Preparation method of red bayberry anthocyanin lipidosome

Abstract

The invention discloses a preparation method of waxberry anthocyanin lipidosome, and belongs to the field of waxberry anthocyanin lipidosome. The preparation method of the waxberry anthocyanin lipidosome comprises the following steps: dissolving anthocyanin, cholesterol and soybean lecithin in an organic solvent, and stirring in an ultrasonic crusher to obtain a mixture; and stirring the mixture, transferring the mixture into a pear-shaped bottle, carrying out rotary evaporation, hydrating, and filtering to obtain the waxberry anthocyanin liposome. The waxberry anthocyanin liposome prepared by the method has better stability; moreover, after the preparation conditions of the waxberry anthocyanin liposome are optimized by using a response surface method, the obtained waxberry anthocyanin liposome has relatively high encapsulation efficiency, so that a theoretical basis is provided for development of a novel anthocyanin dosage form and application of the novel anthocyanin dosage form in a health-care food market.

Description

Technical Field

[0001] The invention relates to the field of preparation of bayberry anthocyanin liposomes, and more specifically to a preparation method of bayberry anthocyanin liposomes and an optimization method for optimizing the preparation conditions of bayberry anthocyanin liposomes by using response surface methodology. Background Art

[0002] As a relatively new microencapsulation technology, liposomes use phospholipids as wall materials and have a stable bilayer structure. Phospholipids are divided into two categories: natural phospholipids and synthetic phospholipids. The structure of liposomes is similar to that of biological membranes and has amphiphilic characteristics. Liposomes perform well in protecting bioactive ingredients, reducing drug loss, improving stability, bioavailability and efficacy, and have therefore been widely used in medicine, genetics, cosmetic formulations, food and nutrition, and other fields. However, the stability of existing anthocyanins is poor.

[0003] In order to solve the above problems, this study aims to improve the stability of anthocyanins by preparing them in the form of liposomes and optimize the preparation process using response surface methodology, so as to provide a theoretical basis for the development of new dosage forms of anthocyanins and their application in the health food market. Summary of the invention

[0004] In view of the problems existing in the prior art, the first purpose of the present invention is to provide a bayberry anthocyanin liposome and a preparation method thereof, wherein the bayberry anthocyanin liposome prepared by the preparation method has good stability; the second purpose is to provide an optimization method for optimizing the preparation conditions of bayberry anthocyanin liposome using response surface methodology, wherein the bayberry anthocyanin liposome prepared after optimization by the method has a high encapsulation rate.

[0005] To solve the above problems, the present invention adopts the following technical solutions.

[0006] A method for preparing bayberry anthocyanin liposomes, the preparation method is as follows: after dissolving anthocyanin, cholesterol and soybean lecithin in an organic solvent, the organic solvent is placed in an ultrasonic crusher and stirred to obtain a mixture; after stirring the mixture, the mixture is transferred into a pear-shaped bottle, and rotary evaporated, hydrated and filtered to obtain bayberry anthocyanin liposomes.

[0007] Furthermore, the mass ratio of the soybean lecithin to cholesterol is 2-6:1; the mass ratio of the soybean lecithin to anthocyanin is 10-40:1.

[0008] Furthermore, the organic solvent is anhydrous ethanol, acetone, a chloroform-methanol mixed solvent, ethyl acetate or isopropanol.

[0009] Furthermore, the organic solvent is preferably anhydrous ethanol.

[0010] Furthermore, the rotary evaporation is performed until a thin film is formed on the wall of the pear-shaped bottle; the temperature of the rotary evaporation is 20-60°C.

[0011] Furthermore, the liquid used for hydration is ultrapure water, and the hydration time is 20 to 40 minutes.

[0012] Furthermore, the pore size of the filter membrane used in the filtration is 0.20 to 0.30 μm.

[0013] The invention also provides a myrica anthocyanin liposome.

[0014] The invention also provides a method for optimizing the preparation conditions of myrica anthocyanin liposomes by using a response surface methodology.

