (RBCL) exposed to chemical oxidizing agents (AAPH) was determined, and the effects of blackcurrant extracts on the properties of the

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membrane of erythrocytes and liposomes (DPPC and RBCL) were examined. Antioxidant activity of the extracts was studied fluorometrically, while effects of the extracts on the properties of membranes were

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examined using calorimetric, IR spectroscopy and fluorimetric methods. In particular, the effects of the extracts on packing order, membrane fluidity and the main phase transition temperature were determined. Additionally, the affinity of extracts to organic or aqueous media was determined on the

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basis of their partition coefficient between octanol and phosphate buffer. The results showed that the tested extracts are rich sources of polyphenols, primarily from the group of flavonoids; in leaves flavonols dominate, while in fruit anthocyanins dominate. Their polyphenolic content was quite stable and only slightly changed within 12 months. The substances used markedly protect the membranes

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against oxidation and to varying degrees modify properties of the hydrophilic regions of membranes. They localize mainly in the area of lipid polar heads of membrane, changing their arrangement, which is

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consistent with their disclosed hydrophilic character. Due to the rich polyphenolic composition and high antioxidant activity, extracts from the leaves and fruits of blackcurrant protect the organism or food products from the harmful effects of free radicals. In addition, they slightly change the properties of biomembrane lipids and lipid vesicles once they incorporate these extracts. This kind of knowledge is not only of pro-health importance, but can also be used in the food, pharmaceuticals or cosmetics industry, for designing liposomal structures as carriers of drugs, dietary supplements and extracts as biologically active substances.

Keywords: Blackcurrant extracts, Erythrocyte membrane, Phase transition, UPLC, Polyphenols, Partition coefficient, FTIR spectroscopy, Membrane fluidity Highlights  Blackcurrant leaves and fruits are a rich source of polyphenols. 2

ACCEPTED MANUSCRIPT  Blackcurrant polyphenols practically do not undergo degradation during storage.  Blackcurrant extracts exhibit high antioxidant activity in relation to biomembrane.  Blackcurrant polyphenols bind to the membrane surface, without altering its structure.

AA – L (+) ascorbic acid AAPH – 2,2’-azobis (2-amidinopropane) dihydrochloride

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BCF – blackcurrant fruits

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Abbreviations:

BCL – blackcurrant leaves

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BHA – butylated hydroxyanisole DPH – 1,6-diphenyl-1,3,5-hexatriene DSC – differential scanning calorimetry

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DPPC – 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine FTIR – Fourier transform infrared spectroscopy

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GAE – gallic acid

Laurdan – 6-dodecanoyl-2-dimethylaminonaphthalene MLV – multilamellar liposomes

TMA-DPH – 1-(4trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate

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Prodan – 6-propionyl-2-dimethylaminonaphthalene RBC – erythrocyte membrane

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RBCL – lipids extracted from erythrocyte membrane SUV – small unilamellar liposomes

1. Introduction Modern medicine cannot provide effective treatment of various dangerous diseases, including civilizational ones. Scientific studies show that a cause of serious diseases, including Alzheimer's disease, Parkinson's disease, cancer and others, is the oxidative stress caused by an excess of free radicals in the organism (Bullon, Newman, & Battino, 2014; Montezano, & Touyz, 2014; Pitocco et al., 2013). The radicals arise in physiological processes and as a result of physicochemical factors of the body. Therefore, intensive research is focused on the search for new efficient protective substances, of plant origin in particular. Numerous studies have shown that plant polyphenols are effective scavengers

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ACCEPTED MANUSCRIPT of free radicals. Another advantage of plant substances is the fact that they have high biological activity (Zhang et al., 2011; Choudhary, & Swarnkar, 2011). These substances also exhibit, apart from antioxidant activity, a number of health-supporting properties:

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anti-inflammatory, anticancer, and antimycosis (Betts et al., 2013; Gonzalez-Vallinas et al., 2013). Thus

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they exhibit a range of protective properties, which allows them to be classified as a group of modern medicines (Kashani et al., 2012). The mechanism responsible for this health protective effect of

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polyphenolic compounds is not yet fully understood; hence intensive studies are being conducted in this field worldwide. In order to clarify the mechanism of action of polyphenolic compounds at the

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molecular and cell level, it is necessary to conduct research also on simple models of well-known structure (Virgili, & Marino, 2008). The first and main place of impact of various physico-chemical

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factors on the organism is the cell membrane and, therefore, it is important to determine the effects of polyphenolic compounds on membranes. Test results indicate that polyphenols can alter the properties of biological membranes by changing both the protein and lipid phase (Pawlikowska-Pawlęga et al.,

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2007; Hendrich, 2006). These compounds interacting with cellular membranes exhibit protective effects in relation to cells, e.g. erythrocytes (Cyboran, Oszmiański, & Kleszczyńska, 2013), hepatocytes (Kim et al., 2012), human colon epithelium and myofibroblast cells (Tomczyk et al., 2013). Modification of membrane properties by these substances can also lead to impaired functioning of cells, including

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inhibition of activity of mitochondrial enzymes (Hodnick, Duval, & Pardini, 1994), aggregation of erythrocyte membrane proteins (Chen et al., 2011), and DNA damage in mice spleen cells (Fan, & Lou,

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2008). Therefore, it is important to determine the molecular mechanism of interaction of these substances with organisms, which will allow their safe and rational use. Especially blackcurrant is of great interest among scientists compared to other plant substances, due to its content of polyphenolic compounds that exhibit a variety of healthy properties. The blackcurrant shrub is commonly cultivated in temperate climates around the world. Its fruits are a rich source of vitamins, macro- and microelements, organic acids, pectins and essential oils, as well as a range of polyphenolic compounds showing positive action on the organism (Mattila et al., 2001; Molan, Liu, & Kruger, 2010; Tabart et al., 2012; Brangoulo & Molan, 2011). The polyphenolic substances contained in fruits and leaves of blackcurrant have a protective action and support the treatment of many diseases. They exhibit anti-inflammatory, antifungal, antioxidant, probiotic and anticancer effects, among others (Szachowicz-Petelska, Dobrzyńska, Skrzydlewska, & Figaszewski, 2012; Cyboran, Bonarska-Kujawa, Oszmiański, & Kleszczyńska, 2011; Kirsch et al., 2009; Molan, Liu, & Kruger, 2010; Jia et al., 2012a; 4

ACCEPTED MANUSCRIPT Bishayee et al., 2011; Sivam, Sun-Waterhouse, Perera, & Waterhouse, 2012). In addition, it has been documented that they improve the metabolism, regulate oxygen economy and improve vision (McDougall, Kulkarni, & Stewart, 2009; Matsumoto et al., 2005; Matsumoto et al., 2006). In our

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previous work (Bonarska-Kujawa et al., 2014; Cyboran, Bonarska-Kujawa, Oszmiański, &

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Kleszczyńska, 2011) we have shown that extracts from the leaves and fruits of blackcurrant in varying degrees modify cells and the membranes of red blood cells. Compounds contained in them do not

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damage the membrane of erythrocytes but strengthen it. In addition, the compounds contained in the extracts induce changes in the shape of erythrocytes, becoming located primarily in the outer monolayer

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of the erythrocyte membrane. We have shown also that they effectively protect the membranes of red blood cells against free radicals induced by UVC radiation and AAPH compound. In order to better

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understand the mechanism responsible for the observed effects on the cells and membranes, in this study we carried out biophysical tests aiming to determine the effect of the extracts mainly on the lipid phase of membrane. Understanding this mechanism will make it possible to extend the range of their activities

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and the use of the substances both in medicine and in the food industry and cosmetics. In the present study we conducted an analysis of the extracts and research was performed defining the biological activity of phenolic compounds contained in the leaves and fruits of blackcurrant in respect of biological membranes and lipid membrane models. Stability of compounds contained therein, and their

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physicochemical properties, are also specified. The main aim of the study was to determine their influence on the different model membranes, and in particular on the membrane lipid phase.

