Mulberry leaf flavone microcapsule as well as preparation and application thereof

Abstract

The invention discloses a mulberry leaf flavone microcapsule which is prepared by taking mulberry leaf flavone extracted from mulberry leaves as a core material, taking mulberry leaf residue polysaccharide re-extracted from mulberry leaf residues left after the extraction of the mulberry leaf flavone and gelatin as a wall material and adopting a complex coacervation method. Before the complex coacervation reaction, the wall material solution needs to be subjected to electric field pulse treatment by adjusting the pH value to 3-3.5, and then subjected to electric field pulse treatment by adjusting the pH value to 6-7; after the wall material and the core material are mixed, a mixed solution needs to be cooled firstly, and then glutamine transaminase is added for a complex coacervation reaction. According to the mulberry leaf flavone microcapsule disclosed by the invention, the mulberry leaf residue polysaccharide and the gelatin are used as composite wall materials to carry out microencapsulation embedding on mulberry leaf flavone, and a flavone delivery system with a slow release function is constructed, so that not only is the reutilization of mulberry leaf residue wastes realized, but also the extracted mulberry leaf residue polysaccharide can be in synergistic interaction with the gelatin; therefore, the obtained mulberry leaf flavone microcapsule is excellent in stability, bioavailability and blood fat reducing effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of mulberry leaf flavonoid products, in particular to a mulberry leaf flavonoid microcapsule, and preparation and application of the microcapsule. BACKGROUND

[0002] As a traditional medicinal and edible plant, mulberry leaves have the medical effects of clearing heat and detoxifying, cooling blood and calming liver, moistening lung and relieving cough, and relieving blood pressure, and can play a therapeutic and conditioning role and have high safety, laying a foundation for its application in the fields of medicine and health care products. One of the main active ingredients of mulberry leaves is mulberry leaf flavonoids, which belong to natural polyphenols. Modern technology has realized the efficient separation and purification of mulberry leaf flavonoids. More than 30 flavonoid components have been separated and identified from mulberry leaves, among which rutin, quercetin, isoquercitrin, kaempferol and their glycoside derivatives are the most common. These components together constitute the core active system of mulberry leaf flavonoids.

[0003] Hyperlipidemia refers to a metabolic abnormality disease in which the total cholesterol (TC) and triglyceride (TG) levels in the blood are too high, or the high-density lipoprotein cholesterol (HDL-C, "good cholesterol") level is too low. With the development of society and the improvement of people's living standards, the phenomenon of lipid metabolism disorder caused by high-sugar and high-fat diet is becoming increasingly serious. Long-term hyperlipidemia can cause lipid deposition in blood vessel walls, leading to atherosclerosis, and thus increasing the risk of complications such as coronary heart disease, cerebral infarction, fatty liver, and diabetes, becoming one of the main risk factors for cardiovascular diseases worldwide. Although clinically commonly used lipid-lowering drugs (such as statins and fibrates) can effectively regulate blood lipids, long-term use may be accompanied by side effects such as liver dysfunction and muscle damage, and some patients have drug tolerance problems. Therefore, finding safe, efficient, and widely available natural lipid-lowering ingredients has become an important research direction for the prevention and adjuvant treatment of hyperlipidemia.

[0004] Existing research shows that mulberry leaf flavonoids can regulate lipid metabolism by inhibiting key enzymes of cholesterol synthesis (such as HMG-CoA reductase), promoting triglyceride decomposition, and increasing HDL-C levels. Animal experiments have shown that even at a higher dose, mulberry leaf flavonoids do not cause significant damage to the liver, kidneys, and other major organs of experimental animals, providing an important safety basis for their clinical application. In addition, mulberry leaves as a basic raw material can be developed into lipid-lowering functional foods (such as mulberry leaf flavonoid capsules and tea drinks) to play a role in making up for the shortcomings of chemical drugs and assisting medical lipid-lowering. However, the traditional extraction process of mulberry leaf flavonoids has problems such as low resource utilization rate and large amounts of waste being discarded. Moreover, existing mulberry leaf flavonoid foods have low bioavailability and blood lipid-lowering effect, so it is necessary to develop new mulberry leaf flavonoid lipid-lowering products to enhance the functional performance of mulberry leaf flavonoids. SUMMARY

[0005] The present application aims at the deficiencies of mulberry leaf flavonoids products and the waste of resources in production, and provides a mulberry leaf flavonoids microcapsule prepared by embedding mulberry leaf flavonoids with mulberry leaf polysaccharides extracted from mulberry leaf residues and gelatin as wall materials, and constructs a flavonoids delivery system with slow-release function, which not only improves the embedding effect of mulberry leaf flavonoids, but also significantly improves the stability, bioavailability and hypolipidemic effect of the microcapsule through in vitro simulated digestion and in vivo animal experiment verification.

[0006] To achieve the above-mentioned object, the mulberry leaf flavonoids microcapsule provided by the present application is prepared by using mulberry leaf flavonoids extracted from mulberry leaves as core materials, and using mulberry residue polysaccharides extracted from the remaining mulberry leaf residues after extracting mulberry leaf flavonoids and gelatin as wall materials, and adopting a complex coacervation method. Before the complex coacervation reaction, the wall material solution needs to be first treated by adjusting the pH to 3-3.5 and then treated by adjusting the pH to 6-7 and electric field pulse treatment; after the mixing of the wall material and the core material, the mixed solution needs to be first cooled, and then glutamine transaminase is added for complex coacervation reaction.

[0007] The present application uses mulberry residue polysaccharides extracted from the remaining mulberry leaf residues after extracting mulberry leaf flavonoids and gelatin as microcapsule wall materials, realizes efficient embedding of mulberry leaf flavonoids, and based on the increase of galacturonic acid content in mulberry residue polysaccharides compared with conventional mulberry polysaccharides and the synergistic effect produced with gelatin, the wall material significantly enhances the protection of the biological activity of mulberry leaf flavonoids, thereby improving the stability of the mulberry leaf flavonoids microcapsule. The in vitro simulated digestion experiment verifies that the microcapsule also realizes the slow-release effect of mulberry leaf flavonoids, thereby improving the bioavailability of flavonoids. Through in vivo hyperlipidemia animal experiments, the hypolipidemic effect of the mulberry leaf flavonoids microcapsule is determined, which provides a basis for the development of natural, safe and efficient hypolipidemic products.

[0008] To ensure that the wall material and the core material complete the embedding by complex coacervation reaction, the wall material solution is treated by adjusting the pH twice in succession and electric field pulse treatment, so as to improve the cross-linking quality of gelatin and mulberry residue polysaccharides.

[0009] As a limitation of the above technical solution, the amount ratio of the core material and the wall material used for preparing the mulberry leaf flavonoids microcapsule is 1:2-3.5 v / v, and / or the amount ratio of gelatin and mulberry residue polysaccharides in the wall material is 1:1-1.5 m / m.

[0010] The amount ratio of the core material and the wall material used for the microcapsule and the amount ratio of gelatin and mulberry residue polysaccharides in the wall material are limited, so as to optimize the performance of the wall material and the microcapsule.

