Mulberry leaf flavone microcapsule as well as preparation method and application thereof

Abstract

The invention relates to a mulberry leaf flavone microcapsule as well as a preparation method and application thereof, and belongs to the technical field of microcapsule drug loading. The preparation method of the mulberry leaf flavone microcapsule comprises the following steps: S1, adding mulberry leaf flavone into a chitosan solution, carrying out ultrasonic treatment, shearing by using a homogenizer, and heating and stirring to obtain a mixed solution; s2, slowly dropwise adding a sodium tripolyphosphate solution into the mixed solution, reacting and curing until the solution becomes milk white liquid from clear and no precipitate is generated, then centrifuging and washing, and collecting particles; and S3, freezing the particles, and performing freeze drying to obtain the mulberry leaf flavone microcapsules. The mulberry leaf flavone microcapsule provided by the invention can be used for developing a novel medicinal preparation in the field of medicines, and can relieve disease symptoms and delay disease progression through the antioxidant and anti-inflammatory characteristics of the mulberry leaf flavone microcapsule; in the field of food, the method can be applied to development of functional food, for example, the method can be added into beverages and health-care products to provide health benefits of antioxidation, blood fat reduction and the like for consumers.

Description

Technical Field

[0001] The present application relates to the technical field of microcapsule drug delivery, and in particular to a mulberry leaf flavonoid microcapsule and a preparation method and application thereof. Background Art

[0002] Mulberry is a perennial deciduous woody plant with a long history of cultivation in China and a wide area, but the main planting areas are mainly in the southern Jiangsu and Zhejiang regions. Mulberry leaves are leaves of the Moraceae plant Morus alba.L., which is one of the traditional Chinese medicinal materials in my country. It can be used as a natural product with both medicinal and edible properties. Mulberry leaves contain a variety of flavonoids, such as rutin, quercetin, kaempferol, isoquercetin, etc. Flavonoids are a class of plant-derived dietary compounds that are secondary metabolites that are abundant in other sources such as fruits, vegetables and herbs. However, the poor stability and low bioavailability of mulberry leaf flavonoids limit their direct application. Therefore, microencapsulation technology that can overcome these limitations has attracted increasing attention.

[0003] These compounds have a variety of pharmacological activities, such as antioxidant, antiviral, antitumor, antibacterial and hypolipidemic activities. In recent years, the concept of promoting human health through dietary intervention has attracted more and more attention from researchers. At the same time, natural products have become a research hotspot for topics related to human nutrition and health due to their advantages such as small side effects, no drug resistance and high safety index. However, mulberry leaf flavonoids have problems such as poor stability and low bioavailability, which greatly limits their practical application. In order to solve these problems, microencapsulation technology has been introduced into the research of mulberry leaf flavonoids as an effective means.

[0004] Microencapsulation technology is a practical and long-lasting drug release method, which can isolate drugs from the external environment, protect drugs, reduce their denaturation, and the outer wall material can play a role in the controlled release process. Microencapsulation technology is an emerging technology applied to the food industry. In recent years, various delivery systems based on proteins, lipids and polysaccharides have been developed to transport bioactive compounds. These systems include liposomes, nanoemulsions and nanomicelles, which aim to improve the properties of bioactive compounds, such as their solubility in water, stability to chemical degradation, biological activity, and the extent to which they are absorbed and utilized by the body. Chitosan, as a natural polycationic polymer, has good biocompatibility and degradability; sodium tripolyphosphate is a commonly used anionic crosslinker. Under specific conditions, chitosan and sodium tripolyphosphate form a gel through ionic interactions, thereby encapsulating the drug inside to form a tiny capsule structure. Based on the excellent safety, biocompatibility, biodegradability and bioadhesion of chitosan, chitosan has been widely used in delivery systems. Positively charged chitosan can be adsorbed onto the negatively charged nanoparticle surface through electrostatic interaction to form a cationic shell, which helps maintain the stability of the nanoparticles under the acidic conditions of gastric fluid. Chitosan has adhesive properties, so the adhesion of chitosan microcapsule particles to the intestine is enhanced. The chitosan coating on the surface of the microcapsule particles can prolong the digestion time of bioactive substances in the gastrointestinal tract and improve their bioavailability. Summary of the invention

[0005] In view of this, the present application provides a mulberry leaf flavonoids microcapsule and a preparation method and application thereof. The present application prepares mulberry leaf flavonoids microcapsule by an ion gel method. The mulberry leaf flavonoids microcapsule can be used to develop new pharmaceutical preparations in the medical field, such as auxiliary therapeutic drugs for chronic diseases such as diabetes and cardiovascular diseases, which can alleviate disease symptoms and delay disease progression through their antioxidant and anti-inflammatory properties; in the food field, it can be applied to the development of functional foods, such as adding to beverages and health products, to provide consumers with health benefits such as antioxidant and lipid-lowering, which can effectively overcome the defects of the above-mentioned prior art.

[0006] The first aspect of the present application provides a method for preparing mulberry leaf flavonoid microcapsules, comprising the following steps:

[0007] S1. Add mulberry leaf flavonoids into the chitosan solution, shear with a homogenizer after ultrasonication, heat and stir to obtain a mixed solution;

[0008] S2, slowly dripping the sodium tripolyphosphate solution into the mixed solution, reacting and solidifying, the solution changes from clear to milky white liquid without precipitation, and then centrifuging, washing and collecting particles;

[0009] S3, freezing the particles and freeze-drying them to obtain mulberry leaf flavonoids microcapsules.

[0010] Preferably, the method specifically comprises the following steps:

[0011] S1. Add mulberry leaf flavonoids to the chitosan solution, ultrasonicate for 10 minutes, shear for 3 minutes using a homogenizer at a rate of 12000 r / min, and stir for 15 minutes at 50°C and 500 r / min to obtain a mixed solution;

[0012] S2. Slowly drip the sodium tripolyphosphate solution into the mixed solution, react and solidify for 1 hour, the solution changes from clear to milky white liquid without precipitation, and then centrifuge at 10000r / min for 30min, wash with ultrapure water for 3 times and collect the particles;

[0013] S3. Freeze the particles at -80°C for 2 hours, and freeze-dry them using a freeze dryer for 14 hours to obtain mulberry leaf flavonoids microcapsules.

[0014] Preferably, the extraction process of mulberry leaf flavonoids is as follows: fresh mulberry leaves are placed in an oven at 55°C, dried for 24 hours, crushed, and sieved through a 40-mesh sieve for later use; a certain amount of mulberry leaf powder is weighed into a beaker, and a 44% ethanol aqueous solution is added to the beaker at a solid-liquid ratio of 1:27, and ultrasonic extraction is performed at 43°C for 30 minutes, and the residue is removed by filtration, and the filtrate is collected and centrifuged in a high-speed centrifuge at 10,000 r / min for 5 minutes to obtain the supernatant, which is concentrated to 10 mL using a rotary evaporator, and finally fixed to 50 mL with anhydrous ethanol for later use.

