Molecular structure and interactions of the flavonols, quercetin, fisetin, kaempferol, and myricetin, with liposomal membranes

Molecular structure and interactions of the flavonols, quercetin, fisetin, kaempferol, and myricetin, with liposomal membranes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular structure and interactions of the flavonols, quercetin, fisetin, kaempferol, and myricetin, with liposomal membranes Artem G. Veiko, Szymon Sekowski, Ewa Olchowik-Grabarek, Agnieszka Z. Wilczewska, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4477073/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The interactions of flavonols with biological membranes underlie their beneficial biochemical effects. In the present work, we performed quantum chemical modeling of the molecular structure and electronic characteristics of some flavonols such as fisetin, kaempferol, and myricetin and compared our findings with those for quercetin obtained earlier. We considered the effects of the flavonols on liposomal membranes, using the methods of fluorescence probe spectroscopy, an electric-kinetical method and differential scanning calorimetry. The AC and B rings in the molecules of all the flavonols studied were located in the same plane. All the flavonols (5–25µM) increased the lipid bilayer order both in the surface zone and the hydrophobic area of the membrane. Quercetin was more effective in changing the liposomal membrane mobility and fisetin modulated markedly the thermotropic behavior of the membrane. Myricetin was located predominantly in the surface zone, whereas quercetin penetrated into the deeper zone of the bilayer. Using the fluorescent probe Laurdan we showed that all the flavonols studied increased the hydration of the lipid bilayer. The incorporation of effector molecules into the liposomal membrane bilayer resulted in an increase in the absolute value of zeta potential and induced an increase in the liposomal diameter. Destabilization and enhanced heterogeneity of liposomal membranes in the presence of all the flavonols studied were revealed. flavonols liposomal membranes fluidity differential scanning calorimetry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Flavonoids, second plant metabolites and one of the most important classes of polyphenolic phytoconstituents, are abundant in human diet. They possess numerous biological activities such as antidiabetic, antihyperlipidemic, antioxidative and freeradical scavenging, cytoprotective, cardioprotective, antiviral and antibacterial, anti-aging, and antiinflammatory properties [1–3]. Application of plant constituents instead of synthetic pharmaceutical agents demonstrated a number of advantages associated with low toxicity, specificity, and a wide spectrum of activities. One of the most common and largest flavonoid subgroups in fruit and vegetables is flavonols which possess high biological and medicinal importance and vary in methylation and hydroxylation modes. Quercetin, myricetin, kaempferol, and fisetin are major dietary flavonols [ 4 ]. The average intake of the flavonol fisetin was determined to be approximately 0.4 mg/day [ 5 ]. The chemical structure of flavonols is characterized by an unsaturated C ring in position C2-C3, a ketone group in position 4 and a hydroxyl group in position 3 of the C ring (the 3-hydroxyflavone backbone) [ 6 ]. It has recently been shown that the biological and antioxidant activities of such flavonoids as luteolin, kaempferol, apigenin and quercetin, are directly proportional to the number of phenolic hydroxyl groups [ 7 ]. A number of the intracellular and extracellular targets of the flavonols was estimated. Fisetin regulates AMP-activated protein kinase (AMPK), nuclear factor-kappa B (NF-kB), epidermal growth factor receptor (EGFR), cyclooxygenase (COX); extracellular signal-regulated kinase (ERKI1/2), metalloproteinase (MMP), prostate-specific antigen (PSA), transcription factor T-cell factor (TCF), TNF-related apoptosis-inducing ligand (TRAIL), and X-linked inhibitor of apoptosis (XIAP) [ 5 , 8 ]. The flavonols quercetin and myricetin (5 µM) were nontoxic to the intestinal epithelial IEC-6 cells, but suppressed the RhoA/ROCK signaling pathway [ 9 ]. The mechanisms for the neuroprotective action of myricetin are prevention of oxidative stress, intracellular Ca 2+ accumulation and apoptosis. Other mechanisms of myricetin health effects include the activation of such signaling cascades as the nuclear factor E2 (Nrf2), extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (Akt), cAMP-response element binding protein (CREB) [ 10 ]. Kaempferol can ameliorate generation of the inflammatory mediators nitric oxide, TNF-a, IL-1β, and IL-8 in macrophages [ 11 ]. Specific (with target proteins and membrane domains) and non-specific (intercalation in lipid bilayer) interactions of polyphenols with cell components are crucial for determination of their beneficial pharmacological and biochemical effects. It has recently been found that kaempferol and myricetin modify the surface charge density and the structure of the model membranes [ 12 ]. It was also demonstrated that phenolic hydroxyl groups in the flavonoids form hydrogen bonds with cell membranes [ 13 ]. The main question dealing with the beneficial health effects of flavonoids is the chemical basis for their biological activity. Since a molecular structure and electronic characteristics play a key role in biochemical activity, the correlations between the structure and the bioactivities of the flavonoids have been widely studied using methods of quantum chemistry [ 14 – 16 ]. Earlier we analyzed the relationship between the molecular optimal conformations and the antioxidative activity of the flavonol quercetin, the flavanol catechin and the flavanone naringenin and evaluated the interaction of these flavonoids with cellular and artificial membranes [ 17 , 18 ]. We showed that the flavonoids considerably inhibited erythrocyte membrane lipid peroxidation and potentiated Ca 2+ ions - induced mitochondrial permeability transition [ 18 ]. The detailed mechanism of interaction of polyphenols with membranes (such as binding affinities, locations in membrane, changes in ordering and motility) has not been fully elucidated. To evaluate the flavonol structure-function relationships, we performed quantum chemical modeling of the optimal geometry and electronic parameters of the flavonols fisetin, kaempferol, and myricetin varying in the number and position of the OH groups, and assessed their interactions with liposomal membranes, using fluorescence probe spectroscopy, an electric-kinetical method and differential scanning calorimetry. We compared our present findings with those for quercetin obtained earlier [ 17 , 18 ]. 2. Materials and methods 2.1. Chemicals The flavonolsquercetin (3,3′,4′,5,7-pentahydroxyflavone),fisetin (3,3′,4′,7-tetrahydroxyflavone), kaempferol (3,4′,5,7-tetrahydroxyflavone), and myricetin (hexahydroxyflavone), as well as 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethyl ammonium phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 6-dodecanoyl-2-dimethylamine-naphthalene (Laurdan), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and other chemicals were from Merck / Sigma-Aldrich (St Louis, MO, USA, or Steinheim am Albuch, Germany), tetrahydrofuran, methanol ethanol, dimethyl sulfoxide, and chloroform were from POCh (Poland). The freshly prepared flavonol solutions (5 mM) in ethanol were used. Ethanol at the concentrations added did not influence the parameters measured. 2.2. Quantum mechanical calculations of molecular geometry and electronic properties of the flavonols The optimal geometries and molecular parameters of the flavonols were evaluated theoretically by both the semi-empirical molecular orbital theory and ab initio calculations by using the HyperChem-8.0 software package (HyperCube, Inc.) [ http://www.hyper.com ] as we described earlier [ 17 ]. The optimized conformations were considered using the Austin Model 1 (AM1) semi-empirical method within unrestricted Hartree-Fock (UHF) formalism and the Polak-Ribiere algorithm [ 19 , 20 ]. 2.3. Liposome preparation Unilamellar liposomes (unilamellar bilayer vesicles) of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC,14:0) were prepared using chloroform/lipid mixtures and an Avanti Polar Lipids Mini-Extruder (Avanti Polar Lipids, Birmingham, AL, USA) as described previously [ 18 ]. The final liposomes concentration was 100 µg/mlin isotonic buffered saline (PBS,145 mM NaCl, 1.9 mM NaH 2 PO 4 , 8,1 mM Na 2 HPO 4 , pH 7.4). 2.4. TMA-DPH, DPH, and Laurdan fluorescence measurements in the lipid bilayer Membrane fluidity and ordering were analyzed by fluorescence anisotropy of TMA-DPH (λex = 340 nm and λem = 430 nm) and DPH (λex = 348 nm and λem = 426 nm)probes as well as probe Laurdan generalized polarization (GP) using a Perkin-Elmer LS 55B spectrofluorimeter (UK) as we described previously [ 18 ]. We used freshly prepared TMA-DPH (in methanol), DPH (in tetrahydrofuran) and Laurdan (in dimethyl sulfoxide) stock solutions, at concentrations of 1 mM. The ratio (r s /r 0 ) was used for describing the TMA-DPH and DPH fluorescence changes, where r s is the probe fluorescence anisotropy in the presence, and r 0 is probe fluorescence anisotropy in the absence of the flavonols. The DPH molecule is located in the hydrophobic area of the liposomal membrane occupied by the hydrocarbon chains and the TMA-DPH dye is located at the aqueous/membrane interface [ 21 ]. The increase in probe fluorescence anisotropy reflects an enhancement in stiffness in the hydrophobic region of the membrane. Laurdan generalized polarization (GP) was calculated using the equation: GP = (I 440 – I 490 )/(I 440 + I 490 ), where I 440 and I 490 are fluorescence intensities recorded at 440 and 490 nm, respectively, after exciting at 350 nm. The fluorophore Laurdan is located at the level of the glycerol backbone of phospholipids and fluorescence parameters are associated with changes in fluorophore dipole moment and provide information about the polarity of the environment, packing order and hydration level in the region of the polar groups of the bilayer. An increase in GP indicates an increase in the packing order in the water-lipid interface of membranes and thus a decrease in the hydration level [ 22 , 23 ]. 2.5. The thermotropic parameters of the liposomal membranes Differential scanning calorimetry (DSC) was applied to analyze the thermal properties of liposomal membranes in the presence of the flavonols using a Mettler Toledo Star DSC system (Mettler Toledo, Switzerland) and Mettler-Toledo STARe software. A sample (6800 µM of DMPC and 500 µM flavonol) in 20 µl of PBC was placed in an aluminum crucible and sealed. The samples were heated from 10°C to 30°C at a rate of 2.5°C/min under argon flow (200 ml/min). An empty sealed crucible was used as a reference. Phase-transition enthalpy (ΔH), temperature of the thermal effect start (T onset ), phase transition temperature (T m , the midpoint of the heat capacity change), and half-width of the transition (ΔT 1/2 ) were determined [ 18 ]. 2.6. Liposomal zeta potential and diameter Electrokinetic parameters of liposomes: zeta potential (сonnected with the mobility of charged particles) and liposomal mean diameter were measured with a Nano ZS Zeta sizer (Malvern, USA) and were analyzed using the Malvern software as we described earlier [ 18 ]. For size measurements DMPC liposomes (100 µg/ml) were dissolved in phosphate buffer, pH 7.4, and for potential measurements liposomes were dissolved in water, pH 6.5. 2.7. Statistics Statistical analysis was performed using the one-way analysis of variance (ANOVA) (GraphPad Prism v.6.0 software, GraphPad Software, Inc., La Jolla, CA, USA) with Tukey’s test. The results of five to six independent experiments were shown as means ± SEM, p < 0.05 was considered statistically significant. 