[0011] As a limitation of the above technical solution, ethanol solution is used to extract mulberry leaf flavonoids from mulberry leaves; as a preferred embodiment, microwave-assisted extraction is used; further preferably, the mulberry leaves are immersed in an ethanol solution with a concentration of 60-80% v / v at a solid-liquid ratio of 1:45-50 m / v, treated in a microwave at 590-610 w and a temperature of 55-60℃ for 4-6 min, and then incubated at 55-60℃ for 20-30 min, and the obtained filtrate is the mulberry leaf flavonoid extract.

[0012] As a limitation of the above technical solution, water is used to extract mulberry residue polysaccharides from mulberry residue, and as a preferred embodiment, ultrasonic-assisted extraction is used; further preferably, the mulberry residue is dried in a hot air fluidized bed at 60-65℃ for 30-40 min, then immersed in purified water at a solid-liquid ratio of 1:20-25 m / v, treated in an ultrasonic bath at 350-400 w and a temperature of 55-60℃ for 40-60 min, and the obtained filtrate is freeze-dried to obtain the mulberry residue polysaccharides.

[0013] Optimizing the extraction conditions of mulberry leaf flavonoids and mulberry residue polysaccharides can improve the performance of the core material and the wall material and improve the quality of the microcapsules.

[0014] As a limitation of the above technical solution, before extracting mulberry leaf flavonoids, the mulberry leaves are subjected to a pretreatment of freezing, high-pressure nitrogen circulation, and high-pressure steam circulation; as a preferred embodiment, the freezing temperature is -30℃ to -20℃, and the freezing time is 4-5 h, and / or the high-pressure nitrogen circulation treatment is to place the mulberry leaves in a nitrogen circulation environment at a pressure of 100-150 MPa for 5-10 min; and / or the high-pressure steam circulation treatment is to place the mulberry leaves in a steam circulation environment at a pressure of 100-150 MPa and a temperature of 55-60℃ for 5-10 min.

[0015] Before extracting mulberry leaf flavonoids, the mulberry leaves are subjected to a pretreatment of freezing, high-pressure nitrogen circulation, and high-pressure steam circulation, which can significantly increase the content of galacturonic acid in the polysaccharides, improve the performance of the wall material, and further optimize the quality of the mulberry leaf flavonoid microcapsules.

[0016] As a limitation of the above technical solution, gelatin and mulberry residue polysaccharides are mixed at a ratio of 1:1-1.5 m / m, and then a gelatin-mulberry polysaccharide solution with a mass concentration of 1-1.5% is prepared as the wall material solution; the pH of the wall material solution is adjusted to 3-3.5, and the solution is placed in an electric field with an intensity of 25 kV / cm and a pulse electric field frequency of 10-12 Hz for 400-500 μs; then the pH of the wall material solution is adjusted to 6-7, and the solution is placed in an electric field with an intensity of 25 kV / cm and a pulse electric field frequency of 10-12 Hz for 400-500 μs to obtain the microcapsule wall material; as a preferred embodiment, 0.5 M hydrochloric acid is used to adjust the pH of the wall material solution.

[0017] As a limitation of the above technical solution, the mulberry leaf flavone extract is taken as the core material, mixed with the wall material in a core material to wall material ratio of 1:2-3.5 v / v, homogenized at 10,000-12,000 rpm / min and 44-48°C, and then cooled to 4°C. Glutamine transaminase is added and incubated for 2 hours. The mulberry leaf flavone microcapsules are obtained by vacuum freeze-drying. Preferably, the amount of glutamine transaminase added is 40-50 mg / L.

[0018] The preparation and treatment of the wall material solution, the core to wall ratio, and the complex coagulation reaction conditions are limited to perfect and optimize the preparation of the microcapsules.

[0019] As a limitation of the above technical solution, the 2-hour retention rate of the mulberry leaf flavone microcapsules in the in vitro simulated gastric juice digestion experiment is more than 80%, and the bioavailability is increased by more than 30% compared with the flavone solution.

[0020] The preparation method of the mulberry leaf flavone microcapsules is provided, which comprises the following preparation steps: a. Extracting mulberry leaf flavone: The mulberry leaves are immersed in an ethanol solution with a concentration of 60-80% v / v at a material to liquid ratio of 1:45-50 m / v. The mixture is treated under microwave at 590-610 W and a temperature of 55-60°C for 4-6 min, and then incubated at 55-60°C for 20-30 min. The filtrate obtained by filtration is the mulberry leaf flavone extract, and the filter residue is the mulberry leaf residue. b. Extracting mulberry leaf polysaccharide: The mulberry leaf residue is dried in a hot air fluidized bed at 60-65°C for 30-40 min, and then immersed in purified water at a material to liquid ratio of 1:20-25 m / v. The mixture is treated under ultrasonic power of 350-400 W and a temperature of 55-60°C for 40-60 min. The filtrate obtained by filtration is subjected to freeze-drying to obtain the mulberry leaf residue polysaccharide. c. Preparing mulberry leaf flavone microcapsules: The gelatin and the mulberry leaf residue polysaccharide obtained in step b are mixed uniformly at a ratio of 1:1-1.5 v / v. A gelatin-mulberry leaf polysaccharide solution with a mass fraction of 1-1.5% is prepared as the wall material solution. The pH of the wall material solution is adjusted to 3-3.5 using 0.5 M hydrochloric acid. The wall material solution is treated in an electric field with an intensity of 25 kV / cm and a pulse electric field frequency of 10-12 Hz for 400-500 μs. The pH of the wall material solution is adjusted to 6-7, and the wall material solution is treated in an electric field with an intensity of 25 kV / cm and a pulse electric field frequency of 10-12 Hz for 400-500 μs to obtain the microcapsule wall material. The mulberry leaf flavone extract is taken as the core material, mixed with the wall material at a core to wall ratio of 1:2-3.5 v / v, homogenized at 10,000-12,000 rpm / min and 44-48°C, and then cooled to 4°C in an ice water bath. 40-50 mg / L of glutamine transaminase is added and incubated for 2 hours. The mulberry leaf flavone microcapsules are obtained by vacuum freeze-drying. As preferred, a pretreatment step s is added before step a, and the concentration of the ethanol solution used in step a is optimized, as follows: s, pretreatment: taking the unground mulberry leaves, first slowly freezing at -30℃~-20℃ for 4~5h, then nitrogen circulation treatment at 100~150MPa for 5~10min, and finally steam circulation treatment at 100~150MPa for 5~10min at 55~60℃; Step a uses 60~67% v / v ethanol solution to extract mulberry flavonoids.

[0021] From the industrial application level, the preparation method of mulberry flavonoids is improved, each step operation is stable and convenient, and through the pretreatment of mulberry leaves, the extraction rate of mulberry flavonoids can be improved, and the monosaccharide composition of mulberry residue polysaccharide can be improved and optimized, especially the content of galacturonic acid is significantly increased, and the mulberry leaves can be treated without grinding, and the concentration of the ethanol solution used for extraction is reduced, which is more beneficial to industrial production and safety.

