A nanoemulsion of total flavonoids from jujube seed, its preparation method and application

Abstract

This invention belongs to the technical field of plant extract nanoemulsion preparation. To address the current limited research on flavonoids in jujube seed, particularly regarding the extraction, separation, and purification of total flavonoids from jujube seed, this invention provides a jujube seed total flavonoid nanoemulsion, its preparation method, and applications. The process involves extracting fatty oil from jujube seed, enzymatically hydrolyzing jujube seed powder, fermenting the jujube seed enzymatic hydrolysate, extracting total flavonoids, purifying the total flavonoids, and preparing the nanoemulsion using a self-emulsification method. The resulting jujube seed total flavonoid nanoemulsion exhibits high purity, a simple process, and is environmentally friendly and safe. It shows promising applications in the preparation of drugs to improve insomnia, depression, and anxiety. It provides excellent encapsulation and delivery capabilities, protecting the total flavonoids from hydrolytic enzymes, gastrointestinal pH, and other environmental conditions, promoting drug absorption, and improving drug bioavailability and brain penetration. It easily crosses intestinal intercellular spaces and the epithelial barrier, increasing the cellular uptake rate of jujube seed total flavonoids and enhancing the brain targeting and effectiveness of in vivo drug administration.

Description

Technical Field

[0001] This invention belongs to the technical field of plant extract nanoemulsion preparation, and particularly to jujube seed extract nanoemulsion, specifically relating to a jujube seed total flavonoid nanoemulsion, its preparation method, and its application. Background Technology

[0002] Ziziphus jujuba var. spinosa is a plant belonging to the genus Ziziphus of the family Rhamnaceae. Ziziphus mauritiana The mature seeds of *L.* are commonly known as "Yunnan Jujube Seed," "Burmese Jujube Seed," or "Imported Sour Jujube Seed." Jujube seed was first recorded in *Diannan Materia Medica* by Lan Mao of the Ming Dynasty: "Abundant in Yunnan. Eating it prevents the dissipation of vital energy, promotes sleep, regulates the interaction between the heart and kidneys, and long-term use can clear the eyes and prolong life." The 2005 edition of *Yunnan Provincial Standards for Traditional Chinese Medicine* describes jujube seed as "sweet and neutral; entering the heart and liver meridians." It has the effects of "calming the mind and soothing the nerves, relieving irritability and astringing sweat," and is used for "insomnia due to deficiency, palpitations, restlessness, and night sweats." As a commonly used folk medicine in Yunnan, jujube seed is used locally to treat insomnia. Currently, more than 110 chemical components have been isolated and identified from jujube seed, including saponins, flavonoids, alkaloids, amino acids, and nucleosides. Among them, jujube seed has a high content of flavonoids. Studies have found that total phenols and total flavonoids in jujube seed can synergistically increase sleep time in mice with sodium pentobarbital, thus improving sleep.

[0003] Flavonoids are widely distributed in the plant kingdom. Their main types include flavones, flavonols, dihydroflavones, biflavones, and isoflavones. They have pharmacological activities such as lowering blood lipids, antidepressant, antitumor, and vasodilator.

[0004] Nanoemulsions are transparent or semi-transparent systems with low viscosity, isotropic properties, and thermodynamic and kinetic stability, formed by combining an aqueous phase, an oil phase, a surfactant, and a co-surfactant in appropriate proportions. These systems typically have particle sizes of 10–100 nm. Nanoemulsions are simple to prepare, highly safe, and can improve the solubility of poorly soluble drugs, enhance drug stability and bioavailability. Due to their unique particle size, they also exhibit sustained-release and targeted effects. Furthermore, depending on the characteristics of the loaded drug and the type of emulsion, different administration methods are available: transdermal, oral, mucosal, and injectable.

[0005] Current research on Ziziphus jujuba var. spinosa is mostly limited to its role as a counterfeit of Ziziphus jujuba var. spinosa, focusing on the identification of its chemical components and comparison with those of Ziziphus jujuba var. spinosa. Research on the flavonoids, which are abundant in Ziziphus jujuba var. spinosa, is scarce, and there are no reports on the extraction, separation, and purification of total flavonoids from Ziziphus jujuba var. spinosa. Furthermore, its subsequent dosage form development and pharmacological effects remain unexplored. Summary of the Invention

[0006] To address the current limited research on flavonoids in jujube seeds, particularly the lack of studies on the extraction, separation, and purification of total flavonoids from jujube seeds, this invention provides a jujube seed total flavonoid nanoemulsion, its preparation method, and its applications. The resulting jujube seed total flavonoid nanoemulsion is high in purity, produced using a simple, green, and safe process. Furthermore, the invention relates to the application of this jujube seed total flavonoid nanoemulsion in the preparation of medicines for improving insomnia, depression, and anxiety.

[0007] To achieve the above objectives, the present invention is implemented by the following technical solution: a method for preparing total flavonoid nanoemulsion from jujube seed, comprising the following steps:

[0008] (1) Extraction of jujube seed fat oil: The jujube seed medicinal material is pulverized into powder of 10-65 mesh. The jujube seed powder is placed in a supercritical CO2 extraction vessel. Petroleum ether is used as an entrainer. Petroleum ether is added according to the material-liquid ratio of petroleum ether to jujube seed 1:0.05g / mL. The jujube seed fat oil is extracted by supercritical CO2 extraction. The CO2 flow rate is controlled at 25-35L / h, the extraction pressure is 20-40Mpa, the extraction temperature is 40-50℃, and the extraction is carried out for 3-5h. The fat oil is collected for later use. The residual petroleum ether in the defatted jujube seed powder is evaporated and stored for later use.

[0009] (2) Enzymatic hydrolysis of jujube kernel powder: Distilled water of 2-3 times the weight of jujube kernel powder was added to the defatted jujube kernel powder in step (1), and a compound enzyme with a final concentration of 0.3-0.4 mg / ml was added to enzymatically hydrolyze and destroy the cell wall of jujube kernel. The enzymatic hydrolysis temperature was controlled at 40-60℃, the pH of the enzymatic hydrolysate was 3-5, and the enzymatic hydrolysis was carried out for 1.5-2 hours. The compound enzyme was a mixture of cellulase, pectinase, papain, β-glucanase and hemicellulase in a mass ratio of 2:1:3:1:1.

[0010] (3) Fermented Jujube Seed Enzymatic Hydrolysate: After complete enzymatic hydrolysis, pasteurize at 65-95℃ for 10-30 min; after natural cooling, add lactic acid bacteria to the jujube seed enzymatic hydrolysate at a mass percentage of 3%-5%, shake on a shaker at 27-32℃ and 80-140r / min for 5-10 min to mix evenly, and then carry out fermentation. Control the fermentation temperature at 26-42℃ and the fermentation time at 48-72h. After the fermentation is completed, obtain the jujube seed fermentation liquid.

[0011] (4) Extraction of total flavonoids from jujube seeds: Add anhydrous ethanol to the fully fermented jujube seed fermentation broth at a material-to-liquid ratio of 15-25:1 to adjust its mass concentration to 70%. Place it in an ultrasonic-microwave synergistic extractor for extraction, controlling the extraction temperature at 55-65℃, ultrasonic power at 300-500W, and microwave power at 500-600W. Extract for 8-12 minutes. Repeat the extraction 3-4 times. Combine the extract filtrates and concentrate under reduced pressure until there is no alcohol odor. Extract the obtained concentrate with ethyl acetate 4-5 times and combine the extracts. Concentrate the ethyl acetate extract under reduced pressure and then evaporate to dryness in a water bath to obtain the extract. The extract yield is 3.5%-4.0%. The total flavonoid content in the extract is considered to be 10.0%-12.0%, and the flavonoid aglycone content is 70%-95% of the total mass of the total flavonoids, indicating that the total flavonoids in jujube seeds have been completely extracted.