3. Results For better understanding of the cellular mechanism(s) of pharmaceutical applications of the flavonols, we compared molecular structures and electronic parameters of the flavonols and their interactions with liposomal membranes. 3.1. Optimal molecular geometry and properties of the flavonols Figure 1 shows the flavonol molecular geometries and excess charges of the atoms calculated by an ab initio method and Table 1 shows the electronic parameters of quercetin, fisetin, kaempferol, and myricetin molecules: dipole moment, heat of formation, E (HOMO) - E (LUMO) Energy, surface area, etc., calculated by the use of the semi-empirical molecular orbital theory [ 17 , 24 ]. The A, C and B rings in the all flavonol molecules studied were located in the same plane (Fig. 1 ). The dipole moment of the flavonol molecules increased in the order: kaempferol (0.195 D) < fisetin (0.617 D) < quercetin (0.986 D) < myricetin (1.224 D) (Table 1 ), which reflects the polarity of the flavonols and the efficiency of their electrostatic interactions. It should be noted that these values depend on the local minima for the molecular geometry optimizations. For example, Aparicio, using DFT calculations, showed the following values: 1.66 D for kaempferol, 2.71 D for quercetin,1.5 D for myricetin [ 25 ]. The surface area and the volume values of the flavonol molecules studied were similar. Table 1 – Quantum chemical parameters and water solubility of kaempferol, fisetin, quercetin, and myricetin Parameters Kaempferol Fisetin Quercetin Myricetin AM1 UHF Number of electrons 106 106 112 118 Total Energy, kcal/mol -91771.70 -91768.75 -99164.77 -106558.6 Binding Energy, kcal/mol -3614.109 -3611.156 -3717.600 -3821.805 Isolated Atomic Energy, kcal/mol -88157.6 -88157.6 -95447.2 -102736.8 Electronic Energy, kcal/mol -550333.8 -547637.9 -601021.9 -653768.3 Heat of Formation, kcal/mol -172.385 -169.432 -216.317 -260.963 Dipole Moment, D 0.195 0.617 0.986 1.224 Ab initio UHF (6-31G) Total Energy, kcal/mol -641697.12 -641698.60 -688649.20 -735602.9 Electronic Kinetic Energy, kcal/mol 642082.6 642118.8 689036.7 735994.3 Nuclear Repulsion Energy, kcal/mol 995016.955 983433.783 1082078.15 1172466.70 E Alpha Orbitals (HOMO), eV -8.2141 -8.318870 -8.238066 -8.321660 E Beta Orbitals (HOMO), eV -8.212995 -8.724547 -8.237996 -8.321773 E Alpha Orbitals (LUMO), eV 1.2821 1.635402 1.229848 1.120749 E Beta Orbitals (LUMO), eV 1.280239 2.486912 1.229325 1.121896 Alpha Orbitals ∆E = E (HOMO) - E (LUMO), eV -9.4962 -9.954272 -9.467914 -9.442409 Beta Orbitals ∆E = E (HOMO) - E (LUMO), eV -9.493234 -11.211459 -9.467321 -9.443669 Surface area (Grid), Å 2 443.65 451.59 453.62 459.99 Volume, Å 3 734.03 743.35 753.52 772.75 Hydration energy, kcal/mol -27.24 -27.24 -32.53 -37.61 Torsion angles, º 180 180 180 180 Water solubility, mg/L 113 [ 26 ] 89 [ 27 ] 0.512 [ 28 ] 0.32 [ 29 ] 3.2. Liposomal membrane ordering and fluidity in the presence of the flavonols. To understand the mechanisms of the flavonol interactions with phospholipid membranes, we evaluated the changes in ordering and fluidity of the DMPC liposomal bilayer in the presence of kaempferol, fisetin, quercetin, and myricetin, using the fluorescent probes, DPH, TMA-DPH, and Laurdan, differently located in the membrane (Figs. 2 – 4 ). We showed that quercetin, kaempferol, fisetin, and myricetin (5–25µM) increased the TMA-DPH and DPH fluorescence anisotropy parameter, reducing lipid bilayer fluidity or enhancing the lipid bilayer order both at the surface zone (at the aqueous/membrane interface) and in the hydrophobic area (the area of the hydrocarbon chains) of the membrane (Fig. 2 ). Quercetin was more effective in changing liposomal membrane fluidity. The flavonols effectively quenched the TMA-DPH and DPH fluorescence in the liposomal lipid bilayer. The plots of TMA-DPH (Fig. 3 a) and DPH (Fig. 3 b) fluorescence quenching in liposomal membranes by the flavonols are presented in Stern-Volmer coordinates. The Stern-Volmer constant values Ksv of the DPH fluorescence quenching decreased in the order: quercetin (1.74 ± 0.28)·10 6 M -1 > kaempferol (0.89 ± 0.16)·10 6 M -1 ≥ myricetin (0.68 ± 0.12)·10 6 M -1 > fisetin (0.22 ± 0.04)·10 6 M -1 , reflecting the accessibility of DPH to the flavonols. The accessibility of the probe TMA-DPH to the flavonols decreased in a different order: myricetin (2.02 ± 0.25)·10 6 M -1 > quercetin (1.53 ± 0.22)·10 6 M -1 > kaempferol (0.71 ± 0.14)·10 6 M -1 > fisetin (0.15 ± 0.02)·10 6 M -1 . We compared the effects of the flavonols on the DPH and TMA-DPH fluorescence parameters with that of the Laurdan probe embedded in the liposomal membrane bilayer. As Fig. 4 showed, all the flavonols significantly reduced Laurdan GP, probably due to growth of the hydration level of the polar group region of the lipid bilayer. According to these observations, kaempferol and quercetin were more effective in the modulation of membrane structure, as was monitored by Laurdan fluorescence. 3.3. Liposomal zeta potential and diameter in the presence of the flavonols. The incorporation of flavonoid molecules into the liposomal membrane bilayer resulted in an increase in an absolute value of the membrane zeta potential in the following order: fisetin ˃ myricetin ˃ quercetin ˃kaempferol (Fig. 5 a). The intercalation of quercetin, kaempferol, and myricetin into the liposomal bilayer increased the diameter of the DPMC liposomes (Fig. 5 b). 3.4. Differential scanning calorimetry measurements of the flavonole effects on the model membranes. The flavonol effects on the thermotropic properties of DMPC liposomes were evaluated by DSC. Figure 6 shows representative thermograms of DMPS membranes in the absence and in the presence of the flavonols studied. The thermogram of pure DMPC bilayer unilamellar liposomes demonstrated a sharp main phase transition peak at a temperature of T m = 25.1 ± 0.3°C and a weak pretransition peak at 15.8 ± 0.3°C. This peak corresponds to a reorganization of individual DPMC molecules. Table 2 shows the thermotropic parameters of the DPMC liposomal membranes in the presence of the flavonols studied. The width of the transition at half-peak height (ΔT 1/2 ) reflects the cooperativity of the membrane thermal transition (Table 2 ). The flavanols caused disappearance of the pretransition peak and significantly changed the parameters of the DMPC membranes melting. All the flavonols studied lowered the temperature and the enthalpy and of the liposomal membrane melting, as well as increased the width of the phase transition peak (Fig. 6 , Table 2 ). We observed the most considerable decrease in the membrane phase transition enthalpy and appearance of a small second transition peak (28.6 ± 0.3°C) in the presence of fisetin. Table 2 Effects of quercetin, myricetin, kaempferol, and fisetin on the parameters of the DMPC membrane phase transition (5 mg of liposomes/ml in PBS, pH 7.4, the DMPC:flavonoid ratio was 6 800 µM:500 µM) Sample ΔH (mJ) T onset (◦C) T m (◦C) ΔT 1/2 (◦C) DMPC 50.49 ± 0.12 24.5 ± 0.3 25.1 ± 0.3 0.75 ± 0.01 DMPC-Quercetin 30.04 ± 0.08 20,9 ± 0.2 22.9 ± 0.2 1.75 ± 0.05 DMPC-Myricetin 30.52 ± 0.12 22.3 ± 0.1 23.9 ± 0.2 1.81 ± 0.02 DMPC-Kaempferol 25.08 ± 0.09 21.5 ± 0.2 23.5 ± 0.2 1.99 ± 0.02 DMPC-Fisetin 8.26 ± 0.12 20.6 ± 0.2 22.3 ± 0.2 2.05 ± 0.03 4. Discussion The complicated chemistry of the flavonols comprises the structure of the molecules, aromatic electron delocalization, proton/electron transfer, the interactions with free radicals, and determines their biological activity [ 8 ]. Numerous works deal with biochemical mechanism(s) of flavonoid-membrane interactions. In an earlier work, Oteiza et al. suggested two possibilities of such interactions depending on the number and distribution of OH-groups, planarity and hydrophilicity of flavonoid molecules: (a) intercalation of non-polar flavonoids into the inner hydrophobic zone of the membrane, and (b) formation of hydrogen bonds at the membrane-water boundary [ 30 ]. Tsuchiya showed that flavonols (kaempferol, quercetin, and myricetin) (1–10 µM) affected deeper areas of liposomal membranes, decreasing membrane fluidity [ 31 , 32 ]. The ability of the flavonols to modify membrane structure, and permeability of artificial and cellular membranes is correlated with their beneficial biochemical effects [ 31 , 32 ]. In cells, the flavonols influence the raft formations and control membrane heterogeneity [ 33 ]. Recently it was shown that flavonol molecules form complexes with membrane phosphatidylcholine in the ratio of 1:1, and the complex formation energy is -37 ± 1 kJ mol − 1 in the case of kaempferol and − 36 ± 1 kJ mol − 1 in the case of myricetin [ 12 ]. All the flavonols studied demonstrated planar molecular geometry. The torsion angle was С3-С2-B1’-B2’ ≈ 180°. Earlier we suggested that the C2 = C3 bond in the C-ring determined the planar geometry of quercetin as well as its semiquinone radicals and the corresponding quinones as optimal forms in vacuum [ 34 ]. These results are in agreement with the findings of Aparacio [ 25 ] and Günther et al. [ 35 ] showing that the quercetin, kaempferol, and myricetin optimized geometry is planar. According to our ab initio calculations, the net negative excess charges of the flavonol C rings (Fig. 1 ) make this part of the molecules attractive for electrophilic attack. It can be noted that the myricetin molecule has the lowest LUMO energy value (the lowest unoccupied molecular orbital, i.e. a free orbital with the lowest energy) and, therefore, acts as an effective electron acceptor when attacked by nucleophiles and possesses the highest dipole moment value (Table 1 ). According to TMA-DPH and DPH fluorescence anisotropy measurements, all the flavonols studied, quercetin, kaempferol, fisetin, and myricetin (5–25µM), modulated the membrane organization and mobility at different depths of the bilayer and quercetin was more effective in changing liposomal membrane properties. Earlier, applying ТМА-DPH and DPH probes, we showed that quercetin at low concentrations of up to 1 µM increased the rigidity of the inner hydrophobic region of the red blood cell membrane and diminished the rigidity of the surface zone of the erythrocyte membrane [ 36 ]. In line with our observations, Włoch et al. have recently shown that methylated flavonoids caused an increase in packing order of polar lipid heads and a decrease in fluidity in erythrocyte and model membranes [ 37 ]. Using the Stern-Volmer constants of fluorescence quenching of DPH (in hydrophobic zone) and TMA-DPH (in surface hydrophilic zone) probes, we concluded that the flavonols quercetin and kaempferol were more predominantly located in the deeper part of the bilayer, and myricetin (possesses 6 hydroxylic groups and the highest dipole moment according to our calculations) was mostly located in the surface zone of the membrane (membrane/water interface). According to this parameter, the accessibility of both the zones of the bilayer to fisetin (possesses 4 hydroxylic groups and low dipole moment) was much lower in comparison with quercetin, myricetin or kaempferol. It has previously been shown that quercetin strongly perturbs cholesterol/sphingolipid enriched domains at cell membranes where signalling platforms are expected to assemble [ 38 ]. Sengupta and coworkers reported that fisetin binds in liposomal membrane area between the polar head and hydrophobic tail of the phospholipids [ 39 ]. According to Laurdan fluorescence, the flavonols increased the bilayer hydration at the hydrophobic-hydrophilic interface of the model membrane. Similarly, Günther et al. suggested a greater possibility of water access to lipid bilayer in the presence of flavonols [ 35 ]. In contrast, Włoch et al. suggested that flavonoids