Interaction between Mimic Lipid Membranes and Acylated and

Article pubs.acs.org/JAFC

Interaction between Mimic Lipid Membranes and Acylated and Nonacylated Cyanidin and Its Bioactivity Paulina Strugała,* Anna Dudra, and Janina Gabrielska Department of Physics and Biophysics, Wrocław University of Environmental and Life Sciences, C.K. Norwida 25, 50-375 Wrocław, Poland ABSTRACT: We investigated the effects of acylated cyanidin-3-O-β-(6″-O-E-p-coumaroyl-sambubioside)-5-O-β-glucoside (C3cs-5G) and nonacylated cyanidin, cyanidin-3,5-di-O-β-glucoside (C3,5G) and cyanidin-3-O-β-glucoside (C3G), on cell-mimic membranes (MM) that reflected the membrane lipid composition of tumor cells. The relationship between structural derivatives of cyanidin (Cy-d), membrane interactivity, their antioxidant activity, and interaction with albumin were characterized. Studies showed that Cy-d caused an increase in packing order mainly in the hydrophilic region of the membranes. Cy-d have shown high antioxidant activity against liposome oxidation induced by AAPH and ability to bind to albumin through a static quenching mechanism. The results showed that glycosylation number and the presence of aromatic acid attached to sugars affected the membrane properties, according to the sequence C3-cs-5G > C3,5G > C3G. It can be stated that Cy-d in the process of interaction with MM caused a rigidifying effect, which is fundamental for understanding their anticancer and antioxidant activity and is one of the possible pharmaceutical mechanisms. KEYWORDS: acylated cyanidin, lipid membrane, antioxidants activity, human albumin, anthocyanins

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INTRODUCTION Anthocyanins, a subclass of dietary flavonoids, can be characterized as intensely colored red, purple, or blue. There are two key factors behind the above-mentioned diversity: pH of the environment exposition as well as the molecular structure, for example, the location numbers of glycoside substituents on the anthocyanidin core.1 Numerous studies and review articles have reported beneficial health effects of consuming on a daily basis fruits and vegetables containing anthocyanins.2−4 It is suggested that flower- and fruit-derived flavonoids are able to play a vital role in modifying the inflammatory process. As a result, they are highly likely to potentially provide benefits to individuals, in particular those prone to atherosclerosis/cardiovascular pathology and cancer.5,6 The most common naturally occurring anthocyanins are the 3-O-glucosides and 3,5-O-diglucosides of malvidin, cyanidin, pelargonidin, delphinidin, petunidin, and peonidin. One of the elements of anthocyanins is an anthocyanidin core structure produced in the process of glycosylation by using glucose or other sugars. Alternatively, aliphatic acids and/or aromatic compounds may be obtained in the process of esterification. The term “acylated” is broadly in use to describe these anthocyanin derivatives.6 Acylated anthocyanins are characterized as more stable and resistible against hydrolysis in comparison with nonacylated anthocyanins. Cyanidin-3-O-glucoside, and other derivatives of cyanidin (Cy-d), which are typically associated with anthocyanins in food, are extremely common in the kingdom of plants. By scavenging free radicals activity, those compounds are able to suppress inflammation, protect against endothelial dysfunction, vascular failure, and myocardial damage, and seem to help in preventing cardiovascular disease.7 Possible beneficial effects of anthocyanins require more information about compounds transportation as well as their bioavailability. The anthocyanins © 2016 American Chemical Society

are bioavailable in conjunction with absorption and metabolism, according to some studies.8 Nevertheless, the number of reports devoted to the process of anthocyanins distributing to tissues through the circulatory system is significantly limited. In view of exerting its favorable effect, it is necessary for anthocyanins to be replaced and allocated into the cells, and such a result is bound to be effected only by binding to human serum albumin.9 The membrane interactions and localization of flavonoids, including their subclasses, such as anthocyanins, are fundamental in an attempt to alter membrane-mediated cell signaling cascades and influence the pharmacological activities, including anticarcinogenic, antioxidant, and antimicrobial of anthocyanins.10 To discover the membrane interaction with flavonoids and anthocyanins, a broad range of techniques has been implemented: fluorescence methods, differential scanning calorimetry, NMR and EPR spectroscopy, and molecular dynamic simulation.11,12 Each technique provided specific information about either alteration of molecular dynamics of membrane or localization of bioactive molecules within the lipid bilayer. To evaluate the membrane interactions of natural compounds, both types of biological membranes and biomimic membranes, prepared on the grounds of authentic or extracted lipids, have been commonly in use.13 To mimic the cellular lipid compositions and plasma membranes of interest, membrane specimens were prepared. One of the most common was biomimic membrane with the unilamellar vesicle (liposome) consisting of two concentric layers of membranous lipids that enclose an aqueous volume. A single lipid bilayer Received: Revised: Accepted: Published: 7414