[0022] In addition, the application also provides the application of the mulberry flavonoid microcapsule as described above, which is used for developing an auxiliary drug for hyperlipidemia and a lipid-lowering functional food.

[0023] Through the hyperlipidemia mouse model experiment, it is verified that the mulberry flavonoid microcapsule can effectively regulate the biochemical level of blood lipids, and compared with the same dose of mulberry flavonoids, it has a significant intervention effect, which provides a theoretical basis for developing a natural, safe and efficient auxiliary drug for hyperlipidemia and a lipid-lowering functional food.

[0024] In summary, the mulberry flavonoids extracted from mulberry leaves are used as core materials, and the mulberry residue polysaccharide extracted from the mulberry residue after extracting the mulberry flavonoids and gelatin are compounded as wall materials, and a flavonoid delivery system, i.e. mulberry flavonoid microcapsule with slow-release function is successfully constructed, which not only realizes efficient embedding of mulberry flavonoids, enhances the protection of the biological activity of mulberry flavonoids, makes the obtained mulberry flavonoid microcapsule significantly improved in stability, biological availability and lipid-lowering effect, but also effectively utilizes the waste mulberry residue after extracting flavonoids, and avoids the waste of mulberry resources. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a graph of the embedding rate of flavonoids of different types of protein-mulberry residue polysaccharide combinations.

[0026] Figure 2 is a graph of the zeta potential results of gelatin and mulberry residue polysaccharide.

[0027] Figure 3 is a graph of the influence of the dosage ratio of gelatin and mulberry residue polysaccharide in the wall material and the pH value on the turbidity of the reaction system.

[0028] Figure 4 Figure 1 is a graph of the embedding rate of each mulberry leaf flavone microcapsule of the examples and the comparative examples.

[0029] Figure 5 Figure 2 is a graph of the particle size and its distribution of the mulberry leaf flavone microcapsule of Example 1.

[0030] Figure 6 Figure 3 is a scanning electron microscope graph of the mulberry leaf flavone microcapsule of Example 1 at 500x and 2000x; a) 500x, b) 2000x.

[0031] Figure 7 Figure 4 is a Fourier infrared absorption spectrum graph of gelatin, mulberry leaf residue polysaccharide, microcapsule wall material, mulberry leaf flavone, and mulberry leaf flavone microcapsule; in the graph, a represents gelatin, b represents mulberry leaf residue polysaccharide, c represents microcapsule wall material (gelatin-mulberry leaf residue polysaccharide), d represents mulberry leaf flavone, and e represents mulberry leaf flavone microcapsule.

[0032] Figure 8 Figure 5 is an X-ray diffraction analysis spectrum graph of gelatin, mulberry leaf residue polysaccharide, microcapsule wall material, mulberry leaf flavone, and mulberry leaf flavone microcapsule; in the graph, a represents gelatin, b represents mulberry leaf residue polysaccharide, c represents microcapsule wall material (gelatin-mulberry leaf residue polysaccharide), d represents mulberry leaf flavone, and e represents mulberry leaf flavone microcapsule.

[0033] Figure 9 Figure 6 is an in-vitro simulated gastrointestinal fluid digestion release curve of the mulberry leaf flavone and mulberry leaf flavone microcapsule prepared in Example 1; in the graph, SGF represents the stomach digestion stage, SIF represents the intestinal digestion stage, MF represents mulberry leaf flavone, and G-MP / MF represents mulberry leaf flavone microcapsule.

[0034] Figure 10 Figure 7 is the regulation effect of the mulberry leaf flavone and mulberry leaf flavone microcapsule prepared in Example 1 on the blood lipid level of high-fat diet-induced mice; in the graph, a represents the total cholesterol level of mice, b represents the triglyceride level of mice, c represents the high-density lipoprotein cholesterol level of mice, d represents the low-density lipoprotein cholesterol level of mice, NC represents the blank control group, MC represents the model group, PC represents the positive control group, MF represents the mulberry leaf flavone group, FL represents the microcapsule low-dose group, FM represents the microcapsule medium-dose group, and FH represents the microcapsule high-dose group. DETAILED DESCRIPTION

[0035] The technical solutions of the present application will be described clearly and completely below in combination with examples. Obviously, the described examples are only some of the examples of the present application, but not all the examples. Based on the examples in the present application, all the other examples obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0036] The experimental methods in the following examples and comparative examples are conventional methods unless otherwise specified; the raw materials or test materials used are typical products purchased on the market unless otherwise specified. The quantitative tests in the following examples and comparative examples are set up in triplicate, and the results are averaged.

[0037] Example

[0038] The preparation of mulberry leaf flavone microcapsules from the extraction of flavones from mulberry leaves and polysaccharides from mulberry leaf residues includes the following steps.

[0039] a. Extracting mulberry leaf flavones: The mulberry leaves treated in step a are immersed in an ethanol solution with a concentration of 60-80% v / v (i.e., the use amount ratio of mulberry leaves to ethanol solution is 1 kg: 45-50 L) according to a solid-liquid ratio of 1:45-50 m / v, treated at a microwave power of 590-610 w and a temperature of 55-60°C for 4-6 min, and then incubated at 55-60°C for 20-30 min. The filtrate obtained by filtration (which can be filtered with a 300-mesh sieve) is the mulberry leaf flavone extract, and the filter residue is the mulberry leaf residue.

[0040] b. Extracting mulberry leaf polysaccharides: The mulberry leaf residue is dried in a hot air fluidized bed at 60-65°C for 30-40 min, and then immersed in purified water according to a solid-liquid ratio of 1:20-25 m / v (i.e., the use amount ratio of mulberry leaf residue to water is 1 kg: 20-25 L), treated at an ultrasonic power of 350-400 w and a temperature of 55-60°C for 40-60 min, and then filtered (which can be centrifugal filtration) to obtain the filtrate. After freeze-drying (laboratory freeze-drying conditions can be freeze-drying at a cold trap temperature of -50°C and a vacuum degree of 200 Pa for 16 h, or using common industrial freeze-drying conditions), the mulberry leaf residue polysaccharides are obtained.

[0041] c. Preparing mulberry leaf flavone microcapsules: Take the gelatin and the mulberry leaf residue polysaccharides prepared in step c, mix them according to a mass ratio of 1:1-1.5 m / m, and then prepare a gelatin-mulberry leaf polysaccharide solution with a mass fraction of 1-1.5% as the wall material solution. Adjust the pH of the wall material solution to 3-3.5 using 0.5 M hydrochloric acid, place it in an electric field with an intensity of 25 kV / cm and a pulse electric field frequency of 10-12 Hz for 400-500 μs, adjust the pH of the wall material solution to 6-7, and then place it in an electric field with an intensity of 25 kV / cm and a pulse electric field frequency of 10-12 Hz for 400-500 μs to obtain the microcapsule wall material. Take the mulberry leaf flavone extract as the core material, mix it with the wall material according to a core-wall ratio of 1:2-3.5 v / v, homogenize the mixture at 10000-12000 rpm / min and 44-48°C for 5 min to obtain a mixed solution, cool it to 4°C in an ice water bath, add 40-50 mg / L transglutaminase (TG) enzyme, and incubate for 2 h. After vacuum freeze-drying, the mulberry leaf flavone microcapsule sample (G-MP / MF) is obtained.