[0015] Furthermore, the optimization method is as follows: the mass ratio of soybean lecithin to cholesterol, the rotary evaporation temperature and the mass ratio of soybean lecithin to anthocyanin are selected as variables, the encapsulation efficiency of bayberry anthocyanin liposomes is selected as the response value, the three-factor three-level BBD method is used to design the preparation experiment, and the experimental data are collected; the fitting equation of the following form is established:

[0016] Y=82.84-0.42A+0.82B+0.61C+0.20AB+0.033AC-0.083BC-2.73A 2 -1.78B 2 -1.78C 2

[0017] Among them, A represents the mass ratio of soybean lecithin to cholesterol, B represents the rotary evaporation temperature, C represents the mass ratio of soybean lecithin to anthocyanin, and Y represents the encapsulation efficiency of bayberry anthocyanin liposomes.

[0018] Compared with the prior art, the advantages of the present invention are:

[0019] 1. This scheme improves the stability of anthocyanins by preparing them in the form of liposomes, and uses response surface methodology to optimize the preparation conditions, so that the optimized bayberry anthocyanin liposomes have a good encapsulation rate, which provides a theoretical basis for the development of new dosage forms of anthocyanins and their application in the health food market. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a full-band scanning image of anthocyanin of the present invention;

[0021] Figure 2 is a standard curve diagram of anthocyanins of the present invention;

[0022] Figure 3 This is a graph showing the effect of the mass ratio of soybean lecithin to cholesterol on the liposome encapsulation efficiency of myrica rubra anthocyanin of the present invention;

[0023] Figure 4 This is a graph showing the effect of the rotary evaporation temperature on the encapsulation efficiency of myrica anthocyanin liposomes of the present invention;

[0024] Figure 5 This is a graph showing the effect of the mass ratio of soybean lecithin to anthocyanin on the encapsulation efficiency of myrica anthocyanin liposomes of the present invention;

[0025] Figure 6 The contour map and response surface diagram of the effects of the mass ratio of soybean lecithin to cholesterol and the rotary evaporation temperature on the encapsulation efficiency of myrica anthocyanin liposomes of the present invention;

[0026] Figure 7 The contour map and response surface diagram of the effects of the mass ratio of soybean lecithin to cholesterol and the mass ratio of soybean lecithin to anthocyanin on the encapsulation efficiency of myrica anthocyanin liposomes of the present invention;

[0027] Figure 8 The contour map and response surface diagram of the effects of the mass ratio of soybean lecithin to anthocyanin and the rotary evaporation temperature on the encapsulation efficiency of the bayberry anthocyanin liposome of the present invention;

[0028] Fig. 9 This is a comparison diagram of the appearance of anthocyanins and myrica anthocyanin liposomes of the present invention;

[0029] Fig.10 It is a transmission electron micrograph of the bayberry anthocyanin liposome of the present invention;

[0030] Fig.11 This is the particle size distribution diagram of the bayberry anthocyanin liposome of the present invention;

[0031] Fig.12 This is a graph showing the pH value of myrica rubra anthocyanin liposomes in Experimental Example 1;

[0032] Fig.13 This is a graph showing the change in particle size of myrica anthocyanin liposomes in Experimental Example 1;

[0033] Fig.14 These are Fourier infrared spectra of anthocyanins, bayberry anthocyanin liposomes, FITC and FITC-labeled bayberry anthocyanin liposomes. DETAILED DESCRIPTION

[0034] Embodiment 1:

[0035] (1) Preparation method:

[0036] Anthocyanins, cholesterol and soybean lecithin were accurately weighed, dissolved in 10 mL of anhydrous ethanol and placed in an ultrasonic disruptor for 2 min to ensure complete dissolution to form a mixture.

[0037] Add 20 mL of ultrapure water and a rotor to a vial and place it on a magnetic stirrer. Use a syringe to inject the above mixture into ultrapure water within 10 minutes and continue stirring. After magnetic stirring for 15 minutes, transfer the solution to a pear-shaped bottle and rotary evaporate until a thin film forms on the bottle wall. Then add 20 mL of preheated ultrapure water and hydrate for 30 minutes. Finally, filter with a 0.22 μm filter membrane to obtain bayberry anthocyanin liposomes; store it in a refrigerator at 4°C.