decreased water content in the hydrophilic region of the bilayer [ 37 ]. The incorporation of negatively charged flavonols into liposomal membranes increased the electrical charge of the membranes and caused an enlargement in zeta potential value, reflecting a strong interaction between the flavonols and liposomes. The interaction depended on the flavonols lipophilicity and charge, and fisetin and myricetin were more effective in influencing zeta potential of the liposomal surface. Zeta potential characterizes the electrostatic interactions between liposomes in suspension and zeta potential value predicts membrane functional activity. Quercetin and kaempferol were most effective in an enlargement of the surface area of the liposomal membrane. It has previously been suggested that flavonoids induced the formation of bridges between adjacent membrane surfaces and aggregation and rigidification of the phospholipid membranes, and that the membrane aggregation was followed either by the production and release of daughter vesicles or formation of endo-buds [ 40 , 41 ]. The interaction of the flavonols with membranes caused membrane destabilization (decreased the enthalpy and transition temperature) and a rise in membrane heterogeneity (increased the width of the transition at half-peak height) (Table 2 ). The complex changes in membrane fluidity, ordering, electrophoretic properties, stability, and surface area depended on the molecular parameters of the flavonols, as well as the number of the OH-groups, and lipophilicity (Table 1 ). The contributions of different forces to flavonoid - membrane interactions underlie the strength of the interactions and localization of the flavonols in the bilayer. Conclusions Flavonols, the main class of the flavonoids, characterized by a complex chemical structure and electronic properties, have numerous biological targets. The AC and B rings in the molecules of all the flavonols studied, quercetin, kaempferol, myricetin, and fisetin, were located in the same plane due to the double C2-C3 bond. In experiments in vitro , all the flavonols (5–25 µM) increased the lipid bilayer order at different depths, reduced the fluidity and increased the hydration. Quercetin was more effective in changing the liposomal membrane mobility and fisetin modulated markedly the thermotropic behavior of the membrane. Quercetin and kaempferol penetrated into the deeper zone of the bilayer and myricetin was located predominantly in the surface zone. We revealed that the flavonols quercetin, kaempferol, myricetin, and fisetin incorporated into the liposomes and increased zeta potential and enlarged the area of the bilayer, as well as led to membrane destabilization and raised membrane heterogeneity. Declarations Funding This study was partially supported by grant No М23Ch-014 (from 01.11.2022) from the Belarusian Republican Foundation for Fundamental Research. AGV was supported by the Program of Fellowships under The Polish National Commission for UNESCO (No 251/E/2020 from 09.10.2020).The thermal analyses were performed in the Centre of Synthesis and Analysis BioNanoTechno of the University of Bialystok. The equipment in the Centre was funded by the EU as a part of the Operational Program Development of Eastern Poland 2007-2013. Projects: POPW.01.03.00-20-034/09-00 and POPW.01.03.00-20-004/11. Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Author Contributions A.G. Veiko: investigation, data curation, software, S. Sekowski: investigation, visualization, supervision,E. Olchowik-Grabarek: investigation, data curation, visualization A. Z. Wilczewska: investigation, data curation, software, visualization, I. Dobrzyńska: investigation, data curation, software, visualization, Anna Roszkowska: investigation, visualization, E.A. Lapshina: data curation, validation, writing-original draft preparation, M. Zamaraeva: conceptualization, methodology, supervision, I.B. Zavodnik: conceptualization, data curation, supervision, writing-reviewing and editing. All the authors approved the final version of the manuscript. Data Availability All the data generated or analyzed during this study have been included in this article. Consent to participate All the authors meet the qualifications for authorship and had an opportunity to read and comment the manuscript. All the authors support publication of the manuscript in Molecular and Cellular Biochemistry. References Khan J, Deb PK, Priya S et al (2021) Dietary flavonoids: cardioprotective potential with antioxidant effects and their pharmacokinetic, toxicological and therapeutic concerns. Molecules 26:4021. https://doi.org/10.3390/molecules26134021. Wang T, Li Q, Bi K (2018) Bioactive flavonoids in medicinal plants: structure, activity and biological fate. Asian J Pharm Sci 13:12–23. https://doi.org/10.1016/j.ajps.2017.08.004. Havsteen BH (2002) The biochemistry and medical significance of the flavonoids. Pharmacology & Therapeutics 96:67–202. https://doi.org/10.1016/S0163-7258(02)00298-X. Mahmud AR, Ema TI, Siddiquee MF et al (2023) Natural flavonols: actions, mechanisms, and potential therapeutic utility for various diseases. Beni-Suef Univ J Basic Appl Sci 12:47. https://doi.org/10.1186/s43088-023-00387-4. Kashyap D, Sharma A, Sak K et al (2018) Fisetin: a bioactive phytochemical with potential for cancer prevention and pharmacotherapy. Life Sci 194:75–87. https://doi.org/10.1016/j.lfs.2017.12.005. Chagas MSS, Behrens MD, Moragas-Tellis CJ et al (2022) Flavonols and flavones as potential anti-inflammatory, antioxidant, and antibacterial compounds. Oxid Med Cell Longev 2022:Article ID 966750. https://doi.org/10.1155/2022/9966750. Tian C, Liu X, Chang Y et al (2021) Investigation of the anti-inflammatory and antioxidant activities of luteolin, kaempferol, apigenin and quercetin. South African Journal of Botany 137:257-264. https://doi.org/10.1016/j.sajb.2020.10.022. Grynkiewicz G and Demchuk OM (2019) New Perspectives for Fisetin. Front Chem 7:697. https://doi.org/10.3389/fchem.2019.00697. Fan J, Li TJ, Zhao XH (2020) Barrier-promoting efficiency of two bioactive flavonols quercetin and myricetin on rat intestinal epithelial (IEC-6) cells via suppressing Rho activation. RSC Adv 10:27249-27258. https://doi.org/10.1039/d0ra04162a. Li J, Xiang H, Huang C, Lu J (2021) Pharmacological actions of myricetin in the nervous system: a comprehensive review of preclinical studies in animals and cell models. Front Pharmacol 12:797298. https://doi.org/10.3389/fphar.2021.797298. Nam SY, Jeong HJ, Kim HM (2017) Kaempferol impedes IL-32-induced monocytemacrophage differentiation. Chem Biol Interact 274:107–115. Laszuk P, Petelska AD (2021) Interactions between phosphatidylcholine and kaempferol or myristicin: langmuir monolayers and microelectrophoretic studies. Int J Mol Sci 22:4729. https://doi.org/10.3390/ijms22094729. Sirk TW, Brown EF, Sum AK, Friedman M (2008) Molecular dynamics study on the biophysical interactions of seven green tea catechins with lipid bilayers of cell membranes. J Agric Food Chem 56:7750–7775. Safe S, Jayaraman A, Chapkin RS et al (2021) Flavonoids: structure–function and mechanisms of action and opportunities for drug development. Toxicol Res 37:147–162. https://doi.org/10.1007/s43188-020-00080-z. Mendoza-Wilson AM, Santacruz-Ortega H, Balandrán-Quintana RR (2011) Spectroscopic and computational study of the major oxidation products formed during the reaction of two quercetin conformers with a free radical. Spectrochim Acta A Mol Biomol Spectrosc 81:481–488. https://doi.org/10.1016/j.saa.2011.06.041. Rasulev BF, Abdullaev ND, Syrov VN, Leszczynski J (2005) A quantitative structure-activity relationship (QSAR) study of the antioxidant activity of flavonoids. QSAR Comb Sci 24:1056–1065. https://doi.org/10.1002/qsar.200430013. Veiko AG, Lapshina EA, Zavodnik IB (2021) Comparative analysis of molecular properties and reactions with oxidants for quercetin, catechin, and naringenin. Mol Cell Biochem 476:4287–4299. https://doi.org/10.1007/s11010-021-04243-w. Veiko AG, Sekowski S, Lapshina EA et al (2020) Flavonoids modulate liposomal membrane structure, regulate mitochondrial membrane permeability and prevent erythrocyte oxidative damage. Biochim. Biophys. Acta– Biomembranes1862:183442. https://doi.org/10.1016/j.bbamem.2020.183442. Erkoç Ş, Erkoç F, Keskin N (2003) Theoretical investigation of quercetin and its radical isomers. J Mol Struct 631:141–146. https://doi.org/10.1016/S0166-1280(03)00237-9. Onishi T (2018) Quantum computational chemistry: modelling and calculation for functional materials. Springer, Singapore. https://doi.org/10.1007/978-981-10-5933-9. Mykytczuk NC, Trevors JT, Leduc LG, Ferroni GD (2007) Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress. Prog Biophys Mol Biol 95:60-82. https://doi.org/10.1016/j.pbiomolbio.2007.05.001. Harris FM, Best KB, Bell JD (2002) Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order. Biochim Biophys Acta 1565:123-128. Sanchez SA, Tricerri MA, Gratton E (2012) Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo. Proc Natl Acad Sci 109:7314–7319. https://doi.org/10.1073/pnas.1118288109. Veiko AG, Lapshina EA, Yukhnevich HG, Zavodnik IB (2023) Study of the interaction of naringenin, apigenin, and menadione with membranes using fluorescent probes and quantum chemistry. J Appl Spectrosc 90:535–542. https://doi.org/10.1007/s10812-023-01564-0. Aparicio S (2010) A systematic computational study on flavonoids. Int J Mol Sci 11:2017-2038. https://doi.org/10.3390/ijms11052017. Deng S, Yang YL, Cheng XX et al (2019) Synthesis, spectroscopic study and radical scavenging activity of kaempferol derivatives: enhanced water solubility and antioxidant activity. Int J Mol Sci 20:975. https://doi.org/ 10.3390/ijms20040975. Skiba M, Gasmi H, Milon N et al (2021)Water solubility and dissolution enhancement of fisetin by spherical amorphous solid dispersion in polymer of cyclodextrin. Austin J Biotechno Bioeng 8:1106. Saija A, Tomaino A, Trombetta D et al (2003)‘In vitro’ antioxidant and photoprotective properties and interaction with model membranes of three new quercetin esters. Eur J Pharm Biopharm 56:167–174. Lin T-C, Yang C-Y, Wu T-H et al (2023) Myricetin nanofibers enhanced water solubility and skin penetration for increasing antioxidant and photoprotective activities. Pharmaceutics 15 :906. https://doi.org/10.3390/pharmaceutics15030906. Oteiza PI, Erlejman AG, Verstraeten SV et al (2005) Flavonoid-membrane interactions: a protective role of flavonoids at the membrane surface? Clin Dev Immunol 12:19-25. https://doi.org/10.1080/10446670410001722168. Tsuchiya H (2011) Effects of red wine flavonoid components on biomembranes and cell proliferation. Int J Wine Res 3:9–17. Tsuchiya H (2015) Membrane interactions of phytochemicals as their molecular mechanism applicable to the discovery of drug leads from plants. Molecules 20:18923-18966. https://doi.org/10.3390/molecules201018923. Tarahovsky YS, Muzafarov EN, Kim YA (2008) Rafts making and rafts braking: how plant flavonoids may control membrane heterogeneity. Mol Cell Biochem 314:65-71. https://doi.org/10.1007/s11010-008-9766-9. Ilyich TV, Veiko AG, Lapshina EA, Zavodnik IB (2018) Quercetin and its complex with cyclodextrin against oxidative damage of mitochondria and erythrocytes: Experimental results in vitro and quantum-chemical calculations. Biophysics 63:537–548. https://doi.org/10.1134/S0006350918040073. Günther G, Berríos E, Pizarro N, et al (2015) Flavonoids in microheterogeneous media, relationship between their relative location and their reactivity towards singlet oxygen. PLoS ONE 10:e0129749. https://doi.org/10.1371/journal.pone.0129749. Veiko AG, Olchowik‐Grabarek E, Sekowski S et al (2023) Antimicrobial activity of quercetin, naringenin and catechin: flavonoids inhibit staphylococcus aureus‐induced hemolysis and modify membranes of bacteria and erythrocytes. Molecules 28:1252. https://doi.org/10.3390/molecules28031252. Włoch A, Strugała-Danak P, Pruchnik H (2021) Interaction of 4′-methylflavonoids with biological membranes, liposomes, and human albumin. Sci Rep 11:16003. https://doi.org/10.1038/s41598-021-95430-8. de Granada-Flor A, Sousa C, Filipe HAL et al (2019) Quercetin dual interaction at the membrane level. Chem Commun 55:1750-1753. https://doi.org/ 10.1039/c8cc09656b. Sengupta B, Banerjee A, Sengupta PK (2004) Investigations on the binding and antioxidant properties of the plant flavonoid fisetin in model biomembranes. FEBS Letters 570:77–81. https://doi.org/10.1016/j.febslet.2004.06.027. Hendrich AB, Flavonoid-membrane interactions: possible consequences for biological effects of some polyphenolic compounds. Acta Pharmacol Sin 27:27–40. https://doi.org/10.1111/j.1745-7254.2006.00238.x. Phan HTT, Yoda T, Chahal B et al (2014) Structure-dependent interactions of polyphenols with a biomimetic membrane system. Biochim Biophys Acta - Biomembranes1838:2670-2677. https://doi.org/10.1016/j.bbamem.2014.07.001. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4477073","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":307534187,"identity":"e7cbd151-0113-4093-9eed-be707b18c941","order_by":0,"name":"Artem G. Veiko","email":"","orcid":"","institution":"Yanka Kupala State University of Grodno","correspondingAuthor":false,"prefix":"","firstName":"Artem","middleName":"G.","lastName":"Veiko","suffix":""},{"id":307534188,"identity":"b3ccdbd3-343a-4876-906b-b4ed48cbc047","order_by":1,"name":"Szymon Sekowski","email":"","orcid":"","institution":"University of Bialystok","correspondingAuthor":false,"prefix":"","firstName":"Szymon","middleName":"","lastName":"Sekowski","suffix":""},{"id":307534189,"identity":"28a2c508-662f-40b2-be0d-b606178c915c","order_by":2,"name":"Ewa Olchowik-Grabarek","email":"","orcid":"","institution":"University of Bialystok","correspondingAuthor":false,"prefix":"","firstName":"Ewa","middleName":"","lastName":"Olchowik-Grabarek","suffix":""},{"id":307534190,"identity":"da24df6f-2263-48b1-a65d-f0e352338c59","order_by":3,"name":"Agnieszka Z. Wilczewska","email":"","orcid":"","institution":"University of Bialystok","correspondingAuthor":false,"prefix":"","firstName":"Agnieszka","middleName":"Z.","lastName":"Wilczewska","suffix":""},{"id":307534191,"identity":"e5548724-96d5-489e-a01d-23b12fbe5227","order_by":4,"name":"Izabela Dobrzyńska","email":"","orcid":"","institution":"University of Bialystok","correspondingAuthor":false,"prefix":"","firstName":"Izabela","middleName":"","lastName":"Dobrzyńska","suffix":""},{"id":307534192,"identity":"6fbefb64-c1fb-4bb0-b870-79aeb557aad1","order_by":5,"name":"Anna Roszkowska","email":"","orcid":"","institution":"University of Bialystok","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Roszkowska","suffix":""},{"id":307534197,"identity":"73d6a6d8-8e4d-4b2f-b134-b3177f35739e","order_by":6,"name":"Elena A. Lapshina","email":"","orcid":"","institution":"Yanka Kupala State University of Grodno","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"A.","lastName":"Lapshina","suffix":""},{"id":307534200,"identity":"1f99354b-b569-4a4f-b9ab-f19477a79594","order_by":7,"name":"Maria Zamaraeva","email":"","orcid":"","institution":"University of Bialystok","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Zamaraeva","suffix":""},{"id":307534203,"identity":"cd49a57a-2279-4d05-ba70-6bb570433092","order_by":8,"name":"Ilya B. Zavodnik","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYLACHgYJAwZmHsYHYJ4ECVqYDUjRwgBUzcMmQZQW+fYzhg/e7rAwZmDnPVb5dU9dYoN0jwFeLYw9OcaGc89ImDEw86Xdlnl2OLFB5gx+LcwMaWnSvG0SNkC/mN2WOHAgsUEiB78WNv5nCC3FEgfqCGvhkUg+BtJiBtLC+OEAM2EtEhKPDxvObZMwBvolWZrhwGHjNpljBXi1yPcnNj5421Zn2MB/9uDHHwfqZPulmzfg1QIH9geAYcED8h0DB36HoQDGH2CK/QHxWkbBKBgFo2AkAACXhz0DTU8R1AAAAABJRU5ErkJggg==","orcid":"","institution":"Yanka Kupala State University of Grodno","correspondingAuthor":true,"prefix":"","firstName":"Ilya","middleName":"B.","lastName":"Zavodnik","suffix":""}],"badges":[],"createdAt":"2024-05-25 13:56:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4477073/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4477073/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58026744,"identity":"46875000-c5a9-47cb-8d7b-acac15b5b669","added_by":"auto","created_at":"2024-06-10 06:50:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":557259,"visible":true,"origin":"","legend":"\u003cp\u003eOptimal molecular geometries and atom excess charges of the flavonols kaempferol, fisetin, quercetin, and myricetin, calculated by the semi-empirical AM1 method and the non-empirical \u003cem\u003eab initio\u003c/em\u003e method with 6-31G basis.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4477073/v1/953df56cc5fc254f0ad4bac0.jpg"},{"id":58026249,"identity":"7a494f34-3d82-44ce-af15-fa1fc01cae59","added_by":"auto","created_at":"2024-06-10 06:42:55","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":92247,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the flavonols on fluorescence anisotropy of TMA-DPH (a) and DPH (b), located in the liposomal membranes. Liposomes (100 µg/ml) were exposed to the probes (1 µM) for 20 min at in PBS, pH 7.4, and then the flavonols were added. r\u003csub\u003e0\u003c/sub\u003eis fluorescence anisotropy in the absence of the flavonoids, while r\u003csub\u003es \u003c/sub\u003eis fluorescence anisotropy in the presence of the flavonols.\u003c/p\u003e\n\u003cp\u003e25 °C\u003c/p\u003e\n\u003cp\u003e* - p \u0026lt; comparison with the liposomes in the absence of the flavonols\u003c/p\u003e\n\u003cp\u003e0.05 in\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4477073/v1/cea6b77ff784ce1fbe466d50.jpg"},{"id":58025553,"identity":"0b42eef9-f8c9-4c59-9844-f4b42ebadfaf","added_by":"auto","created_at":"2024-06-10 06:26:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27665,"visible":true,"origin":"","legend":"\u003cp\u003eStern-Volmer plots of the probes TMA-DPH (a) and DPH (b) fluorescence quenching in liposomal membranes by the flavonols, where F0 and F are fluorescence intensities of the probe in the absence and in the presence of the effectors. Liposomes (100 μg/ml) were exposed to DPH and TMA-DPH (1 μM) for 20 min at 25 ºC in PBS, pH 7.4, and the flavonols were added at 25 ºC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e* - p \u0026lt;0.05 in comparison with liposomes in the absence of the flavonols\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4477073/v1/752cc22f1d49b56e7680d71d.jpg"},{"id":58025847,"identity":"65c69339-ae33-4a68-a479-a48e1f76f1b7","added_by":"auto","created_at":"2024-06-10 06:34:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":64820,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the flavonols quercetin, kaempferol, and myricetin on generalized polarization GP of the probe Laurdan, incorporated into liposomes. Liposomes (100 µg/ml) were exposed to Laurdan (0.3 μM) for 15 min in PBS, pH 7.4, at 25 °C and the effectors were added.\u003c/p\u003e\n\u003cp\u003e* - p \u0026lt;0.05 in comparison with liposomes in the absence of the flavonols\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4477073/v1/b30ef8573749ed15d1c77cd6.jpg"},{"id":58025844,"identity":"526bf6ef-75ea-4f6a-9832-04f4514d4e50","added_by":"auto","created_at":"2024-06-10 06:34:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":24911,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in liposomal membrane zeta potential (a) and diameter (b) in the presence of fisetin, quercetin, myricetin, and kaempferol. For diameter measurements, liposomes (100 µg/ml) were incubated in 10 mM phosphate buffer, pH 7.4, and for zeta potential - in water, pH 6.5, and were exposed to effectors for 5 min at 22ºC.\u003c/p\u003e\n\u003cp\u003e* - p \u0026lt;0.05 in comparison with liposomes the absence of the flavonols\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4477073/v1/81ad0d1a9101706577e853d0.jpg"},{"id":58025556,"identity":"d5c538cf-cb0b-4ebb-a4e8-babca34fbb3e","added_by":"auto","created_at":"2024-06-10 06:26:55","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":23865,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of fisetin, quercetin, myricetin, and kaempferol on thermograms of DMPC membranes (5 mg/ml). The DMPC:flavonoid ratio was 6 800 µM:500 µM, PBS, pH 7.4.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4477073/v1/826b3b30b91d085c29ac3ba2.jpg"},{"id":58054062,"identity":"d20e0202-018f-4271-bee3-e4566ebd36cc","added_by":"auto","created_at":"2024-06-10 13:41:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1455579,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4477073/v1/31e61673-cb14-4cb6-b55e-03d28434ec58.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular structure and interactions of the flavonols, quercetin, fisetin, kaempferol, and myricetin, with liposomal membranes","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFlavonoids, second plant metabolites and one of the most important classes of polyphenolic phytoconstituents, are abundant in human diet. They possess numerous biological activities such as antidiabetic, antihyperlipidemic, antioxidative and freeradical scavenging, cytoprotective, cardioprotective, antiviral and antibacterial, anti-aging, and antiinflammatory properties [1\u0026ndash;3]. Application of plant constituents instead of synthetic pharmaceutical agents demonstrated a number of advantages associated with low toxicity, specificity, and a wide spectrum of activities.\u003c/p\u003e \u003cp\u003eOne of the most common and largest flavonoid subgroups in fruit and vegetables is flavonols which possess high biological and medicinal importance and vary in methylation and hydroxylation modes. Quercetin, myricetin, kaempferol, and fisetin are major dietary flavonols [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The average intake of the flavonol fisetin was determined to be approximately 0.4 mg/day [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe chemical structure of flavonols is characterized by an unsaturated C ring in position C2-C3, a ketone group in position 4 and a hydroxyl group in position 3 of the C ring (the 3-hydroxyflavone backbone) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It has recently been shown that the biological and antioxidant activities of such flavonoids as luteolin, kaempferol, apigenin and quercetin, are directly proportional to the number of phenolic hydroxyl groups [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A number of the intracellular and extracellular targets of the flavonols was estimated. Fisetin regulates AMP-activated protein kinase (AMPK), nuclear factor-kappa B (NF-kB), epidermal growth factor receptor (EGFR), cyclooxygenase (COX); extracellular signal-regulated kinase (ERKI1/2), metalloproteinase (MMP), prostate-specific antigen (PSA), transcription factor T-cell factor (TCF), TNF-related apoptosis-inducing ligand (TRAIL), and X-linked inhibitor of apoptosis (XIAP) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The flavonols quercetin and myricetin (5 \u0026micro;M) were nontoxic to the intestinal epithelial IEC-6 cells, but suppressed the RhoA/ROCK signaling pathway [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The mechanisms for the neuroprotective action of myricetin are prevention of oxidative stress, intracellular Ca\u003csup\u003e2+\u003c/sup\u003e accumulation and apoptosis. Other mechanisms of myricetin health effects include the activation of such signaling cascades as the nuclear factor E2 (Nrf2), extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (Akt), cAMP-response element binding protein (CREB) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Kaempferol can ameliorate generation of the inflammatory mediators nitric oxide, TNF-a, IL-1β, and IL-8 