Liposome for embedding bee pollen phenol amine and preparation method thereof

Abstract

The invention provides a lipidosome for embedding bee pollen phenol amine and a preparation method thereof, and belongs to the technical field of high-value utilization of bee pollen, the preparation method comprises the following steps: placing lecithin, cholesterol, a bee pollen phenol amine extract and an organic solvent in a reaction container, dissolving and uniformly mixing, and then removing the organic solvent to obtain a lipid film; and hydrating the lipid film to obtain a liposome suspension, and carrying out ultrasonic treatment in an ice-water bath to obtain the liposome embedded with bee pollen phenol amine. All the steps are carried out under a dark condition. By preparing and optimizing the lipidosome for embedding bee pollen phenol amine, the problem of poor water solubility of bee pollen phenol amine is successfully solved, and a new thought and method are provided for developing a high-added-value product of bee pollen phenol amine.

Description

Technical Field

[0001] The invention relates to the technical field of high-value utilization of bee pollen, and in particular to a liposome encapsulating bee pollen phenolamine with high water solubility and high stability and a preparation method thereof. Background Art

[0002] Bee pollen is an irregular, oblate mass of pollen grains collected by bees from the stamens of plants and the microsporangium of gymnosperms. Bees add nectar, special glandular secretions and saliva to it, forming a rich nutritional ingredient and biological activity. Compared with other foods or natural products, bee pollen is unique in that it contains the richest variety and highest content of phenolamine compounds (Phenolamides), and is known as the "treasure trove of phenolamine compounds."

[0003] Phenolamine compounds, also known as hydroxycinnamic acids amides, are secondary metabolites of plants. Phenolamine compounds are widely distributed in plants, especially in reproductive organs and seeds. They are important phenolic components in plants. They not only play a key role in plant growth and development and environmental stress response, but also have multiple biological activities such as antioxidant, antiviral, anti-inflammatory, anti-cancer, hypoglycemic and lipid-lowering, cardiovascular protection, tyrosinase inhibition, cholesterol regulation and improvement of intestinal flora. They can be used in health foods, cosmetics, pharmaceuticals and other fields. Phenolamine compounds are usually a combination of amines and hydroxycinnamic acid. The main types of hydroxycinnamic acid are cinnamic acid, coumaric acid, caffeic acid, ferulic acid, etc. Polyamines in aliphatic groups are polar hydrophilic compounds, while hydroxycinnamic acid is characterized by containing an aromatic ring and a C3-structure, which gives it an unsaturated side chain in the lipophilic molecular structure. When polyamines are coupled with hydroxycinnamic acid, the polarity of the resulting metabolites can be significantly reduced, which contributes to the overall translocation, stability and compartmentalization of phenolamine compounds, but makes the phenolamine compounds poorly soluble in water.

[0004] Although phenolamine compounds have rich biological activities, their water insolubility restricts their application potential in the development of high value-added products. In order to further develop bee pollen and improve its economic value, the inventor team used microencapsulation technology to embed bee pollen phenolamines in previous studies, which to a certain extent improved the solubility of bee pollen phenolamines. However, although this solution has a good embedding rate and can effectively prevent the precipitation of phenolamine compounds, the solubility of the obtained microcapsules still cannot fully meet the actual needs. Summary of the invention

[0005] In view of the technical problems existing in the background technology, the present invention provides a highly water-soluble liposome encapsulating bee pollen phenolamine and a preparation method thereof, aiming to solve the technical problem that the solubility of bee pollen phenolamine is insufficient to meet the actual production application.

[0006] The technical solution of the present invention is as follows: A method for preparing a liposome with high water solubility and high stability encapsulating bee pollen phenolamines, comprising the following steps: S1, placing lecithin, cholesterol, bee pollen phenolamine extract and an organic solvent in a reaction container, dissolving and mixing, and then removing the organic solvent to obtain a lipid film; S2, hydrating the lipid film to obtain a liposome suspension, and then ultrasonically treating the suspension to obtain liposomes encapsulating bee pollen phenolamines; The above steps were all carried out under light-proof conditions.