July 8, 2016 September 8, 2016 September 14, 2016 September 14, 2016 DOI: 10.1021/acs.jafc.6b03066 J. Agric. Food Chem. 2016, 64, 7414−7422

Article

Journal of Agricultural and Food Chemistry with incorporated inner and outer leaflets was used to characterized a unilamellar vesicle. Apart from 100 mol % DPPC liposomes (pure lipid), the lipid structure of membrane was diversified by mixing DPPC, POPC, POPE, SOPS, and cholesterol to maintain proper balance of molar ratios. A large number of representative phospholipids were used to prepare tumor biomimic membranes (MM), including POPC:POPE:SOPC:cholesterol (48:24:8:20 mol %).13,14 Although it is commonly known that extremely extended pharmacological spectra are covered by flavonoids, the mode of their interaction with mimic lipid membrane/vesicles has been only partially understood. There is a large number of studies in which it has been shown that flavonoids cause a decrease in membrane fluidity of lipid vesicles.15−17 For example, studies have shown that a large number of compounds (flavones, flavonols, flavanones, flavanonols, anthocyanidins, flavanols, isoflavones, and chalcones) are responsible for decreasing the membrane fluidity by acting at 1−10 μM on the deeper regions of liposomal membranes consisting of POPC, POPE, POPS, and cholesterol, which is said to be a factor of the greatest importance for inhibiting tumor cell proliferation. However, Ajdzanović and others18 reported a EPR spectroscopic study that indicated differences in interactions between membranes and isoflavonoids. According to the observation, genistein was responsible for decreasing the fluidity of erythrocyte membranes near the hydrophilic surfaces. On the contrary, daidzein played a vital role in increasing the fluidity at the membrane deeper regions. It is suggested, on the grounds of comparing membrane interactivity, that 3-hydroxylation of the C ring, nonmodification or 3′,4′-dihydroxylation of the B ring, and 5,7dihydroxylation of the A ring are mainly responsible for producing the most significant membrane interaction to rigidify membranes. The obtained results were proved as a way of comparing between aglycone (quercetin) and glycosides (rutin and isoquercitrin, quercetin 3-O-glucoside). What is more, it was proved that the membrane interactivity of flavonoids can be significantly reduced by glycosylation. Galangin as well as quercetin were responsible for inhibiting the proliferation of tumor cells, while at the same time decreasing the fluidity of their membranes. There are indicators, in the molecular structure of phytochemicals, pointing to their ability to interact with biomembranes. However, in the first place, they depend on research methodology as well as the experimental conditions. This study aims to investigate the interaction of cyanidin derivatives (Cy-d), both acylated cyanidin and nonacylated cyanidin (molecular structure of Cy-d on Figure 1), with lipid tumor biomimic membrane (MM). The potency of selected Cy-d to rigidify the membrane was determined using the liposome membrane and fluorescence measurements. To address the structure-specific activity of Cy-d, its membrane effects were compared to molecular structure and antioxidant efficacy. The other hypothesis that was tested in the study referred to the question if there is a high capacity of the human albumin to bind Cy-d. The aim of the results is to explain the antioxidant action of the studied compounds against mimic tumor lipid membrane and to assess the potential importance of binding Cy-d to albumin in drug pharmacokinetics.