[0042] In the process of establishing the preparation of mulberry leaf flavonoids microcapsules, the screening of different wall materials, Zeta potential, and wall material ratio was tested.

[0043] With flavonoid embedding rate as the index, the spectrophotometric method was used to screen different wall materials constructed by mulberry residue polysaccharide and different proteins including soybean protein, gelatin, casein, whey protein, and zein. The experimental results are shown in Figure 1 The flavonoid embedding rates of different types of protein-mulberry residue polysaccharide (abbreviated as residue polysaccharide in the figure) combinations showed significant differences, and the flavonoid embedding rate of gelatin-mulberry residue polysaccharide combination was the highest.

[0044] Gelatin-mulberry residue polysaccharide as a wall material showed better performance than other proteins in embedding flavonoids. The gelatin molecular chain contains a large number of polar groups such as amino and carboxyl groups, has good molecular flexibility and surface activity, so it can better form a stable and tight microcapsule structure with mulberry residue polysaccharide through hydrogen bonds, electrostatic interactions, and other ways, thereby improving the embedding rate and embedding stability of flavonoids.

[0045] By studying the Zeta potential at different pH values, the interaction between gelatin and mulberry residue polysaccharide was further analyzed. The zeta potential results of gelatin and mulberry residue polysaccharide are shown in Figure 2 With the increase of pH from 2 to 9, the zeta potential of gelatin decreased from +1.73 mV to -6.73 mV, and when the pH was 4.2, the Zeta potential was 0, indicating that the surface charge characteristics of gelatin were regulated by the pH of the solution. This is because there are both amino groups and carboxyl groups in the gelatin molecule, and their interaction is affected by the pH of the solution. The zeta potential of mulberry residue polysaccharide was negative in the entire pH range measured, and the absolute value of its zeta potential showed a gradually increasing trend with the increase of pH, which may be related to the content of uronic acid it contains. The change in zeta potential of gelatin with pH allows it to complex and coagulate with mulberry residue polysaccharide with opposite charges under certain pH conditions.

[0046] Figure 3 is the effect of the ratio of gelatin to mulberry residue polysaccharide in the wall material and pH on the turbidity of the reaction system. During the complex coagulation reaction process, the turbidity of the system increases with the generation of coagulants, and pH is a key factor affecting the occurrence of complex coagulation reaction.

[0047] From Figure 3As can be seen from the figure, when the wall material ratio is 1:1 m / m (i.e. the mass ratio of gelatin to mulberry leaf residue polysaccharide is 1:1), the absorbance values are all higher than those of other ratios. The effect of pH on turbidity shows a trend of first increasing and then decreasing. When the pH increases from 2.0 to 3.0, the turbidity increases sharply to a peak and then decreases, indicating that the electrostatic interaction between proteins and anionic polysaccharides is strongest at pH 3. This pH is selected as the optimal pH for screening wall materials. Therefore, the preferred ratio of gelatin and residue polysaccharide wall materials is 1:1~1.5 m / m, and the pH is 3~3.5.

[0048] In the above preparation process, taking the extraction rate of mulberry leaf flavonoids as an index, single factor experiments and response surface experiments were carried out on ethanol concentration, solid-liquid ratio, ultrasonic time, ultrasonic temperature, etc. to determine the extraction conditions of mulberry leaf flavonoids; taking the extraction rate of mulberry leaf residue polysaccharide and polysaccharide composition analysis as an index, single factor experiments and response surface experiments were carried out on solid-liquid ratio, ultrasonic time, ultrasonic temperature, etc. to determine the extraction conditions of mulberry leaf residue polysaccharide; taking the embedding rate of mulberry leaf flavonoids as an index, single factor experiments were carried out on core-wall ratio, reaction time, reaction temperature, and TG enzyme addition amount to determine the preparation conditions.

[0049] Example 1

[0050] Without pretreatment of mulberry leaves, mulberry leaf flavonoids were directly extracted from mulberry leaves, and mulberry leaf residue polysaccharides were extracted from mulberry leaf residue to prepare mulberry leaf flavonoid microcapsules. The specific process conditions are as follows.

[0051] a. Extract with 70% v / v ethanol solution at a solid-liquid ratio of 1:48 m / v, treat under microwave 595w, temperature 55℃ for 6min, then 60℃ for 20min, filter with 300 mesh, the obtained filtrate is mulberry leaf flavonoid extract, and the obtained filter residue is mulberry leaf residue; b. Dry the mulberry leaf residue at 60℃ hot air for 30min, take purified water, treat at a solid-liquid ratio of 1:20 m / v, ultrasonic power 350w, temperature 60℃ for 50min, filter and freeze-dry (the freeze-drying conditions of laboratory operation can be cold trap temperature-50℃, vacuum degree 200Pa, freeze-drying for 16h, and industrial application is operated according to conventional industrial conditions) to obtain mulberry leaf residue polysaccharide.

[0052] The monosaccharide composition of mulberry leaf polysaccharide is determined by high performance liquid chromatography. The chromatographic column is DionexCarbopacTM PA20 (3*150 mm); the mobile phase A is H2O; the mobile phase B is 15 mM NaOH solution; the mobile phase C is 15 mM NaOH & 100 mM NaAc solution; the flow rate is set to 0.3 mL / min; the column temperature is maintained at 30 DEG C; and an electrochemical detector is used. It is determined that the content of galacturonic acid in the mulberry leaf residue polysaccharide is 27.58%, and the content of impurity protein is 11.23%. The content of galacturonic acid in the mulberry leaf residue polysaccharide extracted from the mulberry leaf residue after extracting mulberry leaf flavones is 25-30%, and the content of impurity protein is 10-13%.

[0053] c. The gelatin and the mulberry leaf polysaccharide are prepared at a mass ratio of 1:1.5 m / m (mass ratio) to prepare a gelatin-mulberry leaf polysaccharide solution with a mass fraction of 1.5% (dissolved in water) and adjusted to pH 3.0, and then subjected to a pulsed electric field with an electric field intensity of 25 kV / cm and a frequency of 11 Hz for 500 μs, and then adjusted to pH 7.0, an electric field intensity of 25 kV / cm, and a frequency of 10 Hz for 400 μs to obtain the microcapsule wall material; the mulberry leaf flavone extract is taken as the core material, mixed at a core-wall ratio of 1:3 v / v (volume ratio), homogenized at 10,000 rpm / min and 44 DEG C for 5 min, and then placed in an ice water bath to cool to 4 DEG C, and then 40 mg / L transglutaminase (TG) is added, and incubated for 2 h, and then subjected to vacuum freeze-drying to obtain the mulberry leaf flavone microcapsule sample.