[0007] In some embodiments of the present invention, the bee pollen phenolamine extract is extracted from apricot bee pollen, and its preparation method is: after the apricot bee pollen is crushed, it is first defatted with petroleum ether, and then ultrasonically extracted with 80% ethanol solution, the supernatant of the obtained extract is taken, vacuum concentrated, and then distilled water and ethyl acetate are added for extraction, and the upper extract is concentrated and freeze-dried to obtain the bee pollen phenolamine extract; wherein the ultrasonic extraction parameters are: solid-liquid ratio 1: (30-40), extraction time 30-50 min, extraction temperature <40 ° C, and ultrasonic power 400-600 W.

[0008] Preferably, in the above preparation method, the concentration of lecithin is 5-15 mg / mL, the mass ratio of bee pollen phenolamine extract to lecithin is 1:(1-6), and the mass ratio of cholesterol to lecithin is 1:(3-6).

[0009] Preferably, in the above preparation method, the organic solvent is anhydrous ethanol. In some embodiments of the present invention, the method for removing the organic solvent is: removing the organic solvent by rotary evaporation at 40-50° C. and under negative pressure.

[0010] Preferably, in the above preparation method, step S2 uses a phosphate buffer solution with a pH of 7.0-8.0 to hydrate the lipid film, and the phosphate buffer contains 0.5-1% (volume percentage) Tween 80.

[0011] Preferably, in the above preparation method, the lecithin is soybean lecithin, and the hydration temperature is 45-55°C; hydration at this temperature can avoid oxidation of the prepared soybean lecithin and can also be better hydrated.

[0012] Preferably, in the above preparation method, the ultrasonic conditions in step S2 are: in an ice-water bath, ultrasonic for 1 s on and 1 s off, for a total treatment of 9-15 min, and the ultrasonic power is 200-300 W. The method of ultrasonicating in an ice-water bath and ultrasonicating for 1 s on and 1 s off can effectively prevent the heat generated by ultrasound from destroying the liposomes, and the ultrasonic time also has a key influence. For example, short-term ultrasonic treatment will lead to uneven liposome particles. When the ultrasonic time is too long, a large amount of heat will be generated, destroying the stable system that has been formed, causing multiple liposomes to aggregate, and there is a risk of demulsification, causing the phenolamine extract to leak from the liposomes. Therefore, under the above preferred conditions, it is easier to obtain complete, uniform and small-sized vesicles.

[0013] The highly water-soluble and highly stable liposomes encapsulating bee pollen phenolamines prepared according to the method of the present invention also fall within the protection scope of the present invention.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) Improving the water solubility of phenolamine compounds is a key direction for developing high value-added products. The bee pollen phenolamine microcapsules previously developed by the inventors still cannot fully and effectively solve the problem of the water solubility of bee pollen phenolamines. The present invention successfully constructs liposomes encapsulating bee pollen phenolamines and significantly improves the solubility of bee pollen phenolamines by optimizing the proportion of liposome components and preparation parameters, which can reach 6.9 times that of phenolamine microcapsules.

