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Investigations on Membrane Perturbation by Chrysin and Its Copper Complex Using Self-Assembled Lipid Bilayers Stalin Selvaraj, Sridharan Krishnaswamy, Venkappayya Devashya, Swaminathan Sethuraman, and Uma Maheswari Krishnan* Centre for Nanotechnology and Advanced Biomaterials (CeNTAB), School of Chemical and Biotechnology, SASTRA University, Thanjavur 613 401, Tamil Nadu, India ABSTRACT: The mechanism of membrane interactions of most of the flavonoids in the presence of transition-metal ions is not well-understood. To understand this phenomenon, the present work aims to synthesize a chrysincopper complex at room temperature and investigate its influence on the electrical characteristics of planar lipid bilayers. The chrysincopper complex was characterized by various spectroscopic techniques and was found to have a metal/ligand ratio of 1:2 and of cationic nature. Its ability to inhibit 1,10 -diphenyl-2-picrylhydrazyl (DPPH) radicals was not significant at alkaline pH because of the involvement of the 5-hydroxy group in coordination with the copper ion compared to its parent flavonoid, chrysin (p < 0.05). The addition of different concentrations (20100 μM) of chrysin and the chrysincopper complex to lipid bilayers decreases the resistance, indicating a strong surface interaction and partial insertion into the bilayer near the lipidwater interface. The dose-dependent reduction in resistance as a result of the chrysincopper complex is more pronounced in comparison to chrysin, implying that the bulkier and charged chrysincopper complex displays greater ability to distort the lipid bilayer architecture. These conclusions were further confirmed by curcumin-loaded liposome permeabilization studies, where both chrysin and its CuII complex increased the fluidity in a dose-dependent manner. However, the extent of fluidization by the chrysincopper complex was nearly twice that of chrysin alone (p < 0.05). The implications of these surface interactions of chrysin and its copper complex on cell membranes were studied using a hypotonic hemolysis assay. Our results demonstrate that, at low concentrations (20 μM), the chrysincopper complex exhibited twice the protection against hypotonic stress-induced membrane disruption when compared to chrysin. However, this stabilizing effect gradually decreased and became comparable to chrysin at higher concentrations. This biphasic behavior of the chrysincopper complex could further be explored for therapeutic applications.
’ INTRODUCTION Chrysin (5,7-dihydroxy flavone) is an isoflavone that is found in Passiflora (passion flower) and in the flowers and honey of many other plant species.1 Chrysin possesses antioxidant, antiinflammatory, vasodilatory, and anticancer properties.29 Chrysin has been widely used by male bodybuilders because it prevents the cytochrome P450-dependent enzyme aromatase from converting androgens to estrogens.10,11 Flavonoids possess high antioxidant potentials because of the presence of hydroxyl and keto groups in their rings and, hence, may offer protection against free-radical-related disorders, such as atherosclerosis, asthma, cancer, diabetes, and neurodegenerative diseases.8,12 However, recent studies have shown the pro-oxidant nature of flavonoids at certain concentrations, thereby raising serious concerns about their safety profile.13 A few studies have also shown that flavonoids in the presence of transition-metal ions can cause oxidative injury.14 Contrarily, a few studies have also highlighted a protective effect for flavonoids against oxidative stress in the presence of metal ions.15 Therefore, it is likely that the microenvironment in the biological system coupled with the nature of r 2011 American Chemical Society
the flavonoids influences the transition between pro- or antioxidant characteristics of flavonoids. The human body contains many transition-metal ions, such as iron and copper, and it becomes important to identify the factors that contribute to promoting the antioxidant or pro-oxidant nature of flavonoids. It is well-known that transition-metal ions can form coordination complexes with flavonoids, and hence, it will be of interest to investigate whether the formation of a flavonoidmetal complex would contribute to their pro-oxidant or antioxidant nature.9 Although a large number of reports are available on the antioxidant properties of flavonoids, very few publications are available on the synthesis and biochemical effects of their metal ion complexes.15 Recently, a few efforts have been directed to synthesize and characterize novel flavonoid derivatives and flavonoidmetal complexes and explore their potential biological effects. Complexes of vanadylrutin,16 ironquercetin,17 Received: July 28, 2011 Revised: September 6, 2011 Published: September 19, 2011 13374