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Figure 1. Structures of cyanidin derivatives (A) cyanidin-3-O-βglucoside, (B) cyanidin-3,5-di-O-β-glucoside, and (C) cyanidin-3-O-β(6″-O-E-p-coumaroyl-sambubioside)-5-O-β-glucoside. (Poland). The probes 1,6-diphenyl-1,3,5-hexatriene (DPH), 3-[p-(6phenyl)-1,3,5-hexatrienyl]propionic acid (DPH-PA), Merocyanine 540 (MC540), and N-phenyl-1-naphthylamine (PNA) were purchased from Molecular Probes (Eugene, Oregon). 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), and 1-stearoyl-2-oleoylphosphatidylserine (SOPS) were obtained from Avanti Polar Lipids (Nederland). Acylated cyanidin, cyanidin-3-O-β-(6″-O-E-p-coumaroyl-sambubioside)-5-O-βglucoside, and nonacylated cyanidin, cyanidin-3,5-di-O-β-glucoside and cyanidin-3-O-β-glucoside, were purchased from Polyphenols (Norwey) and Extrasynthese (France), respectively. Liposome Preparation. The study was carried out using liposome membranes with special lipid composition, consisting of the same ingredients as in the membrane of tumor cells. In our experiments, the cancer-mimic membrane (MM) was prepared according to Tsuchiya and others13 containing 48 mol % POPC, 24 mol % POPE, 8 mol % SOPS, and 20 mol % cholesterol. Liposomes were prepared in accordance with a previously described technique.19 Only minor changes were implemented. A chloroform solution of a lipid mixture (POPC:POPE:SOPS:cholesterol) dried in vacuum under nitrogen for about 1.5 h was prepared. A phosphate buffer of pH 7.4 was added, and the sample was vortexed to obtain a milky suspension of multilamellar vesicles. The final concentration of lipids in the vesicle suspension was 0.1 g/L. Such a suspension was then sonicated for 10 min at 0 °C with a 20 kHz sonicator (Sonic, Italia). The liposomes were the testing facility used as a mimic bilayer structure. Affinity of Cyanidin Derivatives with Cell-Mimic Membranes. The fluorescence experiments were carried out using different fluorescent probes that embedded in the hydrophilic and hydrophobic regions of liposomal MM. We assumed that Cy-d could modify rather the surface layer, and we applied two probes (MC540 and PNA) to monitor the interface of the MM bilayer. Unfortunately, the Cy-d spectra did overlap the Laudran probe absorption spectra; hence two other probes were employed to determine the membrane-rigidifying effect of anthocyanins. The MC540 dye was used according to a method described before by Manrique-Moreno and others.20 The fluorescence intensity of MC540 emission was determined and expressed as relative intensity change in relation to the control as a function of Cy-d concentration. Packing density in the MM was specified on the grounds of anisotropy (A) of PNA, calculated according to the following equation:21

MATERIALS AND METHODS

Materials. Cholesterol, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), and human serum albumin (HSA) (lyophilized powder, essentially fatty acid free) were purchased from Sigma-Aldrich 7415

DOI: 10.1021/acs.jafc.6b03066 J. Agric. Food Chem. 2016, 64, 7414−7422

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Journal of Agricultural and Food Chemistry A=

I − GI⊥ I + GI⊥

bath. The temperature of 295, 300, 305, and 310 K was established for the set of quenching experiments for HSA immersed in a phosphate buffer solution of pH 7.4 and a final concentration 1.5 × 10−5 M. The excitation wavelength was set at 280 nm (excitation of the Trp and Tyr), and the emission spectra were read at 285−460 nm. The length of 5 nm was adapted for excitation as well as emission slits. Our method that consisted of tracking the quenching of natural HSA fluorescence instigated by all Cy-d was implemented successively. The range of final concentration varied from 4 to 50 μM for Cy-d. Three independent replicates (n = 3) were performed in the experiment. Statistical Analysis. The method of mean values ± standard deviation (SD) was implemented in the date analysis. One-way ANOVA and the following Duncan test were both used in the process of analyzing the obtained results. It was presumed that P values C3,5G > C3G, respectively, and the values were substantially (p < 0.05) different from the control. MC540 fluorescence showed the degree of order of lipids in a phospholipid bilayer. The lower was the fluorescence, the greater was the packing order.20 Acylated cyanidin rigidified cell-MM with greater intensity than did nonacylated cyanidin, as it was presented by the relative potencies: C3-cs-5G > C3,5G > C3G (Table 1). On the grounds of fluorescence anisotropy change of the probe PNA, the effect of Cy-d on fluidity of the hydrophobic area of MM was determined. The measurements showed that there were no changes caused by nonacylated Cy-d (C3G and C3,5G) in fluorescence anisotropy at the concentrations used, although slight changes were observed for acylated cyanidin. In this case, the increase relative to control was ca. 6% for the highest 20 μM concentration of C3-cs-5G (p < 0.05) (Table 1). Molecules causing restriction of acyl chain motions are also believed to be responsible for increasing membrane rigidity as well as enhancing the fluorescence anisotropy. Simultaneously, molecular interactions are expected to lead to enhanced