[0012] Total flavonoid purification: The obtained extract was dissolved in hot water and purified using HPD-100 macroporous resin. First, 5-8 column volumes of water were used to remove impurities such as sugars. Then, gradient elution was performed using ethanol of different concentrations. The loading solution concentration was controlled at 7-10 mg / mL, the loading volume at 3.5-6.5 BV, and the pH at 4.30-5.10. The gradient elution ethanol concentration was 10%-60%. The elution volume for each gradient was 4-5 BV, and the elution rate was 3.5-4.5 BV·h. -1 After elution, the ethanol from each elution gradient is recovered, and the mixture is concentrated and dried under reduced pressure in a water bath at 70-80℃ to obtain total flavonoid powder from jujube seed. The yield of total flavonoid fraction from jujube seed is 0.6%-1.0%, and the purity of total flavonoids from jujube seed is calculated to be 75%-90%, with flavonoid aglycone content accounting for 70%-95% of the total flavonoid mass.

[0013] (6) Preparation of nanoemulsion by self-emulsification method: According to the volume ratio of emulsifier polyoxyethylene octylphenol ether-10 (OP-10) to co-emulsifier anhydrous ethanol of 2-3:1, anhydrous ethanol is added to OP-10 and stirred at 60-120r / min for 20-30min at room temperature until a uniform emulsifier / co-emulsifier system is formed.

[0014] The jujube seed fatty oil obtained in step (1) is mixed with ethyl oleate at a volume ratio of 1:1-2 to form the oil phase;

[0015] Slowly add the oil phase to the emulsifier / co-emulsifier system and stir at 60-120 r / min for 20-30 min; add the purified total flavonoid powder of jujube seed in step (5) to the mixture and add purified water dropwise while stirring at 60-120 r / min at room temperature until the solution is clear and transparent, which is the total flavonoid nanoemulsion of jujube seed; wherein: the mass percentage of total flavonoid powder of jujube seed to emulsifier / co-emulsifier system, oil phase and water is 1:8-10:1-2:18-20.

[0016] In step (1), the jujube seed medicinal material is placed in a pulverizer and pulverized for 1-3 seconds at 1500-2000 r / min.

[0017] The extract in step (4) is concentrated under reduced pressure to obtain a concentrated solution. The working pressure for the reduced pressure concentration is 0.07-0.09 MPa, and the water bath temperature is 70-80℃. The ratio of the concentrated solution to ethyl acetate is 1:2-3 for extraction.

[0018] In steps (4) and (5), rutin was used as a reference standard, and the total flavonoid content in the extract was determined by spectrophotometry. Daphne flavonoid, genistein, quercetin, kaempferol and apigenin were used as reference standards, and the total amount of total flavonoid aglycones in the above-mentioned jujube seed was determined by HPLC-DAD chromatography, and the jujube seed flavonoid aglycone content was calculated.

[0019] The total flavonoid nanoemulsion of jujube seed described in step (6) has a particle size of 20-30 nm, a polydispersity index (PDI) of 0.1-0.3, and an encapsulation efficiency of ≥97%.

[0020] This invention also provides a method for preparing a soft capsule of total flavonoids from jujube seed using the nanoemulsion prepared by the method described above. The specific preparation method is as follows: 20-25g of gelatin is added to 20mL of water, the temperature is controlled at 15℃-20℃, and after stirring to accelerate expansion, it is heated to 75℃-80℃ to melt. 1-1.5g of polyethylene glycol and 8-10mL of glycerin are added and mixed well. The mixture is degassed under reduced pressure until the melt is transparent and free of bubbles. The prepared nanoemulsion solution and gelatin solution are pressed into soft capsules using a rotary molding machine. The surface oil layer is washed away with anhydrous ethanol, and the capsules are dried in a ventilated environment at 24℃-27℃ to obtain the soft capsules. The ratio of the nanoemulsion solution to the gelatin solution is 1:1-1.5.

[0021] The working pressure for degassing is 0.06-0.08 MPa.

[0022] The present invention also provides a total flavonoid nanoemulsion soft capsule of jujube seed prepared using the method described above.

[0023] The present invention also provides the application of the aforementioned total flavonoid nanoemulsion soft capsules of jujube seed in medicines for improving insomnia, depression and anxiety.

[0024] The nanoemulsion of total flavonoids from jujube seed prepared in this invention was observed by electron microscopy. The obtained nanoemulsion had a uniform particle size distribution and good dispersion. The particle size of the nanoemulsion was measured by Malvern particle size analyzer and was between 20-30 nm. The polydispersity index (PDI) was 0.1-0.3. A standard curve was established by HPLC with rutin as the standard. After adding a certain amount of methanol to the supernatant of the nanoemulsion of total flavonoids from jujube seed and ultrasonically breaking the emulsion, the encapsulation rate was found to be over 97%.

[0025] This invention also investigated the behavioral indicators related to the improvement of insomnia, depression, and anxiety by total flavonoids from jujube seed nanoemulsion. The results showed that total flavonoids from jujube seed significantly increased the sleep onset rate and shortened the sleep latency time in sodium pentobarbital-induced depressed rats; significantly increased the immobility time in the forced swimming test, activity in the open field test, and sucrose preference in depressed rats; and significantly increased the time spent in the open-arm area in the elevated cruciate maze test. This indicates that the total flavonoids from jujube seed nanoemulsion preparation has significant sedative-hypnotic, antidepressant, and anti-anxiety effects, and can be applied to drugs with different administration methods according to usage needs.

[0026] The total flavonoid nanoemulsion of jujube seed described in this invention can be applied to various drug delivery methods, including oral administration, transdermal administration, nasal administration, and mucosal administration. Furthermore, the refined extract of total flavonoids from jujube seed can be formulated into various dosage forms, including capsules, tablets, granules, drop pills, oral liquids, sugar-coated tablets, film-coated tablets, dispersible tablets, tea bags, and health drinks.

[0027] Compared with existing technologies, this invention uses a compound enzyme to decompose and destroy the effective components of the cell wall of jujube seeds, and combines this with ultrasound-microwave assisted extraction to accelerate the release of total flavonoids from jujube seeds. Furthermore, single-factor experiments and response surface methodology were used to optimize the extraction process of total flavonoids from jujube seeds. Compared with traditional single extraction methods such as water extraction and ultrasound-assisted extraction, this invention greatly improves extraction efficiency and effectively shortens extraction time. It expands the application of synergistic extraction technology for total flavonoids from jujube seeds based on existing extraction processes.

[0028] Supercritical CO2 extraction of jujube seed fatty oil was used as an excipient, which was then mixed with ethyl oleate to prepare the oil phase of a nanoemulsion. This achieved the reuse of jujube seed resources. Jujube seed oil is rich in fatty acids, esters, alcohols, and alkanes, and has certain sedative and hypnotic effects. This further realizes the "pharmaceutical-excipient integration" application of jujube seed oil.

[0029] Flavonoids in jujube seeds mostly exist in glycoside form. To address the poor solubility and low bioavailability of flavonoid glycosides, which lead to slow absorption and insignificant efficacy of flavonoid drugs, this invention utilizes lactic acid bacteria to biotransform the extracted total flavonoids from jujube seeds, converting over 70% of the flavonoid glycosides into their corresponding flavonoid aglycones. This significantly increases the biological activity and bioavailability of the total jujube seed flavonoids, expanding their clinical applications.

[0030] This invention prepares high-precision total flavonoids from jujube seed into nanoemulsion soft capsules. Compared with ordinary dosage forms, these capsules offer significant advantages, providing excellent encapsulation and delivery capabilities, protecting the total flavonoids from the effects of hydrolytic enzymes, gastrointestinal pH, and other environmental conditions. Simultaneously, their low surface tension allows them to easily pass through the hydration layer of the gastrointestinal wall, enabling direct contact between the total flavonoids and gastrointestinal epithelial cells, thereby promoting drug absorption and improving bioavailability. The total flavonoids can easily cross the intercellular spaces and epithelial barrier of the intestinal tract, significantly increasing their cellular uptake rate, thus enhancing their brain-targeting and high efficacy when administered in vivo. Attached Figure Description

[0031] Figure 1 Micrograph of total flavonoids from jujube seed nanoemulsion;

[0032] Figure 2 The response surface optimization diagram of the enzymatic hydrolysis process of total flavonoids in jujube kernel;

[0033] Figure 3 The response surface optimization diagram for the extraction process of total flavonoids from jujube seeds;

[0034] Figure 4 The diagram shows the state of nanoemulsions at different proportions. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0037] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0039] Example 1:

[0040] 1. Preparation of total flavonoid extract from jujube seed:

[0041] (1) Extraction of jujube seed fat oil: The jujube seed medicinal material is pulverized into powder of 10-65 mesh. The jujube seed powder is placed in a supercritical CO2 extraction vessel. Petroleum ether is used as an entrainer. Petroleum ether is added according to the material-liquid ratio of petroleum ether to jujube seed 1:0.05g / mL. The jujube seed fat oil is extracted by supercritical CO2 extraction. The CO2 flow rate is controlled at 25-35L / h, the extraction pressure is 20-40Mpa, the extraction temperature is 40-50℃, and the extraction is carried out for 3-5h. The fat oil is collected for later use. The residual petroleum ether in the defatted jujube seed powder is evaporated and stored for later use.