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Langmuir coppernaringenin,18 coppergenistein,19 etc. have been synthesized and are reported to possess antineoplastic activities. The physiological and therapeutic activities of flavonoids have been mainly attributed to their interactions with the cell membranes. The extent of the interaction of flavonoids with membranes in turn is primarily dependent upon its hydrophobicity and localization in the lipid bilayer.20 Flavonoids are known to localize either in the hydrophobic core of the bilayer or at the lipidelectrolyte interface.20 The membrane interaction and localization might provide interesting insight into the observed biological effects of flavonoids, including chrysin. Although many studies have confirmed the safety of the isoflavone chrysin, it was recently reported that chrysin induces cell cytotoxicity at very low concentrations in normal trout liver cell lines.13 Similarly, another study indicated that the lipid bilayer effectively blocks the entry of chrysin into the cells, which may be the reason for its pro-oxidant behavior.21,22 Therefore, a detailed understanding on the bilayer interaction of chrysin in the presence and absence of transition-metal ions can provide clarity on the molecular level interactions of chrysin with the membrane. The planar lipid bilayer system has been extensively used as an experimental model for membrane interaction studies.23,24 Because the cell membrane is the primary site of oxidative stress as well as flavonoid interactions, the planar lipid bilayer has been chosen as a model system to investigate the influence of chrysin and its copper complex on the membrane integrity. The literature on the bilayer interactions of flavonoids is limited, and there is no information available on the interaction of chrysin or its metal ion complexes with the lipid bilayer. Because copper is one of the common transition-metal ions found in the body, which has also been known to induce oxidative stress, it will be of interest to synthesize the chrysincopper complex and investigate its influence on the membrane integrity. Thus, the aim of the present work is to study the interactions of chrysin and its copper complex and to elucidate the extent of its protective effect on membrane integrity by measuring the change in electrical properties of the bilayer.
’ MATERIALS AND METHODS Materials. Copper(II) acetate (Merck, India) and chrysin (97%, Sigma-Aldrich, St. Louis, MO) were used as such for the synthesis of complexes without any further purification. Methanol (Merck, India) was used as a solvent. Potassium chloride (Merck, India), n-decane, and dimethyl sulfoxide (DMSO, Merck, Germany) were used as such for the bilayer studies. 1,10 -Diphenyl-2-picrylhydrazyl (DPPH) radical (SigmaAldrich, St. Louis, MO) was used for the antioxidant assay. Curcumin, sodium chloride (Merck, India), and TrisHCl buffer (Sigma-Aldrich, St. Louis, MO) were used for membrane permeabilization studies.
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were recorded on an UVvis spectrophotometer (Lambda 25, PerkinElmer, Waltham, MA) using DMSO solvent. Formation of the Bilayer Lipid Membrane (BLM). Phospholipids were extracted from egg yolk using a modified Singleton and Gray method25 and stored in chloroform (50 mg/mL). The planar lipid bilayer was formed using the MuellerRudin method.26 A 2% (w/v) dispersion of phospholipids in n-decane was painted on an aperture with an area of 0.00793 cm2, bifurcating two chambers with a volume of 3.5 mL each. All experiments were carried out in 0.1 M KCl bath medium as higher ionic strengths (>0.1 M KCl), reducing the BLM stability, while lower concentrations displayed very high resistance. All measurements were taken at room temperature (25 °C). The bilayer setup was kept on a vibrationisolated platform and shielded from electrical noise using a Faraday cage. The bilayer formation was monitored for the changes in the electrical parameters (current flow), and a stabilization time of 30 min was allowed before commencing the measurements. The attainment of stability for the lipid bilayer was confirmed by the measured constancy in electrical parameters. Upon the addition of different concentrations (20100 μM) of chrysin or its copper complex to the system, the bath solution was stirred for 5 min to ensure a uniform concentration and the stirrer was switched off during electrical measurements. Electrochemical Measurements. The cyclic voltammetric (CV), chronoamperometric (CA), and admittance spectra experiments were conducted using the electrochemical workstation (model 604C, CH Instruments, Austin, TX). A three-electrode system consisting of a platinum working electrode, a Ag/AgCl (3 M KCl) reference electrode, and a platinum wire as the counter electrode was used for measurements.27 A 1 GΩ resistor was introduced in series to the bilayer capacitor. An interaction time ranging from 20 to 30 min was allowed before measurements of electrical parameters in all cases.