[0015] Preferably, the preparation process of the chitosan solution is as follows: dissolving an appropriate amount of chitosan in 1% acetic acid water, dissolving and stirring at room temperature for 5 hours, shearing at a rate of 10000r / min for 3min, adjusting the pH of the chitosan solution to 5 under strong magnetic stirring, filtering through a micro syringe filter with a pore size of 0.22μm to remove insoluble substances, and obtaining a chitosan solution; and storing the prepared chitosan solution in a refrigerator at 4°C for use.

[0016] Preferably, the preparation process of the sodium tripolyphosphate solution is: weighing an appropriate amount of sodium tripolyphosphate and dissolving it in ultrapure water, magnetically stirring for 2 hours, filtering through a micro syringe filter with a pore size of 0.45 μm to remove insoluble substances to obtain a sodium tripolyphosphate solution; and storing the prepared sodium tripolyphosphate solution in a refrigerator at 4° C. for use.

[0017] Preferably, the mass ratio of chitosan to sodium tripolyphosphate is (3-10):1.

[0018] Preferably, the volume ratio of chitosan to mulberry leaf flavonoids is (1-5):1; the volume ratio of sodium tripolyphosphate to mulberry leaf flavonoids is (1-5):1.

[0019] The second aspect of the present application also provides a mulberry leaf flavonoids microcapsule. The mulberry leaf flavonoids microcapsule prepared by the above method has an average diameter of 542.53±93.95 nm, and the embedding rate of mulberry leaf flavonoids in the mulberry leaf flavonoids microcapsule is 83.39%.

[0020] Preferably, the total release rate of the mulberry leaf flavonoids microcapsules in a 24-hour in vitro release experiment is 72%.

[0021] The third aspect of the present application also provides the application of the above-mentioned mulberry leaf flavonoids microcapsules in the fields of food and medicine.

[0022] Compared with the prior art, this application has the following beneficial effects:

[0023] The present application aims to prepare chitosan-sodium tripolyphosphate microcapsule particles (MLF@CS-TPP) loaded with mulberry leaf flavonoids by ion gel technology to preserve the activity of mulberry leaf flavonoids; and further characterize the morphology, chemical structure and thermal stability of the microcapsules by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The results show that mulberry leaf flavonoids are successfully embedded in the microcapsules. The structure of the microcapsules MLF@CS-TPP is spherical or sub-spherical, with good stability. In the simulated gastrointestinal digestion process, the microcapsules show a lower release rate in the gastric stage, and a higher release rate in the intestinal stage and maintain a higher antioxidant activity, which indicates that it has potential intestinal targeted delivery capabilities. The present application provides a promising strategy for the application of mulberry leaf extracts in the field of functional food and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present application or the prior art, the drawings required for use in the description of the present application or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 SEM images of microcapsules prepared with different CS:TPP mass ratios, including (A) CS:TPP mass ratio of 3:1, (B) CS:TPP mass ratio of 4:1, (C) CS:TPP mass ratio of 6:1, (D) CS:TPP mass ratio of 8:1, and (E) CS:TPP mass ratio of 10:1;

[0026] Figure 2The effects of different addition amounts on the embedding rate of MLF@CS-TPP, including (A) the effect of chitosan addition amount on the embedding rate of MLF@CS-TPP, (B) the effect of sodium tripolyphosphate addition amount on the embedding rate of MLF@CS-TPP;

[0027] Figure 3 Characterization diagram of microcapsule morphology, including (A) SEM: CS-TPP, (B) TEM: CS-TPP, (C) SEM: MLF@CS-TPP, (D) TEM: MLF@CS-TPP;

[0028] Figure 4 Analyze the energy spectrum of chemical elements of CS-TPP;

[0029] Figure 5 Particle size distribution diagram, including (A) CS-TPP, (B) MLF@CS-TPP;

[0030] Figure 6 Zeta potential diagram of CS-TPP and MLF@CS-TPP;

[0031] Figure 7 Fourier transform infrared spectra of MLF@CS-TPP, CS-TPP, MLF, TPP and CS;

[0032] Figure 8 This is the mass loss diagram of mulberry leaf flavonoids, empty and loaded microcapsule particles;

[0033] Fig. 9 It is the release curve of MLF@CS-TPP;

[0034] Fig.10 To simulate the gastrointestinal digestion of MLF and MLF@CS-TPP in vitro, the bioavailability of flavonoids;

[0035] Fig.11 To simulate the clearance rate of DPPH by mulberry leaf flavonoids and MLF@CS-TPP after gastrointestinal digestion;

[0036] Fig.12 To simulate the clearance rate of ABTS by mulberry leaf flavonoids and MLF@CS-TPP after gastrointestinal digestion;

[0037] Fig.13 UV stability diagram of MLF@CS-TPP and MLF;

[0038] Fig.14 This is the ion stability diagram of mulberry leaf flavonoids microcapsules;

[0039] Fig.15 This is a picture of the impact of MLF on mangoes;

[0040] Fig.16 This is a picture of the impact of CS-TPP on mangoes;

[0041] Fig.17 This is a picture of the impact of MLF@CS-TPP on mangoes. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of this application clearer, the technical solutions in this application will be clearly and completely described below in conjunction with the drawings in this application. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0043] Unless otherwise specified, the experimental methods used in the examples of this application are all conventional methods.

[0044] In the following examples, unless otherwise specified, all raw materials can be obtained by commercial purchase or conventional methods.

[0045] It should be noted that MLF@CS-TPP: mulberry leaf flavonoids chitosan microcapsule particles; CS-TPP: empty chitosan microcapsules; MLF: mulberry leaf flavonoids; TPP: sodium tripolyphosphate; CS: chitosan.

[0046] 1. Materials and Methods

[0047] 1.1 Materials and Instruments

[0048] Chitosan ≥ 95% (CS) and sodium tripolyphosphate (TPP) were from Shanghai McLean Biochemical Technology Co., Ltd., porcine bile salt, trypsin, sodium hydroxide (NaOH), and sodium bicarbonate (NaHCO3) were from Shanghai Yuanye Biotechnology Co., Ltd., dialysis bags, rutin standards, and sodium nitrite (Na2NO3) were from Solebold Biochemical Technology Co., Ltd., mulberry leaves were collected in Yazhou District, Sanya City, Hainan Province, China; mangoes were purchased from Nanshan Garden Fruit Wholesale Market, Yazhou District, Sanya City, Hainan Province.

[0049]

[0050] 1.2 Extraction process of mulberry leaf flavonoids

[0051] Fresh mulberry leaves were placed in a 55°C oven, dried for 24 hours, crushed, and passed through a 40-mesh sieve for later use. A certain amount of mulberry leaf powder was weighed into a beaker, and a 44% ethanol aqueous solution was added to the beaker at a solid-liquid ratio of 1:27. Ultrasonic extraction was performed at 43°C for 30 minutes, and the residue was removed by filtration. The filtrate was collected and centrifuged in a high-speed centrifuge at 10,000 r / min for 5 minutes to obtain the supernatant, which was concentrated to 10 mL using a rotary evaporator, and finally fixed to 50 mL with anhydrous ethanol for later use.