[0054] Example 2 The mulberry leaf is pretreated, the mulberry leaf flavones are extracted from the mulberry leaf, and the mulberry leaf polysaccharide is extracted from the mulberry leaf residue to prepare the mulberry leaf flavone microcapsule, that is, a pretreatment step is added on the basis of the preparation process in the example. s. The unground mulberry leaf is slowly frozen at -30 DEG C to -20 DEG C for 4-5 h, subjected to nitrogen circulation treatment at a pressure of 100-150 MPa for 5-10 min, and then subjected to steam circulation treatment at a pressure of 100-150 MPa and a temperature of 55-60 DEG C for 5-10 min; Meanwhile, the concentration of the ethanol solution used in step a for extracting the mulberry leaf flavones is reduced to 60-67% v / v.

[0055] Example 2-1 The mulberry leaf is pretreated, the mulberry leaf flavones are extracted from the mulberry leaf, and the mulberry leaf polysaccharide is extracted from the mulberry leaf residue to prepare the mulberry leaf flavone microcapsule, that is, a pretreatment step is added on the basis of the preparation process in the example.

[0056] s. The unground mulberry leaf is slowly frozen at -30 DEG C to -20 DEG C for 4-5 h, subjected to nitrogen circulation treatment at a pressure of 100-150 MPa for 5-10 min, and then subjected to steam circulation treatment at a pressure of 100-150 MPa and a temperature of 55-60 DEG C for 5-10 min;

[0057] a. The filtrate obtained by the above steps is the mulberry leaf flavonoid extract, and the residue is the mulberry leaf residue.

[0058] b. The mulberry leaf residue is dried in a hot air fluidized bed at 60°C for 30 min, and then extracted with purified water at a material-liquid ratio of 1:20 m / v. The mixture is treated under ultrasonic power of 350 W and at a temperature of 60°C for 50 min. The filtrate obtained by filtration is freeze-dried to obtain the mulberry leaf residue polysaccharide. The galacturonic acid content of the mulberry leaf residue polysaccharide obtained after the pretreatment is 42.53%, and the impurity protein content is 4.27%. The galacturonic acid content of the mulberry leaf residue polysaccharide obtained after the pretreatment is 40-45%, and the impurity protein content is ≤5%.

[0059] c. The gelatin and the mulberry leaf residue polysaccharide are mixed at a ratio of 1:1.2 m / m to prepare a gelatin-mulberry polysaccharide solution with a mass fraction of 1.3%. The pH is adjusted to 3.0, and the mixture is treated under an electric field intensity of 25 kV / cm and a pulse electric field frequency of 12 Hz for 400 μs. The pH is adjusted to 6.5, and the mixture is treated under an electric field intensity of 25 kV / cm and a pulse electric field frequency of 10 Hz for 400 μs to obtain the microcapsule wall material. The flavonoid extract is used as the core material, and the core-wall ratio is 1:3 v / v. The mixture is homogenized at 10,000 rpm / min and at a temperature of 44°C for 5 min. The mixture solution is cooled to 4°C in an ice water bath, and then 40 mg / L glutamine transaminase is added and incubated for 2 h. The mixture is then subjected to vacuum freeze-drying to obtain the mulberry leaf flavonoid microcapsule.

[0060] Example 2-2 The mulberry leaf flavonoid microcapsule is prepared after the pretreatment of the mulberry leaf, and the preparation process conditions are as follows.

[0061] Steps s, a, and b are the same as those in Example 2-1. c. The gelatin and the mulberry leaf residue polysaccharide are mixed at a ratio of 1:1 m / m to prepare a gelatin-mulberry polysaccharide solution with a mass fraction of 1%. The pH is adjusted to 3.0, and the mixture is treated under an electric field intensity of 25 kV / cm and a pulse electric field frequency of 10 Hz for 450 μs. The pH is adjusted to 6.0, and the mixture is treated under an electric field intensity of 25 kV / cm and a pulse electric field frequency of 10 Hz for 450 μs to obtain the microcapsule wall material. The flavonoid extract is used as the core material, and the core-wall ratio is 1:3 v / v. The mixture is homogenized at 10,000 rpm / min and at a temperature of 44°C for 5 min. The mixture solution is cooled to 4°C in an ice water bath, and then 40 mg / L glutamine transaminase is added and incubated for 2 h. The mixture is then subjected to vacuum freeze-drying to obtain the mulberry leaf flavonoid microcapsule.

[0062] Example 2-3 The mulberry leaf flavonoid microcapsule is prepared after the pretreatment of the mulberry leaf, and the preparation process conditions are as follows.

[0063] The operations of steps s, a, and b are the same as in Example 2-1. Gelatin and mulberry residue polysaccharide were mixed at a ratio of 1:1.5 m / m to prepare a 1.5% gelatin-mulberry polysaccharide solution, which was adjusted to pH 3.5, and then subjected to electric field intensity of 25 kV / cm, pulse electric field frequency of 11 Hz, and treatment for 500 μs, and then adjusted to pH 7.0, electric field intensity of 25 kV / cm, pulse electric field frequency of 10 Hz, and treatment for 400 μs to obtain the microcapsule wall material. The flavone extract was used as the core material, and mixed with the wall material at a core / wall ratio of 1:2.5 v / v, homogenized at 10,000 rpm / min and 44°C for 5 min, and then the mixture solution was cooled to 4°C in an ice water bath, 45 mg / L transglutaminase was added, and the mixture was incubated for 2 h, and then subjected to vacuum freeze-drying to obtain the mulberry flavone microcapsules.

[0064] The following comparative examples are used to compare with the mulberry flavone microcapsules of the present application (i.e. examples).

[0065] Comparative Example 1 Compared with Example 1, the difference of the present comparative example is that the electric field pulse treatment is not performed in the preparation of the mulberry flavone microcapsules, and the preparation process conditions are as follows.

[0066] The extraction of mulberry flavone and mulberry residue polysaccharide is the same as in Example 1. Gelatin and mulberry residue polysaccharide were mixed at a ratio of 1:1.5 m / m to prepare a 1.5% gelatin-mulberry polysaccharide solution, which was adjusted to pH 3.5, and then subjected to electric field intensity of 25 kV / cm, pulse electric field frequency of 11 Hz, and treatment for 500 μs, and then adjusted to pH 7.0, electric field intensity of 25 kV / cm, pulse electric field frequency of 10 Hz, and treatment for 400 μs to obtain the microcapsule wall material. The flavone extract was used as the core material, and mixed with the wall material at a core / wall ratio of 1:2.5 v / v, homogenized at 10,000 rpm / min and 44°C for 5 min, and then the mixture solution was cooled to 4°C in an ice water bath, 45 mg / L transglutaminase was added, and the mixture was incubated for 2 h, and then subjected to vacuum freeze-drying to obtain the mulberry flavone microcapsules.

[0067] Comparative Example 2 The present comparative example uses the conventional method to directly extract mulberry polysaccharide from mulberry leaves and uses gelatin as the wall material, and directly extracts mulberry flavone from mulberry leaves as the core material to prepare mulberry flavone microcapsules.

[0068] Comparative Example 2-1 The mulberry flavone microcapsules are prepared without electric field pulse treatment, and the specific preparation process conditions are as follows.

[0069] The extraction of mulberry flavone is the same as in Example 1.