Curcumin Release Measurements Using Unilamellar Liposomes. Curcumin release from unilamellar lipid vesicles (prepared from egg phospholipids) was measured spectrophotometrically at 433 nm. Liposomes were prepared by a thin-film hydration method. A total of 10 mg of phospholipids was purged with nitrogen to remove traces of solvent, suspended in 0.1 mM TrisHCl buffer (pH 7.4), and constantly stirred for 2 h. The suspension was extruded 10 times using an extruder (Liposofast Basic, Avestin, Canada) through a 200 nm polycarbonate membrane to obtain unilamellar vesicles. Chrysin, the chrysincopper complex, and curcumin were dissolved in DMSO to obtain a final concentration of 3.5 mM. Liposomes were incubated with 100 μM curcumin for 20 min in the dark at room temperature and centrifuged at 15 000 rpm to remove unencapsulated curcumin. The pellet was resuspended in 3.5 mL of TrisHCl buffer and incubated with different concentrations (20100 μM) of chrysin or its copper complex individually for 20 min at room temperature. The absorbance was then measured at 433 nm using an UVvis spectrophotometer. The percentage release of curcumin was calculated as follows: percent release of curcumin ¼ ððreleased curcumin ðODÞ=encapsulated curcumin ðODÞÞ 100
Synthesis and Characterization of the ChrysinCopper Complex. Copper(II) acetate (0.233 g) was dissolved in 20 mL of double-distilled water and added slowly to a solution of 0.254 g of chrysin in methanol (5 mL). The mixture was stirred for 4 h at room temperature, filtered under vacuum, washed with water, and air-dried. The formation of the chrysincopper complex was confirmed using a carbon, hydrogen, nitrogen, and sulfur (CHNS) analyzer (Elementar Vario EL III, Germany). The content of metal in the complex was estimated by atomic absorption spectrometry (AAnalyst 400, PerkinElmer, Waltham, MA). The complexation of copper to chrysin was confirmed using electron paramagnetic resonance (EPR) spectroscopy (EMXPlus, Bruker, Germany). Infrared (IR) spectra were recorded using a Fourier transform infrared (FTIR) spectrometer (Spectrum 100, PerkinElmer, Waltham, MA), and ultravioletvisible (UVvis) spectra
DPPH Assay. The reaction mixture containing 3.5 mL of DPPH radical and different concentrations of chrysin or the chrysincopper complex were added to obtain a final concentration of 20, 40, 60, 80, and 100 μM. The reaction mixture was then incubated at room temperature for 45 min with pH 9 using Tris buffer, and the absorbance of the test solutions was measured at 515 nm against a blank sample containing DPPH (negative control) using an UVvis spectrophotometer. Hypotonic Hemolysis Assay. A total of 1 mL of blood was collected from a healthy individual and transferred to 1 mM sodium citrate solution to prevent coagulation. The solution was centrifuged at 3000 rpm for 15 min to obtain packed red blood cells. The supernatant was removed, and the pellet was washed with phosphate-buffered saline 13375
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Table 1. Elemental Analysis Data for Chrysin and the ChrysinCopper Complex
Table 2. Vibrational Frequency Data for Chrysin and Its Copper Complex vibration frequencies (cm1)
elemental analysis (%)
compound chrysin
color
C
H
yellow 71.18 3.481
Cu2+
νCdO
νCOH
νCdC
νCOC
chrysin
1651
1408
1556
1287
chrysincopper complex
1636
1540
1276
compound
calculateda
experimental C
H
Cu2+
70.79 3.93
chrysincopper complex green 59.12 3.546 11.18 62.93 3.16 11.45 a
Assuming a M/L ratio of 1:2.