[0052] 1.3 Determination of flavonoids content in mulberry leaves

[0053] 1.3.1 Drawing of Rutin Standard Curve

[0054] Refer to the method of Mihai et al. (MIHAI B, OLIVIU V, RAMONA P, et al. Chemical Composition, Diuretic, and Antityrosinase Activity of Traditionally Used Romanian Cerasorumstipites [J]. Frontiers in Pharmacology, 2021, 12) and make appropriate adjustments. Take 1 mL of rutin standard solution of different concentrations in a test tube, add 1 mL of 5% NaNO2 solution respectively, and shake well. After 6 minutes, add 1 mL of 10% Al(NO3)3 solution, shake well, let stand for 6 minutes, add 4 mL of 4% NaOH solution, make up to 10 mL with ultrapure water, and let stand for 15 minutes. Use ultrapure water as a blank control and scan at a wavelength of 510 nm using an enzyme reader.

[0055] With the sample concentration (mg / mL) as the horizontal axis and the absorbance value (A) as the vertical axis, a standard curve was drawn to obtain the regression equation y=17.53*x-0.002R 2 =0.9994.

[0056] 1.3.2 Determination of flavonoid content

[0057] The mulberry leaf extract was determined according to the method in 1.3.1 and the flavonoid content was calculated. The formula is as follows:

[0058]

[0059] Where: C MLF : mulberry leaf flavonoids content mg / g; N: dilution multiple; C: standard curve flavonoids content; V: test solution volume; M: mulberry leaf dry sample mass.

[0060] 1.4 Preparation of chitosan microcapsules

[0061] Dissolve an appropriate amount of chitosan in 1% acetic acid water, stir at room temperature for 5 hours, shear the mixed solution at a rate of 10000 r / min for 3 minutes, adjust the pH of the chitosan solution to 5 under strong magnetic stirring, filter with a micro syringe filter (pore size 0.22 μm) to remove insoluble substances. The prepared chitosan solution is stored in a refrigerator at 4°C for later use.

[0062] Weigh an appropriate amount of sodium tripolyphosphate and dissolve it in ultrapure water, stir it magnetically for 2 hours, and filter it through a micro syringe filter (pore size 0.45 μm) to remove insoluble matter. The prepared sodium tripolyphosphate solution is stored in a refrigerator at 4°C for later use.

[0063] Mulberry leaf extract (i.e. mulberry leaf flavonoids) was added to the chitosan solution, and after ultrasonication for 10 minutes, a homogenizer was used to shear at a rate of 12000r / min for 3 minutes, and stirred at 50°C and 500r / min for 15 minutes to obtain a mixed solution. Sodium tripolyphosphate solution was slowly dripped into the mixed solution, and the reaction solidified for 1 hour. The solution changed from clear to milky white liquid and no precipitation was produced. Then, under the condition of centrifugation at 10000r / min for 30 minutes, it was washed repeatedly with ultrapure water for 3 times and the particles were collected. The particles were frozen at -80°C for 2 hours and freeze-dried for 14 hours using a freeze dryer.

[0064] 1.5 Optimization of chitosan microcapsule preparation conditions

[0065] Different factors may lead to preparation failure in the process of preparing chitosan microcapsules. In order to prepare relatively stable microcapsule particles with uniform size, the preparation process of chitosan microcapsules was explored by using different mass ratios of chitosan and sodium tripolyphosphate.

[0066] (1) Preparation of chitosan microcapsules with different CS:TPP mass ratios

[0067] Under the same chitosan microcapsule preparation conditions, the chitosan concentration was controlled to be 4 mg / mL and the sodium tripolyphosphate concentration was 1 mg / mL. This application selected different chitosan to sodium tripolyphosphate mass ratios, which were 3:1, 4:1, 6:1, 8:1, and 10:1. As shown in the following table, chitosan microcapsules were prepared according to the above content, and the morphological characteristics were observed under a scanning electron microscope. (Instrument model: Phenom-WorldBV, Phenom Pro X, Holland).

[0068] Table 1. Effect of CS:TPP mass ratio on the morphology of chitosan microcapsules.

[0069] sample CS:TPP mass ratio (mg / mg) 1 3:1 2 4:1 3 6:1 4 8:1 5 10:1

[0070] 1.6 Single factor test on the conditions affecting the encapsulation efficiency of MLF@CS-TPP

[0071] (1) Effect of chitosan addition on MLF@CS-TPP encapsulation efficiency

[0072] According to the method in 1.5, the chitosan concentration was determined to be 4 mg / ml, the sodium tripolyphosphate addition was 1 mg / ml, and a homogenizer was used to shear at a rate of 12000 r / min for 3 min. The volume ratio of chitosan to mulberry leaf extract was set to 1:1, 2:1, 3:1, 4:1, and 5:1 to prepare microcapsules.

[0073] Table 2 Effect of chitosan addition on MLF@CS-TPP encapsulation efficiency Design table

[0074] sample CS addition ratio 1 1:1 2 2:1 3 3:1 4 4:1 5 5:1

[0075] (2) Effect of sodium tripolyphosphate addition on MLF@CS-TPP encapsulation efficiency

[0076] According to the above method, the volume ratio of chitosan to mulberry leaf extract was determined to be 2:1, and a homogenizer was used to shear at a rate of 12000r / min for 3min. Microcapsules were prepared under the conditions that the volume ratio of sodium tripolyphosphate to mulberry leaf extract was set to 1:1, 2:1, 3:1, 4:1, and 5:1, respectively.

[0077] Table 3 Effect of sodium tripolyphosphate addition on MLF@CS-TPP encapsulation efficiency Design table

[0078] sample TPP addition ratio 1 1:1 2 2:1 3 3:1 4 4:1 5 5:1

[0079] 1.7 Determination of MLF@CS-TPP encapsulation efficiency

[0080] Take a portion of the prepared MLF@CS-TPP suspension, centrifuge at 10000rpm for 30min, take the supernatant and detect the total flavonoid content of the supernatant according to the 1.3 mulberry leaf flavonoid detection method, repeat the test three times for each sample, and calculate the encapsulation efficiency according to the following formula:

[0081] Encapsulation efficiency (%) = [(W1-W2) / W1]*100%

[0082] Wherein W1: the content of flavonoids added to mulberry leaves; W2: the content of flavonoids in the supernatant calculated by the standard curve.

[0083] 1.8 Microcapsule characterization

[0084] 1.8.1 Field emission scanning electron microscopy (FESEM)

[0085] After the microcapsule suspension was centrifuged at 10000r / min for 30min, the precipitated microcapsules were washed three times with ultrapure water, diluted with appropriate ultrapure water, evenly dropped onto a silicon wafer, dried and sprayed with gold for 90s, and then the morphology of CS-TPP and MLF@CS-TPP was observed by scanning electron microscopy (instrument model: Thermofisher, Verios G4 UC, Czech).