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where FS refers to relative fluorescence of the probe oxidized by AAPH in the presence of Cy-d, FC refers to relative fluorescence for control sample oxidized by AAPH excluding Cy-d, and FB refers to relative fluorescence of the blank sample. Five independent replicates (n = 5) were performed in the experiment. Fluorescence Quenching of Human Serum Albumin. To carry out a study of potential interaction between Cy-d and human serum albumin (HSA), the experiment was based on the grounds of the following works.24,25 Only minor changes were implemented. The fluorescence measurements were performed with a fluorimeter (Cary Eclipse, Varian) equipped with 1.0 cm quartz cells and a thermostat 7416


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Journal of Agricultural and Food Chemistry

Affinity of Cyanidin Derivatives To Mimic Lipid Membrane. In the fluorescence quenching method, the DPH probe was used to determine the partition coefficient (given as Kd) of acylated and nonacylated Cy-d to MM. The determined dissociation constant Kd values are shown in Table 2. The high value of dissociation constant inside the lipid

Table 1. Changes in Fluorescence Intensity (for MC540 Probe) and Fluorescence Anisotropy (for PNA Probe) in the Presence of Cyanidin Derivatives at Concentrations of 4, 10, and 20 μM in Tumor Cell-Mimic Lipid Membranes at 310 Ka compound/ concentration [μM] 4 10 20 4 10 20 4 10 20

MC540 intensity change from control C3-cs-5G −0.0957 ± 0.0122* −0.1939 ± 0.0109* −0.3067 ± 0.0257* C3,5G −0.0787 ± 0.0175* −0.1372 ± 0.0108* −0.2172 ± 0.0213* C3G −0.0464 ± 0.0087* −0.0947 ± 0.0158* −0.1759 ± 0.0180*

PNA anisotropy change from control

Table 2. Antioxidant Activity Parameters (IC50) and Partition Coefficients (Kd) for Cyanidin Derivatives into Cell Mimic Membranesa

0.001 ± 0.001 0.010 ± 0.002 0.057 ± 0.003* 0.001 ± 0.001 0.017 ± 0.016 0.002 ± 0.002

a

0.015 ± 0.002 0.006 ± 0.003 −0.053 ± 0.063

compound

Kd [μM]

IC50 [μM]

C3-cs-5G C3,5G C3G

7.25 ± 0.58 9.49 ± 0.28 55.07 ± 1.01

0.98 ± 0.15 1.25 ± 0.03 1.49 ± 0.01

Membrane oxidation was induced by AAPH.