[0015] (2) In the process of preparing liposomes, the present invention found that the stability of the obtained liposomes under the same storage and detection conditions varied greatly. To address this problem, the present invention used high performance liquid chromatography and high performance liquid chromatography tandem mass spectrometry to analyze the components of bee pollen phenolamine extracts, and found that bee pollen phenolamine extracts were extremely sensitive to light and easily underwent photodecomposition reactions, especially under ultraviolet light or strong light. Therefore, the present invention effectively ensured the stability and uniformity of the obtained liposomes by adopting an avoidance method during the preparation process, effectively avoiding the problem of large differences in stability between different batches. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is a high performance liquid chromatogram of the phenolamine extract of apricot bee pollen in an embodiment of the present invention; Figure 2 The figure is a result of analyzing the effect of lecithin concentration on liposome encapsulation efficiency (A) and particle size distribution (B) in the examples of the present invention; Figure 3 The figure is the analysis result of the effect of the mass ratio of PE to lecithin on the liposome encapsulation efficiency (A) and particle size distribution (B) in the embodiment of the present invention; Figure 4 Graph showing the effect of the cholesterol to lecithin mass ratio on liposome encapsulation efficiency (A) and particle size distribution (B) in an embodiment of the present invention; Figure 5 The figure is a result of analyzing the effect of ultrasound time on liposome encapsulation efficiency (A) and particle size distribution (B) in the embodiment of the present invention; Figure 6 The figure is a result of analyzing the effect of hydration temperature on liposome encapsulation efficiency (A) and particle size distribution (B) in the examples of the present invention; Figure 7 The response surface of the interaction of the three factors of hydration temperature, lecithin concentration and the mass ratio of lecithin to PE in the embodiment of the present invention; Figure 8 Figure 2 is a graph showing the particle size (A) and Zeta potential (B) of the phenolamine liposomes in the embodiment of the present invention; Fig. 9 The appearance of PE, blank liposomes and phenolamine liposomes in the embodiments of the present invention; Fig.10 TEM images of blank liposomes and phenolamine liposomes in the embodiments of the present invention; Fig.11 FTIR spectra of PE, blank liposomes and phenolamine liposomes in the embodiments of the present invention; Fig.12 DSC curves of PE, blank liposomes and phenolamine liposomes in the embodiments of the present invention; Fig.13 Graph showing the water solubility test results of PE and phenolamine liposomes in the embodiments of the present invention. DETAILED DESCRIPTION

[0018] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The terms "including" and "having" and any variations thereof herein are intended to cover non-exclusive inclusions.

[0020] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0021] The preparation method of the apricot bee pollen phenolamine extract used in the following examples is as follows: weigh 100 g of apricot bee pollen raw material, mechanically crush it in a wall-breaking machine, and then pass it through a 200-mesh sieve, and ultrasonically defatted it twice with 200 mL of petroleum ether; the defatted bee pollen is ultrasonically assisted extracted with 80% ethanol solution, the ultrasonic power is 500 W, the solid-liquid ratio is 1:30 (w / v), the extraction time is 30 min, the extraction times are 2 times, and the extraction temperature is less than 40 ° C. The extract is filtered to remove the residue to obtain a supernatant, and the supernatant is vacuum concentrated using a rotary evaporator. The concentrate is suspended in distilled water and extracted with ethyl acetate at a volume ratio of 1:1. The upper layer of the extract is rotary evaporated and concentrated, and freeze-dried to obtain a bee pollen phenolamine extract (Phenolamide extract, PE).

[0022] The components of the phenolamine extract of apricot bee pollen were analyzed by high performance liquid chromatography and high performance liquid chromatography tandem mass spectrometry. The high performance liquid chromatography analysis conditions were as follows: Chromatographic column: Hypersil DOLD (250×4.6 mm, 5 μm); mobile phase: phase A is 0.13% formic acid in water, phase B is methanol; injection volume 10 μL, flow rate 0.5 mL / min; column temperature 30 ℃; gradient elution conditions: 0 min, 50% B; 20 min, 63% B; detection wavelength 280 nm; The HPLC-MS / MS analysis conditions were as follows: Mobile phase: Phase A was 0.13% formic acid in water, and phase B was methanol; injection volume was 10 μL, flow rate was 0.5 mL / min; mass spectrometry conditions: ionization mode ESI+; ionization voltage was 20-40 V, scanning mass range was 100-1500, nebulizer pressure was 30-50 psi, drying gas flow rate was 8-10 L / min, drying gas temperature was 350°C, and capillary voltage was 3500 V.