Figure 2. UVvis spectra for chrysin and its copper complex. (Inset) Enlarged region of the spectrum showing the appearance of a new peak at 650 nm for the chrysincopper complex. Figure 1. EPR spectrum for the chrysincopper complex recorded at room temperature. (PBS) (pH 7.4) 3 times and then made up to a final volume of 3.5 mL with PBS to obtain a working solution. Hemolysis of the washed red blood cells was performed using 500 μL of 0.2% sodium chloride solution (negative control). Different concentrations (20100 μM) of the chrysin/chrysincopper complex were added to 3.5 mL of working solution in the presence of 500 μL of 0.2% sodium chloride solution (negative control) and incubated at room temperature for 45 min. The cells were removed by centrifugation at 3000 rpm for 10 min, and the supernatant containing the released hemoglobin was measured at 560 nm using UVvis spectrophotometry.28 Differential Scanning Calorimetry (DSC). The stock solution of the chrysincopper complex (3.5 mM) was prepared using DMSO. A total of 2 mg/mL egg phospholipid in chloroform was purged with nitrogen for 0.5 h to remove the solvent. The lipid was then dispersed in 3.5 mL of TrisHCl buffer (pH 7.4), stirred for 0.5 h, and extruded with polycarbonate membrane with a pore size of 200 nm for 12 cycles to obtain unilamellar liposome vesicles. Then, the liposome dispersion was incubated with 100 μM chrysincopper complex for 20 min. The sample was centrifuged at 10 000 rpm to pellet the liposome, which was freeze-dried. The freeze-dried liposome sample was subjected to analysis by DSC (Q20, TA Instruments, New Castle, DE). The DSC was recorded between 10 and 90 °C at a scan rate of 10 °C/min for 3 cycles. The experiments were repeated with control liposome samples, which were not incubated with the chrysincopper complex. Statistical Analysis. DPPH and membrane permeabilization and stabilization assays were expressed as the mean ( standard deviation (SD) of three values. A comparison between mean values was made using one-way analysis of variation (ANOVA) followed by a Tukey test at a 95% confidence interval (p < 0.05).
’ RESULTS Structural Elucidation of the ChrysinCopper Complex. Table 1 shows the elemental analysis for chrysin and its copper complex. The amount of Cu2+ in the chrysincopper complex was found to be 11.18%, which was in agreement with the theoretical value of 11.45% calculated for a ML2 complex. This suggests that the metal/ligand ratio in the complex is 1:2. The EPR spectrum of the chrysincopper complex reveals a singlet peak, as compared to the hyperfine multiplet pattern reported for free copper ions, suggesting the existence of a copper complex (Figure 1). The IR vibration peaks for chrysin and its copper complex are shown in Table 2. The vibration band for the carbonyl group in chrysin is shifted from 1651 to 1636 cm1 in the complex, indicating coordination of the carbonyl oxygen with copper. The νCOC band of chrysin shows a shift from 1287 to 1277 cm1 in the copper complex, suggesting an interaction of the metal ion with chrysin via the oxygen of the hydroxyl group at position 5 in chrysin. In addition, the bending vibration of OH at 1408 cm1 in chrysin disappears in the complex, indicating bonding via the OH group. The UVvis spectra of chrysin and its copper complex are shown in Figure 2. Both chrysin and the chrysincopper complex show a strong absorption band between 260 and 270 nm because of the A ring (band II, benzoyl system) and a weak band around 330340 nm because of the B ring (band I, cinnamoyl system).16 A change in the intensity of band II and the appearance of a broad band at 650 nm in the case of the copper complex may be attributed to a dππ* transition, implying a square planar geometry for the complex.16,29 From the elemental analysis and spectral data, the structure of the chrysin copper complex is proposed to contain two molecules of chrysin coordinated to CuII through the 4-keto and 5-hydroxy groups, as depicted in Figure 3. 13376
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Figure 3. Proposed structure for the cationic chrysincopper complex.
Figure 4. Percentage reduction in the DPPH concentration in the presence of different concentrations of chrysin and the chrysincopper complex [values are expressed as the mean ( SD; (a) p < 0.05].