[0086] 1.8.2 Field emission transmission electron microscopy (FETEM)

[0087] The microcapsule sample was appropriately diluted with ultrapure water, and the solution was dropped on a standard copper grid covered with a holey carbon film and dried at room temperature, and the morphology of CS-TPP and MLF@CS-TPP was observed using a field emission transmission electron microscope (instrument model: Thermofisher, Talos F200X G2, Czech).

[0088] 1.8.3 Particle size distribution, polydispersity index (PDI) and zeta potential detection

[0089] The particle size distribution and average particle size of the samples were determined by dynamic light scattering using a Malvern laser particle size analyzer. 1 mL of the CS-TPP and MLF@CS-TPP suspension samples were placed in a cuvette and then placed in a Malvern laser particle size analyzer to detect the sample particle size distribution and potential. All measurements were repeated 3 times. (Instrument model: Malvern Instrument, zeta sizer nanoZS90, England).

[0090] 1.8.4 Fourier Transform Infrared Spectroscopy (FTIR)

[0091] After centrifuging the microcapsule suspension at 10,000 r / min for 30 min, the precipitated microcapsules were washed three times with ultrapure water to remove the uncoated mulberry leaf flavonoids extract and excess composite wall materials. After drying at room temperature, mulberry leaf flavonoids extract, chitosan, sodium tripolyphosphate, CS-TPP, and MLF@CSTPP were obtained for FTIR spectral analysis. (Instrument model: Thermofisher, Nicolet is20, Czech) The sample was mixed with KBr at a mass ratio of (1:100) and ground and pressed into tablets. Each sample was heated at 4000–400 cm -1 Within 4cm -1 The infrared spectrum was scanned with 16 resolution scans.

[0092] 1.8.5TGA analysis Thermogravimetric analysis

[0093] Chitosan, sodium tripolyphosphate, mulberry leaf flavonoids, dried CS-TPP and MLF@CS-TPP were weighed in the range of 5-6 mg respectively, purged with nitrogen at a flow rate of 30 mL / min on a comprehensive thermal analyzer (instrument model: NETZSCH Geraetebau GmbH, TG 209F3, Germany), the temperature was controlled in the range of 30-1000°C, and the thermal stability was characterized at a heating rate of 10°C / min. Each sample was analyzed at least 3 times, and the average value was recorded.

[0094] 1.8.6 Determination of release performance of MLF@CS-TPP

[0095] A certain amount of MLF@CS-TPP was placed in a dialysis bag (retained molecular weight 3500), 5 mL of PBS buffer solution was added, and the mixture was placed in a beaker containing 15 mL of PBS buffer solution and shaken at 25 ± 2 °C at 100 r / min. Samples were taken at regular intervals and the flavonoid content was tested. Samples were taken at 0, 0.5, 1, 1.5, 3, 6, 9, 12, 24, 48, 72, and 96 h, and the corresponding PBS buffer solution was supplemented.

[0096]

[0097] Where Mt: the amount of flavonoids released from chitosan at a certain moment; M0: the amount of flavonoids initially embedded in the microcapsule particles.

[0098] 1.8.7 Color Measurement

[0099] At room temperature (25°C), all samples were measured for color analysis using a colorimeter (instrument model: 3NH, SC-10, China), measuring the parameters L* (sample brightness), a* (red / green) and b* (yellow / blue). Before the measurement, the colorimeter was calibrated accordingly, and a white plate was needed to calibrate the equipment. After the calibration, the microencapsulated powder was placed in a transparent glass dish and the measurement was completed. Three different surfaces of the sample were randomly selected to calculate the color parameters L*, a* and b*.

[0100] 1.9 In vitro simulation of gastrointestinal digestion

[0101] With reference to previous studies (Pei Yufang. Effects of Lycium barbarum leaf flavonoids extract on lipid metabolism of HepG2 cells and preparation of microcapsules [D]. Ningxia University, 2023.) and with certain modifications, an in vitro gastrointestinal digestion test of chitosan microcapsules was conducted. In brief, simulated gastric fluid (SGF) was prepared by adding pepsin (0.8 g) to 0.3 mol / L NaCl and adjusting the pH to 3.0 with 3 mol / L HCl. 0.268 g trypsin and 1.714 g bile salts were added to 0.2 mol / L NaHCO3 and the pH was adjusted to 7.0 to prepare simulated intestinal fluid (SIF). The pH value of the digestive fluid plays a vital role in the digestion process. In gastric juice (SGF) with a pH of 3.0, pepsin hydrolyzes the protein in the microcapsules, which may affect the release of mulberry leaf flavonoids. In the small intestinal fluid (SIF) with a pH of 7.0, trypsin and bile salts work synergistically to further decompose the microcapsules and promote the absorption of flavonoids. In addition, the ionic strength and temperature of the digestive fluid also need to be considered, as they may affect the stability and bioavailability of the microcapsules.

[0102] Weigh 20 mg of sample, add 20 mL of prepared SGF and protect from light. Digest in a constant temperature water bath at 37°C for 4 h. Take 3 mL of sample solution at regular intervals and replenish with corresponding liquid. Test the flavonoid content and antioxidant capacity of the sample.

[0103] Weigh 20 mg of sample, add 20 mL of prepared SIF and protect from light. Digest in a constant temperature water bath at 37°C for 4 h. Take 3 mL of sample solution at regular intervals and replenish with corresponding liquid to test its antioxidant capacity.

[0104] The content of mulberry leaf flavonoids was determined according to the above 1.3. The bioavailability index (BI) of mulberry leaf flavonoids was calculated according to the following formula, where CF D is the flavonoid content of mulberry leaves after in vitro digestion, CF A It is the flavonoid content of mulberry leaves before in vitro digestion.

[0105]

[0106] 1.9.1 Analysis of antioxidant activity after simulated gastrointestinal digestion in vitro

[0107] (1) Ability to remove DPPH

[0108] With reference to the method of Song et al. (SONG F, TANG M, WANG H, et al. UHPLC-MS / MS identification, quantification of flavonoid compounds from Areca catechu L. extracts and invitro evaluation of antioxidant and key enzyme inhibition properties involved in hyperglycemia and hypertension [J]. Industrial Crops and Products, 2022, 189), 1 mL of sample solution of different concentrations was taken in a test tube, 1 mL of DPPH was added, 1 mL of anhydrous ethanol and 1 mL of sample solution were added to the blank group, and 1 mL of anhydrous ethanol was added to the control group instead of the sample solution. Under the same conditions, vitamin C (VC) was used as a positive control. The clearance rate was calculated as follows:

[0109]

[0110] Wherein: A1 is the absorbance value of the sample solution; A2 is the corresponding absorbance value measured when anhydrous ethanol replaces the DPPH solution; A0 is the absorbance value of the blank group.