[0042] (2) Enzymatic hydrolysis of jujube kernel powder: Distilled water of 2-3 times the weight of jujube kernel powder was added to the defatted jujube kernel powder in step (1), and a compound enzyme with a final concentration of 0.3-0.4 mg / ml was added to enzymatically hydrolyze and destroy the cell wall of jujube kernel. The enzymatic hydrolysis temperature was controlled at 40-60℃, the pH of the enzymatic hydrolysate was 3-5, and the enzymatic hydrolysis was carried out for 1.5-2 hours. The compound enzyme was a mixture of cellulase, pectinase, papain, β-glucanase and hemicellulase in a mass ratio of 2:1:3:1:1.

[0043] (3) Fermented Jujube Seed Enzymatic Hydrolysate: After complete enzymatic hydrolysis, pasteurize at 65-95℃ for 10-30 min; after natural cooling, add lactic acid bacteria to the jujube seed enzymatic hydrolysate at a mass percentage of 3%-5%, shake on a shaker at 27-32℃ and 80-140r / min for 5-10 min to mix evenly, and then carry out fermentation. Control the fermentation temperature at 26-42℃ and the fermentation time at 48-72h. After the fermentation is completed, obtain the jujube seed fermentation liquid.

[0044] (4) Extraction of total flavonoids from jujube seeds: Add anhydrous ethanol to the fully fermented jujube seed fermentation broth at a material-to-liquid ratio of 15-25:1 to adjust its concentration to 70%, and extract it using an ultrasonic-microwave synergistic extractor. Control the extraction temperature at 55-65℃, the ultrasonic power at 300-500W, and the microwave power at 500-600W. Extract for 8-12 minutes. Repeat the extraction 3-4 times. Combine the extract filtrates and concentrate under reduced pressure until there is no alcohol odor. Extract the concentrate with ethyl acetate 4-5 times and combine the extracts. Concentrate the ethyl acetate extract under reduced pressure and then evaporate to dryness in a water bath to obtain the extract. The extract yield is 3.5%-4.0%. The total flavonoid content in the extract is considered to be 10.0%-12.0%, and the flavonoid aglycone content is 70%-95% of the total mass of the total flavonoids, indicating that the total flavonoids in jujube seeds have been completely extracted.

[0045] 2. Determination of total flavonoid content in jujube seed:

[0046] Accurately weigh 25 mg of dried rutin reference standard to constant weight, place it in a 50 mL volumetric flask, add 30 mL of ethanol, sonicate to dissolve, cool, add ethanol to the mark, and shake well. Accurately measure 20 mL of the rutin ethanol solution into a 50 mL volumetric flask, dilute with water to the mark, and shake well to obtain the rutin reference standard solution. Accurately measure 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL of the rutin reference standard solution into 25 mL volumetric flasks, add 6 mL of water to each, add 1 mL of 5% sodium nitrite solution, shake well, and let stand for 6 min. Add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min. Add 10 mL of 4% sodium hydroxide solution, then add water to the mark, shake well, and let stand for 15 min. Using the corresponding reagents as blanks, immediately measure the absorbance at 500 nm using ultraviolet-visible spectrophotometry. Plot a standard curve with rutin content as the x-axis and absorbance as the y-axis.

[0047] A 0.2 mg / mL sample solution of total flavonoids from jujube seed was prepared following the same procedure as the reference solution. The absorbance was measured at a wavelength of 500 nm, revealing that the total flavonoid content in the jujube seed extract was 11.3%.

[0048] 3. Determination of flavonoid aglycone formation rate:

[0049] Total flavonoid purification: The obtained extract was dissolved in hot water and purified using HPD-100 macroporous resin. First, 5-8 column volumes of water were used to remove impurities such as sugars. Then, gradient elution was performed using ethanol of different concentrations. The loading solution concentration was controlled at 7-10 mg / mL, the loading volume at 3.5-6.5 BV, and the pH at 4.30-5.10. The gradient elution ethanol concentration was 10%-60%. The elution volume for each gradient was 4-5 BV, and the elution rate was 3.5-4.5 BV·h. -1 After elution, the ethanol from each grade of elution is recovered, and the mixture is concentrated and dried under reduced pressure in a water bath at 70-80℃ to obtain total flavonoid powder from jujube seed. The yield of total flavonoid fraction from jujube seed is 0.6%-1.0%, and the purity of total flavonoids from jujube seed is calculated to be 75%-90%, with flavonoid aglycone content accounting for 70%-95% of the total flavonoid mass.

[0050] Accurately weigh the reference standards angelicaflavin, genistein, quercetin, kaempferol, and apigenin, and prepare single reference standard stock solutions with a concentration of 1 mg / mL for each of these standards by dissolving them in methanol. Accurately pipette appropriate amounts of these stock solutions and dissolve them in methanol to prepare mixed reference standard stock solutions of different concentrations. The concentration of angelicaflavin is 100 μg / mL, and the concentration of the other reference standards is 10 μg / mL. Accurately measure appropriate amounts of the above mixed reference standard solutions and dilute them with methanol to prepare mixed reference standard solutions with three concentration gradients: 10-fold, 100-fold, and 1000-fold.

[0051] Take approximately 5 mg of total flavonoid extract from jujube seed, accurately weigh it, place it in a stoppered conical flask, add 10 mL of methanol, weigh it, sonicate it for 20 min, place it at room temperature, replenish the lost weight with methanol, shake well, filter it through a 0.45 μm microporous membrane, collect the filtrate to obtain the test solution, inject it for analysis, record the peak area of ​​the analyte, and calculate that the five flavonoid aglycones account for 87.33% of the total flavonoid mass.

[0052] The chromatographic column was an Apollo C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase was 0.1% formic acid water (A) and acetonitrile (B), with gradient elution (0–26 min, 10%–20% B; 26–30 min, 20%–23% B; 30–43 min, 23%–26% B; 43–45 min, 26%–37% B; 45–47 min, 37% B; 47–54 min, 37%–39% B; 54–63 min, 39%–100% B), and the flow rate was 1.0 ml / min; the column temperature was 25 ℃, and the injection volume was 10 μl; the UV detection wavelengths were 227 nm and 335 nm; the ELSD parameters were: drift tube temperature 95 ℃, and air flow rate 3.0 L / min.

[0053] 4. Preparation of nanoemulsion by self-emulsification method: According to the volume ratio of emulsifier OP-10 to co-emulsifier anhydrous ethanol of 2-3:1, add anhydrous ethanol to OP-10 and stir at 60-120 r / min for 20-30 min at room temperature until a uniform emulsifier / co-emulsifier system is formed.

[0054] The obtained jujube seed fatty oil and ethyl oleate were mixed at a volume ratio of 1:1-2 to form the oil phase;

[0055] Slowly add the oil phase to the emulsifier / co-emulsifier system and stir at 60-120 rpm for 20-30 min. Add purified jujube seed total flavonoid powder to the mixture and add purified water dropwise while stirring at 60-120 rpm at room temperature until the solution is clear and transparent. This is the jujube seed total flavonoid nanoemulsion. The mass percentage of jujube seed total flavonoid powder to the emulsifier / co-emulsifier system, oil phase, and water is 1:8-10:1-2:18-20.