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Additionally, the antioxidant activity of the extracts was determined. This research was conducted on the membranes of red blood cells and on model membranes, i.e. liposomes created from DPPC and lipids extracted from erythrocyte membranes. Using chromatography, spectrophotometry, calorimetry and fluorimetry, the biological activity of the extracts, including antioxidative activity, was determined on the basis of the effects of the substances contained therein on the membranes. 2. Materials Blackcurrant (Ribes nigrum L.) leaves and fruits were harvested from an experimental field of the Garden of Medicinal Plants herbarium of the Medical University of Wroclaw, Poland. Polyphenols were isolated from fruits and leaves by extraction with water containing 200 ppm of SO2, the ratio of solvent to fruits (or leaves) being 3:1. The extracts were absorbed on Purolite AP 400 (UK) for further purification. The polyphenols were then eluted out with 80% ethanol, concentrated and freeze-dried. By

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ACCEPTED MANUSCRIPT means of the above method a mixture of polyphenols was obtained using the method described by Gąsiorowski et al. (1997). The studies were conducted on isolated erythrocyte membranes (RBC), small unilamellar liposomes

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(SUVs) and multilamellar liposomes (MLVs). Pig erythrocyte membranes were obtained from fresh

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blood using the method described by Dodge, Mitchell, & Hanahan (1963). The content of erythrocyte membranes in the samples was determined on the basis of protein concentration, which was assayed

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using the Bradford method (1976), and it was 100 mg/ml. The choice of pig erythrocytes was dictated by the fact that this cell’s percentage content of lipids is closest to that of the human erythrocyte, and the

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blood was readily available. Fresh blood was taken each time to a physiological solution of sodium chloride with heparin added.

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Small unilamellar liposomes (SUV) were composed of lipids extracted from erythrocyte membranes (RBCL) according to the method described by Maddy, Dunn, & Kelly (1972), and of DPPC purchased from Sigma Aldrich, Steinheim, Germany. All lipids were evaporated to dryness under nitrogen.

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Subsequently, a phosphate buffer of pH 7.4 was added and multilamellar liposomes (MLV) were formed by mechanical shaking. Then SUVs were formed using a Sonics VCX750 sonicator (Sonics & Materials, Inc.).

The fluorescent probes Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene), Prodan (6-propionyl-2DPH

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dimethylaminonaphthalene),

(1,6-diphenyl-1,3,5-hexatriene)

trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene

and

p-toluenesulfonate)

TMA-DPH

were

purchased

(1-(4from

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Molecular Probes, Eugene, Oregon, USA. The Folin-Ciocalteu phenol reagent, 2,2’-diazobis (2amidinopropane) dihydrochloride (AAPH), butylated hydroxyanisole (BHA), gallic acid (GAE) and L(+) ascorbic acid (AA) were purchased from Sigma-Aldrich, Inc., Steinheim, Germany.

3. Methods 3.1 UPLC/DAD and UPLC-PDA−Q/TOF-MS methods The content of polyphenols in the extracts of blackcurrant leaves (BCL) and fruits (BCF) was determined by means of liquid chromatography (UPLC/DAD) and the method of UPLC-PDA-Q/TOFMS analysis described by Oszmiański, Kolniak-Ostek, & Wojdyło (2013). Identification of polyphenol in the extracts was carried out using the ACQUITY ultra-performance LC system (UPLC) with binary solvent manager (Waters Corp.; Milford, MA) and a Micromass Q-Tof Micro mass spectrometer (Waters; Manchester, UK) equipped with an electrospray ionization (ESI) 6

ACCEPTED MANUSCRIPT source operating in negative and positive mode. Separations of individual polyphenols were carried out using a UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm, Waters Corp.; Milford, MA) at 30°C. Samples (10 μl) were injected, and elution was completed in 15 min with a sequence of linear gradients and

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isocratic flow rate of 0.45 ml/min. The mobile phase was composed of solvent A (4.5% formic acid, v/v)

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and solvent B (100% acetonitrile). The program began with isocratic elution with 99% A (0−1 min), and then a linear gradient was used for 12 min, lowering A to 0%; from 12.5 to 13.5 min the initial

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composition (99% A) was used and then held constant to re-equilibrate the column. Analysis was carried out using full scan, data-dependent MS scanning from m/z 100 to 1500. Leucine encephalin was used as

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the internal reference compound during ESI-MS accurate mass experiments and was permanently introduced via the LockSpray channel using a Hamilton pump. The lock mass correction was ±1.000 for

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the mass window. All quadrupole time-of-flight mass spectrometry (Q/TOF-MS) chromatograms are displayed as base peak intensity (BPI) chromatograms and scaled to 12 400 counts per second (cps) (=100%). The effluent was led directly to an electrospray source with a source block temperature of

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130°C, desolvation temperature of 350°C, capillary voltage of 2.5 kV, and cone voltage of 30 V. Nitrogen was used as the desolvation gas at a flow rate of 300 l/h. The characterization of single components was carried out via the retention time and the accurate molecular masses. Each compound had a negative and positive mode before and after fragmentation.

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The data obtained from UPLC/MS were subsequently entered into the MassLynx 4.0 ChromaLynx Application Manager software. On the basis of these data, the software is able to scan different samples

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for the characterized substances. The runs were monitored at the following wavelengths: hydroxycinnamates at 320 nm, flavonol glycosides at 360 nm, and anthocyanins at 520 nm. Photodiode detector (PDA) spectra were measured over the wavelength range 200−600 nm in steps of 2 nm. Retention times and spectra were compared with those of pure standards within 200−600 nm. Calibration curves were run for the external standards 3-, and 5-O-caffeoylquinic acids, p-coumaric acid, quercetin-3-O-rutinoside and -3-O-glucoside, peonidin-3-O-rutinoside, delphinidin-3-O-rutinoside, and petunidin-3-O-rutinoside at concentrations ranging from 0.05 to 5 mg/ml. The results were expressed as milligrams per g of dry matter (mg/g).