[0111] (2) Ability to remove ABTS

[0112] Refer to the method of Oyeleke et al. (OYELEKE MB, OWOYELE B V. Saponins and flavonoids from Bacopa floribunda plant extract exhibit antioxidant and anti-inflammatory effects on amyloid beta 1-42-induced Alzheimer's disease in BALB / c mice [J]. J Ethnopharmacol, 2022, 288) and modify it appropriately. Take 200 μL of the sample solution of different concentrations in a test tube, add 3 mL of ABTS working solution, use 3 mL of distilled water for the control group, react at room temperature in the dark for 1 hour, measure the absorbance at 734 nm, and use vitamin C (VC) as a positive control under the same conditions. The clearance rate is calculated as follows:

[0113]

[0114] Where: A1 is the absorbance value of the sample solution; A2 is the corresponding absorbance value measured when distilled water replaces the ABTS working solution; A0 is the absorbance value of the blank group.

[0115] 1.10 UV light stability

[0116] Weigh 10 mg of microcapsule powder particles, add 10 mL of deionized water and mulberry leaf flavonoid extract (1 mg / mL) into a beaker, and irradiate with 365 nm ultraviolet light for 8 h. Use an enzyme marker to detect the change rate of flavonoid content every 1 h.

[0117] 1.11 Ion stability

[0118] The prepared microcapsules were mixed with sodium chloride solutions of different concentrations (0, 100, 200, 300, 400 mM) and stirred for 1 hour before measuring the change rate of flavonoid content.

[0119] 1.12 Method for determining the quality of mango fruit after harvest

[0120] Mango pre-treatment: Select fresh mangoes with roughly the same appearance, 70% to 80% maturity, no mechanical damage and no pests or diseases. Each mango is soaked in 1% sodium hypochlorite and washed with sterile distilled water and then dried naturally.

[0121] There were 13 treatment groups in this experiment, namely, MLF treatment: 1%, 2%, 4%, 8%; CS-TPP treatment: 1%, 2%, 4%, 8%; MLF@CS-TPP treatment: 1%, 2%, 4%, 8%. Water treatment was used as a control. Mangoes were soaked for 30 seconds and then dried at room temperature for 4-5 hours. After drying, they were placed in a storage room at a temperature of 25±2℃. The fruit rot rate was observed and recorded at 1d, 2d, 3d, 4d, 5d, 6d, 7d, 8d, 9d, and 10d. Each group was treated with 15 fruits and repeated three times.

[0122] 1.13 Data processing and analysis

[0123] Each group of experiments was repeated three times, and the results were recorded as mean ± error value. SPSS was used for data processing and significance analysis. P < 0.05 was considered to be significantly different. Different letters represent significant differences, and the same letters represent no significant differences. Origin 2021 software was used to draw the graph.

[0124] 2. Test results and analysis

[0125] 2.1 Effect of the ratio of chitosan to sodium tripolyphosphate on the preparation of microcapsule particles

[0126] from Figure 1It can be seen from Figures A, B, and C that when the CS:TPP mass ratio is 3:1, 4:1, and 6:1, irregularly shaped products and agglomerates are formed, and the adhesion is more serious, the ball formation rate is low, and the viscosity of the chitosan solution is too high, which causes the chitosan molecules to adhere to each other, and it is easy to form irregular products and large agglomerates. This may be because when the concentration of sodium tripolyphosphate is too high, it may be because the ratio of the amino groups in the chitosan solution is not evenly matched, and chitosan microcapsule particles cannot be obtained. When the mass ratio is 6:1 and 10:1 ( Figure 1 In D and E), when the concentration of sodium tripolyphosphate is too low, the relative phosphate groups are too few to effectively attract the amino groups of chitosan with electrostatic attraction. Therefore, the CS:TPP mass ratio is selected to be 8:1.

[0127] 2.2 Determination of encapsulation efficiency of drug-loaded microcapsules

[0128] like Figure 2 As shown in A, the embedding rate of MLF@CS-TPP shows a trend of first increasing and then decreasing with the change of mass ratio. When the amount of chitosan added increases, the contact area between mulberry leaf flavonoids and chitosan increases, which increases the embedding rate of the microcapsules. When the addition ratio of chitosan to mulberry leaf flavonoids is 2:1, the embedding rate of the microcapsules reaches a maximum value of 47.11%. As the amount of chitosan added increases, the viscosity of the solution increases and the fluidity becomes, resulting in a decrease in the effective ion collision between chitosan and mulberry leaf flavonoids, thereby causing its embedding rate to decrease. Therefore, under the premise of ensuring the embedding rate, the optimal addition ratio of chitosan to mulberry leaf flavonoids for preparing MLF@CS-TPP is 2:1.

[0129] like Figure 2 As shown in Figure B, with the increase of sodium tripolyphosphate content in the solution, the anion content in the solution increases, and the contact with the cation ionized by chitosan is more frequent, thereby increasing the embedding rate of the microcapsule. When the addition ratio of sodium tripolyphosphate to mulberry leaf flavonoids is 3:1, the embedding rate reaches a maximum value of 83.39%. When the concentration of sodium tripolyphosphate in the solution is too low, the wall material generated by the reaction is small, and the microcapsule wall formed is thin, which cannot fully embed mulberry leaf flavonoids, resulting in a low encapsulation rate. When the concentration of sodium tripolyphosphate in the solution is too high, the wall material content generated by cross-linking with chitosan increases, resulting in an increase in the thickness of the capsule wall of the microcapsule, which increases the specific gravity of the wall material in the microcapsule, thereby reducing the encapsulation rate. Since the addition ratio of sodium tripolyphosphate to mulberry leaf flavonoids is not much different from 2:1 and 3:1, the addition ratio of sodium tripolyphosphate to mulberry leaf flavonoids is 2:1 as the subsequent experimental condition.

[0130] 2.3 Appearance characteristics of drug-loaded microcapsules

[0131] (1) Field emission scanning electron microscopy

[0132] Depend on Figure 3A. 3C shows that the morphology of CS-TPP and MLF@CS-TPP is regular spheres with uniform and complete shapes and sizes, which can clearly indicate that the microcapsules are successfully prepared.

[0133] (2) Energy spectrum analysis

[0134] Through EDS spectrum ( Figure 4 )Analysis of the element types of microcapsule particles revealed that the elements in CS-TPP include C, N, O, P, and Na, among which C, N, and O are the components of chitosan, and P, Na, and O are the components of sodium tripolyphosphate, which further illustrates that the microcapsule was successfully prepared.

[0135] (3) Field emission transmission electron microscopy

[0136] Depend on Figure 3 B. 3D shows that under TEM observation, the morphology of CS-TPP and MLF@CS-TPP is a regular sphere, which can clearly determine that the microcapsule preparation is successful.

