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.

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.

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 X_RL 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.

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).

Figure 1 showed the Chemical structures of RL and DOPC.

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 X_RL ≤ 0.5, the surface pressures at the collapse (π_coll) increased with the increase of X_RL and reached the maximum for X_RL = 0.5. However, for X_RL > 0.5, π_coll was almost same at 10˚C, and decreased slightly at 25˚C. At 37˚C, the largest values of π_coll of DOPC/RL mixed monolayer were observed for pure DOPC. If two components of mixed monolayer are immiscible, there are two π_coll corresponding to the monolayers of two pure components. Figure 2 showed that there was a single π_coll 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, A_ex, 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. A_ex can be given by Equation (1).

A e x = A 12 − ( X 1 A 1 + X 2 A 2 ) (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 A_ex with the mole fraction of RL at various surface pressures and temperatures.

It can be seen from Figure 3 that at 10˚C, excepting for the case of X_RL > 0.5 and π < 25 mN/m, where A_ex was negative, for all the mole fraction of RL and surface pressures studied, A_ex was positive. At 25˚C, the values of A_ex were all positive regardless the mole fraction of RL and surface pressure. At 37˚C, there was a great fluctuation of A_ex with different X_RL and the most negative deviation of A_ex from ideal was at the range of 0.32 < X_RL < 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 A_ex with different X_RL at 37˚C was observed.

The above-mentioned interaction can be analyzed in more quantitative terms by evaluation of the excess free energy of mixing, Δ G e x ,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, Δ G e x can be expressed as follow:

Δ G e x = ∫ 0 π [ A 12 − ( X 1 A 1 + X 2 A 2 ) ] d π (2)

Negative values of Δ G e x 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 ΔG_ex of DOPC/RL mixed monolayers at various surface pressures at 10˚C, 25˚C and 37˚C. Only for X_RL > 0.5 and π < 25 mN/m, ΔG_ex 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, ΔG_ex 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.

The two-dimensional compressibility C_s of a monolayer at a given molecular area of surface pressure is defined by

C s = ( − 1 / A ) ( d A / d π ) (3)

where A is the area per molecule at a given surface pressure π. C_s can be calculated directly from the slope of the π-A isotherms. In general, C_s 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 C_s 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.