Effect on the Free Radical Scavenging Property. The free
radical scavenging activity of chrysin and the chrysincopper complex was assessed using the standard DPPH assay. Figure 4 shows the percentage inhibition of DPPH exhibited by chrysin and its copper complex in the concentration range of 20100 μM at pH 9.0 (Tris buffer). It is seen that chrysin displays low DPPH inhibitory effects that gradually increase in a dose-dependent manner. In the case of the chrysincopper complex, no significant DPPH inhibitory effect is discernible in the concentration range of 20100 μM (p > 0.05). Interactions of Chrysin and Its Copper Complex with Planar Lipid Bilayers. The lipid bilayer is a well-established organic capacitor with its hydrophilic polar head groups acting as charged plates and the hydrophobic core forming the dielectric medium. Figure 5A shows the CV pattern of the lipid bilayer upon the addition of 0100 μM chrysin, and the CV pattern for the unmodified bilayer displays the characteristic pattern of a parallel plate capacitor, confirming the capacitive nature of the planar lipid bilayer.24 The absence of any anodic or cathodic peak in the cyclic voltammogram of the unmodified lipid bilayer clearly shows the absence of any redox couple in the system. The addition of chrysin did not alter the capacitive nature of the bilayer. The Nyquist plot for admittance of the bilayer under the same conditions shows that the progressive addition of chrysin reduces the resistance in the system, indicated by a reduction in the length of the semicircle (Figure 5B). Simultaneously, the capacitance
Figure 5. (A) Comparison of cyclic voltammograms of the bilayer in the presence of different concentrations of chrysin. Bath medium, 0.1 M KCl; scan voltage, from 0.07 to 0.07 V; scan rate, 0.01 V/s; scan mode, negative; sample interval, 0.001 V; and scan segment, 3. (B) Complex Y plots for different concentrations of chrysin in the BLM system. Frequency, 101 105 Hz; amplitude, 0.005 V; quiet time, 2 s; and bath medium, 0.1 M KCl.
of the bilayer also gradually decreases, as indicated by the decrease in the height of the semicircle. Figure 6A shows the CV pattern of the lipid bilayer upon the addition of 0100 μM chrysincopper complex. The addition of the chrysincopper complex also did not introduce any redox peaks in the system. However, the Nyquist plot for admittance shows a decrease in the resistance as well as capacitance of the bilayer on the progressive addition of the chrysincopper complex (Figure 6B). This behavior is similar to that observed for the parent flavonoid. Figure 6C shows the relative decrease in the resistance (RCT) of the bilayer in the presence of different concentrations (20 100 μM) of chrysin and its copper complex. The change in resistance exhibits a biphasic behavior with an initial steep decrease in both the case of chrysin and its copper complex, followed by a lower magnitude of decrease tending to saturation. However, the chrysincopper complex shows a higher magnitude of decrease in resistance, indicating that it could fluidize the lipid bilayer to a greater extent when compared to its parent flavonoid. 13377
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Figure 7. Chargedischarge profile of the unmodified bilayer in the presence of different concentrations of (A) chrysin and (B) chrysin copper complex and (C) relative change in the charging peak current height in the presence of different concentrations of chrysin and the chrysincopper complex. Scan voltage, 0.07 V; scan mode, positive; step, 4; pulse width, 0.25 s; and sample interval, 0.001 s.
Figure 6. (A) Comparison of cyclic voltammograms of the bilayer in the presence of different concentrations of the chrysincopper complex. Bath medium, 0.1 M KCl; scan voltage, from 0.07 to 0.07 V; scan rate, 0.01 V/s; scan mode, negative; sample interval, 0.001 V; and scan segment, 3. (B) Complex Y plots for different concentrations of the chrysincopper complex in the BLM system. Frequency, 101 105 Hz; amplitude, 0.005 V; quiet time, 2 s; and bath medium, 0.1 M KCl. (C) Relative change in bilayer RCT in the presence of different concentrations of chrysin and the chrysincopper complex. Values obtained from admittance plots.