[0023] Figure 1 The HPLC chromatogram of the phenolamine extract of apricot bee pollen shows that there are 4 main peaks in the HPLC chromatogram of the PE of apricot bee pollen, among which the content of peak 4 is the highest. Table 1 shows the results of the HPLC tandem mass spectrometry analysis of the phenolamine extract of apricot bee pollen.

[0024] Table 1 HPLC-MS / MS analysis results

[0025] From Table 1, we can see that the relative molecular weight of the four main peaks is the same, all 583.2682, and the inferred molecular formula is C 34 H 37 N3O6, the secondary fragment ions are the same, indicating that these four compounds are isomers. The phenolamine compounds in bee pollen are mainly composed of polyamines and hydroxycinnamic acid, and the polyamines mainly include putrescine (C4H 12 N2), spermidine (C7H 19 N3) and spermine (C 10 H 26 N4), etc. The hydroxycinnamic acid substituents are generally coumaric acid (C9H8O3), caffeic acid (C9H8O4) and ferulic acid (C 10 H 10 O4), etc. According to the molecular formula C 34 H 37 The number of N in N3O6 indicates that the polyamine chain is spermidine, and the number of C and H indicates that the compound is trisubstituted spermidine. The structure of the compound was analyzed by fragment information and reference to relevant literature (Elejalde-Palmett et al 2015). The fragment ion peaks in the secondary mass spectra of phenolamine compounds are usually formed by the loss of substituents such as coumaroyl and caffeoyl. The fragment ions in Table 1 are: m / z 147 is the fragment ion of the coumaroyl substituent; m / z 438 is the quasi-molecular ion fragment; m / z 584 loses a coumaroyl group and generates a fragment ion, followed by a coumaroyl group. m / z 292;N 5 and C 3 The bonds between them break, producing fragment ions m / z 204. Combining the fragment ion information and molecular formula, it is speculated that the phenolamine compounds in the apricot bee pollen extract are mainly N 1 - N 5 - N 10 -Tri-p-coumaryl spermidine, peaks 1 to 4 are N 1 - N 5 - N 10 -Different cis-trans isomers of tri-p-coumaryl spermidine (referred to as PA). Studies have shown that phenolamines have a complex structure and exist in a variety of cis-trans isomers. Moreover, the cis-trans isomers have similar structures and polarities, making separation difficult. Currently, there is no commercially available phenolamine standard.N 1 - N 5 - N 10 -(EEE)-tri-p-coumaroyl-spermidine was used as the standard for relative quantification of peaks 1 to 4. The results showed that the content of the corresponding compounds of PE peaks 1 to 4 was 55.66±0.27%.

[0026] In the following examples, the method for determining the liposome encapsulation efficiency is as follows: 1 mL of liposome solution is taken, centrifuged at a speed of 14000 g and a temperature of 4°C for 30 min, the supernatant is aspirated, methanol is added to dilute it to a certain concentration, and the concentration of free phenolamines in the supernatant is determined by liquid phase chromatography (C 游离 ); At the same time, 1 mL of liposome solution was taken, diluted to a certain concentration by adding methanol, and then ultrasonically treated for 30 min to demulsify the liposomes. The total phenolamine concentration (C 总 ).

[0027] .

[0028] In the following examples, the particle size distribution of liposomes was determined using a nanoparticle size potentiometer, specifically: the liposomes were diluted with distilled water to a suitable concentration range, the instrument was balanced for 120 s, and the particle size (d / nm) of the liposomes was measured at 25°C, and the measurement was repeated three times to obtain the particle size and PDI.

[0029] Embodiment 1-21 A method for preparing liposomes encapsulating bee pollen phenolamine extract is provided respectively, and the specific operations are as follows: (1) Wrap the eggplant-shaped rotary evaporation flask with black light-shielding cloth to ensure that the flask is completely protected from light.