[0038] (2) Full-band scanning of anthocyanins

[0039] The appropriate anthocyanin stock solution was scanned in the full band at 185-580nm under the UV-visible spectrophotometer, and the baseline was calibrated with distilled water as the reference. Figure 1 shown.

[0040] (3) Preparation of anthocyanin standard curve

[0041] (a) Accurately weigh 1 mg of anthocyanin and dilute it to a 10 mL volumetric flask with ultrapure water to obtain a 0.1 mg / mL stock solution, which is then diluted to different concentrations. Pipette 2 mL of the prepared solution into a glass cuvette, use ultrapure water as a blank control, and measure the absorbance at 513 nm.

[0042] (b) A standard curve of anthocyanins y=2.2515x+0.001 (R 2 =0.9965, R 2 is the coefficient of determination. The closer the value is to 1, the better the fit between the model and the data is. It is believed that the model can explain most of the data variability), where x represents the concentration of anthocyanins and y represents the absorbance.

[0043] After processing the experimental data with SPSS22.0, Origin 9.0 was used to draw the standard curve of anthocyanins. The standard curve of anthocyanins drawn is as follows: Figure 2 shown.

[0044] (4) Determination of the encapsulation efficiency (EE%) of myrica anthocyanin liposomes by centrifugation-ultraviolet spectrophotometry

[0045] (a) Accurately pipette 0.5 mL of bayberry anthocyanin liposomes and blank liposomes respectively, dilute to 10 mL volumetric flask with ultrapure water, transfer into a 50 mL centrifuge tube, centrifuge at 8000 rpm for 30 min, then take the supernatant, use the blank liposomes as the control group, detect the absorbance at 513 nm, substitute into the anthocyanin standard curve of step (3) to calculate the concentration of free anthocyanins (C1).

[0046] (b) 0.5 mL of bayberry anthocyanin liposomes and blank liposomes were taken respectively and placed in 10 mL volumetric flasks, 4.5 mL of anhydrous ethanol was added to make up the volume, and the flasks were immediately transferred to 50 mL centrifuge tubes after making up the volume, and centrifuged at 8000 rpm for 30 min, the supernatant was taken, and the absorbance value was detected, and then the absorbance value was substituted into the anthocyanin standard curve of step (3) to calculate the concentration of total anthocyanins (C2). The encapsulation efficiency was calculated as follows:

[0047] Encapsulation efficiency (%) = (C2-C1) / C2×100%.

[0048] Embodiment 2:

[0049] 1. Single Factor Experiment

[0050] 1.1 Effect of the mass ratio of soybean lecithin to cholesterol on the encapsulation efficiency of myrica rubra anthocyanin liposomes

[0051] Weigh anthocyanins, cholesterol and soybean lecithin, wherein the mass ratio of soybean lecithin to anthocyanins is 20:1; the mass ratios of soybean lecithin to cholesterol are 2:1, 3:1, 4:1, 5:1 and 6:1, respectively; dissolve them in 10 mL of anhydrous ethanol and place them in an ultrasonic disruptor for 2 minutes to ensure complete dissolution to form a mixture.

[0052] Add 20 mL of ultrapure water and a rotor to a vial and place it on a magnetic stirrer. Use a syringe to inject the above mixture into ultrapure water within 10 minutes and continue stirring. After magnetic stirring for 15 minutes, transfer the solution to a pear-shaped bottle and rotary evaporate at 40°C until a thin film forms on the bottle wall. Then add 20 mL of preheated ultrapure water and hydrate for 30 minutes. Finally, filter with a 0.22 μm filter membrane and then store it in a refrigerator at 4°C.

[0053] Effect of the mass ratio of soybean lecithin to cholesterol on the liposome encapsulation efficiency of myrica rubra anthocyanin Figure 3 shown by Figure 3 It can be seen that with the increase of soybean lecithin, the liposome encapsulation efficiency of bayberry anthocyanin increased, and when the mass ratio of soybean lecithin to cholesterol was 4:1, the liposome encapsulation efficiency of bayberry anthocyanin reached the maximum value, and then gradually decreased and tended to be stable.