membrane attests to the low affinity of the compound to liposome membranes. In our study, the Kd value decreased (i.e., affinity to liposomes increased) according to the sequence: C3G (55.07 ± 1.01 μM) > C3,5G (9.49 ± 0.28 μM) > C3-cs5G (7.25 ± 0.58 μM). The difference in Kd value between the C3-cs-5G and C3,5G and C3G was at least 1 order of magnitude. One may notice that together with the increasing number of sugar moieties within a molecule, the efficacy of DPH quenching increases. The result for C3-cs-5G with the use of PNA probe additionally confirms the ability of the acylated anthocyanin to interact with the hydrophobic region of MM. Van Dijk and others36 showed that glycosylation of both naringenin and eriodictyol at the 7 position led to more significant interactions with the vesicle membrane in comparison with the corresponding aglycons. The explanation for the increase in affinity upon increased amount of sugar moieties of anthocyanins is likely to be found in decreasing torsion on the line: the exocyclic phenyl ring and the rest of the structure that promotes a more dimensional structure.37 An important parameter determining the affinity of molecules to the liposome membrane is their spatial structure. Hendrich38 suggested that flavanons are more hydrophobic as compared to flavonons (of the same degree of hydroxylation), and their affinity to membranes is higher. This observation was in line with earlier suggestions by some authors concerning the effect of spatial structure of a molecule on its hydrophobicity. Antioxidant Efficiency of Cyanidin Derivatives. While testing the antioxidant ability of Cy-d in proportion to the AAPH-mediated peroxidation of lipids, it was our aim to prepare cell MM. The degree of oxidation was established in connection with the fluorescence intensities of the probe as a function of oxidation time in the presence of Cy-d, or in the control sample, relative to the initial value of fluorescence intensity. Cy-d concentration was responsible for 50% reduction in lipid peroxidation (IC50) and has been assumed as the measure of the compound’s antioxidant activity. To give insight into relative fluorescence intensity examples, observed as kinetic curves of the probe DPH-PA in the presence of Cy-d for lipid membranes, Figure 2 was attached. With increasing Cy-d concentration, simultaneously, the degree of fluorescence is increasing comparably to the proportion of oxidation observed in lipid membrane. According to the results, acylated cyanidin was 1.3 and 1.5 times more antioxidant-active than C3,5G and C3G, respectively (Table 2). The rank order in lipid peroxidation inhibition was C3-cs-5G > C3,5G > C3G, suggesting the importance of the presence of the substituent

Negative values indicate a decrease in fluorescence intensity/ anisotropy as compared to the control, while positive values indicate an increase in fluorescence intensity/anisotropy as compared to the control. Means labeled with an asterisk (*) are significantly (p < 0.05) different from control. a

membrane fluidity. That was proved by decreasing the anisotropic emission.28 A number of studies have shown that there is a relation between cell membrane fluidity and extent of cancerous changes in the cell.29,30 Measurements of microscopic viscosity of biological membranes of melanoma cells done by Inbar and Shinitzky31 indicated that the viscosity of healthy leucocytes is twice as high as that of cancer-cell membranes. Other studies showed that such a relationship applies not only for leucocytes but also for other tumors.32 Because viscosity is inversely related to fluidity, the conclusion has been drawn that membranes of cancerous cells are more fluid than their healthy counterparts. Tsuchiya and others focused on the estimation of the membrane interaction of flavonoids with the use of cancer MM systems by application of fluorescence polarization techniques. The drawn conclusion was that various flavonols (e.g., quercetin, galangin, luteolin, and apigenin) tend, in concentration 10 μM, to rigidify the cell membrane.13 Studies in vitro conducted on diversified amount of cancer cell lines have shown that anthocyanins exhibit antiproliferation activity. An anticancerogenic action was also shown, for example, with respect to melanoma cell lines,33 S180 liver cancer,34 or colon cancer.35 Jing and others35 reported that a dose of 25 μg/mL chokeberry anthocyanins was responsible for 50% limitation of the carcinoma cell line, clearly showing no effects on growing normal colonic NCM460 cells. These authors stated that the antiproliferation potential of anthocyanins in relation to cancer cells is closely related to the kind of aglicone, sugar residue, and phenolic acids, place and degree of glycosylation, and acylation. One can assume that Cy-d, acylated and nonacylated, used in our experiments, by causing a rigidifying effect on tumor cell MM induced anticancer activity. A larger MM tumor effect in our research has been demonstrated for anthocyanin acylated with p-cumaric acid in the 3 position of the C ring (Figure 1). It can be concluded from our study that a glycosyl group at the 3 and 5 positions of the C and A ring and acylation at the carbon 6 of the glycosyl moiety is expected to be the key factor in such a noticeable membrane interaction. 7417


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Figure 2. Relative fluorescence intensity of DPH-PA probe as a function of oxidation time of cell-mimic membranes for AAPH radicals in the presence of (A) C3-cs-5G, (B) C3,5G, and (C) C3G at chosen concentrations. The relative change in fluorescence intensity F/F0 is a measure of the degree of lipid peroxidation (F0, fluorescence anisotropy in control sample; F, fluorescence anisotropy of samples in the presence of cyanidin derivatives).