Panels A and B of Figure 7 show the CA plot for the bilayer recorded after the addition of 0100 μM chrysin and its copper
complex, respectively. The typical chargedischarge pattern for the lipid bilayer recorded at a constant potential of 0.07 V and negative scan shows the absence of any leakage current through the bilayer. This indicates the presence of a compact, stable lipid bilayer with no defects, and the presence of a transportation lag spike in the CA plot is characteristic of a capacitor. Figure 7C shows the relative decrease in the height of the transportation lag spike in the presence of 20100 μM chrysin and the chrysin copper complex. Initially, the addition of chrysin (20 μM) increases the transportation lag spike, while progressive additions of chrysin decreased its height. On the other hand, in the case of the chrysincopper complex, the transportation lag spike gradually decreased with increasing concentrations of the complex. This behavior correlates with our observation that the chrysincopper 13378
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Figure 8. Influence of different concentrations of the chrysin/chrysincopper complex on the curcumin release from the lipid vesicles [values are expressed as the mean ( SD; (star symbol) p < 0.05].
complex fluidizes the bilayer to a greater extent when compared to chrysin. Curcumin Release Studies. Figure 8 shows the percentage of curcumin released from the liposomes after 20 min of incubation with different concentrations of chrysin (20100 μM) or its copper complex. It is seen that the amount of curcumin released in the presence of the chrysincopper complex is higher at each concentration when compared to chrysin (p < 0.05). Because the release of any molecule from a liposome is favored when the acyl chains are more fluidized, it is clear that the chrysincopper complex produces more fluidization of the lipid bilayer architecture, which is in conformity with the conclusions drawn from the electrophysiological experiments. DSC. The DSC results show that the phase transition of plain liposomes occurs at 47.91 °C. The phase transition temperature shifts to 44.41 °C after incubation with the chrysincopper complex. This lowering in the phase transition is associated with a disruption in membrane packing.20 Membrane Stabilization Studies. Figure 9 shows the membrane stabilization effects of chrysin and its copper complex on human erythrocytes subjected to hypotonic stress. Chrysin and its copper complex display membrane stabilization effects against saline-induced hypotonic stress. However, the chrysincopper complex exhibits nearly 72.56 ( 8.38% stabilization at 20 μM concentration when compared to 32.99 ( 6.51% for chrysin at the same concentration. As the concentration increases, the stabilization effect of chrysin remains nearly static, while that for the copper complex reduces and becomes comparable to chrysin at concentrations greater than 80 μM (p < 0.05).
’ DISCUSSION Metalflavonoid complexes have elicited great interest in recent years for their potential therapeutic applications. The structure of the complex depends upon the chelating sites available in the flavonoids. It has been reported that the most easily available chelating sites in isoflavones are the 4CdO and 5OH groups30 (Figure 1). The spectroscopic data of the chrysincopper complex shows the involvement of the 4-keto and 5-hydroxy groups of chrysin in the formation of the copper complex. The 7-OH group is not usually involved in coordination
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Figure 9. Percentage inhibition of hypotonic hemolysis in the presence of different concentrations of chrysin and the chrysincopper complex (values are expressed as the mean ( SD).
with the metal. The M/L ratio of 1:2 in the chrysincopper complex confirmed from elemental as well as spectroscopic data is in concurrence with earlier reports on copper complexes with genistein, hesperitin, naringenin, and apigenin.18,31 The proposed structure of the chrysincopper complex is in agreement with the structure of another isoflavonemetal complex, namely, the genisteincopper complex, suggested by Dowling et al.19 (Tables 1 and 232 and Figure 3). Two major types of antioxidant mechanisms have been described for flavonoids based on if they are classified as either chain-breaking antioxidants or preventive antioxidants.12 While the former class removes free radicals by electron transfer or hydrogen atom transfer, the latter inhibits free radicals by sequestering transition-metal ions by chelation. In either case, the structure of the flavonoids has a major contribution. Three major structural factors have been reported to contribute to the antioxidant activity of flavonoids: (a) the presence of an ortho-dihydroxy catechol ring, which aids in a higher degree of metal chelation, (b) a 2,3 double bond in conjugation with the 4-keto group, facilitating electron delocalization and, hence, radical scavenging property, and (c) the presence of hydroxyl groups at both the third and fifth carbon, which enables the formation of a stable quinonic structure upon oxidation.12 The absence of a 3-OH group is reported to reduce