[0054] 1.2 Effect of rotary evaporation temperature on the encapsulation efficiency of myrica anthocyanin liposomes

[0055] Anthocyanin, cholesterol and soybean lecithin were weighed, wherein the mass ratio of soybean lecithin to cholesterol was 4:1, and the mass ratio of soybean lecithin to anthocyanin was 20:1; then they were dissolved with 10 mL of anhydrous ethanol and placed in an ultrasonic crusher for 2 min to ensure complete dissolution to form a mixture.

[0056] Add 20 mL of ultrapure water and a rotor to a vial and place it on a magnetic stirrer. Use a syringe to inject the above mixture into ultrapure water within 10 minutes and continue stirring. After magnetic stirring for 15 minutes, transfer the solution to a pear-shaped bottle, and then rotary evaporate at rotary evaporation temperatures of 20°C, 30°C, 40°C, 50°C, and 60°C until a thin film forms on the bottle wall, then add 20 mL of preheated ultrapure water and hydrate for 30 minutes. Finally, filter with a 0.22 μm filter membrane and store in a refrigerator at 4°C.

[0057] Effect of rotary evaporation temperature on the encapsulation efficiency of myrica rubra anthocyanin liposomes Figure 4 As shown by Figure 4 It can be seen that the encapsulation efficiency of myrica anthocyanin liposomes gradually increases at 20-40°C, reaches the maximum at 40°C, and then gradually decreases. Too high a temperature will lead to the degradation of anthocyanins, and too low a temperature will take a long time to form a film and be uneven, so the optimal rotary evaporation temperature is 40°C.

[0058] 1.3 Effect of the mass ratio of soybean lecithin to anthocyanin on the encapsulation efficiency of bayberry anthocyanin liposomes

[0059] Anthocyanin, cholesterol and soybean lecithin were weighed, wherein the mass ratios of soybean lecithin to anthocyanin were 5:1, 10:1, 20:1, 30:1 and 40:1 respectively; the mass ratio of soybean lecithin to cholesterol was 4:1; the above substances were dissolved in 10 mL of anhydrous ethanol and placed in an ultrasonic disruptor for 2 minutes to ensure complete dissolution to form a mixture.

[0060] Add 20 mL of ultrapure water and a rotor to a vial and place it on a magnetic stirrer. Use a syringe to inject the above mixture into ultrapure water within 10 minutes and continue stirring. After magnetic stirring for 15 minutes, transfer the solution to a pear-shaped bottle and rotary evaporate at 40°C until a thin film forms on the bottle wall. Then add 20 mL of preheated ultrapure water and hydrate for 30 minutes. Finally, filter it with a 0.22 μm filter membrane and store it in a refrigerator at 4°C.

[0061] The effect of the mass ratio of soybean lecithin to anthocyanin on the encapsulation efficiency of bayberry anthocyanin liposomes is shown in the figure Figure 5 As shown by Figure 5 It can be seen that when the mass ratio of soybean lecithin to anthocyanin is 10N20:1, the encapsulation efficiency of bayberry anthocyanin liposomes gradually increases; when the mass ratio of soybean lecithin to anthocyanin is 20:1, the encapsulation efficiency of bayberry anthocyanin liposomes reaches the maximum value and then gradually decreases.

[0062] 2. Optimization of preparation conditions of bayberry anthocyanin liposomes by response surface methodology

[0063] Based on previous studies, the mass ratio of soybean lecithin to cholesterol, rotary evaporation temperature, and mass ratio of soybean lecithin to anthocyanin were determined to be the main factors affecting the liposome encapsulation efficiency of bayberry anthocyanin. The variable range was set as follows: the mass ratio of soybean lecithin to cholesterol was 2-6:1, the rotary evaporation temperature was 20-60℃, and the mass ratio of soybean lecithin to anthocyanin was 10-40:1.