[0030] (2) Accurately weigh a certain amount of soybean lecithin, cholesterol, and PE, add them to an eggplant-shaped rotary evaporator, add anhydrous ethanol, and sonicate in a water bath until they are completely dissolved. Then adjust the water bath temperature to 45°C and, under vacuum, rotary evaporate to remove the anhydrous ethanol. Continue vacuuming for 1 hour to completely remove the ethanol and form a uniform lipid film on the inner wall.

[0031] (3) Add an appropriate amount of pH 7.4 phosphate buffer solution (containing 0.5% Tween 80) to the lipid film to hydrate the film for 1 h to form a liposome suspension. Then, perform ultrasonication in an ice-water bath (ultrasonication for 1 s, stop for 1 s, power of 200 W) to finally obtain liposomes encapsulating bee pollen phenolamines, which were then stored at 4°C in the dark.

[0032] In the above embodiments, the amounts of PE, soybean lecithin and cholesterol as well as the hydration temperature and ultrasonic time in step (3) are specifically shown in Table 2.

[0033] Table 2 Reaction parameter statistics in Examples 1-21

[0034] The encapsulation efficiency and particle size distribution of the liposomes prepared in each example were tested, and the following conclusions were obtained based on the test results: 1. Effect of lecithin concentration on liposomes.

[0035] Figure 2 The results of the tests in Examples 1-5 are analyzed. Different letters in the figure indicate that different concentrations of lecithin have significant differences in sample encapsulation efficiency and particle size distribution ( P <0.05). Figure 2 It can be seen that with the increase of lecithin concentration, the encapsulation efficiency and average particle size of liposomes first increased and then decreased. When the lecithin concentration was 10 mg / mL, the encapsulation efficiency of phenolamines in liposomes reached a maximum value of 57.42%. At this concentration, the average particle size was 73.5 nm, and the PDI was lower than 0.3. When the lecithin concentration continued to increase, its encapsulation efficiency decreased. This may be because the addition of too much lecithin would increase the viscosity of the liposome suspension, leading to the agglomeration of the liposomes, thereby reducing the encapsulation efficiency of the liposomes, increasing the average particle size and causing slight precipitation.

[0036] Effect of the mass ratio of PE to lecithin on liposomes. Figure 3 The results of the tests in Examples 2 and 6-9 are analyzed. Different letters in the figure indicate that different mass ratios of PE to lecithin have significant differences in sample encapsulation efficiency and particle size distribution ( P <0.05). Figure 3 It can be seen that with the increase of PE mass, the encapsulation efficiency shows a trend of first increasing and then decreasing. When the mass ratio of PE to lecithin is 1:5, the encapsulation efficiency increases from 48.7% to 75.81%. After continuing to add PE, the encapsulation efficiency did not increase, but showed a downward trend. It can be seen that too low or too high a core material addition is not conducive to the formation of liposomes; with the increase of PE addition, the average particle size of the liposome increases accordingly, and reaches the maximum value when the ratio is 1:1. This may be due to the fact that the hydrophobic substance is encapsulated in the hydrophobic part of the phospholipid bilayer of the liposome. When the addition amount is too high, the bilayer will expand.

[0037] 3. Effect of the mass ratio of cholesterol to lecithin on liposomes.

[0038] Figure 4 The results of the tests in Examples 8 and 10-13 are analyzed. Different letters in the figure indicate that different mass ratios of cholesterol to lecithin have significant differences in sample encapsulation efficiency and particle size distribution (P <0.05). Figure 4 It can be seen that the encapsulation rate of liposomes shows a trend of first increasing and then decreasing with the increase of the amount of cholesterol added. This may be because when the cholesterol content is too high, the proportion of lecithin is small, which makes it difficult to form a lipid membrane and the formed liposome has a smaller loading space. Excessive cholesterol may cause the phospholipid bilayer to be arranged loosely, thereby reducing the loading amount of fat-soluble substances in the phospholipid bilayer; when the mass ratio of cholesterol to lecithin is 1:4, the encapsulation rate increases from 45.8% to 69.22%. At this time, the average particle size of the liposome is 76.62nm, and the PDI is lower than 0.3.