3.2 Folin-Ciocalteu method Total phenolic content (TPC) was determined using Folin-Ciocalteu (F-C) reagent, adapted from Singleton & Rossi (1965). The standard curve was prepared for gallic acid. The results were expressed as 7

ACCEPTED MANUSCRIPT mg gallic acid equivalents (GAE) per 1 g of dry matter. TPC in the BCL and BCF extracts was investigated twice at a yearly interval. Extracts were stored at a temperature of -15°C, and before dissolving they were left for 15 min at room temperature in the dark. Stability of polyphenols in the

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extracts was estimated based on the change in their amount after a year of storage, calculated using the

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formula TPC = [(TPC0 - TPC12)/TPC0] 100%, where TFC0 is the initial amount and TFC12 the amount

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after 12 months.

3.3 Affinity for organic/aqueous media

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The partition coefficient (P) between octanol and phosphate buffer (pH=7.4) for the polyphenolic compounds contained in the extracts was estimated by the spectrophotometric method described by

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Nenadis, Zafiropoulou, & Tsimidou (2003). The extracts were added to 3 ml of octanol and incubated for 30 min at 37°C. Then spectra of solutions were taken using a Cary 300 spectrophotometer (Varian), 200-380 nm (UV). The absorbance A0, corresponding to the maximum concentration of tested

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compounds in the organic phase, represented by octanol, was read at the wavelengths at which absorption maxima occurred. Then phosphate buffer was added to the octanol in a 1: 1 ratio (v:v), stirred for a minute using a shaker (2500 rpm) and incubated for 30 min (t = 37°C). After incubation the samples were centrifuged for 5 min (2500 rpm), and then absorbance of the organic phase at the same

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wavelength was measured.

Partition coefficient P was calculated based on the formula P=Ax/(A0 – Ax) where: A0 is the absorbance

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corresponding to the maximum concentration of extract in the organic phase (represented by octanol), and Ax is the absorbance corresponding to the concentration of extracts that remained in the organic phase after 30 min of incubation with phosphate buffer (measured in the organic phase). The partition coefficient was expressed as log P. With an increasing negative value of log P, the hydrophilic nature of the compounds increased, and thus their affinity for aqueous media compared to lipid media.

3.4 Fluidity and packing arrangement of the membranes The effect of extracts of BCL and BCF on packing arrangement and fluidity of lipids in the erythrocyte membrane (ghosts) and model lipid membrane (RBCL and DPPC liposomes) was investigated using the fluorimetric method described in our previous work (Bonarska-Kujawa, Pruchnik, & Kleszczyńska, 2012), with minor modifications. The SUVs of RBCL or DPPC were formed in the presence of fluorescent probes. Control samples contained only lipid (at 1 mg/ml concentration) or erythrocyte 8

ACCEPTED MANUSCRIPT membranes (at 0.1 mg/ml protein concentration) in suspension with a fluorescence probe, whose concentration in the samples was 10 M, an appropriate compound at a concentration in the range from 0.005 to 0.05 mg/ml being added to the remaining samples.

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Fluorescence intensity was measured with the Laurdan, Prodan and DPH probes. For liposomes formed

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of DPPC the measurements were made above and below the main phase transition (Laurdan, Prodan and DPH). For erythrocyte membranes and for liposomes formed of RBCL the measurements were made at

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37°C (DPH) and at a different temperature (Laurdan). These fluorescent probes were chosen because each of them becomes incorporated in a different region of the lipid bilayer. The active part

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(fluorophore) of the DPH probe is localized in the hydrophobic and Laurdan and Prodan in hydrophilic regions of the bilayer. Such differentiated incorporation of the probes gives an insight into the structural

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changes caused by incorporation of BCL and BCF (Harris, Best, & Bell, 2002; Lakowicz, 2006; Parasassi, Krasnowska, Bagatolli, & Gratton, 1998). The excitation and emission wavelengths were as follows: for the DPH probe, ex = 360 nm, em = 425 nm, for Laurdan and Prodan ex = 360 nm, and the

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emitted fluorescence was recorded at two wavelengths of 440 and 490 nm. On the basis of the measured fluorescence intensity of probes, the values of fluorescence anisotropy (A) for DPH and generalized polarization (GP) for Laurdan and Prodan were calculated using the formulae described by Lakowicz (2006) and Parasassi, Krasnowska, Bagatolli, & Gratton (1998).

A

( I II  GI ) (1); ( I II  2GI )

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Fluorescence anisotropy (A) for the DPH probe was calculated using the formula:

where III and I are fluorescence intensities observed in directions parallel and perpendicular, respectively, to the polarization direction of the exciting wave. G is an apparatus constant dependent on the emission wavelength. Generalized polarization (GP) for Laurdan and Prodan probes was calculated with the formula: GP 

( Ib  I r ) (2); ( Ib  I r )

where Ib is fluorescence intensity at  = 440 nm, and Ir is fluorescence intensity at  = 490 nm. 3.5 Phase transition of lipid membranes In the calorimetric studies the effect of BCL and BCF on the pretransition (Tp) and main transition (Tm) temperature of DPPC was analyzed. For that purpose differential scanning calorimetry (DSC) was used.

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ACCEPTED MANUSCRIPT Samples for DSC consisted of multilamellar vesicles (MLVs) made of DPPC modified with BCL and BCF (DPPC+BCL or DPPC+BCF) (Tien, 1974). The measurements were made with a calorimeter of Mettler Toledo Thermal Analysis System D.S.C. 821e (2ºC/min scanning rate). The lipids were

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dissolved in chloroform and then evaporated for approx. 2 h under nitrogen to dryness. The obtained

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film was admixed with BCL or BCF dissolved in phosphate buffer of pH 7.4 and liposomes were formed by mechanical shaking at a temperature above the main phase transition. The lipid concentration

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in the samples was 25 mg/ml. The prepared dispersions of pure DPPC (control sample) and DPPC with

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the compounds added were encapsulated in 40 μl volumes and left for 48 h at 4ºC before measurement.

3.6 Hydration of lipid membrane

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The experiments were performed with DPPC multilamellar liposomes (MLVs). IR spectra were taken of a 70 mM liposome suspension. Mixtures of lecithin dissolved in chloroform (1 mg/ml) and extract (BCL or BCF) dissolved in methanol were dried under nitrogen for a few minutes and under a vacuum for

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another 2 h. Dried lipids were resuspended in a phosphate buffer of pH 7.4. Extract concentration was 0.05 mg/ml. The preparations were intensively shaken with a vortex mixer under nitrogen at 45°C. The measurements were performed using a Thermo Nicolet 6700 spectrometer with ZnSe crystal at room temperature. Each single spectrum was obtained from 128 records at 2 cm-1 resolution in the range 700-

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4000 cm-1. Preliminary elaboration of a spectrum was done using the EZ OMNIC v 8.0 program, also by Thermo Nicolet. After filtering the noise out from the spectrum of the object studied, the spectrum of the