[0064] 2.1 Response surface methodology optimization steps

[0065] Step 1: Based on the single factor experiment, the mass ratio of soybean lecithin to cholesterol, rotary evaporation temperature and the mass ratio of soybean lecithin to anthocyanin were selected as variables, and the encapsulation efficiency of bayberry anthocyanin liposomes was selected as the response value. The three-factor three-level BBD method was used to optimize the preparation process. The relevant parameters and level change values ​​are shown in Table 1.

[0066] Table 1. Response surface test factor level table

[0067]

[0068] Step 2: The preparation of bayberry anthocyanin liposomes was carried out according to the design scheme of step 1, and the results obtained are shown in Table 2 (in Table 2, X1 represents soybean lecithin: cholesterol; X2 represents soybean lecithin: anthocyanin; X3 represents rotary evaporation temperature).

[0069] Table 2 Central composite design and results

[0070]

[0071]

[0072] Step 3: After a detailed analysis of the data in Table 2, a response model was established with the mass ratio A of soybean lecithin to cholesterol, the rotary evaporation temperature B, and the mass ratio C of soybean lecithin to anthocyanin as independent variables; and the encapsulation efficiency Y of bayberry anthocyanin liposomes as the response value; and the BBD evaluation parameter optimization was used to obtain the fitting equation for predicting the maximum encapsulation efficiency.

[0073] The fitting equation for predicting the maximum encapsulation efficiency is as follows:

[0074] Y=82.84-0.42A+0.82B+0.61C+0.20AB+0.033AC-0.083BC-2.73A 2 -1.78B 2 -1.78C 2

[0075] Among them, A represents the mass ratio of soybean lecithin to cholesterol, B represents the rotary evaporation temperature, C represents the mass ratio of soybean lecithin to anthocyanin, and Y represents the encapsulation efficiency of bayberry anthocyanin liposomes.

[0076] The F value of the model is 213.58, indicating that the model is significant and can correctly explain and predict the experimental results.

[0077] The variance analysis was performed on the fitting equation for predicting the maximum encapsulation efficiency to determine the influence of single factors on the encapsulation efficiency of bayberry anthocyanin liposomes, and also to test the validity of the regression equation; the analysis results are shown in Table 3.

[0078] Table 3 Analysis of variance of regression model

[0079]

[0080] Note: *: indicates extremely significant difference when P < 0.001; **: indicates significant difference when 0.01 < P < 0.05.

[0081] 2.2 Optimization response surface diagram and contour curve diagram of preparation conditions of bayberry anthocyanin liposomes

[0082] The response surface diagram and contour diagram were drawn using the binomial fitting model using Design-Expert8.0.6 software; the contour diagram of the effects of the mass ratio of soybean lecithin to cholesterol and the rotary evaporation temperature on the encapsulation efficiency of bayberry anthocyanin liposomes is shown in Figure 2. Figure 6 As shown in part (a); the response surface diagram of the effect of the mass ratio of soybean lecithin to cholesterol and the rotary evaporation temperature on the encapsulation efficiency of bayberry anthocyanin liposomes is shown in Figure 6 As shown in part (b).

[0083] The contour diagram of the effect of the mass ratio of soybean lecithin to cholesterol and the mass ratio of soybean lecithin to anthocyanin on the encapsulation efficiency of bayberry anthocyanin liposomes is shown in Figure 2. Figure 7 As shown in part (c); the response surface diagram of the effect of the mass ratio of soybean lecithin to anthocyanin and the mass ratio of soybean lecithin to cholesterol on the encapsulation efficiency of bayberry anthocyanin liposomes is shown in Figure 7 As shown in part (d).

[0084] The contour diagram of the effect of the mass ratio of soybean lecithin to anthocyanin and the rotary evaporation temperature on the encapsulation efficiency of bayberry anthocyanin liposomes is shown in Figure 2. Figure 8 As shown in part (e); the response surface diagram of the effect of the mass ratio of soybean lecithin and anthocyanin and the rotary evaporation temperature on the encapsulation efficiency of bayberry anthocyanin liposomes is shown in Figure 8 As shown in part (f).