[0056] Observation of nanoemulsion morphology: A drop of nanoemulsion sample was placed on a glass slide, covered with a coverslip, and observed and photographed under an electron microscope. The micrographs are shown below. Figure 1 As shown, the obtained nanoemulsions exhibited a uniform particle size distribution and good dispersion.

[0057] 5. Determination of nanoemulsion particle size and polydispersity index (PDI):

[0058] The nanoemulsion sample was diluted at a ratio of 1:10 (v / v) to avoid multiple scattering effects. It was then equilibrated at room temperature (25±0.5°C) for 60 seconds. After the instrument was cycled 30 times and stabilized, the particle size of the nanoemulsion was measured to be 22.75±0.16 nm and the PDI value was 0.18±0.08.

[0059] 6. Determination of nanoemulsion encapsulation efficiency: Accurately measure 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of 1 mg / mL rutin standard solution into 10 mL volumetric flasks, add a certain amount of methanol, sonicate to dissolve, dilute to the mark, mix thoroughly, and take 20 μL of each for high-performance liquid chromatography (HPLC). Measure at a wavelength of 254 nm. Perform linear regression of concentration (x) against peak area (y).

[0060] The nanoemulsion was precisely transferred to a 10 mL volumetric flask, and a certain amount of methanol was added for ultrasonic demulsification. The solution was then diluted to the mark with methanol to obtain a buckwheat flavonoid nanoemulsion solution with a concentration of 100.0 μg / mL. 20 μL of this solution was injected into a high-performance liquid chromatography (HPLC) sample and measured at a wavelength of 254 nm. The encapsulation efficiency of the total flavonoid nanoemulsion from jujube seed was found to be 98.35 ± 0.04%.

[0061] Chromatographic column: Agilent ODS C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: methanol: water: glacial acetic acid (50:50:0.5, v / v); flow rate: 1.0 mL / min; detection wavelength: 254 nm; column temperature: 30℃; injection volume: 20 μL.

[0062] Example 2: Optimization Experiment of Enzymatic Hydrolysis Process of Total Flavonoids in Jujube Seed

[0063] 1. Single-factor investigation of the enzymatic hydrolysis process of total flavonoids in jujube kernel

[0064] (1) Investigation of the composition of complex enzymes: An appropriate amount of Ziziphus jujuba seed was pulverized in a pulverizer, passed through a 16-mesh sieve, and then placed in a supercritical CO2 extraction vessel. A certain amount of petroleum ether was added as an entrainer to extract the fatty oil of Ziziphus jujuba seed. The fatty oil was collected, the residual petroleum ether was evaporated, and it was stored for later use. The defatted Ziziphus jujuba seed powder was then mixed with 3 times the amount of distilled water and enzymes with different compositions at a final concentration of 0.4 mg / ml. These enzymes were cellulase, pectinase, papain, β-glucanase, hemicellulase, cellulase-pectinase complex enzyme (2:1), cellulase-papain complex enzyme (2:3), cellulase-β-glucanase (2:1), cellulase-hemicellulase complex enzyme (2:1), and cellulase-pectinase-papain-β-glucanase-hemicellulase complex enzyme (2:1:3:1:1). The enzymes were enzymatically hydrolyzed at 40℃ and pH 4 for 2 h. The total flavonoid extract of Ziziphus jujuba seed was then prepared according to the subsequent operation steps. The total flavonoid content in the extract was determined by spectrophotometry. The results are shown in Table 1. Considering the total flavonoid content under different enzyme composition conditions, the cellulase-pectinase-papain-β-glucanase-hemicellulase complex enzyme (2:1:3:1:1) was selected as the optimal enzyme composition.

[0065] Table 1: Composition of the complex enzyme

[0066]

[0067] (2) Investigation of the amount of compound enzyme: Take an appropriate amount of Ziziphus jujuba seed, grind it in a pulverizer, pass it through a 16-mesh sieve, and place it in a supercritical CO2 extraction vessel. Add a certain amount of petroleum ether as an entrainer to extract the fatty oil of Ziziphus jujuba seed. Collect the fatty oil, evaporate the residual petroleum ether, and store it for later use. Add 3 times the amount of distilled water and a complex enzyme (1:1:1:1:1) of cellulase-pectinase-papainase-β-glucanase-hemicellulase) with a final concentration of 0.2, 0.3, 0.4, 0.5 and 0.6 mg / ml to the defatted Ziziphus jujuba seed powder. Enzymatic hydrolysis was carried out at 40℃ and pH 4 for 2 h. Prepare the total flavonoid extract of Ziziphus jujuba seed according to the subsequent operation steps. The content of total flavonoids in the extract was determined by spectrophotometry. The results are shown in Table 2. Considering the content of total flavonoids under different compound enzyme amounts, the final concentration of 0.3-0.4 mg / ml was selected as the optimal amount of compound enzyme added.

[0068] Table 2: Results of Complex Enzyme Amount

[0069]

[0070] (3) Investigation of enzymatic hydrolysis time: An appropriate amount of Ziziphus jujuba seed was crushed in a pulverizer, passed through a 16-mesh sieve, and placed in a supercritical CO2 extraction vessel. A certain amount of petroleum ether was added as an entrainer to extract the fatty oil of Ziziphus jujuba seed. The fatty oil was collected, the residual petroleum ether was evaporated, and it was stored for later use. The defatted Ziziphus jujuba seed powder was mixed with 3 times the amount of distilled water and a complex enzyme (1:1:1:1:1) with a final concentration of 0.4 mg / ml cellulase-pectinase-papain-β-glucanase-hemicellulase. Enzymatic hydrolysis was carried out at 40℃ and pH 4 for 10 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, and 3 h, respectively. The total flavonoid extract of Ziziphus jujuba seed was prepared according to the operation steps. The content of total flavonoids in the extract was determined by spectrophotometry. The results are shown in Table 3. Considering the content of total flavonoids under different enzymatic hydrolysis time conditions, the optimal enzymatic hydrolysis time was selected as 1.5-2.5 h.

[0071] Table 3: Results of Enzymatic Hydrolysis Time

[0072]

[0073] (4) Investigation of enzymatic hydrolysis temperature: Take an appropriate amount of Ziziphus jujuba seed, grind it in a pulverizer, pass it through a 16-mesh sieve, and place it in a supercritical CO2 extraction vessel. Add a certain amount of petroleum ether as an entrainer to extract the fatty oil of Ziziphus jujuba seed. Collect the fatty oil, evaporate the residual petroleum ether, and store it for later use. Add 3 times the amount of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml cellulase-pectinase-papain-β-glucanase-hemicellulase to the obtained defatted Ziziphus jujuba seed powder. Enzymatic hydrolysis was carried out at 20℃, 40℃, 60℃, 80℃, and 100℃, with a pH of 4, for 2 h. Prepare total flavonoid extract of Ziziphus jujuba seed according to the operation steps. Measure the total flavonoid content in the extract by spectrophotometry. The results are shown in Table 4. Considering the total flavonoid content under different enzymatic hydrolysis temperature conditions, the optimal extraction temperature was selected as 40-60℃.

[0074] Table 4: Results of Enzymatic Hydrolysis Temperature

[0075]

[0076] (5) Investigation of pH for enzymatic hydrolysis: An appropriate amount of Ziziphus jujuba seed was pulverized in a pulverizer, passed through a 16-mesh sieve, and then placed in a supercritical CO2 extraction vessel. A certain amount of petroleum ether was added as an entrainer to extract the fatty oil of Ziziphus jujuba seed. The fatty oil was collected, the residual petroleum ether was evaporated, and it was stored for later use. The defatted Ziziphus jujuba seed powder was mixed with 3 times the amount of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml cellulase-pectinase-papain-β-glucanase-hemicellulase. Enzymatic hydrolysis was performed for 2 h at pH 1, 3, 5, 7, 9, and 11 and at a temperature of 40℃. The total flavonoid extract of Ziziphus jujuba seed was prepared according to the operation steps. The total flavonoid content in the extract was determined by spectrophotometry. The results are shown in Table 5. Considering the different enzymatic hydrolysis pH conditions, the optimal enzymatic hydrolysis pH was selected as 3-5.

