The preparation and purification of apigenin

The apigenin was extracted from the leaves of A. nitida. Afterward, its apigenin was obtained by hydrolysis and recrystallization.[13] The structural characteristics of apigenin were identified by various methods such as ultraviolet (UV), Fourier transform-infrared (FT-IR), nuclear magnetic resonance (NMR), mass spectrum, and differential scanning calorimetry (DSC) analysis to certify the obtained crystal is apigenin of high purity.

The synthesis of the complex of soy lecithin and apigenin

A total of 100 mg of apigenin and 200 mg of soy lecithin were dissolved in 50 mL tetrahydrofuran and the mixture was magnetically stirred at 25°C for 3 h. Then, the solvent was removed by vacuum concentration to obtain the yellow substance of soy lecithin and apigenin (SLA).

The manufacture of a physical mixture of soybean lecithin and apigenin

Hundred milligram of apigenin were fully mixed with 200 mg of soy lecithin to obtain the physical mixture.

The ultraviolet and Fourier transform infrared analyses

One milligram of soy lecithin, apigenin, SLA, physical mixture of SLA (PMSLA) was dissolved in 10 mL of methanol separately for the UV spectra analysis. The UV spectra analysis was conducted under the wavelength of 220–500 nm on a scanning UV spectrophotometer (UV-2500PC, Shimadzu, Japan).

The sample for FT-IR analysis was prepared by 1 mg of sample and 150 mg of dried KBr. In brief, after being fully mixed and crushed to powder, the powder were pressed into a 1 mm thick disk for the analysis. Fourier transformed IR spectrophotometer (VECTOR22, Bruker, Germany) was used to carry out the experiment. 4000–400 cm⁻¹ was set as the measuring range. All data processing is done by OPUS software (Bruker, Germany).

Scanning electron microscopy

The testing samples of soy lecithin, apigenin, SLA, PMSLA were adhered on a copper testing stub before a layer of gold was evenly sputtered on them. The experimental condition was set as 10 kV and low vacuum. A SU1510 (Hitachi, Japan) scanning electron microscope was used to observe the microstructure of samples.

Differential scanning calorimetry

The temperature was increased from 30°C to 300°0 at the speed of 10°0eeda protected by nitrogen. The type of DSC equipment (DSC60, Shimadzu, Japan). The recorded data was processed by software (Shimadzu, Japan).

Spectra analyses

The UV spectra of lecithin, apigenin, SLA, and PMSLA are shown in Figure 1. No difference was observed between the spectrum of SLA and PMSLA. The same as that of apigenin, characteristic absorption peaks were observed at 268 and 334 nm in these two samples. This suggested that no change happened in the absorption generated and conjugated structures existed in the structure of apigenin that causes these absorption peaks. It could be observed from infrared spectra of these four [Figure 2]. Judging from the fact that the similarity of characteristic absorption peaks of SLA and PMSLA, the figures of these two were resulted from the pilling up of lecithin and formononetin. The characteristic absorption peak of apigenin is inhibited by the peaks of lecithin, and the fact between 500/cm and 1750/cm can be demonstrated. In the meantime, the broad peaks of -OH in the wavelength of 3500–2800 cm⁻¹ have changed into narrow peaks with high intensity, a conclusion could be reached that no hydrogen bond was formed during the synthesis of SLA and that the conjugated structure of both lecithin and apigenin remained intact in the complex.

Open in a separate window

Open in a separate window

Figure 1

Ultraviolet spectra of lecithin (2), apigenin (1), their physical mixture (3) and complex (4)

The UV data indicated that the conjugated groups of apigenin which caused the absorption peaks remained unchanged. Judging from the similarity of characteristic absorption peaks of SLA and PMSLA, the figures of these two were resulted from the pilling up of lecithin and apigenin. Meanwhile, some characteristic absorption peaks of apigenin were subdued by that of lecithin and thus not occurred in the absorption peak of SLA. This indicated that the basic chemical of both apigenin and lecithin remained unchanged in SLA and PMSLA.

Scanning electron microscopy analysis

The SEM images of lecithin, apigenin, SLA, and PMSLA are shown in Figure 3, the surface of lecithin showed irregular structure while that of apigenin appeared as crystals; Both the irregular structure of lecithin and the crystals of apigenin were observed in the sample of PMSLA. This indicated that lecithin and apigenin in PMSLA were simply mixed. The SEM image of SLA showed the irregular structure of lecithin. This indicated that apigenin was successfully incubated in the structure of lecithin.

Open in a separate window

Figure 2

Infrared spectra of lecithin (1), apigenin (4), their physical mixture (2) and complex (3)

Thermal characteristics

The DSC thermograms of lecithin, apigenin, SLA and PMSLA are shown in Figure 4. The thermogram of the apigenin showed an endothermal peak at around 355°C. It was at this temperature that apigenin started to melt. The characteristic absorption peaks of apigenin were completely disappeared in the thermogram of PMSLA. This might be resulted from the fact that the characteristics of apigenin were covered by that of lecithin in the melting process. The endothermal peak of apigenin was disappeared in the thermograms of the complex of the two. This showed that crystal structures were absent in the complex and that the complex existed in the form of irregularity. Overall, the results suggested that apigenin was evenly dispersed in the structure of lecithin and that the two may be connected by hydrogen bonding and van der Waals force. No obvious endothermal peak was observed in that of lecithin and SLA, the possible reason is that the polarity end of the apigenin and the lecithin were interacted with each other and that the regularity decreased between the fatty hydrocarbon chain of phospholipid molecules in lecithin with the interaction of apigenin.

Open in a separate window

Figure 3

Scanning electron micrographs of lecithin (1), apigenin (2), their physical mixture (3) and complex (4)

Financial support and sponsorship

The project supported by the National Science Foundation of China (31401663).

The project supported by the National Science Foundation of China (31401663).