[0085] Depend on Figure 6-Figure 8 It can be seen that the mass ratio of soybean lecithin to cholesterol and the mass ratio of soybean lecithin to anthocyanin are the main factors affecting the encapsulation efficiency. With the increase of each factor, the encapsulation rate first increases and then decreases. The optimal process conditions and encapsulation rate prediction values ​​were obtained through regression equation analysis.

[0086] 2.3 Verification Experiment

[0087] The response surface analysis adopted the model equation: the mass ratio of soybean lecithin to cholesterol was 3.93:1, the mass ratio of soybean lecithin to anthocyanin was 22.21:1, and the rotary evaporation temperature was 41.65℃; the predicted encapsulation efficiency value was 82.9998%; multiple experimental operations of bayberry anthocyanin liposomes with a mass ratio of soybean lecithin to cholesterol of 4:1, a mass ratio of soybean lecithin to anthocyanin of 22:1, and a rotary evaporation temperature of 42℃ were set up, and the encapsulation efficiency of the bayberry anthocyanin liposomes prepared in these experiments was tested, and the average encapsulation efficiency was measured to be 82.23±1.01%; the result was not much different from the encapsulation efficiency value predicted by using the model equation, which shows that the mathematical model is reliable and predictable.

[0088] Test Example 1:

[0089] Each set of experiments in this test case was carried out in parallel three times, and the results were expressed as mean ± standard deviation. Design-Expert 8.0.6 software was used for response surface design and statistical analysis, SPSS22.0 was used to process the experimental data, and Origin9.0 was used for graphics.

[0090] (1) Detection of the appearance of myrica anthocyanin liposomes

[0091] The appearance of anthocyanins Fig. 9 As shown, the left side is anthocyanin liquid at room temperature, which is bright red and transparent; the right side is the prepared bayberry anthocyanin liposome, which is dark red and a completely opaque emulsion.

[0092] (2) Internal structure of bayberry anthocyanin liposomes

[0093] (a) The prepared myrica anthocyanin liposomes were measured using a particle size analyzer.

[0094] The determination method is as follows: 1 mL of the prepared bayberry anthocyanin liposomes is pipetted into a 10 mL volumetric flask, and ultrapure water is added to make up to 10 mL. The particle size distribution is detected by a particle size analyzer equipped with a He / Ne laser (K=633 nm), the measurement range is 0.1-1000 μm, and the scattering angle is 90 b″. The sample is placed in a test dish (the refractive index of water is set to 1.33, and the buffer system is ultrapure water), and the test is repeated three times at room temperature and the results (particle size and polydispersity index PDI) are recorded.

[0095] The experimental results are as follows: the particle size of the prepared bayberry anthocyanin liposomes is 186.6±3.47 nm; the encapsulation efficiency is 82.23±1.01%; and the polydispersity index (PDI) is 0.174±0.024.

[0096] (b) Microscopic morphology of myrica anthocyanin liposomes observed using transmission electron microscopy

[0097] The negative staining method was used for observation. The specific operation was as follows: take the copper mesh with tweezers, use a pipette to absorb the bayberry anthocyanin liposomes and drop them on the surface of the copper mesh, use absorbent paper to absorb the excess bayberry anthocyanin liposomes, add a drop of uranyl acetate for negative staining, place it on filter paper, dry it at room temperature, place it in the sample rod, and observe it under a transmission electron microscope.

[0098] The microscopic morphology observed by this method is as follows Fig.10 As shown; the particle size distribution of bayberry anthocyanin liposomes is shown Fig.11 As shown (wherein, part (1) shows the intensity particle size distribution; part (2) shows the statistical diagram of the intensity particle size distribution measured three times). Figure 10-11 It can be seen that the bayberry anthocyanin liposomes have a spherical structure and a smooth surface, and there is a certain distance between the bayberry anthocyanin liposome particles, and there is a more obvious hollow structure; the particle size of the bayberry anthocyanin liposome is about 200nm, which is basically consistent with the particle size measurement result of the (a) scheme. However, the particle size distribution of the bayberry anthocyanin liposome is not very uniform. Related studies have found that the use of some mechanical methods, such as ultrasound, homogenization, extrusion and other mechanical methods can reduce the particle size and make it more evenly distributed. In subsequent experiments, homogenization, ultrasound and other methods are used to reduce the particle size according to actual conditions, so as to obtain liposomes with more application value.