[0070] b, 10 kg of mulberry leaves were taken, purified water was taken, and the material-liquid ratio was 1:20 m / v, the ultrasonic power was 350 w, the temperature was 60°C, and the treatment time was 50 min. The filtrate was filtered and freeze-dried (the freeze-drying conditions were the same as in the example) to obtain mulberry leaf polysaccharides. The galacturonic acid content in the polysaccharides was 8.7%, and the impurity protein content was 15.96%. The galacturonic acid content in the mulberry leaf polysaccharides extracted by the conventional method was usually 5-10%, and the impurity protein content was usually 12-18%.

[0071] c, the gelatin and the mulberry leaf polysaccharides were mixed at a ratio of 1:1.2 m / m to prepare a gelatin-mulberry leaf polysaccharide solution with a mass fraction of 1.3%, which was used as the microcapsule wall material; the mulberry leaf flavone extract was used as the core material, and the core-wall ratio was 1:3 v / v. After homogenization at 10,000 rpm / min and 44°C for 5 min, the mixed solution was cooled to 4°C in an ice water bath, 40 mg / L of glutamine transaminase was added, and the solution was incubated for 2 h. Finally, the mulberry leaf flavone microcapsules were obtained by vacuum freeze-drying.

[0072] Comparative Example 2-2 The mulberry leaf flavone microcapsules were prepared under the condition of electric field pulse treatment, and the specific preparation process conditions were as follows.

[0073] The same mulberry leaf flavone and mulberry leaf polysaccharides as in Comparative Example 2-1 were taken (i.e., the operations and conditions of steps a and b were the same as in Comparative Example 2-1).

[0074] The gelatin and the mulberry leaf polysaccharides were mixed at a ratio of 1:1.2 m / m to prepare a gelatin-mulberry leaf polysaccharide solution with a mass fraction of 1.3% and a pH of 3.5. The solution was treated under the condition of an electric field intensity of 25 kV / cm and a pulse electric field frequency of 10 Hz for 500 μs, and then the pH of the gelatin-mulberry leaf polysaccharide solution was adjusted to 6.8. The solution was treated under the condition of an electric field intensity of 25 kV / cm and a pulse electric field frequency of 10 Hz for 500 μs to obtain the microcapsule wall material. The mulberry leaf flavone extract was used as the core material, and the core-wall ratio was 1:3 v / v. After homogenization at 10,000 rpm / min and 44°C for 5 min, the mixed solution was cooled to 4°C in an ice water bath, 40 mg / L of glutamine transaminase was added, and the solution was incubated for 2 h. Finally, the mulberry leaf flavone microcapsules were obtained by vacuum freeze-drying.

[0075] Comparative Example 2-3 The mulberry leaf flavone microcapsules were prepared by adding CaCl2 without electric field pulse treatment, and the specific preparation process conditions were as follows.

[0076] The same mulberry leaf flavone and mulberry leaf polysaccharides as in Comparative Example 2-1 were taken (i.e., the operations and conditions of steps a and b were the same as in Comparative Example 2-1).

[0077] Gelatin and mulberry leaf polysaccharide are prepared in a proportion of 1:1.2 m / m to prepare a gelatin-mulberry leaf polysaccharide solution with a mass fraction of 1.3%, and then CaCl2 with a mass fraction of 1.2% of the solution is added to obtain a microcapsule wall material; the mulberry leaf flavone extract is taken as the core material, mixed with the wall material in a core / wall ratio of 1:3 v / v, homogenized at 10000 rpm / min and 44°C for 5 min, and then the mixed solution is cooled to 4°C in an ice water bath, 40 mg / L of glutamine transaminase is added, and incubated for 2 h, and then vacuum freeze-dried to obtain the mulberry leaf flavone microcapsule.

[0078] The monosaccharide composition of the mulberry leaf residue polysaccharide prepared in Examples 1 and 2-1 and the mulberry leaf polysaccharide of Comparative Example 2-1 is analyzed (determination method: high performance liquid chromatography), and the results are shown in Table 1.

[0079]

[0080] As shown in the above table, the mulberry leaf residue polysaccharide of Examples 1 and 2-1 and the mulberry leaf polysaccharide of Comparative Example 2-1 have a large difference in galacturonic acid content.

[0081] The embedding rate of the mulberry leaf flavone microcapsule of the examples and the comparative examples is determined, and the results are shown in Table 2. Figure 4 The mulberry leaf residue polysaccharide can significantly improve the embedding rate when it is used as the wall material together with gelatin.

[0082] The mulberry leaf flavone microcapsule obtained by the present application (specifically, the mulberry leaf flavone microcapsule prepared in Example 1) is subjected to physicochemical property and structure characterization analysis.

[0083] (1) Particle size analysis The particle size and its distribution of the microcapsule are one of the important indicators for measuring the quality of the microcapsule. Figure 5 As shown in Table 3, the average particle size of the mulberry leaf flavone microcapsule prepared by the present application is 482.8 nm, the particle size distribution is uniform and the range is relatively concentrated, showing a normal distribution, and it belongs to a nano microcapsule. The particle size dispersion index (PDI) of the microcapsule is 0.461. PDI is a dimensionless statistical parameter used to characterize the dispersion degree of the particle size distribution, and the range is 0~1. The smaller the value, the higher the uniformity and the stronger the concentration of the particle size distribution.

[0084] (2) Scanning electron microscope morphology analysis Figure 6 The scanning electron microscope results of the mulberry leaf flavone microcapsule at 500 times and 2000 times, respectively, are shown in Figures 1 and 2. As shown in Figure 1, the microcapsule has an irregular broken block shape and a relatively loose structure under 500 times. When the magnification is increased to 2000 times, the microcapsule has a good appearance, a regular spherical shape, and is complete and continuous, which indicates that the embedding effect is good.

[0085] (III) Fourier Transform Infrared Spectroscopy Measurement and Analysis Fourier transform infrared spectroscopy can be used to determine the molecular structure of a substance and identify its characteristic chemical bonds. Figure 7 Fourier transform infrared (FTIR) spectra of gelatin (a), mulberry leaf residue polysaccharide (b), microcapsule wall material (c), mulberry leaf flavonoids (d), and mulberry leaf flavonoid microcapsules (e). Gelatin (a) is at 3420.1 cm⁻¹. -1 1667.8 cm -1 The peak at point b shows a characteristic peak, representing OH / NH stretching and (C=O) stretching; mulberry leaf residue polysaccharide (i.e., the mulberry leaf residue polysaccharide of Example 1) is located at 3418.7 cm⁻¹. -1 2928.2 cm -1 1637.4 cm -1 The nearby absorption peaks are OH, CH, and COO, respectively. - Stretching vibration, 1154.5 cm -1 The nearby absorption peak is due to CH stretching and angle-shifting vibrations, at 764.3 cm⁻¹. -1 The symmetric stretching vibration of the D-glucopyranose ring is shown. c is the infrared spectrum of the microcapsule wall material prepared by mixing gelatin and mulberry leaf residue polysaccharide (i.e., the microcapsule wall material of Example 1), with the gelatin OH / NH stretched at 3420.1 cm⁻¹. -1 The redshift reached 3432.5 cm. -1 At this location, the polysaccharide content of mulberry leaf residue was 1637.4 cm. -1 Represents carboxylic acid COO - The vibration peak shifted to 1654.7 cm⁻¹. -1 The presence of this feature indicates the electrostatic interaction between gelatin and mulberry leaf residue polysaccharides.