[0039] 4. Effect of ultrasound time on liposomes.

[0040] Figure 5 The results of the tests in Examples 11 and 14-17 are analyzed. Different letters in the figure indicate that different ultrasonic times have significant differences in the encapsulation efficiency and particle size distribution of the samples ( P <0.05). Figure 5 It can be seen that with the increase of ultrasonic time, the encapsulation efficiency of liposomes first decreased, then increased, and then slowly decreased. The average particle size showed a trend of first decreasing and then slowly increasing. When the ultrasonic time was 12 min, the system tended to be stable, with the smallest average particle size and the highest encapsulation efficiency. At this time, the encapsulation efficiency of liposomes was 65.07%, the particle size was 67.46 nm, and the PDI was lower than 0.3.

[0041] 5. Effect of hydration temperature on liposomes.

[0042] Figure 6 This is the analysis of the test results of Examples 17-21. Different letters in the figure indicate that different hydration temperatures have significant differences in sample encapsulation efficiency and particle size distribution ( P <0.05). Figure 6 It can be seen that with the increase of hydration temperature, the encapsulation efficiency of liposomes shows a trend of first increasing and then decreasing. The reason may be the phase transition temperature of phospholipids. If the hydration temperature is lower than the phase transition temperature of phospholipids, it will not cause glass transition. Too high hydration temperature will lead to oxidation of soybean lecithin, thereby affecting its encapsulation efficiency and particle size; among them, when the hydration temperature is 50℃, the encapsulation efficiency is 72.10%, the average particle size under this condition is 96.54 nm, and the PDI is lower than 0.3.

[0043] Embodiment 22 In this case, Design Expert software is used to design and analyze the response surface experiment to obtain the optimal liposome preparation process. The specific operations are as follows: According to the analysis results of Examples 1-21, the encapsulation efficiency of liposomes was used as an evaluation index, and three factors that had a significant impact on the encapsulation efficiency were selected: hydration temperature (A), lecithin concentration (B), and the mass ratio of lecithin to PE (C). The ultrasonic time was fixed at 12 min and the mass ratio of cholesterol to lecithin was 1:4. A response surface experiment was performed. The experimental factors and levels are shown in Figure 3. The design and results of the response surface experiment are shown in Table 4.

[0044] Table 3 Response surface design experimental factors and levels

[0045] With the encapsulation efficiency as the response value, 17 experiments were carried out (see Table 4). The response surface results analyzed the experimental data in Table 3 and obtained a binomial regression equation with Y (encapsulation efficiency) as the dependent variable and A (hydration temperature, °C), B (lecithin concentration, mg / mL), and C (lecithin to PE mass ratio) as independent variables: Y=85.23-1.45A+14.27B-3.63C+2.87AB+0.50AC+8.56BC+0.33A 2 -15.16B 2 -18.31C 2 .

[0046] Table 4 Design and results of response surface experiment

[0047] The variance analysis of the response surface results is shown in Table 5. The fitted model is significant ( P <0.05), the lack of fit was not significant ( P =0.0883>0.05), model determination coefficient R 2 =0.9966, indicating that the model method is reliable and the regression equation fits well, which can be used to analyze and predict the optimal process of PA-LP. The results of the variance analysis model showed that the hydration temperature (A), lecithin concentration (B) and the mass ratio of lecithin to PE (C) had a significant effect on the encapsulation efficiency ( P <0.05). In addition, according to the fitting results of the quadratic regression equation, the interaction between the other two factors was analyzed by fixing the level of one factor. The strength of the interaction can be judged by the shape of the contour lines. When the contour lines are circular, it means that the interaction between the two factors is not significant, and when they are elliptical, the interaction is significant. The results are as follows Figure 7 As shown in the figure, the interaction between hydration temperature and lecithin concentration, and between lecithin concentration and the mass ratio of lecithin to PE in the model established in this example is significant ( P <0.05), but the interaction between hydration temperature and the mass ratio of lecithin to PE was not significant ( P >0.05), the order of interaction is BC>AB>AC.