[0099] (3) Study on the storage stability of bayberry anthocyanin liposomes

[0100] We investigated the changes in pH and particle size of bayberry anthocyanin liposomes during two weeks of storage. The prepared bayberry anthocyanin liposomes were stored in a 4°C refrigerator, and the changes in particle size and pH were measured using a Malvern particle size analyzer and a pH meter on days 0, 1, 3, 5, 7, 12, and 15.

[0101] The pH value changes of bayberry anthocyanin liposomes during the study period are shown in the figure below. Fig.12 As shown in the figure; the change of particle size of bayberry anthocyanin liposomes is shown in Fig.13 shown.

[0102] Depend on Fig.12 It can be seen that the pH value of myrica rubra anthocyanin liposomes decreased slightly on the first day, and then gradually increased until the 12th day, but was still less than the initial value; on the 15th day, the pH value rose rapidly.

[0103] Depend on Fig.13It can be seen that: within two weeks, the particle size of the bayberry anthocyanin liposomes showed a slow increase trend, and its change trend within 0-12 days was opposite to the pH change trend. Within 0-5 days, the particle size of the bayberry anthocyanin liposomes increased slowly, basically at around 200nm, and then continued to increase. By the 15th day, the particle size reached 234nm, an overall increase of 47.4nm. The continuous increase in particle size is due to the sedimentation and aggregation of the bayberry anthocyanin liposomes during storage. Therefore, the bayberry anthocyanin liposomes we used for the experiment were freshly prepared each time to prevent changes in their characteristics and physiological indicators and reduce experimental errors.

[0104] (3) Infrared spectroscopy analysis of anthocyanins and myrica anthocyanin liposomes

[0105] The specific operation process of infrared spectroscopy analysis of anthocyanins and bayberry anthocyanin liposomes is as follows: Pour the prepared bayberry anthocyanin liposome liquid into a culture dish, seal it with plastic wrap, and freeze it in a -80℃ refrigerator overnight. After taking it out, poke a hole in the sealing film with a syringe needle and place it in a freeze dryer for freeze drying for 24 hours. Accurately weigh 2 mg of the dried liposome freeze-dried powder, mix it with 100 times the dry potassium bromide powder, grind it thoroughly under an infrared lamp with an agate mortar for 5 minutes, put it into a tableting mold for tableting, and then place it in a Fourier transform infrared spectrometer for detection, with a scanning range of 4000-~400cm -1 , resolution 4cm -1 .

[0106] The infrared spectroscopic analysis of anthocyanins, myrica anthocyanin liposomes, FITC and FITC-labeled myrica anthocyanin liposomes was as follows: Fig.14 shown.

[0107] like Fig.14 As shown, the characteristic peak of anthocyanin (C3G) is between 4000-3000 cm -1 The characteristic absorption at 2920.27cm is due to the stretching frequency of the OH group; -1 There is a weak CH2 asymmetric absorption peak at 1443.68 cm -1 There is a deformation absorption of CH3 at 728.61cm, and this band overlaps with the CH2 scissor vibration; -1 The existence of a peak at the point proves that four or more consecutive CH2 molecules have appeared, and swing absorption has occurred.

[0108] The characteristic peak of myrica anthocyanin liposome (C3G-LP) is at 1741.35 cm -1 There is a strong C=O stretch at 1466.72 cm -1The asymmetric deformation vibration of CH3 is shown at 1377.87cm. -1 The symmetric vibration of CH3 is shown here.

[0109] The characteristic peak of fluorescein isothiocyanate (FITC) is at 1260.97 cm -1 and 1217.40cm -1 The relative weak CO stretching is shown at , which belongs to the carboxylic acid group.