sugar and p-cumaric acid in A and C ring structures. Research reports suggest that the molecular structure of flavonoids, their spatial structure, kind of aglycone, or the presence of acyl groupings to a large extent determine the antioxidant activity of the substances.39 Previous studies have asserted that as far as the process of glycosylation of anthocyanidins is progressing, their antioxidant activity is usually regressing. Nevertheless, it is also possible to improve activity, which is determined by the type of anthocyanidin as well as the method selected during the experiment.40 However, other authors concluded that anthocyanidin glycosylation increases the antioxidant capacity.41 Additionally, glycosylation at C-3 and C-5 of the anthocyanidin skeleton has shown a petrifying result in the chemiluminescence intensity in malvidin (lipid peroxidation).42 Jing and others43 proved that the underlying cause for increasing activity of 3-glucoside anthocyanins and not for their aglycons lies in the electron-donating effect of the 3-bulky sugar group.40 These authors explained the increased activity of anthocyanins with sugar substituents in the following way: the hydroxyl or glycosylated substituent around the C-3 (C ring) and C-3′ (B ring) carbon skeleton should be positive for high activity because they are both hydrogen-bond acceptors and hydrogen-

bond donors. Additionally, the presence of additional electrondonating and/or hydrophobic groups in the glycosylation might enhance the radical scavenging activity consistent with previous studies.44 Studies have shown that anthocyanins have strong abilities as antioxidants in vitro. Quenching free radicals and terminating the chain reaction, which is the cause of oxidative damage, are listed among others.39 For example, Tsuda and others45 reported that the cyanidin had a stronger antioxidant activity as compared to alfa tocopherol in the liposome and rabbit erythrocyte membrane systems. In addition, Tsuda and others46 investigated inhibitory effects in connection with lipid peroxidation by UV light irradiation of cyanidin 3-O-glucoside and their aglycones, cyanidin chloride. On the basis of the obtained results, they suggested that anthocyanidins had stronger antioxidative activity as compared to the glycoside derivative in reducing the ability to form malondialdehyde from UVB irradiation in lipid membrane. Jankowski and others47 observed that anthocyanin dyes provided in small doses from Aronia melanocarpa to rats are responsible for reducing pancreatic swelling and decreasing lipid peroxidation. Various studies have also presented evidence that anthocyanins have a much higher antioxidant potential than other known, reference 7418


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Figure 3. Emission spectra of HSA in the presence of various concentrations of C3-cs-5G (A), C3,5G (B), and C3G (C), and Stern−Volmer plots of F0/F against concentration of cyanidin derivatives (HSA = 1.5 × 10−5 M, λex = 280 nm, T = 310 K).

antioxidants such as β-carotene or vitamin C.48 In summary, we can say that demonstrated antioxidant properties of Cy-d (C3cs-5G > C3,5G > C3G) in vitro in relation to MM may, causing inactivation of RFT, indirectly affect the inhibition of inflammatory processes of the body that are the breeding ground of a number of serious medical conditions. They may, when acting on cancer-modified cell membranes, be causing them to stiffen up and thus positively influence their cellular homeostasis. Fluorescence Quenching Mechanism of HSA and Determination of Binding Parameters. The fluorescence spectra of HSA in the absence and presence of different

compounds of C3-cs-5G, C3,5G, and C3G were recorded. The effect of Cy-d on HSA fluorescence intensity is presented in Figure 3. The fluorescence intensity of HSA is decreasing in the presence of all Cy-d, as it was presented, which indicated that these compounds might interact with main plasma proteins. The data referring to fluorescence quenching were interpreted on the grounds of the Stern−Volmer equation, to shed light on the fluorescence quenching mechanism stimulated by Cy-d:49 F0 = 1 + Kqτ0[Q] = 1 + KSV[Q] F 7419

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Table 3. Quenching (Ksv) and Binding (Kb) Constants and Thermodynamic Parameters (n, ΔG, ΔH, and ΔS) for the Cyanidin Derivatives (C3-cs-5G, C3,5G, C3G) and Human Serum Albumin at Different Temperaturesa compound

T (K)

Ksv (×104 M−1)

Kb (×104 M−1)

n

ΔG (kJ/M)

ΔH (kJ/M)

ΔS (J/M/K)