[0048] Table 5 Regression model and variance analysis

[0049] Note: * indicates significant difference ( P <0.05,** indicates extremely significant difference( P <0.01), NS indicates no significance.

[0050] The optimal process conditions were analyzed by Design-expert software: hydration temperature of 53°C, lecithin concentration of 10.56 mg / mL, and the mass ratio of lecithin to PE of 5.14. The encapsulation efficiency of liposomes prepared under the optimal process conditions was 83.02%, close to the theoretical value of 84.10%, indicating that the model prediction results were good and suitable for optimizing the process parameters for preparing PA-LP.

[0051] Further, the phenolamine liposomes prepared under the optimal process conditions were characterized as follows: (1) Particle size distribution and Zeta potential determination.

[0052] After the liposomes were diluted with distilled water, 1 mL was taken and placed in a potential cup for measurement using a nanoparticle size potential analyzer. The Zeta potential value of the liposomes was obtained by measuring three times at 25°C as above.

[0053] The particle size and Zeta potential of liposomes were measured as follows: Figure 8 As shown. The average particle size of the liposomes is 101.57 nm, and the PDI is 0.225, indicating that the liposome system is evenly dispersed. The Zeta potential of the liposomes is -39.53 mV, indicating that the surface of the prepared liposomes is negatively charged. This is because lecithin contains negatively charged substances, such as phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, which make the liposomes negatively charged, thereby preventing aggregation between vesicles.

[0054] (2) Observation of surface and microscopic morphology.

[0055] Weigh equal amounts of PE, blank liposomes (prepared in the same way as phenolamine liposomes, except that no PE was added), and phenolamine liposomes, dissolve them in distilled water, and observe their dissolution state after leaving them for 24 hours. Fig. 9 As shown: the PE suspension directly dissolved in water has precipitation, indicating that PE has poor water solubility, while the phenolamine liposomes show better solubility, dispersibility and uniform state. The blank liposomes are transparent and uniform milky white suspension, and the phenolamine liposomes are light yellow suspension observed with the naked eye.

[0056] The microstructure of liposomes was observed by transmission electron microscopy (TEM). Fig.10 As shown: blank liposomes and phenolamine liposomes are round or oval, with a relatively rounded surface and similar particle sizes; compared with blank liposomes, phenolamine liposomes are dark black, indicating that PE has been successfully embedded or encapsulated in the liposome phospholipid bilayer membrane.

[0057] (3) Fourier transform infrared (FTIR) analysis.

[0058] PE, blank liposomes, and phenolamine liposomes were mixed with 10% sucrose, and then the mixture was freeze-dried and pressed using KBr. The scanning range was set to 4000 cm −1 Up to 400 cm −1 Each spectrum was acquired by 64 scans with a resolution of 4 cm −1 .

[0059] FTIR results are as follows Fig.11 Shown: PE at 1513.92 cm -1 and 1442.82 cm -1 The characteristic peak due to the amide bond is shown at 2928.92 cm -1 and 831.56 cm -1 The characteristic peak due to -CH2 stretching vibration appeared at 1730 cm -1 The characteristic peak of C=O stretching vibration is shown at 1260 cm -1 The peak at 925 cm represents the characteristic phosphate group (PO2-) of lipid molecules. -1 The peak at 1513.92 cm represents the asymmetric stretching vibration of the polar part of phosphatidylcholine; in phenolamine liposomes, -1 and 1442.82cm -1 The characteristic peak at disappeared, which may be caused by the interaction between liposomes and phenolamines through hydrogen bonds, van der Waals forces, etc.; while the structures of blank liposomes and phenolamine liposomes did not change, indicating that the addition of PE did not change the overall structure of liposomes.