[0086] (iv) X-ray diffraction analysis X-ray diffraction analysis is a method for studying the structural information and molecular properties of crystalline materials. Figure 8XRD analysis spectrum of a gelatin, b mulberry leaf residue polysaccharide, c microcapsule wall material, d mulberry leaf flavone and e mulberry leaf flavone microcapsule, respectively. Among them, a gelatin has sharp diffraction peaks, indicating that it has a crystal structure; b mulberry leaf residue polysaccharide (i.e. mulberry leaf residue polysaccharide of example 1) has a wide peak-shaped diffraction spectrum characteristic near 20°, indicating that it has an amorphous structure characteristic. The microcapsule wall material prepared by mixing gelatin and mulberry leaf polysaccharide (i.e. the microcapsule wall material of example 1) has diffraction peaks near 22.94°, 28.18° and 30.9°, indicating that after the complex coagulation reaction of gelatin and mulberry leaf residue polysaccharide, the crystalline properties and ability are affected by the electrostatic interaction. This shows that the protein molecules and polysaccharide molecular chains are closely adsorbed, which promotes the interaction between them, and then forms a protein-polysaccharide amorphous compound. d mulberry leaf flavone (i.e. mulberry leaf flavone of example 1) has diffraction peaks near 23.18° and 31.3°, indicating that the mulberry leaf flavone exists in the form of crystal. e mulberry leaf flavone microcapsule (i.e. mulberry leaf flavone of example 1) has basically the same diffraction peak position as the mulberry leaf flavone, only the peak intensity has slight changes, indicating that after the mulberry leaf flavone is embedded in the microcapsule complex, the crystal strength of the system increases, thereby proving that the mulberry leaf flavone is successfully embedded.

[0087] (V), in vitro simulation of gastrointestinal fluid digestion analysis In vitro digestion experiment is to simulate the environment of human digestive tract by using enzymes and chemicals, to verify whether the microcapsule can protect flavones from damage by gastric acid, gastric juice digestive enzymes or oxidative environment, effectively control the release rate of core material, and realize targeted release, release and utilization in the process of intestinal juice digestion, and improve the bioavailability of core material.

[0088] The in vitro digestion release curve of the mulberry leaf flavone and the mulberry leaf flavone microcapsule prepared by the present application is as follows Figure 9The release rate of mulberry leaf flavone microcapsules was 16.28% and the retention rate was 83.72% at 2 h in the simulation of gastric juice digestion, and the release was slow; the release rate of mulberry leaf flavone was 38.4% and the retention rate was 61.6% at 2 h; according to the retention rate in the gastric juice digestion, it was concluded that the bioavailability of the mulberry leaf flavone microcapsules was increased by 35.9% compared with the mulberry leaf flavone, which might be because the complex coagulation process of the wall material was carried out in an acidic environment, and the protein and polysaccharide were combined through electrostatic interaction due to the opposite charges, forming a stable structure, so that the microcapsules could maintain structural stability in the acidic environment, reduce the loss caused by the gastric juice digestion, and increase the intestinal juice digestion utilization rate. In the process of intestinal juice digestion, the release rate of the mulberry leaf flavone microcapsules gradually increased, and compared with the cumulative release rate of the mulberry leaf flavone microcapsules and the mulberry leaf flavone at 6 h in the digestion, it was concluded that the release rates of the mulberry leaf flavone microcapsules and the mulberry leaf flavone were similar in the process of intestinal juice digestion, which might be because the protein was hydrolyzed by trypsin, resulting in the destruction of the complex wall material, so that the core material was released, and with the prolongation of the intestinal juice digestion time, the release rate continued to increase. Therefore, to some extent, the microcapsules effectively delayed the release of flavones through the embedding structure, achieved the slow-release effect, and thus improved the stability and bioavailability of the flavones.

[0089] (Six), evaluation of mulberry leaf flavone microcapsules on lowering blood lipid A hyperlipidemia mouse model was established by high-fat diet induction, and the mulberry leaf flavone microcapsules of Example 1 were used for intervention. It was found that the mulberry leaf flavone microcapsules could effectively regulate the blood lipid level of the mice, and the total cholesterol TC, total triglyceride TG, and low-density lipoprotein cholesterol LDL-C were decreased, and the high-density lipoprotein cholesterol HDL-C was increased.

[0090] The intervention experiment results are shown in Table 1. Figure 10 NC represents a blank control group, i.e., a healthy mouse fed with drinking water; MC represents a model group, i.e., a hyperlipidemia mouse without intervention; PC represents a positive control group, i.e., a hyperlipidemia mouse fed with a lipid-lowering drug simvastatin, and the feeding amount is 10 mg / kg; MF represents a mulberry leaf flavone group, i.e., a hyperlipidemia mouse fed with mulberry leaf flavone, and the feeding amount is 128 mg / kg of body weight; FL represents a microcapsule low-dose group, i.e., a hyperlipidemia mouse fed with mulberry leaf flavone microcapsules, and the feeding amount is 64 mg / kg of body weight; FM represents a microcapsule medium-dose group, i.e., a hyperlipidemia mouse fed with mulberry leaf flavone microcapsules, and the feeding amount is 128 mg / kg of body weight; and FH represents a microcapsule high-dose group, i.e., a hyperlipidemia mouse fed with mulberry leaf flavone microcapsules, and the feeding amount is 250 mg / kg of body weight.

[0091] Obviously, through the hyperlipidemia mouse experiment, the blood lipid-lowering effect of the mulberry leaf flavone microcapsules of the present application is clear.

[0092] In summary, the application uses mulberry leaf residue polysaccharide and gelatin as a composite wall material to microencapsulate and embed mulberry leaf flavones, and constructs a slow-release system of mulberry leaf flavones, which not only improves the embedding effect of mulberry leaf flavones, but also makes the obtained mulberry leaf flavone microcapsules have good stability, bioavailability and blood lipid-lowering effect, and realizes the reuse of mulberry leaf residue waste.

[0093] Please note that the technical features of the above embodiments can be combined in any way, and in order to make the description simple, not all possible combinations of the technical features in the above embodiments are described, however, as long as the combination of the technical features does not exist contradictory, it should be considered as the scope of the description. The above embodiments only express several implementation manners of the application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which are all within the protection scope of the application. Therefore, the protection scope of the patent of the application should be subject to the appended claims.

Claims

1. A mulberry leaf flavonoid microcapsule, characterized in that: Mulberry leaf flavonoids extracted from mulberry leaves were used as the core material, and mulberry leaf residue polysaccharide extracted from the mulberry leaf residue remaining after mulberry leaf flavonoid extraction was combined with gelatin as the wall material. Mulberry leaf flavonoid microcapsules were prepared by composite coagulation method. Before the composite coagulation reaction, the wall material solution needs to be subjected to first pH adjustment to 3~3.5 electric field pulse treatment, and then pH adjustment to 6~7 electric field pulse treatment. After the wall material and core material are mixed, the mixed solution needs to be cooled first, and then transglutaminase is added to carry out a composite coagulation reaction.