the antioxidant nature of the flavonoid.12 Chrysin lacks both the catechol ring as well as the 3-OH and, hence, exhibits minimal radical scavenging activity because of the contribution from only the 2,3 double bond in conjugation with the 4-keto group. Scheme 1 shows the mechanism involved in the free radical scavenging activity of chrysin. The phenolate form of chrysin enhances the electron-transfer reactions and, hence, contributes to better free radical scavenging effects when compared to its corresponding protonated form. In the case of the chrysincopper complex, the involvement of the 5-OH group in the formation of the complex makes it unavailable for participation in the free radical scavenging reactions and is responsible for the insignificant values observed for DPPH inhibition (Figure 4). Several studies have reported that flavonoids possess two important physiological functions: anticancer9,13,15 and antioxidant activities.24 The anticancer and antioxidant activities of 13379
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Scheme 1. Proposed Mechanism for Scavenging of Free Radicals by Chrysin in Alkaline Medium
the flavonoids have been attributed to their structure, chelation ability with metal ions, and membrane interactions.20 Studies have also shown that flavonoids promote cell death at lower concentrations and retard the same in higher doses.12,13 The interaction of flavonoids with biological membranes depends upon the polarity and hydroxyl group positions in the flavonoid core ring.20 However, the exact mechanism of action of flavonoids on cell membranes is yet to be completely understood. The molecular level interactions of the flavonoids at the membrane interface could manifest itself as changes in the fluidity of the membrane, which in turn could alter the cell functions. Similarly, the interaction of the flavonoidmetal complex at the membrane interface could provide valuable insight into the probable role of these molecules in alleviating or deteriorating cellular functions. The electrical characteristics of the lipid bilayer depend upon the nature of the phospholipids as well as the constituents present in the bathing fluid in the bilayer compartment.25 The self-assembly of the lipid bilayer driven by the presence of the aqueous medium in the bilayer chamber leads to the formation of a compact, tightly packed structure with the acyl chains forming a hydrophobic core minimizing contact with the aqueous medium and the polar head groups facing outward, favoring maximum interactions with the hydrophilic exterior.25 This arrangement is analogous to a parallel plate capacitor, with the polar head groups forming the charged plates and the acyl chains forming the dielectric layer. However, the entire structure is in a dynamic state with the lateral and flipflop motions of the phospholipid head groups and acyl chains strongly governed by their gelliquid phase transitions.20 The phase transition of the lipid bilayer is strongly influenced by numerous factors, such as temperature, ionic concentration and composition of the aqueous medium, length of the acyl chains and degree of unsaturation in the phospholipid, type of phospholipids, etc.33 The decrease in the capacitance of the lipid bilayer observed from the CV data and Nyquist plots upon addition of chrysin may be attributed to either an increase in the thickness of the bilayer or a decrease in the membrane area24 (Figure 5).
Chrysin is a relatively hydrophobic molecule with poor solubility in water, and hence, it can partition into the hydrophobic region of the lipid bilayer. However, the presence of polar groups, such as the 4-keto, 5-OH, and 7-OH groups, restricts the complete insertion of chrysin into the lipid bilayer. The polar groups in chrysin tend to interact with the polar head groups of the lipid bilayer. As the surface concentration of chrysin increases, the molecular stress at the membraneliquid interface increases, leading to partial insertion of chrysin molecules into the outer leaflet of the bilayer. These interactions lead to a distortion in the lipid arrangement and result in a curvature of the bilayer that is reflected in a decrease in membrane capacitance (Figure 5A). A similar mode of interaction with bilayers has been proposed by Henry et al. for the HIV-1 Tat peptide using molecular dynamics (MD) simulations.34 The surface interaction and partial insertion of chrysin in the bilayer leads to the creation of greater disordered regions that serve to enhance permeability to ions by providing low resistance paths through the bilayer. This is observed as a reduction in the resistance in the Nyquist plots for admittance of the bilayer as well as a reduction in the transportation lag spike in the presence of chrysin during CA measurements (Figures 5B, 6C, and 7A). However, the integrity of the bilayer is not completely compromised by the addition of chrysin because the decrease in the resistance tends to saturate beyond concentrations of 60 μM chrysin and the bilayer architecture is not completely destructed even upon the addition of 100 μM chrysin. The surface interaction of the chrysincopper complex is expected to be more pronounced because of its cationic nature. Therefore, more lateral movement of the phospholipid head groups is likely to be favored to facilitate better interaction with the strongly polar chrysincopper complex. This surface interaction will cause more distortion in the lipid arrangement, leading to greater fluidity of the bilayer (panels A and B of Figure 6A).35,24 Similar observations were predicted for the interaction of benzyl alcohol with planar lipid bilayers.36 The 13380