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buffer solution was subtracted in order to remove the OH band of water and the baseline was corrected. In spectra thus prepared we examined four bands located in the range 3000–2800 cm-1 from vibrations of CH2 and CH3 groups of alkyl chains, at 1780–1700 cm-1, which correspond to carbonyl group (C=O) vibrations, at 1300–1000 cm-1, which correspond to vibrations of the phosphate group (PO2), and at 1000–940 cm-1, corresponding to choline group (N-CH3) vibrations. The frequencies of methylene and methyl groups of alkyl chains depend on mobility (fluidity) of the chains and increase e.g. with increasing temperature or during transition from the gel to the liquidcrystalline state. The increase in the wavenumber of these bands (3000–2800 cm-1) testifies to an increase in liquidity of the hydrophobic part of the membrane. The carbonyl group and even more the phosphate groups form hydrogen bonds with water. The carbonyl group can bind one molecule of water, while the phosphate group can bind a few. Hence the carbonyl and phosphate bands of phospholipids are the sum of vibrations of C=O or PO2 groups that are at different levels of hydration (Blume, Hubner, & 10

ACCEPTED MANUSCRIPT Messner, 1988; Wong, Siminovitch, & Mantsch, 1988; Lewis, McElhaney, Pohle, & Mantsch, 1994; Attar et al., 2000). Vibrations of C=O and PO2 groups which do not have water bonds are represented by the wavenumbers ≈ 1740 cm-1 and ≈ 1260 cm-1. Each bound water molecule moves these values by

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about 20 cm-1 in the direction of smaller values. The changes observed in phosphate (1300–1000 cm-1)

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and carbonyl (1760–1700 cm-1) bands testify, therefore, to changes in the degree of hydration of carbonyl and phosphate groups. The frequency of vibration of the methyl groups of the choline part

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(trimethyl ammonium) depends on the medium and grows slightly when its viscosity decreases. The wavenumber of vibration of completely dry lipid is slightly more than two units less than that of

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completely hydrated lipid membrane (of a liposome). The half width of the band depends on the

3.7 Antioxidant activity of the extracts

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"stiffness" throughout the polar head, i.e. on possible mobility along the skeleton -N-C-C-O-P-O-.

Antioxidant activity of BCL and BCF extracts was determined using the fluorimetric method described

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in our previous work (Bonarska-Kujawa, Pruchnik, & Kleszczyńska, 2012) with minor modifications. These studies were carried out on membranes of erythrocytes at 0.1 mg/ml protein concentration in the sample and small unilamellar liposomes formed from RBCL at 1 mg/ml concentration in the sample. The TMA-DPH probe at concentration of 10 µM in the sample was used in these experiments.

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Excitation and emission wavelengths of the TMA-DPH probe were λex = 362 nm and λem = 428 nm. Suspensions of erythrocyte membranes or RBCL liposomes (3 ml) were treated with the chemical

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oxidation inductor AAPH (60 µl at 1 mM) for 30 min at 37°C. Free radicals, released in the process of AAPH decomposition, caused quenching of TMA-DPH fluorescence and decreased fluorescence intensity. Relative fluorescence, i.e. the ratio of AAPH-oxidized fluorescence probe to the initial fluorescence of the probe, was adopted as a measure of the extent of lipid oxidation. The percentage of lipid oxidation inhibition was calculated from the following formula: % of oxidation inhibition 

( Fx  Fu )  100 % (3); ( Fk  Fu )

where: Fx = relative fluorescence of sample oxidized by AAPH, for 30 min in the presence of the compounds, Fu = relative fluorescence of the control sample, oxidized by AAPH, without the compounds, measured after 30 min.

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ACCEPTED MANUSCRIPT Fk = relative fluorescence of the blank sample, not subjected to oxidation procedure, measured after 30 min. Antioxidant activity of the extracts was evaluated in relation to the antioxidant activity of the synthetic

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antioxidant BHA, which is often used as a preservative in food and animal feed but also in cosmetics

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and petroleum products (Sacchez-Gallego et al., 2011), and also in relation to ascorbic acid (AA). As a measure of antioxidant activity of used substances the value of their concentration which causes 50%

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inhibition of lipid peroxidation was adopted (IC50).

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3.8 Statistical analysis

Statistical analysis of the results was performed using the STATISTICA 9.0 (StatSoft PL) software. Depending on the experiment, the following tests were used: the t-test (for independent samples) and

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Dunnett’s post-hoc ANOVA test at significance level α = 0.01 or α = 0.05. The t-test was used to estimate the differences between mean values of measured parameters. Dunnett’s test was used to

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estimate the differences between mean value of measured parameters of control samples and mean value of the parameters for samples modified with the extracts. All the experiments were done in at least five replicates, the results being presented as means ± standard deviation (α = 0.05). 4. Results and discussion

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4.1 UPLC-DAD and UPLC-PDA−Q/TOF-MS methods Quantitative and qualitative contents of polyphenolic compounds in BCL and BCF extracts are given in

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Table 1. Chromatograms that served for identification of polyphenols occurring in the extracts, using the UPLC-DAD method, are published in our previous papers. For BCL they are presented by Oszmiański, Wojdyło, Gorzelany, & Kapusta (2011), and for BCF by Wojdyło, Oszmiański, Milczarek, & Wietrzyk (2013).

Table 1. Content and characterization of phenolic compounds of the blackcurrant leaf (BCL) and fruit (BCF) preparations, using their spectral characteristics in UPLC-DAD (tr – retention time, λmax) and negative and positive ions (MS) in UPLC-PDA−Q/TOF-MS. [M_H]/MS2 (m/z)

BCFb mg/g

BCLc mg/g

[nm]

MS (m/z)

1

p-Coumaroylglucose

2.26

312

325

145

0.31

0.35

2

Neochlorogenic acid

2.49

324

353

191

1.37

1.33

3

Acetyl-caffeoylquinic acid

2.80

312

359

161

-

1.00

No. Phenolic compounds

tr

λmax

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ACCEPTED MANUSCRIPT Caffeoylglucose