[0110] The characteristic peak of the mixture of fluorescein isothiocyanate and myrica rubra anthocyanin liposomes (FITC-C3G-LP) is at 1738.38 cm -1 There is a saturated C=O stretch at 2924.41cm -1 and 2042.14cm -1 The infrared absorption at 1467.15 cm -1 The asymmetric deformation vibration of CH3 is shown here.

[0111] After anthocyanins were prepared into bayberry anthocyanin liposomes, the characteristic peak of anthocyanins was found to be 2920.27 cm -1 Move to the low wave number direction, shift to 2928.18cm -1 ; 28.61cm -1 The characteristic peak at 721.59 cm-1 shifts to a higher wavelength. -1 After anthocyanins were encapsulated in liposomes, the FTIR spectrum of C3G-LP was similar to that of liposomes, with almost no characteristic peaks of anthocyanins, which indicated that anthocyanins had been successfully encapsulated in liposomes.

[0112] After mixing fluorescein isothiocyanate with myrica rubra anthocyanin liposomes, it was found that FITC -1 and 1217.40cm -1 The CO stretch at the liposomes disappeared, indicating that FITC had been successfully loaded into the anthocyanin liposomes. In addition, the slight change in the CH2 band indicated that anthocyanin had no effect on the bilayer interior, which strongly supported the encapsulation efficiency results.

Claims

1. A method for preparing myrica rubra anthocyanin liposomes, characterized in that: The preparation method is as follows: anthocyanin, cholesterol and soybean lecithin are dissolved in an organic solvent, and then placed in an ultrasonic crusher and stirred to obtain a mixture; after stirring the mixture, the mixture is transferred to a pear-shaped bottle, and rotary evaporated, hydrated and filtered to obtain bayberry anthocyanin liposomes.

2. The method for preparing myrica anthocyanin liposome according to claim 1, characterized in that: The mass ratio of the soybean lecithin to cholesterol is 2-6:1; the mass ratio of the soybean lecithin to anthocyanin is 10-40:

1.

3. The method for preparing myrica anthocyanin liposome according to claim 1, characterized in that: The organic solvent is anhydrous ethanol, acetone, a chloroform-methanol mixed solvent, ethyl acetate or isopropanol.

4. The method for preparing myrica anthocyanin liposome according to claim 1, characterized in that: The rotary evaporation is performed until a thin film is formed on the wall of the pear-shaped bottle; the temperature of the rotary evaporation is 20-60°C.

5. The method for preparing myrica anthocyanin liposome according to claim 1, characterized in that: The liquid used for hydration is ultrapure water, and the hydration time is 20 to 40 minutes.

6. The method for preparing myrica rubra anthocyanin liposome according to claim 1, characterized in that: The pore size of the filter membrane used in the filtration is 0.20-0.30 μm.

7. A myrica anthocyanin liposome, characterized in that: The liposome is prepared by the method for preparing myrica anthocyanin liposome according to any one of claims 1 to 6.

8. A method for optimizing the preparation conditions of myrica rubra anthocyanin liposomes by response surface methodology, characterized in that: Used to optimize the preparation conditions of the method for preparing bayberry anthocyanin liposomes described in any one of claims 1-6.

9. The method for optimizing the preparation conditions of myrica rubra anthocyanin liposomes by response surface methodology according to claim 8, characterized in that: The optimization method is as follows: the mass ratio of soybean lecithin to cholesterol, rotary evaporation temperature and the mass ratio of soybean lecithin to anthocyanin are selected as variables, the encapsulation efficiency of bayberry anthocyanin liposomes is selected as the response value, the three-factor three-level BBD method is used to design the preparation experiment, and the experimental data are collected; the fitting equation of the following form is established: <h2 style=";text-align:left;direction:ltr">Y = 82.84 - 0.42A + 0.82B + 0.61C + 0.20AB + 0.033AC - 0.083BC - 2.73A<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> -1.78B<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> -1.78C<h2 style=";text-align:left;direction:ltr"> 2 Among them, A represents the mass ratio of soybean lecithin to cholesterol, B represents the rotary evaporation temperature, C represents the mass ratio of soybean lecithin to anthocyanin, and Y represents the encapsulation efficiency of bayberry anthocyanin liposomes.

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