Flavonoids are widely distributed in plants and exhibit a wide range of biological activities, such as anticancer , anti-inflammatory [2,3], anti-virus , anticoagulant [5,6], anti-atherosclerosis , and inhibitory effects on low-density lipoprotein (LDL) oxidation [7-10]. Among others, the flavonoids are known as potent cell growth inhibitors , multidrug resistance modulators  and inhibitors of different types of kinases [13,14]. Most of biological activities of flavonoids arise from their resistance to oxidation including scavenging free radicals, chelating transition metal ions, and protecting the lipid from peroxidization . The aglucon shows more excellent anti-oxidation compared to glucoside, because the former exhibits strong lipotropy and can partition preferentially into the hydrophobic core of the bio-membrane . These results were confirmed by protecting LDL  and the complex of carotenoids and unsaturated fatty acids from oxidation [18,19] by flavonoids. Unfortunately, the hydrophilic property of flavonoids glucosides due to polyhydroxyl and saccharide groups, compresses their anti-oxidative activity. Esterification of the hydroxyl by fatty acids is a solution to improve the hydrophobic of flavonoids. The lipophilic rutin laurate (RL) was obtained by acylation of rutin with lauric acid using the immobilized Candida antarctica lipase B (Novozym 435) as catalyst in present paper. RL was amphiphilic molecule with rutin moiety as the polar group and alkyl chain as the hydrophobic group (see in Figure 1(a)). We expect that RL will inset into bio-membrane with alkyl chain locating at the hydrophobic core. The hydrophobic interaction between the alkyl chain steadies the location of RL in the bio-membrane. Furthermore, the anti-oxidation groups keep retentive during the esterification of rutin. Thus, more prominent antioxidative activity may be expected for RL.
Biological membranes are involved in a variety of cellular functions [20,21], such as cells adjust structure and physio-properties of their membranes (e.g. permeation, fluidity) during absorption and metabolism of pharmaceuticals. The interaction of biological membranes with partially or completely miscible biomolecules is a topic of importance, because it can be applied in broad fields, such as chemopharmacodynamic study of drug, control of medicines release, biosensors, and bioreceptor .
The monolayer at the air/water interface is a simple and ideal model membrane system from which the membrane property and the lipid interaction within monolayer can be easily measured under various conditions [23,24]. However, few papers have reported the interaction of flavonoids with lipid monolayer , more with the lipid bilayers [21,22]. This is probably because flavonoids cannot spread at the air/water interface, as other conjugated aromatic molecule. However, amphiphilic flavonoid esters which were obtained by esterification could spread at air/water interface to form monolayer [20,23].
In this paper, monolayer of L-α dioleoylphosphatidylcholine (DOPC) was used as the model membrane to study the molecular interaction with RL, which was derived by esterification of the C4”’ OH of rhamnose moiety with lauric acid. The characteristics such as miscibility, thermodynamic stability and compressibility of DOPC/RL mixed monolayers were investigated by Langmuir film balance to yield quantitative information on the nature of the molecular interaction, which provided valuable insights to reveal the antioxidative activity mechanism of flavonoids. The effects of temperature of the subphase on the DOPC-RL interactions were investigated.
2. MATERIALS AND METHODS
DOPC (99%) was purchased from Sigma-Aldrich LLC. Its formula is reported in Figure 1(b). Rutin (≥95%) was purchased from Beijing Biochemical Reagent Company, China, and was used as obtained. Novozym 435 (immobilized lipase B from Candida Antarctica) (enzyme activity: 10470PLU/g) was obtained from Novo-Nordisk Co., China. Lauric acid was analytical reagent and from Chengdu Kelong Chemical Plant, China. The tert-amyl alcohol was chemically pure and purchased from Beijing Chemical Plant, China. Other reagents were purchased from local reagent company.
2.2. Preparation of RL
RL was synthesized as follows: Novozym 435 (340 mg) was added to a solution of rutin (762 mg) and lauric acid (1253 mg) in tert-amyl alcohol (85 mL). The suspension was heated to 60˚C and stirred at 300 rpm with a mechanical stirrer. In order to control water content in the reaction medium, after 24 h of reaction, molecular sieves 4Å were added to the reactor with 100 g/L. Molecular sieves were activated by heating at 150˚C for 24 h before used. Finally, the reaction was stopped and the enzyme filtered off. The solvent was removed in vacuum. The residue was chromatographed on silica gel [ethyl acetate/ethanol/water, 15:1:1 (v/v/v)] to give yellow powder. Then the powder was crystallized with methanol and pure RL was obtained.
2.3. Film Balance Measurements
Film balance measurements were performed using a KSV trough (KSV Instrument Ltd.) with a Wilhelmy type microbalance using a platinum plate. RL was dissolved in a mixture of chloroform and methanol with a volume ratio of 9:1. DOPC was dissolved in chloroform. The concentration of each solution was 1 μmol/mL. Mixed DOPC/RL solutions were successively prepared with RL molar fractions XRL of 0.167, 0.25, 0.5, 0.75 and 0.833. 100 μL of the mixed solutions and the pure components were randomly spread on the aqueous subphase. Following the evaporation of the solvent (15 min), the monolayers were compressed at a rate of 5 mm/min. Surface-pressure vs molecular-area isotherms were recorded by film balance measurements. The experiments were performed at subphase temperatures of 37˚C, 25˚C and 10˚C.