2.87

320

341

179

7.52

-

5

p-Coumaroylglucose

2.96

313

325

145

-

0.40

6

Caffeoylglucose

3.15

320

341

179

-

1.29

7

p-Coumaroyl quinic acid

3.34

312

337

191

1.37

-

8

(+)Catechin

3.38

278

289

-

4.73

8.38

9

Caffeoylglucose

3.54

320

341

179

0.61

-

10

Chlorogenic acid

3.68

324

353

191

0.21

15.41

11

p-Coumaroylglucose

3.87

313

325

145

3.19

1.03

12

Caffeoylquinic acid

4.10

324

353

191

-

1.10

525

465

a

a

72.62

-

611

a

303

a

176.30

-

771

301

-

1.48

RI P

14

Delphinidin 3-O-rutinoside

4.50

521

15

Quercetin glucoside-rutinoside

4.52

350

SC

303

16

Feruoyl-glucose

4.70

327

355

191

1.40

-

17

Myricetin-pentoside

4.76

349

449

316

0.60

2.60

515

a

285

a

22.85

-

324

353

191

-

1.12

278

289

-

4.73

2.62

313

337

191

-

0.89

a

287

a

18

Cyanidin 3-O-glucoside

4.93

19

Cryptochlorogenic acid

4.94

20

(-)Epicatechin

4.95

21

p-Coumaroylquinic acid

5.20

Kaempferol-glucoside-rutinoside

5.24

24

Petunidin-3-O-rutinoside

25

Kaempferol-glucoside-acetate

26

Myricetin glucoside

27

Myricetin rutinoside

28

Peonidin-3-O-rutinoside

29

Myricetin glucoside acetate

30

Quercetin rutinoside

31

Quercetin glucoside

32

449

514

595

148.07

-

5.34

340

755

285

-

0.78

5.66

533

625a

317a

4.90

-

5.96

346

651

285

5.99

350

479

316

2.58

-

6.17

350

625

316

19.46

5.10

6.38

533

609a

301a

1.98

-

6.76

350

521

316

1.75

1.52

7.36

351

609

301

1.99

28.60

7.53

351

463

301

1.62

8.63

Quercetin glucoside acetate

8.19

352

505

301

0.39

61.05

33

Kaempferol rutinoside

8.46

340

593

285

0.24

8.30

34

Quercetin glucoside acetate

8.56

349

505

301

-

3.01

35

Kaempferol glucoside

8.66

340

447

285

0.11

8.46

36

Isorhamnetin rutinoside

8.80

351

623

315

0.09

-

37

Kaempferol glucoside acetate

9.55

346

489

285

-

27.30

38

Kaempferol glucoside acetate

9.70

346

489

285

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Cyanidin-3-O-rutinoside

23

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22

Total a

4.22

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Delphinidin 3-O-glucoside

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13

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4

1.64

-

1.22

480.99

194.61

positive ions (MS), b chromatograms were published earlier by Wojdyło et al., 2013; c chromatograms were published earlier by Oszmiański et al., 2011;

The quantitative and qualitative analysis of polyphenol composition of the extracts BCL and BCF revealed that they differ not only in the quantity but also the type of polyphenolic compounds. According to the study, the extract from blackcurrant fruit contains more than twice the amount of polyphenolic compounds (481 mg/g) of the leaf extract (194 mg/g). In addition, in the fruit extract 13

ACCEPTED MANUSCRIPT anthocyanin glycosides dominate, mainly cyanidin and delphinidin rutinosides, while flavonol glycosides dominate in the leaves, especially quercetin (Table 1). The above results are similar to those obtained by us in our previous works using UPLC-DAD and UPLC-ESI-MS methods (Oszmiański,

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Wojdyło, Gorzelany, & Kapusta, 2011; Wojdyło, Oszmiański, Milczarek, & Wietrzyk) and by other

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authors. Derivatives of anthocyanins in blackcurrant fruit were identified by other authors (Slimestad & Solheim, 2002; Jia et al., 2012b; Anttonen & Karjalainen, 2006), as was the presence of quercetin and

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kaempferol derivatives in the leaves (Raudseppa et al., 2010; Heabc et al., 2010; Tabat et al., 2006).

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4.2 Folin-Ciocalteu method

The calculated percentage change in the content of polyphenols in the extracts was as follows: for the

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blackcurrant fruit extract TPC = 15 ± 4.2% and for the extract from leaves TPC = 10 ± 3.1%. The results obtained indicate that the compounds contained in both the extracts, when stored in appropriate conditions (lyophilized mixture, -15°C), showed good stability. The total amount of polyphenolic

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compounds in leaves decreased by approx. 10% per year, while in BCF a decline to 15% in relation to the initial value was observed. A decrease in the content of polyphenolic compounds during storage was observed by other authors for extract from blackcurrant buds (Tabart et al., 2007), whose durability was evaluated on the basis of the decline in antioxidant activity. The extract from blackcurrant fruit, being

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rich in anthocyanins, is less stable, as these compounds during storage easily succumb to degradation under the influence of many factors such as temperature, oxygen or light (Patras, Brunton, O’Donnell, &

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Tiwari, 2010), and polymerization of the compounds with other polyphenols (Ochoa, Kesseler, Vullioud, & Lozano, 1999).

4.3 Affinity for organic/aqueous media Using spectrophotometric methods, the affinity of the polyphenols contained in BCL and BCF for the lipid/water phase was determined on the basis of the partition coefficient (P) between octanol and phosphate buffer. Affinity is expressed in the form of log P, whose values were for BCL extract -0.286 ± 0.015 and for BCF -0.435 ± 0.08. The obtained values indicate that polyphenols contained in the extracts have a higher affinity for the aqueous phase (represented by phosphate buffer), demonstrating their hydrophilic nature. In addition, the results indicate that the compounds contained in BCF have a somewhat more hydrophilic nature than those of BCL (greater negative value of log P).

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ACCEPTED MANUSCRIPT Literature data indicate that individual phenolic compounds in the extracts studied also show hydrophilic character. The BCL extract contains mainly flavonol glycosides and chlorogenic acid. The presence of sugar residues increases the polarity of flavonols, and also chlorogenic acid shows a more hydrophilic

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character compared to other phenolic acids (Rastija, Nikolić, & Masand, 2013), whereas BCF extract

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contains mainly anthocyanin glycosides (cyanidin-3-0-rutinoside and delphinidin-3-0-rutinoside), which exhibit higher hydrophilicity than the compounds of BCL. As shown by Vather, Martin, Au, & McGhie

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(2006), anthocyanins containing rutinose may have even twice as small partition coefficients (octanol/water) than those containing glucose. The hydrophilic character of the extract compounds is

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confirmed in other assays of this study.

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4.4 Fluidity and packing arrangement of the membranes

The effect of blackcurrant extracts on fluidity of the lipid phase of RBC and RBCL liposome membranes was studied on the basis of fluorescence anisotropy measured with the fluorescent probe

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DPH. The results of fluorescence anisotropy for such a membrane are presented in Table 2.

Table 2. Values of fluorescence anisotropy (A) of the DPH probe for the erythrocyte membrane (RBC)

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and liposomes of RBCL modified by blackcurrant extracts (BCL – leaves, BCF – fruits) at 37°C.

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Statistical analysis was conducted using the Dunnett test. Statistically significant differences between results of control and modified samples are denoted, respectively: a level α = 0.01, b level α = 0.05.

DPH anisotropy ± SD Concentration [mg/ml]

BCF

BCL

RBC

RBCL

RBC

RBCL

0.245 ± 0.013

0.202 ± 0.002

0.245 ± 0.013

0.202 ± 0.002

0.005

0.229 ± 0.007

0.201 ± 0.002

0.231 ± 0.016

0.201 ± 0.002

0.0075

0.227 ± 0.008b

0.200 ± 0.001

0.225 ± 0.013b

0.201 ± 0.002

b

0.200 ± 0.002

Control

0.01

0.230 ± 0.013

0.201 ± 0.002

0.223 ± 0.016

0.025

0.229 ± 0.008

0.202 ± 0.002

0.229 ± 0.008

0.05

0.232 ± 0.005

b

0.203 ± 0.005

0.221 ± 0.005

a

0.199 ± 0.002 0.196 ± 0.004b

15

ACCEPTED MANUSCRIPT A lack of significant changes for extracts is observed in the hydrophobic region where the unspecific probe DPH localizes. This means that the polyphenolic compounds contained in the extracts do not modify membrane fluidity in the acyl chains area of membrane lipids.