in macrophages [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSpecific (with target proteins and membrane domains) and non-specific (intercalation in lipid bilayer) interactions of polyphenols with cell components are crucial for determination of their beneficial pharmacological and biochemical effects. It has recently been found that kaempferol and myricetin modify the surface charge density and the structure of the model membranes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It was also demonstrated that phenolic hydroxyl groups in the flavonoids form hydrogen bonds with cell membranes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The main question dealing with the beneficial health effects of flavonoids is the chemical basis for their biological activity. Since a molecular structure and electronic characteristics play a key role in biochemical activity, the correlations between the structure and the bioactivities of the flavonoids have been widely studied using methods of quantum chemistry [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEarlier we analyzed the relationship between the molecular optimal conformations and the antioxidative activity of the flavonol quercetin, the flavanol catechin and the flavanone naringenin and evaluated the interaction of these flavonoids with cellular and artificial membranes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We showed that the flavonoids considerably inhibited erythrocyte membrane lipid peroxidation and potentiated Ca\u003csup\u003e2+\u003c/sup\u003e ions - induced mitochondrial permeability transition [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The detailed mechanism of interaction of polyphenols with membranes (such as binding affinities, locations in membrane, changes in ordering and motility) has not been fully elucidated.\u003c/p\u003e \u003cp\u003eTo evaluate the flavonol structure-function relationships, we performed quantum chemical modeling of the optimal geometry and electronic parameters of the flavonols fisetin, kaempferol, and myricetin varying in the number and position of the OH groups, and assessed their interactions with liposomal membranes, using fluorescence probe spectroscopy, an electric-kinetical method and differential scanning calorimetry. We compared our present findings with those for quercetin obtained earlier [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e \u003cp\u003eThe flavonolsquercetin (3,3\u0026prime;,4\u0026prime;,5,7-pentahydroxyflavone),fisetin (3,3\u0026prime;,4\u0026prime;,7-tetrahydroxyflavone), kaempferol (3,4\u0026prime;,5,7-tetrahydroxyflavone), and myricetin (hexahydroxyflavone), as well as 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethyl ammonium phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 6-dodecanoyl-2-dimethylamine-naphthalene (Laurdan), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and other chemicals were from Merck / Sigma-Aldrich (St Louis, MO, USA, or Steinheim am Albuch, Germany), tetrahydrofuran, methanol ethanol, dimethyl sulfoxide, and chloroform were from POCh (Poland). The freshly prepared flavonol solutions (5 mM) in ethanol were used. Ethanol at the concentrations added did not influence the parameters measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Quantum mechanical calculations of molecular geometry and electronic properties of the flavonols\u003c/h2\u003e \u003cp\u003eThe optimal geometries and molecular parameters of the flavonols were evaluated theoretically by both the semi-empirical molecular orbital theory and \u003cem\u003eab initio\u003c/em\u003e calculations by using the HyperChem-8.0 software package (HyperCube, Inc.) [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.hyper.com\u003c/span\u003e\u003cspan address=\"http://www.hyper.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e] as we described earlier [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The optimized conformations were considered using the Austin Model 1 (AM1) semi-empirical method within unrestricted Hartree-Fock (UHF) formalism and the Polak-Ribiere algorithm [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Liposome preparation\u003c/h2\u003e \u003cp\u003eUnilamellar liposomes (unilamellar bilayer vesicles) of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC,14:0) were prepared using chloroform/lipid mixtures and an Avanti Polar Lipids Mini-Extruder (Avanti Polar Lipids, Birmingham, AL, USA) as described previously [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The final liposomes concentration was 100 \u0026micro;g/mlin isotonic buffered saline (PBS,145 mM NaCl, 1.9 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 8,1 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, pH 7.4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. TMA-DPH, DPH, and Laurdan fluorescence measurements in the lipid bilayer\u003c/h2\u003e \u003cp\u003eMembrane fluidity and ordering were analyzed by fluorescence anisotropy of TMA-DPH (λex\u0026thinsp;=\u0026thinsp;340 nm and λem\u0026thinsp;=\u0026thinsp;430 nm) and DPH (λex\u0026thinsp;=\u0026thinsp;348 nm and λem\u0026thinsp;=\u0026thinsp;426 nm)probes as well as probe Laurdan generalized polarization (GP) using a Perkin-Elmer LS 55B spectrofluorimeter (UK) as we described previously [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We used freshly prepared TMA-DPH (in methanol), DPH (in tetrahydrofuran) and Laurdan (in dimethyl sulfoxide) stock solutions, at concentrations of 1 mM. The ratio (r\u003csub\u003es\u003c/sub\u003e/r\u003csub\u003e0\u003c/sub\u003e) was used for describing the TMA-DPH and DPH fluorescence changes, where r\u003csub\u003es\u003c/sub\u003e is the probe fluorescence anisotropy in the presence, and r\u003csub\u003e0\u003c/sub\u003e is probe fluorescence anisotropy in the absence of the flavonols. The DPH molecule is located in the hydrophobic area of the liposomal membrane occupied by the hydrocarbon chains and the TMA-DPH dye is located at the aqueous/membrane interface [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The increase in probe fluorescence anisotropy reflects an enhancement in stiffness in the hydrophobic region of the membrane. Laurdan generalized polarization (GP) was calculated using the equation:\u003c/p\u003e \u003cp\u003eGP = (I\u003csub\u003e440\u003c/sub\u003e \u0026ndash; I\u003csub\u003e490\u003c/sub\u003e)/(I\u003csub\u003e440\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;I\u003csub\u003e490\u003c/sub\u003e),\u003c/p\u003e \u003cp\u003ewhere I\u003csub\u003e440\u003c/sub\u003e and I\u003csub\u003e490\u003c/sub\u003e are fluorescence intensities recorded at 440 and 490 nm, respectively, after exciting at 350 nm. The fluorophore Laurdan is located at the level of the glycerol backbone of phospholipids and fluorescence parameters are associated with changes in fluorophore dipole moment and provide information about the polarity of the environment, packing order and hydration level in the region of the polar groups of the bilayer. An increase in GP indicates an increase in the packing order in the water-lipid interface of membranes and thus a decrease in the hydration level [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. The thermotropic parameters of the liposomal membranes\u003c/h2\u003e \u003cp\u003eDifferential scanning calorimetry (DSC) was applied to analyze the thermal properties of liposomal membranes in the presence of the flavonols using a Mettler Toledo Star DSC system (Mettler Toledo, Switzerland) and Mettler-Toledo STARe software. A sample (6800 \u0026micro;M of DMPC and 500 \u0026micro;M flavonol) in 20 \u0026micro;l of PBC was placed in an aluminum crucible and sealed. The samples were heated from 10\u0026deg;C to 30\u0026deg;C at a rate of 2.5\u0026deg;C/min under argon flow (200 ml/min). An empty sealed crucible was used as a reference. Phase-transition enthalpy (ΔH), temperature of the thermal effect start (T\u003csub\u003eonset\u003c/sub\u003e), phase transition temperature (T\u003csub\u003em\u003c/sub\u003e, the midpoint of the heat capacity change), and half-width of the transition (ΔT\u003csub\u003e1/2\u003c/sub\u003e) were determined [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Liposomal zeta potential and diameter\u003c/h2\u003e \u003cp\u003eElectrokinetic parameters of liposomes: zeta potential (сonnected with the mobility of charged particles) and liposomal mean diameter were measured with a Nano ZS Zeta sizer (Malvern, USA) and were analyzed using the Malvern software as we described earlier [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. For size measurements DMPC liposomes (100 \u0026micro;g/ml) were dissolved in phosphate buffer, pH 7.4, and for potential measurements liposomes were dissolved in water, pH 6.5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistics\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using the one-way analysis of variance (ANOVA) (GraphPad Prism v.6.0 software, GraphPad Software, Inc., La Jolla, CA, USA) with Tukey\u0026rsquo;s test. The results of five to six independent experiments were shown as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eFor better understanding of the cellular mechanism(s) of pharmaceutical applications of the flavonols, we compared molecular structures and electronic parameters of the flavonols and their interactions with liposomal membranes.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Optimal molecular geometry and properties of the flavonols\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the flavonol molecular geometries and excess charges of the atoms calculated by an \u003cem\u003eab initio\u003c/em\u003e method and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the electronic parameters of quercetin, fisetin, kaempferol, and myricetin molecules: dipole moment, heat of formation, E (HOMO) - E (LUMO) Energy, surface area, etc., calculated by the use of the semi-empirical molecular orbital theory [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The A, C and B rings in the all flavonol molecules studied were located in the same plane (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The dipole moment of the flavonol molecules increased in the order: kaempferol (0.195 D)\u0026thinsp;\u0026lt;\u0026thinsp;fisetin (0.617 D)\u0026thinsp;\u0026lt;\u0026thinsp;quercetin (0.986 D)\u0026thinsp;\u0026lt;\u0026thinsp;myricetin (1.224 D) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which reflects the polarity of the flavonols and the efficiency of their electrostatic interactions. It should be noted that these values depend on the local minima for the molecular geometry optimizations. For example, Aparicio, using DFT calculations, showed the following values: 1.66 D for kaempferol, 2.71 D for quercetin,1.5 D for myricetin [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The surface area and the volume values of the flavonol molecules studied were similar.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u0026ndash; Quantum chemical parameters and water solubility of kaempferol, fisetin, quercetin, and myricetin\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKaempferol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFisetin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQuercetin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMyricetin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eAM1 UHF\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of electrons\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e118\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Energy, kcal/mol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-91771.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-91768.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-99164.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-106558.