C3-cs-5G

295 300 305 310 295 300 305 310 295 300 305 310

3.696 3.594 3.580 3.333 1.494 1.223 1.189 1.082 1.818 1.771 1.667 1.402

35.424 15.856 9.716 4.290 9.064 5.394 3.046 1.709 0.406 0.160 0.085 0.074

1.225 1.149 1.104 1.030 1.641 1.586 1.580 1.496 0.853 0.757 0.699 0.706

−31.339 −29.856 −29.121 −27.492 −39.290 −38.662 −37.857 −36.988 −20.381 −18.405 −17.122 −17.016

−103.730

−245.530

−84.761

−153.917

−87.910

−230.361

C3,5G

C3G

a

Standard deviations (mean value of three independent experiments) were lower than 10%.

where F0 and F refer to fluorescence intensities of HSA prior to and in succession with addition of the Cy-d quencher, Kq refers to a bimolecular quenching constant, τ0 refers to the lifetime of the fluorophore in the absence of quencher (the fluorescence lifetime of a biopolymer is calculated as 5 × 10−9 s),25 [Q] refers to the concentration of the quencher, and KSV refers to the Stern−Volmer quenching constant (KSV = Kq·τ0). On the basis of eq 4, the Stern−Volmer quenching constant (KSV) for the ligand−protein complex was determined by the linear regressions of the plots of F0/F versus [Q]. The plots were linear in the 4−50 μM range of concentration for all of the compounds (Figure 3). There are two types of fluorescence quenching: static (a ground-state complex created between the fluorophore and the quencher is observed) or dynamic (collisional encounters between the fluorophore and the quencher are observed). The key factor that allows making a distinction between dynamic and static quenching is differing dependence on temperature. An increase of temperature is usually responsible for the dissociation of infirmly bound complexes. As a result, the static quenching is reduced.50 As shown in our results (Table 3), the Ksv of all Cy-d decreased with increasing temperature, which proved that static quenching materialized. Moreover, the values of Kq for binding of C3-cs-5G, C3,5G, and C3G to HSA, for example, at 310 K, were calculated to be 6.667 × 1012, 2.164 × 1012, and 2.804 × 1012 M−1 s−1, respectively. These values are significantly more substantial than the diffusion-limited rate constant of the biomolecule (1.0 × 1010 M−1 s−1).49 That is proof of basic static quenching, as a result of complex formation between HSA and Cy-d. It is possible to show the relation between the apparent binding constant (Kb) and number of binding sites (n) by implementing the following equation: ⎛F − F⎞ ⎟ = log K + n log[Q] log⎜ 0 b ⎝ F ⎠

C3-cs-5G was greater than that of C3,5G and C3G, which was confirmed by the results of the fluorescence quenching. The global binding constants of anthocyanins to HSA (in 310 K) were included from 4.290 × 104 to 0.074 × 104 M−1 for studied Cy-d, which is in direct proportion to values obtained for nonacylated Cy-d bound to HSA in the available research papers.9,51 The presence of glycosyl and/or acid substituents in the C ring of Cy-d has an impact on the binding to HSA. Additional sugars (sambubiose) and p-coumaric acid on the C ring increased the interaction with HSA. Binding constants of Cy-d to HSA decreased in the order: C3-cs-5G > C3,5G > C3G. Also, in other studies, the glycosylation of pelargonidin increased the affinity for HSA.52 It is bound to conclude that the presence of sugar substitution has an influence on the binding parameters to HSA. Furthermore, the fact of the matter is that the values of n at the experimental temperatures were almost on the same level, that is 1, proving that the only existence of a single binding site in HSA for all anthocyanins could be detected, as shown in Table 3. It is possible to distinguish four types of noncovalent interactions in the process of drug−protein binding, including hydrogen bonds, hydrophobic effects, electrostatic interactions, and van der Waals forces.53 It is possible to calculate the thermodynamic parameters in the process of binding using van’t Hoff’s equation:54 ln Kb =

−ΔH ΔS + RT R

(6)