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We also investigated, using the Laurdan probe, the packing order in the hydrophilic part of RBC and

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liposomes composed of RBCL. The calculated values of general polarization (GP) at 37°C decreased with increasing extract concentrations (Fig. 1), which is indicative of increasing disorder in the

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hydrophilic part of the lipid layer. Though the changes induced by BCL and BCF extracts in liposome membranes are smaller than those in erythrocyte membranes, the conviction remains that the compounds

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become incorporated into erythrocyte and liposome membranes, concentrating mainly in the hydrophilic

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layers.

Fig. 1. Values of generalized polarization (GP) of Laurdan probe for RBC and liposomes composed of RBCL modified by blackcurrant extracts (BCL, BCF) at 37°C. Statistically significant differences between results of control and modified samples are denoted, respectively: a level α = 0.01, b level α = 0.05.

Additionally, the fluorimetric measurements were carried out for erythrocyte membranes and RBCL liposomes for a selected concentration of 0.01 mg/ml of extracts and different temperatures (results not reported). The results show that GP values, calculated on the basis of the emission spectrum of Laurdan

16

ACCEPTED MANUSCRIPT for RBC and RBCL liposomes, depend on temperature. The observed fall in the values of GP for both the membranes indicates a growth in disorder in the hydrophilic part of both the membranes. Using the DPH, Laurdan and Prodan probes, we also investigated the effect of the extracts at 0.05 mg/ml

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concentration on fluidity in the hydrophobic part (using the DPH probe) and degree of order in the

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hydrophilic part (using Laurdan and Prodan probes) of the model membrane composed of DPPC. The calculated values of DPH anisotropy (A) and general polarization (GP) of the Laurdan and Prodan

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probes for SUVs of DPPC are presented in Fig. 2.

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Fig. 2. Values of DPH fluorescence anisotropy (A) and general polarization (GP) of Prodan (B) and Laurdan (C) in SUVs composed of DPPC in absence and presence of extracts (0.05 mg/ml) as a function of temperature.

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The results obtained in studies carried out in the one-component membranes (DPPC) confirm earlier results from fluorimetric studies. Similarly as in the case of RBC and RBCL membranes, the extracts did

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not cause significant changes in fluorescence anisotropy of the DPH probe in the DPPC membrane (Fig. 2A), which means that they do not change the fluidity of the hydrophobic interior of the DPPC membrane. In addition, the observed increase in GP of both Laurdan and Prodan in the liquid-crystalline phase of DPPC, particularly in the presence of BCL extract (Figs. 2B and 2C), confirms the previously reported changes in the hydrophilic area of the membrane. The results obtained in studies using fluorescent probes indicate that the compounds contained in BCL and BCF concentrate mainly in the hydrophilic area of the lipid bilayer, causing an increase in the disorder of the polar heads of lipids, while probably not penetrating deep into the hydrophobic region of the membrane, as the unchanged fluidity indicates. Such localization (in the hydrophilic area of membrane) of individual polyphenolic compounds and those from various plant extracts is confirmed in the works of Cyboran, Oszmiański, & Kleszczyńska (2013) and Bonarska-Kujawa, Pruchnik, & Kleszczyńska (2012). 17

ACCEPTED MANUSCRIPT 4.5 Phase transition of lipid membranes We also examined the impact of extracts on phase transitions of multilamellar liposomes (MLVs) formed with DPPC using differential scanning calorimetry (DSC). Fig. 3 shows curves for selected

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concentrations of extracts (in the range 0.05–10 mg/ml).

Fig. 3. DSC heating curves for MLVs DPPC liposomes containing different concentrations of extract of blackcurrant leaves (A) and fruits (B). The curves are normalized for the amount of DPPC; scan rate

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2°C/min.

Table 3. Temperature values of: (Tm) main phase transition, (T1/2) peak half-width of main phase BCF extracts. Concentration [mg/ml] 0.00 0.05 0.10 0.50

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transition, (Tp) pretransition for DPPC liposomes, in the presence of selected concentrations of BCL and

TEMPERATURE OF PHASE TRANSITIONS BCF BCL

Tm [0C] 41.2 40.7 40.8 40.8

T1/2 [0C] 0.7 0.8 0.8 0.9

Tp [0C] 34.9 34.3 34.8 35.0

Tm [0C] 41.2 40.8 40.7 40.5

T1/2 [0C] 0.7 0.9 0.8 0.9

Tp [0C] 34.9 34.4 34.5 34

Increasing concentration of the extracts caused a very slight decrease (Tm < 0.5°C for BCF and Tm< 1°C for BCL at maximum concentration of 0.5 mg/ml) in the phase transition temperature (Tm) and increase in the peak half-width (Fig. 3, Table 3). Observed changes were greatest for BCL extract. The results for the single-component DPPC membrane demonstrate that blackcurrant extract practically has 18

ACCEPTED MANUSCRIPT no influence on the phase transition temperature of the membrane lipids, and thus it does not modify their acyl chain area, which corresponds to results obtained with RBC and RBCL membranes.

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4.6 Hydration of lipid membrane

The results obtained showed that the tested compounds do not cause any changes in the absorption

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spectrum, in terms of the frequency corresponding to the vibrations of the CH2 and CH3 groups of DPPC acyl chains (2800–3000 cm-1, results not shown). Furthermore, obtained results showed that the

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compounds used cause changes in the carbonyl, choline and phosphate band of the lipid molecules (Fig.

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4).

Fig. 4. DPPC IR spectra, including A) carbonyl band, B) phosphate band C) choline band, of pure DPPC

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liposomes and liposomes with extracts of blackcurrant leaves (BCL) and fruits (BCF). The maximum in the carbonyl band (DPPC) in membranes modified with BCL or BCF is shifted in the direction of lower frequency (Fig. 4A). The changes in the position of the maximum in the carbonyl band suggest that compounds contained in the extracts slightly increase the degree of hydration of carbonyl groups. In addition, in the carbonyl band region an additional maximum appears at 1716 cm-1, which was not seen in the case of unmodified DPPC liposomes. That change in the shape of the spectrum appears to be due to direct interaction between polyphenol compounds and the carbonyl group of lipids, and may be the result of e.g. hydrogen bond formation between OH groups of polyphenols and oxygen of the ester bond of lipids (Pawlikowska-Pawlęga et al., 2012). In the part of the spectrum which corresponds to vibrations of the phosphate groups (Fig. 4B), also visible is a shift of the maximum in the direction of lower frequency, both in the range of asymmetric 19

ACCEPTED MANUSCRIPT vibrations of PO2 (1270–1190 cm-1) and for the frequency corresponding to the symmetric vibrations of PO2 groups (1088 cm-1). The observed changes were significantly greater in the presence of BCL and suggest that the phosphate group region undergoes a small additional hydration under the influence of

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the extracts. In addition, in the band of N-C (970 cm-1) vibrations of choline groups there was observed a

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reduction in the half-width of the peak (Fig. 4C). As in the case of the phosphate band, this effect is much stronger for BCL. The narrowing of the choline bandwidth indicates that, as a result of the

