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

doi:10.21203/rs.3.rs-4477073/v1

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

https://doi.org/10.21203/rs.3.rs-4477073/v1

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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

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 Ca²⁺ 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 Ca²⁺ 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].

2.1. 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 NaH₂PO₄, 8,1 mM Na₂HPO₄, 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/r₀) was used for describing the TMA-DPH and DPH fluorescence changes, where r_s is the probe fluorescence anisotropy in the presence, and r₀ 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 = (I₄₄₀ – I₄₉₀)/(I₄₄₀ + I₄₉₀),

where I₄₄₀ and I₄₉₀ 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.

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), Å²	443.65	451.59	453.62	459.99
Volume, Å³	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. 3a) and DPH (Fig. 3b) 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)·10⁶ M^-1> kaempferol (0.89 ± 0.16)·10⁶ M^-1≥ myricetin (0.68 ± 0.12)·10⁶ M^-1> fisetin (0.22 ± 0.04)·10⁶ 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)·10⁶ M^-1> quercetin (1.53 ± 0.22)·10⁶ M^-1> kaempferol (0.71 ± 0.14)·10⁶ M^-1> fisetin (0.15 ± 0.02)·10⁶ 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. 5a). The intercalation of quercetin, kaempferol, and myricetin into the liposomal bilayer increased the diameter of the DPMC liposomes (Fig. 5b).

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

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^− 1in the case of kaempferol and − 36 ± 1 kJ mol^− 1in 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.

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.

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.

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

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Chemicals

2.2. Quantum mechanical calculations of molecular geometry and electronic properties of the flavonols

2.3. Liposome preparation

2.4. TMA-DPH, DPH, and Laurdan fluorescence measurements in the lipid bilayer

2.5. The thermotropic parameters of the liposomal membranes

2.6. Liposomal zeta potential and diameter

2.7. Statistics

3. Results

3.1. Optimal molecular geometry and properties of the flavonols

3.2. Liposomal membrane ordering and fluidity in the presence of the flavonols.

3.3. Liposomal zeta potential and diameter in the presence of the flavonols.

3.4. Differential scanning calorimetry measurements of the flavonole effects on the model membranes.

4. Discussion

Conclusions

Declarations

References

Additional Declarations

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