[0137] 2.4 Particle size and potential analysis of microcapsules

[0138] After obtaining the optimal preparation conditions and encapsulation efficiency, the particle size and potential of the microcapsules need to be further analyzed to understand their physical properties. CS-TPP and MLF@CS-TPP were measured using dynamic dispersion spectroscopy (DLS). Figure 5 , Figure 6 As shown in Table 4, the particle sizes of CS-TPP and MLF@CS-TPP are 613.68±84.53nm and 542.53±93.95nm, respectively. In water, CS-TPP and MLF@CS-TPP are both positively charged, with Zeta potentials of 50.7±1.31mV and 33.23±1.56mV, respectively. The system is stable, with PDI values ​​between 0.232 and 0.272, indicating that the particle size is well controlled and has good uniformity.

[0139] Table 4 Particle size and Zeta potential of CS-TPP and MLF@CS-TPP

[0140]

[0141] 2.5 Fourier transform infrared spectroscopy analysis of microcapsules

[0142] Fourier transform spectroscopy is mainly used to analyze the structure of different chemical substances. When the physical and chemical environment of the functional group changes, the position and intensity of its characteristic absorption peak will also change accordingly. Figure 7 As shown, the characteristic peak of chitosan is 3357.7 cm -1 (-OH, -NH2 stretching vibration peak), 2867.5cm-1 (CH stretching vibration), 1646.2cm -1 (C=O amide I, N-acetylglucosamine), 1592.9 cm -1 (NH bending vibration (amide II, N-acetylated residue), 1416 cm -1 (NH stretching vibration, amide II (glucosamine), 1379.4 cm -1 (NH stretching vibration, amide III), 1148 cm -1 (COC stretching vibration), 1061.2cm -1 (CO stretching vibration), 893.2 cm -1 (Pyranose ring).

[0143] Sodium tripolyphosphate shows 1209.4cm -1 To 1162cm -1 The spectral bands are related to the stretching vibrations of sulfate ion PO and PO2 groups.

[0144] For the blank chitosan microcapsule particles, the peaks of amide I and II shifted to 1630 cm -1 and 1586.9cm -1 This indicates that there is an electrostatic interaction between the phosphate groups in TPP and the amines in chitosan. This is mainly due to the ionic cross-linking reaction between the amino groups of chitosan in the microcapsule particles and the phosphate groups of TPP, which enhances the molecular-molecular interaction of the microcapsule particles and forms a microcapsule structure. In addition, the 1209cm -1 The peak at (stretching vibration of PO group) shifts to 1148.2 cm in CS-TPP microcapsules. -1 , indicating the cross-linking effect between chitosan and TPP.

[0145] At 3284.5cm -1 The OH stretching vibration of hydrogen bonds between MLF molecules is at 2916.9 cm. The absorption peak is very strong, indicating that there are phenolic hydroxyl groups in MLF or hydroxyl groups on sugar. -1 -CH2- asymmetric stretching vibration appears at 1577cm -1 There is a C=O stretching vibration peak at 1037cm -1 There is a stretching vibration of the carbon-oxygen single bond in the primary alcohol at 998-562cm -1 The absorption peak at is caused by the position of the substituent on the benzene ring. Different flavonoid samples have different hydroxyl substitution positions, so the peak positions are different.

[0146] At 2800cm -1 and 2725.2cm-1 The spectral band appears at , indicating that after adding mulberry leaf flavonoids extract, the characteristic absorption peak of MLF@CS-TPP appears red-shifted, which can confirm the presence of the extract in the microcapsule.

[0147] 2.6 TGA thermogravimetric analysis

[0148] Thermogravimetric analysis is widely used to evaluate the thermal stability of microcapsule particles. The thermodynamic properties of MLF@CS-TPP were analyzed to show whether the thermal stability of MLF was improved. CS-TPP, MLF, and MLF@CS-TPP were studied at 30-800℃. Figure 8 As shown in the figure, the weight loss before 100℃ is attributed to the evaporation of bound water in the material. From the TGA curve, it can be seen that the weight loss of MLF@CS-TPP is divided into two obvious stages. The first stage has obvious weight loss in the range of 100-256℃, which is due to the preferential decomposition of MLF attached to the outer surface of MLF@CS-TPP. At 256℃, the remaining weight of MLF@CS-TPP is 68.23%. The second stage weight loss is at 256-700℃, and the weight loss comes from the decomposition of the microcapsule wall material. The weight change of MLF@CS-TPP corresponds to that of MLF. At the same temperature, the weight loss of MLF@CS-TPP is lower than that of MLF. The results show that the thermal stability of MLF is improved due to the encapsulation of microcapsules. After loading, the microcapsule particles require more energy to evaporate water and the load, and the loaded microcapsule particles require a higher temperature to decompose, reflecting the improvement of the thermal stability of the load by chitosan microcapsules, which is consistent with the experimental results.

[0149] 2.7 Color Measurement

[0150] Evaluating the color of microcapsules by measuring the parameters L*, a*, b*, chromaticity, and color change (ΔE) can provide useful data for potential use as food additives. The table shows the color parameters of five different samples (CS, TPP, CS-TPP, MLF@CS-TPP, MLF) measured by a colorimeter. Overall, the different samples have significant differences in brightness (L*), red / green (a*), and yellow / blue (b*) as well as color difference metrics. Comparing CS-TPP with MLF@CS-TPP, it is found that the brightness of MLF@CS-TPP is significantly lower than that of CS-TPP, while the brightness value of MLF is the lowest. At the same time, there are significant differences in the color difference values ​​of CS-TPP, MLF@CS-TPP, and MLF, which indicates that the addition of MLF in the preparation process of MLF@CS-TPP will have a significant effect on the color difference value, which may be caused by the excessive concentration of mulberry leaf flavonoids in the microcapsules.

[0151] Table 5 Color analysis of different materials

[0152] sample <![CDATA[L * ]]> <![CDATA[ * ]]> <![CDATA[b * ]]> △E CS <![CDATA[91.71±0.01 b ]]> <![CDATA[-1.552±0.004 c ]]> <![CDATA[8.748±0.01 b ]]> <![CDATA[82.794±0.01 b ]]> TPP <![CDATA[96.312±0.01 a ]]> <![CDATA[-0.454±0.01 b ]]> <![CDATA[0.592±0.01 e ]]> <![CDATA[87.084±0.02 a ]]> CS-TPP <![CDATA[86.216±0.04 c ]]> <![CDATA[-3.308±0.01 e ]]> <![CDATA[7.172±0.04 c ]]> <![CDATA[77.486±0.04 c ]]> MLF@CS-TPP <![CDATA[46.796±0.02 d ]]> <![CDATA[-2.498±0.02 d ]]> <![CDATA[18.854±0.02 a ]]> <![CDATA[41.698±0.01 d ]]> MLF <![CDATA[6.776±1.36 e ]]> <![CDATA[12.512±1.19 a ]]> <![CDATA[4.974±0.09 d ]]> <![CDATA[5.826±1.6 e ]]>

[0153] Superscript letters (ae) indicate significant differences in the same column (p < 0.05).