[0077] Table 5: Results of pH enzymatic hydrolysis

[0078]

[0079] 2. Response Surface Optimization of Total Flavonoid Extraction from Jujube Seeds: A t-test was performed on the total flavonoid content of jujube seeds under the above-mentioned conditions, indicating that the main factors affecting the extraction rate of total flavonoids from jujube seeds were the amount of compound enzyme, hydrolysis time, and hydrolysis temperature. Based on the Box-Behnken central composite design principle, a three-factor, three-level response surface design was designed using Design Expert 8.0.6 software, with the amount of compound enzyme, hydrolysis time, and hydrolysis temperature as independent variables, to further optimize the enzymatic hydrolysis process of total flavonoids from jujube seeds. In Design Expert software, the model was further subjected to typicality analysis, and the range values ​​of the amount of compound enzyme, hydrolysis time, and hydrolysis temperature were set. The software automatically predicted the optimal extraction conditions. The optimized response surface graph of the enzymatic hydrolysis process of total flavonoids from jujube seeds is shown below. Figure 2 As shown, taking all factors into consideration, the following extraction conditions were selected: final concentration of the compound enzyme 0.4 mg / ml; enzymatic hydrolysis temperature 40℃; enzymatic hydrolysis time 110 min; and the predicted total flavonoid content was 11.81%.

[0080] Example 3: Optimization Experiment of Total Flavonoid Extraction Process from Jujube Seed

[0081] 1. Single-factor investigation of the extraction process of total flavonoids from jujube seed

[0082] (1) Investigation of the concentration of ethanol as the extraction solvent: An appropriate amount of Ziziphus jujuba seed was pulverized in a pulverizer, passed through a 16-mesh sieve, and then placed in a supercritical CO2 extraction vessel. A certain amount of petroleum ether was added as an entrainer to extract the fatty oil of Ziziphus jujuba seed. The fatty oil was collected, the residual petroleum ether was evaporated, and it was stored for later use. The defatted Ziziphus jujuba seed powder was mixed with 3 times the amount of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml. Enzymatic hydrolysis was carried out at 40℃ and pH 4 for 2 h. After complete enzymatic hydrolysis, pasteurization was performed (65-95℃, 10-30 min). After cooling, 4% lactic acid bacteria were inoculated into the Ziziphus jujuba seed hydrolysate, and fermentation was carried out at 37℃ with shaking for 60 h. Anhydrous ethanol was added to the fully fermented jujube seed fermentation broth to adjust its concentration to 50%, 60%, 70%, 80%, and 90% ethanol, with a material-to-liquid ratio of 1:20. The broth was then placed in an ultrasonic-microwave synergistic extraction apparatus and extracted for 10 min at an ultrasonic power of 400 W, a microwave power of 550 W, and an extraction temperature of 60℃, for three extractions. The total flavonoid extract of jujube seed was prepared according to subsequent procedures. The total flavonoid content in the extract was determined spectrophotometrically. The results are shown in Table 6. Considering the total flavonoid content under different ethanol concentrations, the optimal ethanol concentration was selected as 60%-70%.

[0083] Table 6

[0084]

[0085] (2) Investigation of the material-liquid ratio: Take an appropriate amount of Ziziphus jujuba seed, grind it in a pulverizer, pass it through a 16-mesh sieve, and place it in a supercritical CO2 extraction vessel. Add a certain amount of petroleum ether as an entrainer to extract the fatty oil of Ziziphus jujuba seed. Collect the fatty oil, evaporate the residual petroleum ether, and store it for later use. Add 3 times the amount of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml cellulase-pectinase-papain-β-glucanase-hemicellulase) to the obtained defatted Ziziphus jujuba seed powder. Enzymatic hydrolysis was carried out at 40℃ and pH 4 for 2 h. After complete enzymatic hydrolysis, pasteurization was carried out (65-95℃, 10-30 min). After cooling, 4% lactic acid bacteria were added to the Ziziphus jujuba seed enzymatic hydrolysate, and fermentation was carried out at 37℃ with shaking for 60 h. Anhydrous ethanol was added to the fully fermented jujube seed broth to adjust its concentration to 70%, with solid-liquid ratios of 1:5, 1:10, 1:15, 1:20, 1:25, and 1:30, respectively. The mixture was placed in an ultrasonic-microwave synergistic extraction apparatus and extracted for 10 min at an ultrasonic power of 400 W, a microwave power of 550 W, and an extraction temperature of 60℃, for three extractions. The total flavonoid extract of jujube seed was prepared according to subsequent procedures. The total flavonoid content in the extract was determined spectrophotometrically. The results are shown in Table 7. Considering the total flavonoid content under different solid-liquid ratios, the optimal solid-liquid ratio was selected as 1:15-25.

[0086] Table 7

[0087]

[0088] (3) Examination of ultrasonic power: An appropriate amount of Ziziphus jujuba seed was pulverized in a pulverizer, passed through a 16-mesh sieve, and then placed in a supercritical CO2 extraction vessel. A certain amount of petroleum ether was added as an entrainer to extract the fatty oil of Ziziphus jujuba seed. The fatty oil was collected, the residual petroleum ether was evaporated, and it was stored for later use. The defatted Ziziphus jujuba seed powder was mixed with 3 times the amount of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml. Enzymatic hydrolysis was carried out at 40℃ and pH 4 for 2 h. After complete enzymatic hydrolysis, pasteurization was performed (65-95℃, 10-30 min). After cooling, 4% lactic acid bacteria were added to the Ziziphus jujuba seed hydrolysate, and fermentation was carried out at 37℃ with shaking for 60 h. Anhydrous ethanol was added to the fully fermented jujube seed broth to adjust its concentration to 70%, and the material-to-liquid ratio to 1:20. The broth was then placed in an ultrasonic-microwave synergistic extraction apparatus and extracted for 10 min for three times under ultrasonic powers of 200 W, 300 W, 400 W, 500 W, and 600 W, a microwave power of 550 W, and an extraction temperature of 60℃. The total flavonoid extract from the jujube seed was prepared according to subsequent procedures. The total flavonoid content in the extract was determined spectrophotometrically. The results are shown in Table 8. Considering the total flavonoid content under different ultrasonic power conditions, the optimal ultrasonic power was selected as 300-500 W.

[0089] Table 8

[0090]

[0091] (4) Microwave power test: Take an appropriate amount of Ziziphus jujuba seed, grind it in a pulverizer, pass it through a 16-mesh sieve, and place it in a supercritical CO2 extraction vessel. Add a certain amount of petroleum ether as an entrainer to extract the fatty oil of Ziziphus jujuba seed. Collect the fatty oil, evaporate the residual petroleum ether, and store it for later use. Add 3 times the amount of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml cellulase-pectinase-papain-β-glucanase-hemicellulase) to the obtained defatted Ziziphus jujuba seed powder. Enzymatic hydrolysis was carried out at 40℃ and pH 4 for 2 h. After complete enzymatic hydrolysis, pasteurization was carried out (65-95℃, 10-30 min). After cooling, 4% lactic acid bacteria were added to the Ziziphus jujuba seed hydrolysate, and fermentation was carried out at 37℃ with shaking for 60 h. Anhydrous ethanol was added to the fully fermented jujube seed broth to adjust its concentration to 70%, and the material-to-liquid ratio to 1:20. The broth was then placed in an ultrasonic-microwave synergistic extraction apparatus and extracted for 10 min for three times under the following conditions: ultrasonic power of 400 W, microwave power of 450 W, 500 W, 550 W, 600 W, and 650 W, and extraction temperature of 60℃. The total flavonoid extract of jujube seed was prepared according to subsequent procedures. The total flavonoid content in the extract was determined spectrophotometrically. The results are shown in Table 9. Considering the total flavonoid content under different microwave power conditions, the optimal microwave power was selected as 500-600 W.