ΔG° = ΔH − T ΔS = −RT ln Kb

(7)

where ΔH, ΔG, and ΔS refer to enthalpy change, free enthalpy change, and entropy change, respectively. R refers to the gas constant 8.314 J mol−1 K−1. The values of parameters ΔG, ΔH, and ΔS obtained at different temperatures are presented in Table 3. The link between HSA and Cy-d shows a negative value of ΔG, ΔH, and ΔS. The values of ΔG in the above-mentioned binding processes were negative, proving that the character of the binding of C3-cs-5G, C3,5G, and C3G with HSA was spontaneous. What is more, when ΔH < 0, ΔS < 0, van der Waals forces and hydrogen bonds are the main forces; when ΔH > 0, ΔS > 0, hydrophobic effects are significant in the interaction; when ΔH < 0, ΔS > 0, it is the electrostatic forces.55 In our experiments, the negative values of ΔH and ΔS suggest that hydrogen bonds and van der Waals forces are

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The values of n and Kb were obtained by plotting log [(F0 − F)/F] versus log[Q]. The results for Cy-d as Q (as the quencher) at four different temperatures (295, 300, 305, and 310 K) are given in Table 3. It was observed that the values of Kb decreased when the temperature rose, suggesting that the complex of acylated and nonacylated Cy-d with HSA has a tendency to be unstable as the temperature goes up. The Kb values of C3-cs-5G were larger than those of C3,5G and C3G. That may suggest that the strength of the binding affinity of 7420



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crucial for understanding the interaction of all of the Cy-d tested with human serum albumin. In conclusion, it is worth noticing that the results of the study have proved that the acylated cyanidin (C3-cs-5G) and nonacylated ones (C3,5G and C3G) showed high biological activities. It was demonstrated that, by using fluorescence probes, which became embedded at different depths within the bilayer of tumor cell mimic membranes, all of the Cy-d studied were responsible for increasing the packing order of the hydrophilic region of the membrane. C3-cs-5G also showed the greatest affinity to the membrane and caused a decrease in fluidity of the membrane hydrophobic interior. It can be concluded that a glycosyl group at positions 3 and 5 of the C and A ring, and acetylation at carbon 6 of the glycosyl moiety, should be considered as the key factor for the noticeable lipid membrane interaction and biological activity. This conclusion was confirmed by the results of antioxidant activity of Cy-d toward MM, oxidized by the AAPH compound that followed the sequence C3-cs-5G > C3,5G > C3G. We suggest that membrane rigidification by Cy-d is the underlying cause leading to the suppression of lipid peroxidation. The process happens in a way of reducing the diffusion of free radicals in the lipid MM and thus reducing their reaction efficiencies. In addition, the tumor lipid MM rigidifying the effects of studied compounds can be fundamental for their anticancer activity. Studied Cy-d were able to bind to human serum albumin and quench its fluorescence. The process of binding of all of the Cyd to HSA was a static quenching mechanism. The key factors behind it were van der Waals and hydrogen-bonding forces. That kind of research (using human serum albumin) is significant in pharmacology and pharmacokinetics because the binding process affects the drug distribution and its pharmacological behavior. The results of this pioneering research indicated a strict connection between antioxidant activity that depends on the molecular structure of cyanidin derivatives and structural changes in the membranes, as well as a possible mechanism of their anticancer activity.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +48 71 320 5167. Fax: +48 71 320 5167. E-mail: paulina. [email protected]. Funding

This work was supported by the statutory activities of the Department of Physics and Biophysics of Wroclaw University of Environmental and Life Sciences. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED C3-cs-5G, cyanidin-3-O-β-(6″-O-E-p-coumaroyl-sambubioside)-5-O-β-glucoside; C3,5G, cyanidin-3,5-di-O-β-glucoside; C3G, cyanidin-3-O-β-glucoside; DPH, 1,6-diphenyl-1,3,5-hexatriene; DPH-PA, 3-[p-(6-phenyl)-1,3,5-hexatrienyl]propionic acid; HSA, human serum albumin; MC540, merocyanine 540; PNA, N-phenyl-1-naphthylamine; POPC, 1-palmitoyl-2oleoylphosphatidylcholine; POPE, 1-palmitoyl-2-oleoylphosphatidylethanolamine; SOPS, 1-stearoyl-2-oleoylphosphatidylserine; DPPC, 1,2-dipalmitoylphosphatidylcholine 7421


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Interaction between Mimic Lipid Membranes and Acylated and

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