3. RESULTS AND DISCUSSION
3.1. 1H-NMR of RL
1H-NMR (d6-DMSO) spectra of the final products showed the following shifts and related chemical species: δ7.53 (2H, m, H2’ and H6’), 6.78 (1H, d, J = 9 Hz, H5’), 6.28 (1H, s, H8), 6.11 (1H, s, H6), 5.32 (1H, d, J = 7.4 Hz, H1”), 4.66 (1H, t, J = 9 Hz, H4”’), 4.48 (1H, s, H1”’), 3.74-3.04 (9H, m, H2”-H6”, H2”’-H5”’), 2.21-2.13 (2H, m, CH2α, fatty chain), 1.5 (2H, m, CH2β, fatty chain), 1.25-1.13 (16H, m, -CH2-, fatty chain), 0.95 (1H, t, J = 7.0, CH3, fatty chain), 0.85 (1H, d, J = 7.0, CH3, rhamnose).
Selected 13C-NMR data (d6-DMSO): δ177.5 (C4), 174 (C=O ester), 168.3 (C7), 157.7 (C9 or C2), 150.2 (C4’), 133 (C3), 122.5 (C6’), 116.7 (C5’), 77.9 (C3”), 75.3 (C5”), 74.7 (C4”’), 71.6 (C4” or C2”’), 70.9 (C2”’ or C4”), 69.3 (C3”’), 67 (C5”’), 34.7 (fatty chain), 31.8 (fatty chain), 29.7 (fatty chain), 25.4 (fatty chain), 25 (fatty chain), 24.5 (fatty chain), 23.7 (fatty chain), 23.1 (fatty chain), 18.5 (fatty chain), 15.2 (fatty chain), 12.1 (fatty chain).
3.2. Miscibility of DOPC/RL Mixed Monolayer
Figure 1 showed the Chemical structures of RL and DOPC.
Figure 1. Chemical structures of RL (a) and DOPC (b).
Figure 2 showed the surface pressure vs. mean molecular area (π-A) isotherms of DOPC/RL monolayers at various mole fraction at 10˚C (a), 25˚C (b) and 37˚C (c), respectively. The RL mole fraction of each curve is reported in the figures.
The isotherms of pure RL in Figure 2 showed RL formed monolayer with liquid-phase at the air/water interface. The intermolecular hydrogen bonds between molecules of water and rutin leaded almost three of aromatic nucleus of rutin formed a large flat at air/water interface. Compared with the large polar groups, the alkyl chains are relatively short. Near to the collapse of the monolayers, the polar groups arranged tightly, while alkyl chains were still outside the range that was prerequisite for interaction between them and then alkyl chains were orientation disorder. Thus, the isotherm of RL monolayers with liquid-expanded phase was observed. Pure DOPC also formed the liquid-phase monolayer. The two cis-double bonds and a cone-like structure of DOPC led to the mismatch of molecular shapes and loose array of hydrophobe alky chains.
From the isotherms of DOPC/RL mixed monolayers, it was observed that at 10˚C and 25˚C, for the mole fraction of RL XRL ≤ 0.5, the surface pressures at the collapse (πcoll) increased with the increase of XRL and reached the maximum for XRL = 0.5. However, for XRL > 0.5, πcoll was almost same at 10˚C, and decreased slightly at 25˚C. At 37˚C, the largest values of πcoll of DOPC/RL mixed monolayer were observed for pure DOPC. If two components of mixed monolayer are immiscible, there are two πcoll corresponding to the monolayers of two pure components. Figure 2 showed that there was a single πcoll for all the mole fraction and temperatures studied. Thus, the two components are miscible throughout the mixture composition range.
The nature of the molecular interaction between the two components can be obtained quantitatively from a plot of the excess molecular area of mixing, Aex, which is the difference in molecular area between the ideal value and the measured value, as a function of the mole fraction at a given pressure. Aex can be given by Equation (1).
where A12 is molecular area of the mixed monolayer; A1 and A2 are the molecular areas of the monolayer of component 1 and component 2 at a given surface pressure π, respectively; and X1 and X2 are their corresponding mole fractions. With mixtures which both components exhibit ideal miscibility or complete immiscibility, the excess area is zero and the plot of A12 versus X1 gives a straight line. Any deviation from the straight line indicates miscibility and non-ideality. With positive excess areas meaning greater cohesion between like molecules than the unlike components, that is, the interactions between the two components are repulsive, and negative excess areas indicating attractive forces between the unlike ones [24-26].
Figure 3 showed the variation of Aex with the mole fraction of RL at various surface pressures and temperatures.
Figure 2. Surface pressure vs mean molecular area of DOPC/RL mixed monolayers at 10˚C (a), 25˚C (b) and 37˚C (c).