[0092] Table 9

[0093]

[0094] (5) Investigation of extraction temperature: Take an appropriate amount of Ziziphus jujuba seed, grind it in a pulverizer, pass it through a 16-mesh sieve, and place it in a supercritical CO2 extraction vessel. Add a certain amount of petroleum ether as an entrainer to extract the fatty oil of Ziziphus jujuba seed. Collect the fatty oil, evaporate the residual petroleum ether, and store it for later use. Add 3 times the amount of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml cellulase-pectinase-papain-β-glucanase-hemicellulase) to the obtained defatted Ziziphus jujuba seed powder. Enzymatic hydrolysis was carried out at 40℃ and pH 4 for 2 h. After complete enzymatic hydrolysis, pasteurization was carried out (65-95℃, 10-30 min). After cooling, 4% lactic acid bacteria were inoculated into the Ziziphus jujuba seed hydrolysate, and fermentation was carried out at 37℃ with shaking for 60 h. Anhydrous ethanol was added to the fully fermented jujube seed broth to adjust its concentration to 70%, and the material-to-liquid ratio to 1:20. The broth was then placed in an ultrasonic-microwave synergistic extraction apparatus and extracted for 10 min for 3 times at ultrasonic power of 400 W, microwave power of 550 W, and extraction temperatures of 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃. The total flavonoid extract of jujube seed was prepared according to subsequent procedures. The total flavonoid content in the extract was determined spectrophotometrically. The results are shown in Table 10. Considering the total flavonoid content under different extraction temperatures, the optimal extraction temperature was selected as 55-65℃.

[0095] Table 10

[0096]

[0097] (6) Investigation of extraction time: An appropriate amount of Ziziphus jujuba seed was pulverized in a pulverizer, passed through a 16-mesh sieve, and then placed in a supercritical CO2 extraction vessel. A certain amount of petroleum ether was added as an entrainer to extract the fatty oil of Ziziphus jujuba seed. The fatty oil was collected, the residual petroleum ether was evaporated, and it was stored for later use. The defatted Ziziphus jujuba seed powder was mixed with 3 times the amount of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml. Enzymatic hydrolysis was carried out at 40℃ and pH 4 for 2 h. After complete enzymatic hydrolysis, pasteurization was performed (65-95℃, 10-30 min). After cooling, 4% lactic acid bacteria were inoculated into the Ziziphus jujuba seed hydrolysate, and fermentation was carried out at 37℃ with shaking for 60 h. Anhydrous ethanol was added to the fully fermented jujube seed broth to adjust its concentration to 70%, and the material-to-liquid ratio to 1:20. The broth was then placed in an ultrasonic-microwave synergistic extraction apparatus and extracted for 6 min, 8 min, 10 min, 12 min, and 14 min, respectively, at an ultrasonic power of 400 W, a microwave power of 550 W, and an extraction temperature of 60 ℃, for three extractions. The total flavonoid extract of jujube seed was prepared according to subsequent procedures. The total flavonoid content in the extract was determined spectrophotometrically. The results are shown in Table 11. Considering the total flavonoid content under different extraction time conditions, the optimal extraction time was selected as 8-12 min.

[0098] Table 11

[0099]

[0100] (7) Examination of extraction times: An appropriate amount of Ziziphus jujuba seed was pulverized in a pulverizer, passed through a 16-mesh sieve, and then placed in a supercritical CO2 extraction vessel. A certain amount of petroleum ether was added as an entrainer to extract the fatty oil of Ziziphus jujuba seed. The fatty oil was collected, the residual petroleum ether was evaporated, and it was stored for later use. The defatted Ziziphus jujuba seed powder was mixed with 3 times the amount of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml. Enzymatic hydrolysis was carried out at 40℃ and pH 4 for 2 h. After complete enzymatic hydrolysis, pasteurization was performed (65-95℃, 10-30 min). After cooling, 4% lactic acid bacteria were inoculated into the Ziziphus jujuba seed hydrolysate, and fermentation was carried out at 37℃ with shaking for 60 h. Anhydrous ethanol was added to the fully fermented jujube seed broth to adjust its concentration to 70%, and the material-to-liquid ratio to 1:20. The broth was then placed in an ultrasonic-microwave synergistic extraction apparatus and extracted for 10 min at an ultrasonic power of 400 W, a microwave power of 550 W, and an extraction temperature of 60 ℃. Extraction was repeated 1, 2, 3, 4, and 5 times. The total flavonoid extract of jujube seed was prepared according to subsequent procedures. The total flavonoid content in the extract was determined spectrophotometrically. The results are shown in Table 12. Considering the total flavonoid content under different extraction times, the optimal number of extractions was selected as 3-4 times.

[0101] Table 12

[0102]

[0103] 2. Response Surface Optimization of Total Flavonoid Extraction Process from Jujube Seed: A t-test was conducted on the extraction rate of total flavonoids from jujube seed under the above-mentioned conditions, indicating that the main factors affecting the extraction rate were ultrasonic power, microwave power, and extraction time. Based on the Box-Behnken central composite design principle, a 4-factor, 3-level response surface design was designed using Design Expert 8.0.6 software, with ultrasonic power, microwave power, and extraction time as independent variables, to further optimize the extraction process of total flavonoids from jujube seed. In Design Expert software, the model's typicality was further analyzed, setting the ultrasonic power, microwave power, extraction temperature, and the range of extraction temperature. The software automatically predicted the optimal extraction conditions. The optimized response surface diagram of the total flavonoid extraction process from jujube seed is shown below. Figure 3 As shown; considering all factors, the selected extraction conditions were: ultrasonic power 490 W; microwave power 600 W; extraction time 12 min. The predicted total flavonoid content was 11.86%.

[0104] Example 4: Optimization Experiment of Preparation Process of Total Flavonoids Nanoemulsion from Jujube Seed

[0105] 1. Single-factor investigation of the preparation process of total flavonoids nanoemulsion from jujube seed

[0106] (1) Investigation of the ratio of emulsifier OP-10 to co-emulsifier anhydrous ethanol: OP-10 and anhydrous ethanol were weighed in ratios of 1:1, 2:1, 3:1 and 4:1. Jujube seed oil and ethyl oleate were mixed in a 1:1 ratio to prepare the oil phase. The oil phase was slowly added to the emulsifier / co-emulsifier and stirred evenly (60~120 r / min, 20~30 min). Jujube seed total flavonoid powder was added to the mixture, and purified water was added dropwise while stirring at room temperature (60~120 r / min) until the solution was clear and transparent to form a stable emulsion, thus preparing jujube seed total flavonoid nanoemulsion. The changes in coagulation time, coagulation color and coagulation texture under different concentration ratios were observed. Considering the state of the nanoemulsion under different ratios, the optimal ratio was selected as 2-3:1.

[0107] (2) Investigation of the ratio of jujube seed oil to ethyl oleate: OP-10 and anhydrous ethanol were weighed in a ratio of 2:1. Jujube seed oil and ethyl oleate were mixed in ratios of 1:1, 1:2, 1:3, and 1:4 to prepare the oil phase. The oil phase in a ratio of 1:9 was slowly added to the emulsifier / co-emulsifier and stirred evenly (60~120 r / min, 20~30 min). Jujube seed total flavonoid powder was added to the mixture, and purified water was added dropwise at room temperature while stirring (60~120 r / min) until the solution was clear and transparent to form a stable emulsion, thus preparing jujube seed total flavonoid nanoemulsion. The changes in coagulation time, coagulation color, and coagulation texture under different concentration ratios were observed. Considering the state of the nanoemulsion under different ratios, the optimal ratio was selected as 1:1-2.

[0108] (3) Investigation of the ratio of total flavonoids from jujube seed to emulsifier / co-emulsifier: OP-10 and anhydrous ethanol were weighed in a ratio of 2:1. Jujube seed oil and ethyl oleate were mixed in a ratio of 1:1 to prepare the oil phase. The oil phase was slowly added to the emulsifier / co-emulsifier and stirred evenly (60~120 r / min, 20~30 min). Total flavonoids from jujube seed powder with emulsifier / co-emulsifier ratios of 7:1, 8:1, 9:1, 10:1, and 11:1 were added to the mixture, and purified water was added dropwise at room temperature while stirring (60~120 r / min) until the solution was clear and transparent to form a stable emulsion, thus preparing jujube seed total flavonoid nanoemulsion. The changes in coagulation time, coagulation color, and coagulation texture under different concentration ratios were observed. Considering the state of the nanoemulsion under different ratios, the optimal ratio was selected as 8-10:1.