2. The mulberry leaf flavonoid microcapsules according to claim 1, characterized in that: The ratio of core material to wall material used in the preparation of mulberry leaf flavonoid microcapsules is 1:2~3.5 v / v, and / or the ratio of gelatin and mulberry leaf residue polysaccharide in the wall material is 1:1~1.5 m / m.

3. The mulberry leaf flavonoid microcapsules according to claim 1, characterized in that: Mulberry leaf flavonoids are extracted from mulberry leaves using an ethanol solution; preferably, microwave-assisted extraction is used; more preferably, mulberry leaves are immersed in an ethanol solution with a concentration of 60-80% v / v at a material-to-liquid ratio of 1:45-50 m / v for extraction, and treated under microwave conditions of 590-610w and 55-60℃ for 4-6 min, and then kept at 55-60℃ for 20-30 min. The filtrate obtained by filtration is the mulberry leaf flavonoid extract.

4. The mulberry leaf flavonoid microcapsules according to claim 1, characterized in that: Mulberry leaf residue polysaccharide is extracted from mulberry leaf residue using water. Preferably, ultrasonic-assisted extraction is employed. Further preferred methods include drying the mulberry leaf residue in a hot air fluidized bed at 60-65℃ for 30-40 minutes, then immersing it in purified water at a material-to-liquid ratio of 1:20-25 m / v for extraction, treating it with ultrasonic power of 350-400 W and temperature of 55-60℃ for 40-60 minutes, filtering to obtain the filtrate, and freeze-drying it to obtain mulberry leaf residue polysaccharide.

5. The mulberry leaf flavonoid microcapsules according to claim 1, characterized in that: Preferably, before extracting mulberry leaf flavonoids, the mulberry leaves undergo a treatment involving freezing, high-pressure nitrogen circulation, and finally high-pressure steam circulation. The freezing temperature is -30℃ to -20℃, and the freezing time is 4 to 5 hours. Alternatively, the high-pressure nitrogen circulation treatment involves placing the mulberry leaves under nitrogen circulation at 100 to 150 MPa for 5 to 10 minutes; and / or the high-pressure steam circulation treatment involves placing the mulberry leaves under steam circulation at 100 to 150 MPa and 55 to 60℃ for 5 to 10 minutes.

6. The mulberry leaf flavonoid microcapsules according to any one of claims 1 to 5, characterized in that: Gelatin and mulberry leaf residue polysaccharide were mixed at a ratio of 1:1 to 1.5 m / m, and a gelatin-mulberry leaf polysaccharide solution with a mass concentration of 1 to 1.5% was prepared as the wall material solution. The pH of the wall material solution was adjusted to 3 to 3.5, and the solution was placed in an environment with an electric field strength of 25 kV / cm and a pulse electric field frequency of 10 to 12 Hz for 400 to 500 μs. The pH of the wall material solution was then adjusted to 6 to 7, and the solution was placed in an environment with an electric field strength of 25 kV / cm and a pulse electric field frequency of 10 to 12 Hz for 400 to 500 μs to obtain the microcapsule wall material. Preferably, 0.5 M hydrochloric acid was used to adjust the pH of the wall material solution.

7. The mulberry leaf flavonoid microcapsules according to claim 6, characterized in that: Mulberry leaf flavonoid extract was used as the core material and mixed with the wall material at a ratio of 1:2~3.5 v / v. The mixture was homogenized at 10000~12000 rpm / min and 44~48℃. After the mixed solution was cooled to 4℃, transglutaminase was added and kept warm for 2 h. Then, it was freeze-dried under vacuum to obtain mulberry leaf flavonoid microcapsules. Preferably, the amount of transglutaminase added was 40~50 mg / L.

8. The mulberry leaf flavonoid microcapsules according to claim 7, characterized in that: The 2-hour retention rate of mulberry leaf flavonoid microcapsules, as determined in an in vitro simulated gastric juice digestion experiment, reached over 80%, and the bioavailability was more than 30% higher than that of flavonoid solutions.

9. The method for preparing mulberry leaf flavonoid microcapsules according to any one of claims 1 to 8, characterized in that: The preparation steps include the following: a. Extraction of mulberry leaf flavonoids: Mulberry leaves are immersed in an ethanol solution with a concentration of 60-80% v / v at a material-to-liquid ratio of 1:45-50 m / v. The solution is then treated with microwave at 590-610w and 55-60℃ for 4-6 minutes, and then kept at 55-60℃ for 20-30 minutes. The filtrate obtained is the mulberry leaf flavonoid extract, and the filter residue is the mulberry leaf residue. b. Extraction of mulberry leaf polysaccharides: The mulberry leaf residue is dried in a hot air fluidized bed at 60-65℃ for 30-40 min, and then extracted by immersion in purified water at a material-to-liquid ratio of 1:20-25 m / v. The residue is then treated with ultrasonic power of 350-400w and temperature of 55-60℃ for 40-60 min. The filtrate is obtained by filtration and freeze-drying to obtain mulberry leaf residue polysaccharides. c. Preparation of mulberry leaf flavonoid microcapsules: Take gelatin and mulberry leaf residue polysaccharide obtained in step b, mix them at a ratio of 1:1~1.5 m / m, and then prepare a gelatin-mulberry leaf polysaccharide solution with a mass fraction of 1~1.5% as the wall material solution. Adjust the pH of the wall material solution to 3~3.5 using 0.5M hydrochloric acid, and place it in an environment with an electric field strength of 25kV / cm and a pulse electric field frequency of 10~12Hz for 400~500μs. Then adjust the pH of the wall material solution to 6~7. The microcapsule wall material was obtained by treating the microcapsule in an environment with an electric field strength of 25 kV / cm and a pulse electric field frequency of 10~12 Hz for 400~500 μs. Mulberry leaf flavonoid extract was used as the core material and mixed at a core-to-wall ratio of 1:2~3.5 v / v. The mixture was homogenized at 10000~12000 rpm / min and 44~48℃ to obtain a mixed solution. The solution was cooled to 4℃ in an ice-water bath, and 40~50 mg / L transglutaminase was added and kept warm for 2 h. The solution was then freeze-dried under vacuum to obtain mulberry leaf flavonoid microcapsules. Preferably, a pretreatment step s is added before step a, and the concentration of the ethanol solution used in step a is optimized, as follows: s. Pretreatment: Take uncrushed mulberry leaves, first freeze them slowly at -30℃~-20℃ for 4~5 hours, then circulate nitrogen at 100~150MPa for 5~10 minutes, and finally circulate steam at 100~150MPa at 55~60℃ for 5~10 minutes. Step a involves extracting mulberry leaf flavonoids using a 60-67% v / v ethanol solution.

10. The application of the mulberry leaf flavonoid microcapsules according to any one of claims 1 to 8, characterized in that: It is used to develop adjuvant drugs for hyperlipidemia and lipid-lowering functional foods.

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