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBinding Energy, kcal/mol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-3614.109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3611.156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3717.600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3821.805\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolated Atomic Energy, kcal/mol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-88157.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-88157.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-95447.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-102736.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectronic Energy, kcal/mol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-550333.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-547637.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-601021.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-653768.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeat of Formation, kcal/mol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-172.385\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-169.432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-216.317\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-260.963\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDipole Moment, D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.195\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.986\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.224\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAb initio\u003c/em\u003e UHF (6-31G)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Energy, kcal/mol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-641697.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-641698.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-688649.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-735602.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectronic Kinetic Energy, kcal/mol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e642082.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e642118.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e689036.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e735994.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNuclear Repulsion Energy, kcal/mol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e995016.955\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e983433.783\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1082078.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1172466.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE Alpha Orbitals (HOMO), eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.2141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-8.318870\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.238066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-8.321660\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE Beta Orbitals (HOMO), eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.212995\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-8.724547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.237996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-8.321773\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE Alpha Orbitals (LUMO), eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2821\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.635402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.229848\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.120749\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE Beta Orbitals (LUMO), eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.280239\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.486912\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.229325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.121896\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlpha Orbitals ∆E\u0026thinsp;=\u0026thinsp;E (HOMO) - E\u0026nbsp;(LUMO), eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-9.4962\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-9.954272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-9.467914\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-9.442409\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBeta Orbitals\u003c/p\u003e \u003cp\u003e∆E\u0026thinsp;=\u0026thinsp;E (HOMO) - E\u0026nbsp;(LUMO), eV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-9.493234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-11.211459\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-9.467321\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-9.443669\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurface area (Grid), \u0026Aring;\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e443.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e451.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e453.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e459.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVolume, \u0026Aring;\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e734.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e743.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e753.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e772.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHydration energy, kcal/mol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-27.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-27.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-32.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-37.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTorsion angles, \u0026ordm;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater solubility, mg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e113 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.512 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.32 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Liposomal membrane ordering and fluidity in the presence of the flavonols.\u003c/h2\u003e \u003cp\u003eTo understand the mechanisms of the flavonol interactions with phospholipid membranes, we evaluated the changes in ordering and fluidity of the DMPC liposomal bilayer in the presence of kaempferol, fisetin, quercetin, and myricetin, using the fluorescent probes, DPH, TMA-DPH, and Laurdan, differently located in the membrane (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe showed that quercetin, kaempferol, fisetin, and myricetin (5\u0026ndash;25\u0026micro;M) increased the TMA-DPH and DPH fluorescence anisotropy parameter, reducing lipid bilayer fluidity or enhancing the lipid bilayer order both at the surface zone (at the aqueous/membrane interface) and in the hydrophobic area (the area of the hydrocarbon chains) of the membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Quercetin was more effective in changing liposomal membrane fluidity. The flavonols effectively quenched the TMA-DPH and DPH fluorescence in the liposomal lipid bilayer. The plots of TMA-DPH (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and DPH (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) fluorescence quenching in liposomal membranes by the flavonols are presented in Stern-Volmer coordinates. The Stern-Volmer constant values Ksv of the DPH fluorescence quenching decreased in the order: quercetin (1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28)\u0026middot;10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e-1\u003c/sup\u003e\u0026gt; kaempferol (0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16)\u0026middot;10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e-1\u003c/sup\u003e\u0026ge; myricetin (0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12)\u0026middot;10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e-1\u003c/sup\u003e\u0026gt; fisetin (0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04)\u0026middot;10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e-1\u003c/sup\u003e, reflecting the accessibility of DPH to the flavonols. The accessibility of the probe TMA-DPH to the flavonols decreased in a different order: myricetin (2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25)\u0026middot;10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e-1\u003c/sup\u003e\u0026gt; quercetin (1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22)\u0026middot;10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e-1\u003c/sup\u003e\u0026gt; kaempferol (0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14)\u0026middot;10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e-1\u003c/sup\u003e\u0026gt; fisetin (0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02)\u0026middot;10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe compared the effects of the flavonols on the DPH and TMA-DPH fluorescence parameters with that of the Laurdan probe embedded in the liposomal membrane bilayer. As Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e showed, all the flavonols significantly reduced Laurdan GP, probably due to growth of the hydration level of the polar group region of the lipid bilayer. According to these observations, kaempferol and quercetin were more effective in the modulation of membrane structure, as was monitored by Laurdan fluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Liposomal zeta potential and diameter in the presence of the flavonols.\u003c/h2\u003e \u003cp\u003eThe incorporation of flavonoid molecules into the liposomal membrane bilayer resulted in an increase in an absolute value of the membrane zeta potential in the following order: fisetin ˃ myricetin ˃ quercetin ˃kaempferol (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The intercalation of quercetin, kaempferol, and myricetin into the liposomal bilayer increased the diameter of the DPMC liposomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Differential scanning calorimetry measurements of the flavonole effects on the model membranes.\u003c/h2\u003e \u003cp\u003eThe flavonol effects on the thermotropic properties of DMPC liposomes were evaluated by DSC. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows representative thermograms of DMPS membranes in the absence and in the presence of the flavonols studied. The thermogram of pure DMPC bilayer unilamellar liposomes demonstrated a sharp main phase transition peak at a temperature of T\u003csub\u003em\u003c/sub\u003e= 25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C and a weak pretransition peak at 15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C. This peak corresponds to a reorganization of individual DPMC molecules. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the thermotropic parameters of the DPMC liposomal membranes in the presence of the flavonols studied. The width of the transition at half-peak height (ΔT\u003csub\u003e1/2\u003c/sub\u003e) reflects the cooperativity of the membrane thermal transition (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The flavanols caused disappearance of the pretransition peak and significantly changed the parameters of the DMPC membranes melting.\u003c/p\u003e \u003cp\u003eAll the flavonols studied lowered the temperature and the enthalpy and of the liposomal membrane melting, as well as increased the width of the phase transition peak (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We observed the most considerable decrease in the membrane phase transition enthalpy and appearance of a small second transition peak (28.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C) in the presence of fisetin.