[0109] (4) Investigation of the ratio of total flavonoids from jujube seed to emulsifier / co-emulsifier: OP-10 and anhydrous ethanol were weighed in a ratio of 2:1. Jujube seed oil and ethyl oleate were mixed in a ratio of 1:1 to prepare the oil phase. The oil phase was slowly added to the emulsifier / co-emulsifier and stirred evenly (60~120 r / min, 20~30 min). Total flavonoids powder from jujube seed with ratios of 1:1, 2:1, 3:1, and 4:1 to the oil phase were added to the mixture, and purified water was added dropwise at room temperature while stirring (60~120 r / min) until the solution was clear and transparent to form a stable emulsion, thus preparing jujube seed total flavonoid nanoemulsion. The changes in coagulation time, coagulation color, and coagulation texture under different concentration ratios were observed. Considering the state of the nanoemulsion under different ratios, the optimal ratio was selected as 1-2:1.

[0110] (5) Investigation of the ratio of total flavonoids from jujube seed to aqueous phase: OP-10 and anhydrous ethanol were weighed in a ratio of 2:1. Jujube seed oil and ethyl oleate were mixed in a ratio of 1:1 to prepare the oil phase. The oil phase was slowly added to the emulsifier / co-emulsifier and stirred evenly (60~120 r / min, 20~30 min). Jujube seed total flavonoid powder was added to the mixture, and purified water with ratios of 1:16, 1:18, 1:20, 1:22, and 1:24 was added dropwise at room temperature while stirring (60~120 r / min) until the solution was clear and transparent to form a stable emulsion, thus preparing jujube seed total flavonoid nanoemulsion. The changes in coagulation time, coagulation color, and coagulation texture under different concentration ratios were observed. Considering the state of the nanoemulsion under different ratios, the optimal ratio was selected as 1:18-20.

[0111] 2. Response Surface Optimization of the Preparation Process of Jujube Seed Total Flavonoids Nanoemulsion: A t-test was conducted on the extraction rate of jujube seed total flavonoids under the above-mentioned conditions. The results showed that the main factors affecting the coagulation time, color, and texture of the jujube seed total flavonoids nanoemulsion were the emulsifier / co-emulsifier ratio, the oil phase ratio, and the water phase ratio. Based on the Box-Behnken central composite design principle, a three-factor, three-level response surface design was designed using Design Expert 8.0.6 software, with the emulsifier / co-emulsifier ratio, oil phase ratio, and water phase ratio as independent variables, to further optimize the preparation process of jujube seed total flavonoids nanoemulsion. In Design Expert software, the model was further subjected to typicality analysis, and the range values ​​of the emulsifier / co-emulsifier ratio, oil phase ratio, and water phase ratio were set. The software automatically predicted the optimal emulsification conditions. Considering all factors, the selected emulsification ratio should be 1:8-10:1-2:18-20 for jujube seed total flavonoids to emulsifier / co-emulsifier, oil phase, and water.

[0112] Example 5: Preparation of Jujube Seed Total Flavonoid Nanoemulsion: An appropriate amount of jujube seed was pulverized in a grinder, passed through a 16-mesh sieve, and then placed in a supercritical CO2 extraction vessel. A certain amount of petroleum ether was added as an entrainer to extract the jujube seed fatty oil. The fatty oil was collected, the residual petroleum ether was evaporated, and the oil was stored for later use. The obtained defatted jujube seed powder was then mixed with three times the amount of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.1 mg / ml. Enzymatic hydrolysis was performed at 60℃ and pH 6 for 1 h. After complete hydrolysis, pasteurization (80℃, 20 min) was used for sterilization. After cooling, 4% lactic acid bacteria were inoculated into the jujube seed hydrolysate, and fermentation was carried out at 37℃ with shaking for 60 h. Anhydrous ethanol was added to the fully fermented jujube seed fermentation broth to adjust its concentration to 70% ethanol, with a material-to-liquid ratio of 1:20. The mixture was placed in an ultrasonic-microwave synergistic extraction apparatus and extracted for 10 min at an ultrasonic power of 400 W, a microwave power of 550 W, and an extraction temperature of 60℃. This extraction was repeated three times. The resulting filtrate was concentrated under reduced pressure until no alcohol odor remained. The concentrate was then extracted with ethyl acetate, and the extracts were combined. After concentration under reduced pressure, the extract was evaporated to dryness in a water bath to obtain an extract. The extract was dissolved in hot water and purified using HPD-100 macroporous resin. First, 5-8 column volumes of water were used to remove impurities such as sugars, followed by gradient elution with 10%-60% ethanol. After elution, the ethanol was recovered, and the extract was concentrated and dried under reduced pressure in a water bath at 70℃-90℃ to obtain refined jujube seed total flavonoid powder. OP-10 and anhydrous ethanol were weighed in a 2:1 ratio. Jujube seed oil and ethyl oleate were mixed in a 1:1 ratio to prepare the oil phase. Slowly add the oil phase to the emulsifier / co-emulsifier at a ratio of 9:1 and stir until homogeneous (60-120 r / min, 20-30 min). Add jujube seed total flavonoid powder at a ratio of 10:1 to the mixture, and add purified water at a ratio of 1:2 (60-120 r / min) dropwise while stirring at room temperature until the solution becomes clear and transparent to form a stable emulsion, thus preparing jujube seed total flavonoid nanoemulsion.

[0113] Example 6: Preparation of Jujube Seed Total Flavonoid Nanoemulsion: Three times the volume of distilled water and a complex enzyme (2:1:3:1:1) of cellulase-pectinase-papain-β-glucanase-hemicellulase) with a final concentration of 0.4 mg / ml were added to defatted jujube seed powder. Enzymatic hydrolysis was carried out at 40℃ and pH 4 for 2 h. Fermentation was not carried out with lactic acid bacteria; the remaining methods were the same as those described in Example 5.

[0114] Example 7: Preparation of total flavonoid nanoemulsion from jujube seed: Three times the volume of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml cellulase-pectinase-papain-β-glucanase-hemicellulase) were added to the defatted jujube seed powder. Enzymatic hydrolysis was performed at 40℃ and pH 4 for 2 h. Anhydrous ethanol was added to the fully fermented jujube seed broth to adjust its concentration to 50% ethanol, with a material-to-liquid ratio of 1:10. The broth was placed in an ultrasonic-microwave synergistic extraction apparatus and extracted for 5 min at an ultrasonic power of 200 W, a microwave power of 250 W, and an extraction temperature of 40℃. This extraction was repeated three times. The remaining methods were the same as described in Example 5.

[0115] Example 8: Preparation of total flavonoid nanoemulsion from jujube seed: Three times the volume of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml cellulase-pectinase-papain-β-glucanase-hemicellulase) were added to the defatted jujube seed powder. Enzymatic hydrolysis was performed at 40℃ and pH 4 for 2 h. The resulting extract was dissolved in hot water, purified using HPD-100 macroporous resin (without removing sugars or other impurities), and then directly eluted with a gradient of 10%-60% ethanol. The remaining methods were the same as described in Example 5.

[0116] Example 9: Preparation of total flavonoid powder from jujube seed: Three times the volume of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml cellulase-pectinase-papain-β-glucanase-hemicellulase) were added to the defatted jujube seed powder. Enzymatic hydrolysis was performed at 40℃ and pH 4 for 2 h. The powder was concentrated and dried to obtain refined total flavonoid powder from jujube seed. Nanoemulsion was not prepared; the remaining methods were the same as in Example 5.

[0117] Example 10: Preparation of total flavonoid nanoemulsion from jujube seed: Three times the volume of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml cellulase-pectinase-papain-β-glucanase-hemicellulase) were added to the defatted jujube seed powder. Enzymatic hydrolysis was carried out at 40℃ and pH 4 for 2 h. The remaining methods were the same as in Example 5.