Figure 3. Excess area, Aex, as a function of the mole fraction of RL in DOPC/RL mixed monolayers at various surface pressures and temperatures. (a) 10˚C, (b) 25˚C, (3) 37˚C. ■ 5 mN/m, ● 10 mN/m, ▲ 15 mN/m, ▼ 20 mN/m, ◆ 25 mN/m, ◄ 30 mN/m.
It can be seen from Figure 3 that at 10˚C, excepting for the case of XRL > 0.5 and π < 25 mN/m, where Aex was negative, for all the mole fraction of RL and surface pressures studied, Aex was positive. At 25˚C, the values of Aex were all positive regardless the mole fraction of RL and surface pressure. At 37˚C, there was a great fluctuation of Aex with different XRL and the most negative deviation of Aex from ideal was at the range of 0.32 < XRL < 0.35. This difference from RL may be due to its shorter alkyl chain. At some mole fraction of RL and surface pressure, DOPC and RL molecular shapes mismatched, and the cohesion between RL and RL, DOPC and DOPC were greater than RL and DOPC. At high temperature, the lipid monolayers stayed in a disorder state, thus the fluctuation of Aex with different XRL at 37˚C was observed.
3.3. Thermodynamic Stability of DOPC/RL Monolayer
The above-mentioned interaction can be analyzed in more quantitative terms by evaluation of the excess free energy of mixing, ,at a given π. This parameter is also an indicator of the type of interaction between the component [27,28]. For a two-component mixed monolayer at constant surface pressure π and temperature, can be expressed as follow:
Negative values of indicate the presence of attractive interaction. The existence of a minimum will indicate a mixture composition of the greatest thermodynamic stability in comparison with pure component monolayer. On the other hand, positive values suggest that the interactions between the two components are repulsive and the mixing is non-spontaneous process.
Figure 4 showed ΔGex of DOPC/RL mixed monolayers at various surface pressures at 10˚C, 25˚C and 37˚C. Only for XRL > 0.5 and π < 25 mN/m, ΔGex were negative. It implied that only at these conditions, the two components of DOPC/RL monolayers were miscible and mixed spontaneously. This result was consistent with the above miscibility studies. With the increase of surface pressures, ΔGex was more positive. This can be explained that high pressure resulted to short intermolecular distance, thus the repulsive force between DOPC and RL molecules was more prominent and the system was more unstable.
3.4. Compressibility of DOPC/RL Monolayer
The two-dimensional compressibility Cs of a monolayer at a given molecular area of surface pressure is defined by
where A is the area per molecule at a given surface pressure π. Cs can be calculated directly from the slope of the π-A isotherms. In general, Cs depends on the state of the monolayer, being lower for more condensed films, indicating low interfacial elasticity [29-32].
Figure 5 showed the compressibility of DOPC/RL mixed monolayers versus the mole fraction of RL at 10˚C, 25˚C and 37˚C.
For all surface pressures studied, DOPC/RL mixed monolayer became more compressible as the mole fraction of RL increase. This suggested that RL made DOPC monolayer more elastic and fluid. Larger values of Cs occurred at lower surface pressures, e.g. π = 5 mN/m. This was because at low surface pressures, the monolayers were in their expanded state, thus their compressibility was higher as they were more fluid.
It can be summarized from the above experimental results, RL was miscible with DOPC throughout the mixture composition range and increase the compressibility and fluid of DOPC monolayer at all the experimental temperature, and at different temperatures and different molar fractions of RL, it showed different forms of intermolecular forces of RL and DOPC. At lower temperature (10˚C), attracting intermolecular forces occurred at higher RL mole fraction, while at higher temperature (37˚C) it occurred at lower RL mole fraction. At 25˚C, mutually exclusive forces occurred at all the RL mole fraction. This was most likely due to the effect of temperature on the molecular configuration of RL and DOPC.
Figure 4. Excess free energy of mixing ΔGex as a function of XRS in the DOPC/RS (A) and XRL in the DOPC/RL (B) mixed monolayers. 10˚C, (b) 25˚C, (c) 37˚C. ■ 5 mN/m, ● 10 mN/m, ▲ 15 mN/m, ▼ 20 mN/m, ◆ 25 mN/m, ◄ 30 mN/m.
Figure 5. Surface compressibility Cs as a function of mole fraction of RL of DOPC/RL mixed monolayer at 10˚C (a), 25˚C (b) and 37˚C (c). ■ 5 mN/m, ● 10 mN/m, ▲ 15 mN/m, ▼ 20 mN/m, ◆ 25 mN/m, ◄ 30 mN/m.
The two components of DOPC/RL mixed monolayer were miscible throughout the mixture composition range and at three experimental temperatures of 10˚C, 25˚C and 37˚C. At all experimental conditions, RL increased the compressibility and elasticity of the DOPC monolayer. Both the temperature and the composition of the membrane affected the form of intermolecular forces in the mixed monolayer.
We thank Dacheng Wu, Zongliang Du and Ruixia Li for their helpful assistance in the experiment.
The work was supported by the National Undergraduate Training Program for Innovation and Entrepreneurship (201910438015) and Natural Science Foundation of Shandong Province [ZR2016HM47].
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