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interaction of BCL and BCF with the membrane, a restriction on the choline group mobility is imposed. The observed changes suggest, therefore, that the compounds contained in the extracts interact with

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polar heads of the lipids by limiting their mobility in the area of choline groups and introducing additional water molecules in the polar region of the DPPC membrane. The FTIR studies were

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confirmed by the fluorimetric and calorimetric tests, which registered slight changes only in the polar area of DPPC and their absence in the area of hydrocarbon chains of lipids. In addition, BCL extract causes more changes on the surface of the membrane, which is also in line with the previously

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evidenced less hydrophilic nature of this extract. 4.7 Antioxidant activity of extracts

Examples of relative fluorescence intensity kinetic curves of the probe TMA-DPH in the presence of

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BCL and BCF extracts are shown in Fig. 5. With the increase in the concentration of the antioxidant, the intensity of fluorescence grows, in proportion to the degree of membrane lipid oxidation inhibition. The

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polyphenol compounds contained in the extracts inhibit the oxidation process within membranes (the probe), because they bind free radicals.

20

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ACCEPTED MANUSCRIPT

Fig. 5. Relative fluorescence intensity of TMA-DPH probe in the absence and presence of blackcurrant

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leaf (A, C) and fruit (B, D) extracts in erythrocyte membranes (A, B) and liposome membranes created

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on RBCL (C, D).

Based on the kinetics of the oxidation curves obtained for various concentrations of BCL and BCF, the

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results are given in Fig. 6.

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concentration responsible for 50% oxidation inhibition of the membrane lipids (IC 50) was found. The

Fig. 6. Values of IC50 for erythrocyte membranes (RBC) and liposomes (RBCL) for blackcurrant extracts (BCL and BCF). The results obtained indicate that the compounds contained in the extracts protect erythrocyte and lipid membranes against oxidation. Both the extracts show similar antioxidant activity on the lipid phase, while the antioxidant activity of the BCL extract is about 30% higher than that of BCF towards lipids in the membrane of red blood cells (Fig. 6). In our previous works it was found that BCL extract is a weaker antioxidant than apple and strawberry leaf extracts (Cyboran et al., 2011), but it is more effective in protecting the erythrocyte membrane against oxidation induced by UVC than BCF extract. In 21

ACCEPTED MANUSCRIPT fluorimetric studies, using the DPH-PA probe, we found that BCL extract is a weaker scavenger of free radicals which arise in the process of homolytic disintegration of AAPH compared to BCF extract, when tested on erythrocytes (Bonarska-Kujawa et al., 2014). Research carried out using the same inducer, but

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with a different fluorescent probe (TMA-DPH), showed, in contrast, that the extract from the leaves is a

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more efficient antioxidant. These differences result from different locations of these probes in the membrane: the DPH-PA probe emits fluorescence from the hydrophilic part of the membrane, while the

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TMA-DPH probe is located deeper at the hydrophilic-hydrophobic interface. The results obtained lead to the conclusion that the two fluorescent probes, which monitor the membrane at different depths of its

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hydrophilic region, signal different activities of the polyphenols of BCL and BCF. The compounds of the leaf extract induce greater changes in the packing arrangement of the hydrophilic region of the

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membrane (Fig. 1). And apparently for that reason they better protect the membrane at the TMA-DPH probe level. This reasoning is supported by the more hydrophobic nature of the leaf extract components. In addition, small differences in antioxidant activity of BCL and BCF extracts in relation to lipid phase

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(RBCL) are correlated with similar changes induced by the compounds in the hydrophilic area of the membrane (Figs. 1 and 6). The higher antioxidant activity of the BCL than BCF extract was also demonstrated in research using various standard in vitro tests (e.g. DPPH) on cells (Tabart et al., 2012). The compounds present in leaves quercetin-3-0-rutinoside and chlorogenic acid at a concentration of 50

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µm inhibit the oxidation of emulsified methyl linoleate (oil-in-water emulsion) by 79% and 35%, whereas the cyanidin-3-rutinoside in fruits does so by 21%, and delphinidin-3-rutinoside has a

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prooxidative action (Kahkonen & Heinonen, 2003). Antioxidant activity of the extracts, which are a mixture of different compounds, depends on many factors, including the method of measurement, the inducer of free radicals, and the nature and quantity of the substances contained therein. It was found that blackcurrant juice rich in anthocyanins was the most effective antioxidant in an in vitro test (DPPH) compared to other fruit juices; however, with regard to yeast cells it showed the lowest antioxidant activity (Slatnar et al., 2012). Antioxidant activity, as research shows, does not always depend on the total amount of phenolic compounds contained in extracts (Tabrat et al., 2012; Saeed, Khan, & Shabbir, 2012; Slatnar et al., 2012). This fact was confirmed by the results of our research, which showed that the BCL extract has higher antioxidant activity, although it contains half the amount of polyphenolic compounds compared to BCF. This is possible because from published research on lipid models it is known that antioxidant activity of phenols depends not only on their ability to donate hydrogen, which plays a key role in assaying their antioxidant 22

ACCEPTED MANUSCRIPT activity in tests such as DPPH, but also many other factors, e.g. their ability to chelate transition metal ions, affinity for the lipid phase, and interaction with emulsifiers and/or proteins (Schwarz et al., 2001). The study has also shown that the tested extracts more effectively protect the membranes of red blood

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cells against free radicals than AA (IC50 = 20.1 ± 1.15 µg/ml) and BHA (IC50 = 36.4 ± 1.5 µg/ml). The

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results indicate that the protection of erythrocytes by the extracts is seven times higher than that shown by BHA and over four times higher than that of AA. This is possible because the synthetic antioxidant

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BHA, as research by other authors shows, is a hydrophobic compound (Fujisawa, Atsumi, Kadoma, & Sakagami, 2002) that is incorporated into the hydrophobic interior of the erythrocyte membrane

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(Sacchez-Gallego et al., 2011). Its presence mainly in the area of hydrocarbon chains restricts its ability to capture free radicals generated by AAPH in the aqueous environment. The hydrophilic extracts in the

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immediate vicinity of free radicals prevent their further propagation, sweeping them away effectively. In addition, the compounds contained in the extracts, due to the presence of numerous hydroxyl groups involved in scavenging free radicals, ensure better protection than the universal, hydrophilic ascorbic

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acid. Single polyphenols contained in the extracts also show better protection of human low-density lipoprotein against oxidation than ascorbic acid (Kahkonen & Heinonen, 2003). The antioxidant activity of the extracts of blackcurrant leaves evidenced here also suggests the potentially wide range of their use in industry. These extracts can be used as effective scavengers of free

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radicals, not only for protection of living organisms, e.g. in the form of dietary supplements or additives

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to enhance cosmetics but also as a natural equivalent of synthetic antioxidants in various industries.