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of quercetin, myricetin, kaempferol, and fisetin on the parameters of the DMPC membrane phase transition (5 mg of liposomes/ml in PBS, pH 7.4, the DMPC:flavonoid ratio was 6 800 \u0026micro;M:500 \u0026micro;M)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eΔH (mJ)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT\u003csub\u003eonset\u003c/sub\u003e(◦C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003csub\u003em\u003c/sub\u003e(◦C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eΔT\u003csub\u003e1/2\u003c/sub\u003e(◦C)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e50.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMPC-Quercetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e30.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e20,9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e22.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMPC-Myricetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e30.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e22.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMPC-Kaempferol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMPC-Fisetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e22.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe complicated chemistry of the flavonols comprises the structure of the molecules, aromatic electron delocalization, proton/electron transfer, the interactions with free radicals, and determines their biological activity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Numerous works deal with biochemical mechanism(s) of flavonoid-membrane interactions. In an earlier work, Oteiza et al. suggested two possibilities of such interactions depending on the number and distribution of OH-groups, planarity and hydrophilicity of flavonoid molecules: (a) intercalation of non-polar flavonoids into the inner hydrophobic zone of the membrane, and (b) formation of hydrogen bonds at the membrane-water boundary [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Tsuchiya showed that flavonols (kaempferol, quercetin, and myricetin) (1–10 µM) affected deeper areas of liposomal membranes, decreasing membrane fluidity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The ability of the flavonols to modify membrane structure, and permeability of artificial and cellular membranes is correlated with their beneficial biochemical effects [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In cells, the flavonols influence the raft formations and control membrane heterogeneity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Recently it was shown that flavonol molecules form complexes with membrane phosphatidylcholine in the ratio of 1:1, and the complex formation energy is -37 ± 1 kJ mol\u003csup\u003e− 1\u003c/sup\u003ein the case of kaempferol and − 36 ± 1 kJ mol\u003csup\u003e− 1\u003c/sup\u003ein the case of myricetin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll the flavonols studied demonstrated planar molecular geometry. The torsion angle was С3-С2-B1’-B2’ ≈ 180°. Earlier we suggested that the C2 = C3 bond in the C-ring determined the planar geometry of quercetin as well as its semiquinone radicals and the corresponding quinones as optimal forms in vacuum [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These results are in agreement with the findings of Aparacio [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and Günther et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] showing that the quercetin, kaempferol, and myricetin optimized geometry is planar. According to our \u003cem\u003eab initio\u003c/em\u003e calculations, the net negative excess charges of the flavonol C rings (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) make this part of the molecules attractive for electrophilic attack. It can be noted that the myricetin molecule has the lowest LUMO energy value (the lowest unoccupied molecular orbital, i.e. a free orbital with the lowest energy) and, therefore, acts as an effective electron acceptor when attacked by nucleophiles and possesses the highest dipole moment value (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to TMA-DPH and DPH fluorescence anisotropy measurements, all the flavonols studied, quercetin, kaempferol, fisetin, and myricetin (5–25µM), modulated the membrane organization and mobility at different depths of the bilayer and quercetin was more effective in changing liposomal membrane properties. Earlier, applying ТМА-DPH and DPH probes, we showed that quercetin at low concentrations of up to 1 µM increased the rigidity of the inner hydrophobic region of the red blood cell membrane and diminished the rigidity of the surface zone of the erythrocyte membrane [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In line with our observations, Włoch et al. have recently shown that methylated flavonoids caused an increase in packing order of polar lipid heads and a decrease in fluidity in erythrocyte and model membranes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUsing the Stern-Volmer constants of fluorescence quenching of DPH (in hydrophobic zone) and TMA-DPH (in surface hydrophilic zone) probes, we concluded that the flavonols quercetin and kaempferol were more predominantly located in the deeper part of the bilayer, and myricetin (possesses 6 hydroxylic groups and the highest dipole moment according to our calculations) was mostly located in the surface zone of the membrane (membrane/water interface). According to this parameter, the accessibility of both the zones of the bilayer to fisetin (possesses 4 hydroxylic groups and low dipole moment) was much lower in comparison with quercetin, myricetin or kaempferol. It has previously been shown that quercetin strongly perturbs cholesterol/sphingolipid enriched domains at cell membranes where signalling platforms are expected to assemble [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Sengupta and coworkers reported that fisetin binds in liposomal membrane area between the polar head and hydrophobic tail of the phospholipids [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. According to Laurdan fluorescence, the flavonols increased the bilayer hydration at the hydrophobic-hydrophilic interface of the model membrane. Similarly, Günther et al. suggested a greater possibility of water access to lipid bilayer in the presence of flavonols [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In contrast, Włoch et al. suggested that flavonoids decreased water content in the hydrophilic region of the bilayer [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe incorporation of negatively charged flavonols into liposomal membranes increased the electrical charge of the membranes and caused an enlargement in zeta potential value, reflecting a strong interaction between the flavonols and liposomes. The interaction depended on the flavonols lipophilicity and charge, and fisetin and myricetin were more effective in influencing zeta potential of the liposomal surface. Zeta potential characterizes the electrostatic interactions between liposomes in suspension and zeta potential value predicts membrane functional activity. Quercetin and kaempferol were most effective in an enlargement of the surface area of the liposomal membrane. It has previously been suggested that flavonoids induced the formation of bridges between adjacent membrane surfaces and aggregation and rigidification of the phospholipid membranes, and that the membrane aggregation was followed either by the production and release of daughter vesicles or formation of endo-buds [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe interaction of the flavonols with membranes caused membrane destabilization (decreased the enthalpy and transition temperature) and a rise in membrane heterogeneity (increased the width of the transition at half-peak height) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The complex changes in membrane fluidity, ordering, electrophoretic properties, stability, and surface area depended on the molecular parameters of the flavonols, as well as the number of the OH-groups, and lipophilicity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The contributions of different forces to flavonoid - membrane interactions underlie the strength of the interactions and localization of the flavonols in the bilayer.\u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003eFlavonols, the main class of the flavonoids, characterized by a complex chemical structure and electronic properties, have numerous biological targets. The AC and B rings in the molecules of all the flavonols studied, quercetin, kaempferol, myricetin, and fisetin, were located in the same plane due to the double C2-C3 bond. In experiments \u003cem\u003ein vitro\u003c/em\u003e, all the flavonols (5–25 µM) increased the lipid bilayer order at different depths, reduced the fluidity and increased the hydration. Quercetin was more effective in changing the liposomal membrane mobility and fisetin modulated markedly the thermotropic behavior of the membrane. Quercetin and kaempferol penetrated into the deeper zone of the bilayer and myricetin was located predominantly in the surface zone. We revealed that the flavonols quercetin, kaempferol, myricetin, and fisetin incorporated into the liposomes and increased zeta potential and enlarged the area of the bilayer, as well as led to membrane destabilization and raised membrane heterogeneity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was partially supported by grant No М23Ch-014 (from 01.11.2022) from the Belarusian Republican Foundation for Fundamental Research. AGV was supported by the Program of Fellowships under The Polish National Commission for UNESCO (No\u0026nbsp;251/E/2020 from 09.10.2020).The thermal analyses were performed in the Centre of Synthesis and Analysis BioNanoTechno of the University of Bialystok. The equipment in the Centre was funded by the EU as a part of the Operational Program Development of Eastern Poland 2007-2013. Projects: POPW.01.03.00-20-034/09-00 and POPW.01.03.00-20-004/11.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.G. Veiko: investigation, data curation, software,\u0026nbsp;S. Sekowski: investigation, visualization, supervision,E. Olchowik-Grabarek: investigation,\u0026nbsp;data curation, visualization\u0026nbsp;A. Z. Wilczewska:\u0026nbsp;investigation, data curation, software, visualization, I. Dobrzyńska:\u0026nbsp;investigation, data curation, software, visualization, Anna Roszkowska:\u0026nbsp;investigation, visualization,\u0026nbsp;E.A. Lapshina: data curation, validation, writing-original draft preparation,\u0026nbsp;M. Zamaraeva: conceptualization, methodology, supervision, I.B. Zavodnik: conceptualization, data curation, supervision, writing-reviewing and editing.\u003c/p\u003e\n\u003cp\u003eAll the authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data generated or analyzed during this study have been included in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors meet the qualifications for authorship and had an opportunity to read and comment the manuscript. All the authors support publication of the manuscript in Molecular and Cellular Biochemistry.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKhan J, Deb PK, Priya S et al (2021) Dietary flavonoids: cardioprotective potential with antioxidant effects and their pharmacokinetic, toxicological and therapeutic concerns. Molecules 26:4021. https://doi.org/10.3390/molecules26134021.\u003c/li\u003e\n\u003cli\u003eWang T, Li Q, Bi K (2018) Bioactive flavonoids in medicinal plants: structure, activity and biological fate. Asian J Pharm Sci 13:12\u0026ndash;23. https://doi.org/10.1016/j.ajps.2017.08.004.\u003c/li\u003e\n\u003cli\u003eHavsteen BH (2002) The biochemistry and medical significance of the flavonoids. Pharmacology \u0026amp; Therapeutics 96:67\u0026ndash;202. https://doi.org/10.1016/S0163-7258(02)00298-X.