[0154] 2.8 In vitro release performance of drug-loaded microcapsules

[0155] like Fig. 9 As shown in the figure, the release of MLF@CS-TPP in vitro under the condition of pH 7.4 simulating physiological fluid showed a general trend of first fast and then slow. The rapid release in the early stage was caused by the unencapsulated mulberry leaf flavonoids and their high content in the microcapsules. After 3 hours, the cumulative release reached 67.8%, and the release rate began to slow down. After 24 hours, the cumulative release reached 72%, and the release rate tended to be flat. The results show that microcapsules prepared with ion gel can effectively slow down the release rate of mulberry leaf flavonoids.

[0156] 2.9 In vitro simulated gastrointestinal digestion

[0157] In order to better understand the relationship between extract type and carrier mixture, the present application conducted in vitro simulated digestion of microcapsules and evaluated the bioavailability index of mulberry leaf flavonoids. Fig.10 It can be seen that the bioavailability of mulberry leaf flavonoids in SGF gradually increases with time. This is because gastric protein promotes the release of mulberry leaf flavonoids under strong acid conditions. At the same time, MLF@CS-TPP also shows the same change. This is due to the direct release of mulberry leaf flavonoids attached to the surface of the microcapsule in the solution and the hydrolysis of the microcapsule surface. When the gastric juice digestion reaches the 4th hour, the bioavailability of mulberry leaf flavonoids and MLF@CS-TPP reaches the maximum of 11.92% and 80.9% respectively. The bioavailability of mulberry leaf flavonoids is lower than that of MLF@CS-TPP. This may be because the shell formed by chitosan and sodium tripolyphosphate protects mulberry leaf flavonoids and reduces direct contact with gastric acid. The results show that MLF@CS-TPP has better stability and can provide a certain protection for flavonoids in gastric juice, reduce the decomposition of flavonoids, and improve the bioavailability in gastric juice.

[0158] In SIF, trypsin is a proteolytic enzyme that can hydrolyze some chitosan microcapsule particles. At the same time, because the pH of SIF is relatively low, the bioavailability of its flavonoids is relatively high. Among them, the bioavailability of MLF@CS-TPP is much better than that of mulberry leaf flavonoids, indicating that the microencapsulation technology can achieve a sustained release effect of mulberry leaf flavonoids in the gastric digestion stage, and effectively release and be absorbed and utilized by the human body in the intestinal digestion stage.

[0159] The results show that in the simulated gastrointestinal digestion, the bioavailability of mulberry leaf flavonoids gradually increases with the digestion stage. Under acidic and weakly alkaline conditions, the interaction between unencapsulated mulberry leaf flavonoids and MLF@CS-TPP and pepsin, trypsin and α-amylase found that the bioavailability of MLF@CS-TPP increased rapidly and was higher than that of unencapsulated mulberry leaf flavonoids. This may be due to the fact that in the primary stage of gastrointestinal digestion, pH will cause different enzyme treatments to have different degrees of contact with flavonoids, thereby promoting the change of flavonoid content in the digestive juice, thereby increasing the bioavailability of MLF@CS-TPP. In the gastric digestion stage, the bioavailability reached the maximum value at 4h, indicating that the combined chemical bonds of mulberry leaf flavonoids were broken by the combined action of strong acid and pepsin, causing the release of mulberry leaf flavonoids in the microcapsule. In the intestinal digestion stage, the change in bioavailability is likely to come from the hydrolysis phenomenon in the weakly alkaline environment.

[0160] 2.10 Analysis of antioxidant activity after in vitro simulated digestion

[0161] Depend on Fig.11 and Fig.12 It can be seen that in the simulated gastric digestion stage, the DPPH scavenging ability of MLF@CS-TPP gradually increased with time, while the DPPH scavenging ability of MLF was relatively low. MLF was affected by strong acid, resulting in a decrease in its activity. Due to the shell protection of CS-TPP, MLF@CS-TPP can be slowly released in the gastric fluid environment, and its scavenging ability is different. At the same time, in the simulated gastric digestion stage, mulberry leaf flavonoids and MLF@CS-TPP have weak ABTS scavenging abilities. The scavenging ability of mulberry leaf flavonoids to ABTS does not change significantly with the time of gastric digestion stage, while MLF@CS-TPP is protected by the shell of CS-TPP, which allows MLF to be slowly released in the gastric fluid environment and can always maintain a high antioxidant capacity. In the simulated intestinal digestion stage, the DPPH scavenging ability of MLF@CS-TPP gradually increased with time, which is related to its sustained release effect. When MLF reached the maximum value at 1h, it gradually decreased. This may be because MLF was affected by intestinal fluid, resulting in a decrease in activity. At the same time, in the simulated intestinal digestion stage, the ABTS scavenging abilities of MLF@CS-TPP and MLF were as high as 69.9% and 69.8%, respectively, indicating that there was no significant effect on the ABTS scavenging abilities of MLF@CS-TPP and MLF under this environment.

[0162] The results show that the free radical scavenging ability of MLF@CS-TPP and MLF is significantly higher in the intestinal digestion stage than in the gastric digestion stage. In the simulated gastric digestion stage, the lower antioxidant capacity of MLF@CS-TPP is related to pH value, solubility, enzyme contact degree, and the amount of mulberry leaf flavonoids released by MLF@CS-TPP in the simulated gastric digestion stage. In the simulated intestinal digestion stage, it may be because the weak alkaline environment is conducive to the release of flavonoids, which leads to the significantly higher antioxidant capacity of MLF@CS-TPP and MLF than in the simulated gastric digestion stage. In addition, there is also the possibility that digestive enzymes can affect different soluble chemical structures, so that the active substances are released on the surface of the microcapsules, thereby exerting a higher antioxidant capacity.

[0163] 2.11 Microcapsule stability

[0164] 2.11.1 Photostability

[0165] The photostability of MLF@CS-TPP was evaluated under UV irradiation. Fig.13 As shown. The retention rate of mulberry leaf flavonoids in MLF@CS-TPP slowly decreased to 76.85% after 8 hours of irradiation, and most of the mulberry leaf flavonoids were retained in the microcapsule particles. The results show that MLF@CS-TPP is stable in nature and has a good protective effect on mulberry leaf flavonoids under ultraviolet light irradiation. The reason may be that the dense and compact structure of MLF@CS-TPP effectively prevents ultraviolet radiation from penetrating the microcapsule particles and contacting the encapsulated mulberry leaf flavonoids.

[0166] 2.11.2 Ion stability

[0167] Microcapsule particles will experience different ionic strengths when used in the food industry. Different concentrations of NaCl solution were added to study the effect of ionic strength on the stability of microcapsule particles. Fig.14 As shown in the figure, with the increase of NaCl ion concentration, the retention rate of mulberry leaf flavonoids in the solution becomes lower. It is possible that the interaction between microcapsule particles and flavonoids (such as electrostatic effect, van der Waals force, etc.) is weakened in a high salt environment. At the same time, the presence of high concentration of NaCl ions causes the change of microcapsule ion structure, resulting in a decrease in retention rate.