5. Conclusion

The polyphenolic substances contained in blackcurrant fruits and leaves exhibit high antioxidant activity in regard to the biological membrane. That activity depends not only on the percentage of polyphenols in the extract, but also on their kind. This applies in particular to the less known and used extracts from the leaves of blackcurrant. The leaf polyphenols, mainly glycosides of flavonols, although present in quantities of less than half of those of anthocyanin glycosides present in the fruits, show antioxidant activity about 30% higher than that of polyphenols from the fruit. In addition, both the extracts more effectively protect the biological membrane against free radicals than synthetic, commonly used antioxidants such as ascorbic acid and BHA. Not only fruits but also blackcurrant leaves are a source of valuable polyphenolic substances that can effectively protect the body against oxidative stress, preventing the development of dangerous diseases. 23

ACCEPTED MANUSCRIPT Studies have also shown a good shelf life of the extracts; they are only slightly degraded during appropriate storage over a long period of time. In the study, it was found that the used extracts have hydrophilic properties, showing a higher affinity for the aqueous phase (determined on the basis of the

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partition coefficient octanol/phosphate buffer). Hydrophilic properties of the extracts were confirmed in

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biophysical studies, in which for the first time the nature of the interaction between the substances contained in the extracts and the biological membrane was examined. The membrane is the first and

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most important place of contact between various substances and the organism. The effects of the extractmembrane interaction, established on the basis of membrane fluidity tests, packing order of the polar

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heads of lipids, phase transitions and hydration, helped to determine the localization of polyphenolic substances in the membrane, which indicates that the polyphenols present in the extracts in principle do

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not penetrate the hydrophobic region, but bind to the membrane surface, changing its physical parameters.

These biophysical studies allow us to conclude that over the tested range of concentrations the extracts

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of blackcurrant fruits and leaves can be safely used, as they do not show any side effects, do not change the structure of the membrane, and position themselves on its surface without interfering in its function. Such location explains their good antioxidant properties that arise from a kind of barrier created on the surface of the membrane, which is able to reduce free radicals. The results presented indicate that

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blackcurrant fruit and leaf extracts can be safely used as substitutes for synthetic antioxidants, e.g. in the food industry and cosmetics. Their addition will not only affect the sensory qualities of products, such as

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color or smell, but also enrich them with substances of high antioxidative performance.

Acknowledgements

This work was sponsored by the Ministry of Science and Education, scientific project no. N N312 422340 and N N304 173840.

24

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1. Anttonen, M. J., & Karjalainen, R. O. (2006). High-performance liquid chromatography analysis of black currant (Ribes nigrum L.) fruit phenolics grown either conventionally or organically. Journal of Agricultural Food Chemistry, 54, 7530-7538. 2. Attar, M., Kates, M., Khalil, M. B., Carrier, D., & Wong, P. T. T. (2000). A Fourier transform infrared study of the interaction between germ-cell specific sulfogalactosylglycerolipid and dimyristoylglycerophosphocholine. Chemistry and Physics of Lipids, 106, 101-114. 3. Betts, J. W., Wareham, D. W., Haswell, S. J., & Kelly, S. M. (2013). Antifungal synergy of theaflavin and epicatechin combinations against Candida albicans. Journal of Microbiology and Biotechnology, 23(9), 1322-1326. 4. Bishayee, A., Mbimba, T., Thoppil, R., Haznagy-Radnai, E., Sipos, P., Darvesh, A. S., Folkesson, H. G., & Hohmann, J. (2011). Anthocyanin-rich black currant (Ribes nigrum L.) extracts afford chemoprevention against diethylnitrosamine-induced hepatocellular carcinogenesis in rats. Journal of Nutritional Biochemistry, 22, 1035-1046. 5. Blume, A., Hubner, W., & Messner, G. (1988). Fourier transform infrared spectroscopy of 13C=O-labeled phospholipids hydrogen bonding to carbonyl groups. Biochemistry, 27, 82398249. 6. Bonarska-Kujawa, D., Cyboran, S., Żyłka, R., Oszmiański, J., & Kleszczyńska, H. (2014). Biological Activity of Blackcurrant Extracts (Ribes nigrum L.) in relation to erythrocyte membranes. BioMed Research International, ID 783059, 13. 7. Bonarska-Kujawa, D., Pruchnik, H., & Kleszczyńska H. (2012). Interaction of selected anthocyanins with erythrocytes and liposome membranes. Cellular & Molecular Biology Letters, 17, 289-308. 8. Bullon, P., Newman, H. N., & Battino, M. (2014). Obesity, diabetes mellitus, atherosclerosis and chronic periodontitis: a shared pathology via oxidative stress and mitochondrial dysfunction? Periodontology 2000, 64(1). 139-153. 9. Bradford, M. (1976). Rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. 10. Brangoulo, H. L., & Molan, P. C. (2011). Assay of antioxidant capacity of foods using an iron(II)-catalysed lipid peroxidation for greater nutritional relevance. Food Chemistry, 125, 1126-1130. 11. Chen, R., Wang, J. B., Zhang, X-Q., Ren, J., & Zeng, C-M. (2011). Green tea polyphenols epigallocatechin-3-gallate (EGCG) inducted intermolecular cross-linking of membrane proteins. Archives of Biochemistry and Biophysics, 507, 343-349. 12. Choudhary, R. K., & Swarnkar, P. L. (2011). Antioxidant activity of phenolic and flavonoid compounds in some medicinal plants of India. Natural Product Research, 25,1101-1109. 13. Cyboran, S., Oszmiański, J., & Kleszczyńska, H. (2013). Modification of the lipid phase of biological and model membranes by bilberry leaf extract. Food Biophysics, 8, 321-333.

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ACCEPTED MANUSCRIPT Figure captions Fig. 1. Values of generalized polarization (GP) of Laurdan probe for RBC and liposomes composed of RBCL modified by blackcurrant extracts (BCL, BCF) at 37°C. Statistically significant differences

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Fig. 2. Values of DPH fluorescence anisotropy (A) and general polarization (GP) of Prodan (B) and

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Laurdan (C) in SUVs composed of DPPC in absence and presence of extracts (0.05 mg/ml) as a function

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Fig. 3. DSC heating curves for MLV DPPC liposomes containing different concentrations of extract of

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blackcurrant leaves (A) and fruits (B). The curves are normalized for the amount of DPPC; scan rate 2°C/min.

Fig. 4. DPPC IR spectra, including A) carbonyl band, B) phosphate band C) choline band, of pure DPPC

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liposomes and liposomes with extracts of blackcurrant leaves (BCL) and fruits (BCF). Fig. 5. Relative fluorescence intensity of TMA-DPH probe in the absence and presence of blackcurrant leaf (A, C) and fruit (B, D) extracts in erythrocyte membranes (A, B) and liposome membranes created

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Fig. 6. Values of IC50 for erythrocyte membranes (RBC) and liposomes (RBCL) for blackcurrant

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extracts (BCL and BCF).

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ACCEPTED MANUSCRIPT Highlights  Blackcurrant leaves and fruits are rich source of polyphenols.  Blackcurrant polyphenols practically do not undergo degradation during storage.

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 Blackcurrant extracts exhibit high antioxidant activity in relation to biomembrane.

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 Blackcurrant polyphenols bind to membrane surface, without altering its structure.

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