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Langmuir decrease in capacitance and resistance of the bilayer as well as the transportation lag spike observed during chronoamperometry studies (Figure 7B) in the presence of the chrysincopper complex also supports the above mechanism. The magnitude of decrease in the membrane parameters is much higher in the case of the chrysincopper complex when compared to the parent flavonoid. This can be due to greater surface interactions of the charged complex as well as the larger size of the complex when compared to chrysin (Figure 6C). The partial insertion of the bulkier complex will cause significant structural reorganization of the lipids and, hence, experience greater fluidization.37 A membrane permeabilization study using fluorophoreloaded liposomes is a simple and elegant method to determine the influence of various parameters on membrane permeability and integrity.20 The experiments carried out with curcuminloaded liposomes incubated with chrysin and the chrysin copper complex confirm the conclusions drawn from the electrochemical studies, with more amount of curcumin released from liposomes incubated with the chrysincopper complex (Figure 8). This implies that, while both chrysin and its copper complex fluidize the acyl chains of the lipids causing the release of curcumin, the magnitude of fluidization caused by the chrysin copper complex is significantly higher than chrysin. Similar experiments have been carried out with calcein-loaded liposomes to evaluate the fluidization capability of genistein.38 DSC is one of the well-known techniques to monitor the membrane interaction and alteration of membrane fluidity by different pharmacologically active components. The interaction of the chrysincopper complex with the phospholipid bilayer had resulted in a reduction in the phase transition temperature, implying fluidization of the lipid acyl chains. This inference is in concurrence with the curcumin release studies.20,39 The interactions of chrysin and the chrysincopper complex with cellular membranes could provide insight into their role in stabilizing or destabilizing cellular integrity. Erythrocyte membranes provide a more realistic model for probing molecular interactions and membrane integrity.28 Stabilization against hypotonic stress-induced hemolysis is a good indicator of the protective effect of a molecule against disruption of the bilayer architecture. Flavonoids have generally been shown to exhibit membrane-protective effects against hypotonic hemolysis.28 However, the stabilization effect is dependent upon the localization of the molecule in the membrane.28 Both chrysin and its copper complex exhibit protection against hypotonic hemolysis. Chrysin shows a membrane stabilization effect that is significantly lower than that exhibited by its copper complex at concentrations below 80 μM. However, the membrane stabilization effect of the chrysincopper complex gradually reduces with increasing concentrations and becomes comparable to chrysin at concentrations of 80 μM and above. The initial stabilization effect of chrysin and its copper complex may be attributed to their strong surface interactions. The cationic copper complex shows greater ability to preserve the membrane integrity, owing to its strong electrostatic interactions with the membrane surface. However, at higher concentrations, a greater degree of fluidization caused by the copper complex counters the stabilizing effect, and hence, a decrease is observed in the membrane stabilization value. At concentrations of 80 μM and above, no significant difference is observed between chrysin and its copper complex. Thus, strong surface interactions and partial penetration into the outer leaflet of the membrane seem to promote a better stabilizing effect by reducing osmotic forces against hypotonic hemolysis (Figure 9).
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’ CONCLUSION Chrysin is a flavonoid with mild antioxidant properties. In this study, we have used a lipid bilayer model to understand the molecular interactions of flavonoids at the membranewater interface. Chrysin forms a coordination complex with copper at room temperature, having a metal/ligand ratio of 1:2. The complex exhibits a strong surface interaction with the polar head groups of the phospholipids in the bilayer. Electrochemical investigations of chrysin and its copper complex, reported here for the first time, reveal that the copper complex displays a greater membrane fluidizing effect in a dose-dependent manner, as indicated by the liposomal permeabilization, erythrocyte stabilization, and DSC studies. It could stabilize membranes at lower concentrations (