[0168] 2.12 Postharvest storage quality of mango fruit

[0169] After the mangoes are picked, the appearance of the mangoes changes most obviously during storage. First, the color changes from green to yellow, the skin changes from hard to soft, and black spots appear, causing the mangoes to rot and deteriorate. Figure 15-17As shown in the figure, after treatment with different concentrations of MLF, the changes over time were compared with CK. MLF1% and 2% showed significant black spots on the 10th day and the surface color changed from green to yellow. In the same period, CK had already shown large areas of black spots and obvious decay. For MLF4% and 8%, the appearance changes were not much different from the first day. The skin color slightly turned yellow, but was still mainly green. In the CS-TPP treatment, the fruit in the CS-TPP1% treatment group had yellow skin on the 7th day and obvious color changes on the surface of the skin on the 10th day, and black spots also appeared. In comparison, the fruit treated with CS-TPP2% had the same situation, but the preservation effect was better than CS-TPP1%. The high concentration treatment did not change significantly. The overall preservation effect of mango fruit treated with MLF@CS-TPP was obvious. From the appearance, there was no obvious change in the skin color and no black spots. Based on the above results, MLF@CS-TPP treatment has a significant effect on the preservation of mango, and the preservation capacity is: MLF@CS-TPP>MLF>CS-TPP>CK.

[0170] In summary, the present application successfully prepared mulberry leaf flavonoids microcapsules using ion gel technology and studied the structure, morphological characteristics, stability and in vitro release of the microcapsules. The present application shows that the microcapsules can successfully embed mulberry leaf flavonoids, with an embedding rate as high as 83.39%. The obtained microcapsules are spherical, with a smooth surface and an average diameter of 542.53±93.95nm. In a 24-hour in vitro release experiment, the microcapsules can achieve controlled release, and the release rate is appropriate, with a total release rate of 72%, and good release. After the in vitro digestion process, mulberry leaf flavonoids can still maintain antioxidant activity well and effectively scavenge free radicals, which helps to improve its bioavailability.

[0171] In addition, the results of this application have important application value in the fields of food and medicine. In the food industry, mulberry leaf flavonoid microcapsules can be used to develop functional foods with high antioxidant activity, providing consumers with healthy food options. In the field of medicine, it can be used as a drug carrier or active ingredient for the prevention and treatment of oxidative stress-related diseases. At the same time, this study also provides new ideas and methods for the comprehensive utilization of mulberry leaves and the development of the agricultural industry, which helps to increase the economic added value of mulberry leaves and promote the sustainable development of agriculture.

[0172] This application successfully improves the stability and biological activity of mulberry leaf flavonoids through microencapsulation technology, providing an important theoretical and practical basis for its application development, and is expected to have broad application prospects in the fields of food, medicine and agriculture.

[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing mulberry leaf flavonoid microcapsules, characterized in that: The following steps are involved: S1. Add mulberry leaf flavonoids into the chitosan solution, shear with a homogenizer after ultrasonication, heat and stir to obtain a mixed solution; S2, slowly dripping the sodium tripolyphosphate solution into the mixed solution, reacting and solidifying, the solution changes from clear to milky white liquid without precipitation, and then centrifuging, washing and collecting particles; S3, freezing the particles and freeze-drying them to obtain mulberry leaf flavonoids microcapsules.

2. The method for preparing mulberry leaf flavonoid microcapsules according to claim 1, characterized in that: The specific steps include: S1. Add mulberry leaf flavonoids to the chitosan solution, ultrasonicate for 10 minutes, shear for 3 minutes using a homogenizer at a rate of 12000 r / min, and stir for 15 minutes at 50°C and 500 r / min to obtain a mixed solution; S2. Slowly drip the sodium tripolyphosphate solution into the mixed solution, react and solidify for 1 hour, the solution changes from clear to milky white liquid without precipitation, and then centrifuge at 10000r / min for 30min, wash with ultrapure water for 3 times and collect the particles; S3. Freeze the particles at -80°C for 2 hours, and freeze-dry them using a freeze dryer for 14 hours to obtain mulberry leaf flavonoids microcapsules.

3. The method for preparing mulberry leaf flavonoid microcapsules according to claim 1 or 2, characterized in that: The extraction process of mulberry leaf flavonoids is as follows: fresh mulberry leaves are placed in an oven at 55°C, dried for 24 hours, crushed, and sieved through a 40-mesh sieve for later use; a certain amount of mulberry leaf powder is weighed into a beaker, and a 44% ethanol aqueous solution is added to the beaker according to a solid-liquid ratio of 1:27, and ultrasonic extraction is performed at 43°C for 30 minutes, and the residue is removed by filtration, and the filtrate is collected and centrifuged in a high-speed centrifuge at 10,000 r / min for 5 minutes to obtain the supernatant, which is concentrated to 10 mL using a rotary evaporator, and finally fixed to 50 mL with anhydrous ethanol for later use.

4. The method for preparing mulberry leaf flavonoid microcapsules according to claim 1 or 2, characterized in that: The preparation process of the chitosan solution is as follows: dissolving an appropriate amount of chitosan in 1% acetic acid water, dissolving and stirring at room temperature for 5 hours, shearing at a rate of 10000r / min for 3 minutes, adjusting the pH of the chitosan solution to 5 under strong magnetic stirring, filtering through a micro syringe filter with a pore size of 0.22μm to remove insoluble substances, and obtaining a chitosan solution; and storing the prepared chitosan solution in a refrigerator at 4°C for use.

5. The method for preparing mulberry leaf flavonoid microcapsules according to claim 1 or 2, characterized in that: The preparation process of the sodium tripolyphosphate solution is as follows: weighing an appropriate amount of sodium tripolyphosphate and dissolving it in ultrapure water, magnetically stirring for 2 hours, filtering through a micro syringe filter with a pore size of 0.45 μm to remove insoluble substances to obtain a sodium tripolyphosphate solution; and storing the prepared sodium tripolyphosphate solution in a refrigerator at 4° C. for later use.

6. The method for preparing mulberry leaf flavonoid microcapsules according to claim 1 or 2, characterized in that: The mass ratio of chitosan to sodium tripolyphosphate is (3-10):

1.

7. The method for preparing mulberry leaf flavonoid microcapsules according to claim 1 or 2, characterized in that: The volume ratio of chitosan to mulberry leaf flavonoids is (1-5):1; the volume ratio of sodium tripolyphosphate to mulberry leaf flavonoids is (1-5):

1.

8. A mulberry leaf flavonoid microcapsule, characterized in that: The mulberry leaf flavonoids microcapsules prepared by the method described in any one of claims 1 to 7 have an average diameter of 542.53±93.95 nm, and the embedding rate of mulberry leaf flavonoids in the mulberry leaf flavonoids microcapsules is 83.39%.

9. The mulberry leaf flavonoids microcapsule according to claim 8, characterized in that: The total release rate of the mulberry leaf flavonoids microcapsules in a 24-hour in vitro release experiment was 72%.

10. Application of the mulberry leaf flavonoids microcapsules according to claim 8 in the fields of food and medicine.

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