\u003c/li\u003e\n\u003cli\u003eMahmud AR, Ema TI, Siddiquee MF et al (2023) Natural flavonols: actions, mechanisms, and potential therapeutic utility for various diseases. Beni-Suef Univ J Basic Appl Sci 12:47. https://doi.org/10.1186/s43088-023-00387-4.\u003c/li\u003e\n\u003cli\u003eKashyap D, Sharma A, Sak K et al (2018) Fisetin: a bioactive phytochemical with potential for cancer prevention and pharmacotherapy. Life Sci 194:75\u0026ndash;87. https://doi.org/10.1016/j.lfs.2017.12.005.\u003c/li\u003e\n\u003cli\u003eChagas MSS, Behrens MD, Moragas-Tellis CJ et al (2022) Flavonols and flavones as potential anti-inflammatory, antioxidant, and antibacterial compounds. Oxid Med Cell Longev 2022:Article ID 966750. https://doi.org/10.1155/2022/9966750.\u003c/li\u003e\n\u003cli\u003eTian C, Liu X, Chang Y et al (2021) Investigation of the anti-inflammatory and antioxidant activities of luteolin, kaempferol, apigenin and quercetin. South African Journal of Botany 137:257-264. https://doi.org/10.1016/j.sajb.2020.10.022.\u003c/li\u003e\n\u003cli\u003eGrynkiewicz G and Demchuk OM (2019) New Perspectives for Fisetin. Front Chem 7:697. https://doi.org/10.3389/fchem.2019.00697.\u003c/li\u003e\n\u003cli\u003eFan J, Li TJ, Zhao XH (2020) Barrier-promoting efficiency of two bioactive flavonols quercetin and myricetin on rat intestinal epithelial (IEC-6) cells \u003cem\u003evia\u003c/em\u003e suppressing Rho activation. RSC Adv 10:27249-27258. https://doi.org/10.1039/d0ra04162a.\u003c/li\u003e\n\u003cli\u003eLi J, Xiang H, Huang C, Lu J (2021) Pharmacological actions of myricetin in the nervous system: a comprehensive review of preclinical studies in animals and cell models. Front Pharmacol 12:797298. https://doi.org/10.3389/fphar.2021.797298.\u003c/li\u003e\n\u003cli\u003eNam SY, Jeong HJ, Kim HM (2017) Kaempferol impedes IL-32-induced monocytemacrophage differentiation. Chem Biol Interact 274:107\u0026ndash;115.\u003c/li\u003e\n\u003cli\u003eLaszuk P, Petelska AD (2021) Interactions between phosphatidylcholine and kaempferol or myristicin: langmuir monolayers and microelectrophoretic studies. Int J Mol Sci 22:4729. https://doi.org/10.3390/ijms22094729.\u003c/li\u003e\n\u003cli\u003eSirk TW, Brown EF, Sum AK, Friedman M (2008) Molecular dynamics study on the biophysical interactions of seven green tea catechins with lipid bilayers of cell membranes. J Agric Food Chem 56:7750\u0026ndash;7775.\u003c/li\u003e\n\u003cli\u003eSafe S, Jayaraman A, Chapkin RS et al (2021) Flavonoids: structure\u0026ndash;function and mechanisms of action and opportunities for drug development. Toxicol Res 37:147\u0026ndash;162. https://doi.org/10.1007/s43188-020-00080-z.\u003c/li\u003e\n\u003cli\u003eMendoza-Wilson AM, Santacruz-Ortega H, Balandr\u0026aacute;n-Quintana RR (2011) Spectroscopic and computational study of the major oxidation products formed during the reaction of two quercetin conformers with a free radical. Spectrochim Acta A Mol Biomol Spectrosc 81:481\u0026ndash;488. https://doi.org/10.1016/j.saa.2011.06.041.\u003c/li\u003e\n\u003cli\u003eRasulev BF, Abdullaev ND, Syrov VN, Leszczynski J (2005) A quantitative structure-activity relationship (QSAR) study of the antioxidant activity of flavonoids. QSAR Comb Sci 24:1056\u0026ndash;1065. https://doi.org/10.1002/qsar.200430013.\u003c/li\u003e\n\u003cli\u003eVeiko AG, Lapshina EA, Zavodnik IB (2021) Comparative analysis of molecular properties and reactions with oxidants for quercetin, catechin, and naringenin. Mol Cell Biochem 476:4287\u0026ndash;4299. https://doi.org/10.1007/s11010-021-04243-w.\u003c/li\u003e\n\u003cli\u003eVeiko AG, Sekowski S, Lapshina EA et al (2020) Flavonoids modulate liposomal membrane structure, regulate mitochondrial membrane permeability and prevent erythrocyte oxidative damage. Biochim. Biophys. Acta\u0026ndash; Biomembranes1862:183442. https://doi.org/10.1016/j.bbamem.2020.183442.\u003c/li\u003e\n\u003cli\u003eErko\u0026ccedil; Ş, Erko\u0026ccedil; F, Keskin N (2003) Theoretical investigation of quercetin and its radical isomers. J Mol Struct 631:141\u0026ndash;146. https://doi.org/10.1016/S0166-1280(03)00237-9.\u003c/li\u003e\n\u003cli\u003eOnishi T (2018) Quantum computational chemistry: modelling and calculation for functional materials. Springer, Singapore. https://doi.org/10.1007/978-981-10-5933-9.\u003c/li\u003e\n\u003cli\u003eMykytczuk NC, Trevors JT, Leduc LG, Ferroni GD (2007) Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress. Prog Biophys Mol Biol 95:60-82. https://doi.org/10.1016/j.pbiomolbio.2007.05.001.\u003c/li\u003e\n\u003cli\u003eHarris FM, Best KB, Bell JD (2002) Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order. Biochim Biophys Acta 1565:123-128.\u003c/li\u003e\n\u003cli\u003eSanchez SA, Tricerri MA, Gratton E (2012) Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo. Proc Natl Acad Sci 109:7314\u0026ndash;7319. https://doi.org/10.1073/pnas.1118288109.\u003c/li\u003e\n\u003cli\u003eVeiko AG, Lapshina EA, Yukhnevich HG, Zavodnik IB (2023) Study of the interaction of naringenin, apigenin, and menadione with membranes using fluorescent probes and quantum chemistry. J Appl Spectrosc 90:535\u0026ndash;542. https://doi.org/10.1007/s10812-023-01564-0.\u003c/li\u003e\n\u003cli\u003eAparicio S (2010) A systematic computational study on flavonoids. Int J Mol Sci 11:2017-2038. https://doi.org/10.3390/ijms11052017.\u003c/li\u003e\n\u003cli\u003eDeng S, Yang YL, Cheng XX et al (2019) Synthesis, spectroscopic study and radical scavenging activity of kaempferol derivatives: enhanced water solubility and antioxidant activity. Int J Mol Sci 20:975. https://doi.org/ 10.3390/ijms20040975.\u003c/li\u003e\n\u003cli\u003eSkiba M, Gasmi H, Milon N et al (2021)Water solubility and dissolution enhancement of fisetin by spherical amorphous solid dispersion in polymer of cyclodextrin. Austin J Biotechno Bioeng 8:1106.\u003c/li\u003e\n\u003cli\u003eSaija A, Tomaino A, Trombetta D et al (2003)\u0026lsquo;In vitro\u0026rsquo; antioxidant and photoprotective properties and interaction with model membranes of three new quercetin esters. Eur J Pharm Biopharm 56:167\u0026ndash;174.\u003c/li\u003e\n\u003cli\u003eLin T-C, Yang C-Y, Wu T-H et al (2023) Myricetin nanofibers enhanced water solubility and skin penetration for increasing antioxidant and photoprotective activities. \u003cem\u003ePharmaceutics 15\u003c/em\u003e:906. https://doi.org/10.3390/pharmaceutics15030906.\u003c/li\u003e\n\u003cli\u003eOteiza PI, Erlejman AG, Verstraeten SV et al (2005) Flavonoid-membrane interactions: a protective role of flavonoids at the membrane surface? Clin Dev Immunol 12:19-25. https://doi.org/10.1080/10446670410001722168.\u003c/li\u003e\n\u003cli\u003eTsuchiya H (2011) Effects of red wine flavonoid components on biomembranes and cell proliferation. Int J Wine Res 3:9\u0026ndash;17.\u003c/li\u003e\n\u003cli\u003eTsuchiya H (2015) Membrane interactions of phytochemicals as their molecular mechanism applicable to the discovery of drug leads from plants. Molecules 20:18923-18966. https://doi.org/10.3390/molecules201018923.\u003c/li\u003e\n\u003cli\u003eTarahovsky YS, Muzafarov EN, Kim YA (2008) Rafts making and rafts braking: how plant flavonoids may control membrane heterogeneity. Mol Cell Biochem 314:65-71. https://doi.org/10.1007/s11010-008-9766-9.\u003c/li\u003e\n\u003cli\u003eIlyich TV, Veiko AG, Lapshina EA, Zavodnik IB (2018) Quercetin and its complex with cyclodextrin against oxidative damage of mitochondria and erythrocytes: Experimental results \u003cem\u003ein vitro\u003c/em\u003e and quantum-chemical calculations. Biophysics 63:537\u0026ndash;548. https://doi.org/10.1134/S0006350918040073.\u003c/li\u003e\n\u003cli\u003eG\u0026uuml;nther G, Berr\u0026iacute;os E, Pizarro N, et al (2015) Flavonoids in microheterogeneous media, relationship between their relative location and their reactivity towards singlet oxygen. PLoS ONE 10:e0129749. https://doi.org/10.1371/journal.pone.0129749.\u003c/li\u003e\n\u003cli\u003eVeiko AG, Olchowik‐Grabarek E, Sekowski S et al (2023) Antimicrobial activity of quercetin, naringenin and catechin: flavonoids inhibit staphylococcus aureus‐induced hemolysis and modify membranes of bacteria and erythrocytes. Molecules 28:1252. https://doi.org/10.3390/molecules28031252.\u003c/li\u003e\n\u003cli\u003eWłoch A, Strugała-Danak P, Pruchnik H (2021) Interaction of 4\u0026prime;-methylflavonoids with biological membranes, liposomes, and human albumin. Sci Rep 11:16003. https://doi.org/10.1038/s41598-021-95430-8.\u003c/li\u003e\n\u003cli\u003ede Granada-Flor A, Sousa C, Filipe HAL et al (2019) Quercetin dual interaction at the membrane level. Chem Commun 55:1750-1753. https://doi.org/ 10.1039/c8cc09656b.\u003c/li\u003e\n\u003cli\u003eSengupta B, Banerjee A, Sengupta PK (2004) Investigations on the binding and antioxidant properties of the plant flavonoid fisetin in model biomembranes. FEBS Letters 570:77\u0026ndash;81. https://doi.org/10.1016/j.febslet.2004.06.027.\u003c/li\u003e\n\u003cli\u003eHendrich AB, Flavonoid-membrane interactions: possible consequences for biological effects of some polyphenolic compounds. Acta Pharmacol Sin 27:27\u0026ndash;40. https://doi.org/10.1111/j.1745-7254.2006.00238.x.\u003c/li\u003e\n\u003cli\u003ePhan HTT, Yoda T, Chahal B et al (2014) Structure-dependent interactions of polyphenols with a biomimetic membrane system. Biochim Biophys Acta - Biomembranes1838:2670-2677. https://doi.org/10.1016/j.bbamem.2014.07.001.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"flavonols, liposomal membranes, fluidity, differential scanning calorimetry","lastPublishedDoi":"10.21203/rs.3.rs-4477073/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4477073/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe interactions of flavonols with biological membranes underlie their beneficial biochemical effects. In the present work, we performed quantum chemical modeling of the molecular structure and electronic characteristics of some flavonols such as fisetin, kaempferol, and myricetin and compared our findings with those for quercetin obtained earlier. We considered the effects of the flavonols on liposomal membranes, using the methods of fluorescence probe spectroscopy, an electric-kinetical method and differential scanning calorimetry. The AC and B rings in the molecules of all the flavonols studied were located in the same plane. All the flavonols (5\u0026ndash;25\u0026micro;M) increased the lipid bilayer order both in the surface zone and the hydrophobic area of the membrane. Quercetin was more effective in changing the liposomal membrane mobility and fisetin modulated markedly the thermotropic behavior of the membrane. Myricetin was located predominantly in the surface zone, whereas quercetin penetrated into the deeper zone of the bilayer. Using the fluorescent probe Laurdan we showed that all the flavonols studied increased the hydration of the lipid bilayer. The incorporation of effector molecules into the liposomal membrane bilayer resulted in an increase in the absolute value of zeta potential and induced an increase in the liposomal diameter. Destabilization and enhanced heterogeneity of liposomal membranes in the presence of all the flavonols studied were revealed.\u003c/p\u003e","manuscriptTitle":"Molecular structure and interactions of the flavonols, quercetin, fisetin, kaempferol, and myricetin, with liposomal membranes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-10 06:26:50","doi":"10.21203/rs.3.rs-4477073/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d923b32f-a141-483f-8329-3b75ae616d03","owner":[],"postedDate":"June 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-10T13:33:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-10 06:26:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4477073","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4477073","identity":"rs-4477073","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}