[0060] (4) Differential Scanning Calorimetry (DSC) Analysis.

[0061] The thermal stability of the phenolamine liposome samples was analyzed using a differential scanning calorimeter. Before the analysis, the samples of PE, blank liposomes, and phenolamine liposomes were sealed in an aluminum pan, and an empty pan was used as a reference; in a nitrogen atmosphere, the instrument was heated from 25°C to 170°C at a heating rate of 5°C / min. The results are shown in Figure 2. Fig.12As shown, compared with the phenolamine extract, the temperature of the phenolamine liposomes increased to 88.9°C, which was higher than that of the blank liposomes, indicating that the temperature stability of the phenolamine extract was improved by encapsulating it in liposomes.

[0062] (5) Water solubility test.

[0063] 10 mg of PE, phenolamine microcapsules containing the same PE content (preparation method is the same as that of Chinese Patent 202111403777.9), and phenolamine liposomes containing the same PE content were dissolved in 5 mL of distilled water, stirred thoroughly, and centrifuged at 3000×g for 5 min. The supernatant was filtered through a 0.22 μm filter and then the content of phenolamine compounds was determined by high performance liquid chromatography. The results are as follows: Fig.13 As shown. Fig.13 It can be seen that PE is completely insoluble in water. After being made into liposomes, the water solubility of phenolamine compounds is significantly improved, which is 6.9 times that of phenolamine microcapsules.

[0064] In summary, the present invention solves the problem of poor water solubility of bee pollen phenolamines, and provides a new idea and method for the development and application of bee pollen phenolamines.

[0065] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A method for preparing liposomes encapsulating bee pollen phenolamines, characterized in that: The following steps are involved: S1, placing lecithin, cholesterol, bee pollen phenolamine extract and an organic solvent in a reaction container, dissolving and mixing, and then removing the organic solvent to obtain a lipid film; S2, hydrating the lipid film to obtain a liposome suspension, and then sonicating in an ice water bath to obtain liposomes encapsulating bee pollen phenolamines; The above steps were all carried out under light-proof conditions.

2. The preparation method according to claim 1, characterized in that: The bee pollen phenolamine extract is extracted from apricot bee pollen, and its preparation method is as follows: after the apricot bee pollen is crushed, it is first defatted with petroleum ether, and then ultrasonically extracted with 80% ethanol solution, the supernatant of the obtained extract is taken, vacuum concentrated, and then distilled water and ethyl acetate are added for extraction, and the upper extract is concentrated and freeze-dried to obtain the bee pollen phenolamine extract; wherein the ultrasonic extraction parameters are: solid-liquid ratio 1: (30-40), extraction time 30-50 min, extraction temperature <40°C, and ultrasonic power 400-600 W.

3. The preparation method according to claim 1, characterized in that: The concentration of the lecithin is 5-15 mg / mL, and the mass ratio of the bee pollen phenolamine extract to the lecithin is 1:(1-6).

4. The preparation method according to claim 1 or 3, characterized in that: The mass ratio of cholesterol to lecithin is 1:(3-6).

5. The preparation method according to claim 1, characterized in that: The organic solvent is anhydrous ethanol.

6. The preparation method according to claim 5, characterized in that: The method for removing the organic solvent is: removing the organic solvent by rotary evaporation at 40-50° C. and under negative pressure.

7. The preparation method according to claim 1, characterized in that: Step S2: hydrate the lipid film using a phosphate buffer solution with a pH of 7.0-8.0, wherein the phosphate buffer contains 0.5-1% Tween 80.

8. The preparation method according to claim 7, characterized in that: The hydration temperature is 45-55°C.

9. The preparation method according to claim 1, characterized in that: The ultrasonic conditions in step S2 are: ultrasonication for 1 s on and 1 s off, for a total treatment of 9-15 min, and an ultrasonic power of 200-300 W.

10. A liposome encapsulating bee pollen phenolamine, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 9.

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