[0118] Example 11: Preparation of Jujube Seed Total Flavonoid Nanoemulsion: Three times the volume of distilled water and a complex enzyme (2:1:3:1:1) with a final concentration of 0.4 mg / ml cellulase-pectinase-papain-β-glucanase-hemicellulase) were added to the defatted jujube seed powder. Enzymatic hydrolysis was performed at 40℃ and pH 4 for 2 h. OP-10 and anhydrous ethanol were weighed in a 1:1 ratio. The oil phase was slowly added to the emulsifier / co-emulsifier at a 4:1 ratio and stirred until homogeneous. Jujube seed total flavonoid powder was added to the mixture in a 5:1 ratio, and the remaining steps were the same as in Example 5.

[0119] Example 12: Preparation of total flavonoid nanoemulsion from jujube seed: Defatted jujube seed powder was mixed with three times the volume of distilled water and a complex enzyme (2:1:3:1:1) of cellulase-pectinase-papain-β-glucanase-hemicellulase at a final concentration of 0.4 mg / ml. Enzymatic hydrolysis was carried out at 40℃ and pH 4 for 2 h.

[0120] Weigh out OP-10 and anhydrous ethanol in a 1:1 ratio. Slowly add the oil phase to the emulsifier / co-emulsifier in a 4:1 ratio and stir until homogeneous. Add jujube seed total flavonoid powder in a 5:1 ratio to the mixture, and follow the same procedure as in Example 5.

[0121] Example 13: Study on the antidepressant, anxiety and insomnia effects of total flavonoids from jujube seed

[0122] 1. Animal grouping, drug administration, and model replication: 120 healthy male SD rats were randomly divided into 7 groups of 12 rats each after 7 days of acclimatization. These groups included a blank control group, a model group, a positive control group (venlafaxine hydrochloride 35 mg / kg), and the total flavonoids from jujube seed (0.1 g / kg) group (Examples 5-12). Drug administration was performed simultaneously with model establishment. A CUMS rat depression model was established, with rats housed individually. A single stressor was randomly selected daily, with the same stimulus used no more than 4 times cumulatively. Examples included heat stimulation at 50 ℃ for 10 min, swimming in 4 ℃ ice water for 5 min, ultrasound stimulation at 60 Hz for 3 h, tail clamping for 2 min, 24-hour day-night reversal, 10 foot shocks (10 s each), water deprivation for 24 h, restraint for 3 h, and fasting for 24 h, etc., avoiding repetition, and continuing for 4 weeks. Rats in the normal control group consumed normal food and water without any stimulation; rats in the normal control group and model group were administered equal volumes of physiological saline by gavage.

[0123] 2. Behavioral Testing: Sugar Water Preference Test: Before the experiment, each rat was given a 1% sucrose solution and drinking water for sugar water preference training. After training, the rats were fasted and deprived of water for 12 hours. Then, two identical, pre-weighed water bottles (one bottle of 1% sucrose solution and one bottle of drinking water) were placed at the same height in each rat cage, and the sugar water consumption was measured over 4 hours. The sugar water preference degree was calculated. The sugar water preference test was conducted on day 28 of the experiment. Sugar water preference degree = sugar water consumption / (sugar water consumption + normal water consumption) × 100%. The experimental results are shown in Table 13.

[0124] Table 13: CUMS Depressed Rats' Sugar Water Preference Experiment ( (n = 10)

[0125]

[0126] Note: Compared with the blank group, ##P <0.01; compared with the model group, ** P <0.01, * P <0.05.

[0127] Compared with the control group, the sucrose preference rate in the model group was significantly lower, which was statistically significant. P <0.01, the model is valid; compared with the model group, the sucrose preference rate increased in the positive drug group, and the difference was statistically significant. P <0.05); Compared with the model group, the total flavonoid groups of jujube seed in Examples 5-12 all significantly increased the sugar water preference rate; among them, the total flavonoid group of jujube seed in Example 10 showed the most significant increase in sugar water preference rate ( P The result was <0.01, indicating that the total flavonoids in jujube seed have certain significance in improving depression.

[0128] Open field test: The open field test was conducted on day 28 of the experiment. The test was performed in an open field box measuring 50 cm long, 10 cm wide, and 50 cm high, with the bottom divided into 25 equal small squares. The experiment was conducted in a quiet environment from 2:00 PM to 7:00 PM. First, the rats were placed in the central square to acclimatize for 1 minute. Then, the number of squares the rats crossed and the number of times they stood upright were recorded over 4 minutes. The experimental results are shown in Table 14.

[0129] Table 14: Open field test of CUMS depressed rats ( (n = 10)

[0130]

[0131] Note: Compared with the blank group, ## P <0.001, ### P <0.001; compared with the model group, ** P <0.01, * P <0.05.

[0132] Compared with the control group, the model group had a significantly reduced number of grid crossings and upright times, and the difference was statistically significant. P <0.001; P <0.01, the model is valid; compared with the model group, the positive drug group showed a significant increase in the number of grid crossings and the number of upright standing times, and the difference was statistically significant. P <0.01; P <0.01); Compared with the model group, the total flavonoid groups of jujube seed in Examples 5-12 all significantly increased the number of grid crossings and the number of upright times (P <0.05); while the effect of the total flavonoids group of jujube seed in Example 10 was more significant ( P The total flavonoids in jujube seed (<0.01) significantly enhanced the rats' activity level, indicating that the total flavonoids in jujube seed have a certain depressive effect on rats.

[0133] Forced swimming experiment: The experimental setup consisted of a cylindrical plexiglass container (50 cm high, 20 cm in diameter), with a water depth of 30 cm and a temperature of 25 ± 1℃. On day 28, each rat underwent 15 minutes of forced swimming training. On day 29, the forced swimming experiment was conducted. Each rat was first allowed 2 minutes of acclimatization, and the immobility time (s) within the following 4 minutes was recorded. The immobile state was defined as the rat ceasing to struggle in the water and floating, with only minor limb movements to keep its head afloat. The results are shown in Table 15.

[0134] Table 15: Forced swimming test in CUMS depressed rats ( (n = 10)

[0135]

[0136] Note: Compared with the blank group, ### P <0.001; compared with the model group, ** P <0.01, * P <0.05.

[0137] Compared with the control group, the model group had a significantly longer immobile swimming time, and the difference was statistically significant. P <0.001, the model is valid; compared with the model group, the swimming immobility time in the positive drug group was significantly reduced, and the difference was statistically significant. P <0.01); Compared with the model group, the total flavonoids in jujube seed groups in Examples 5-12 significantly reduced the immobile swimming time ( P <0.05); while the effect of the total flavonoids group of jujube seed in Example 10 was more significant ( P <0.01), indicating a significant improvement in activity level, suggesting that total flavonoids from jujube seed have a certain significance in improving depression.

[0138] Elevated cross maze experiment: The elevated cross maze consisted of two closed arms and two open arms. Each arm was 50 cm long and 10 cm wide, with a 10 cm × 10 cm wide platform where the closed and open arms met. The edge of the open arm was 1 cm high, and the edge of the closed arm was 40 cm high. The apparatus was 50 cm above the ground, and the platform was black. Rats were placed into the maze from the central area facing the closed arms, and their activities within the maze were recorded over 5 minutes. The number of times the rats entered the open arms and the time spent in the open arms were observed. Each rat was thoroughly cleaned after the test to avoid odor residue affecting the experimental results. The results are shown in Table 16.

[0139] Table 16: CUMS Depressed Rats Elevated Cross Maze Test ( (n = 10)

[0140]

[0141] Note: Compared with the blank group, ### P <0.001; compared with the model group, *** P <0.01, ** P <0.01, * P <0.05.

[0142] Compared with the model group, the number of times rats entered the open arm and the time spent in the open arm were significantly reduced in the model group, and the differences were statistically significant. P <0.001; P <0.001, the model is established; compared with the model group, the number of times rats entered the open arm and the time spent in the open arm were significantly increased in the positive drug group, and the difference was statistically significant. P <0.01; P <0.01); Compared with the model group, the total flavonoid groups of jujube seed in Examples 5-12 all significantly increased the number of times they entered the open arm and the residence time in the open arm ( P <0.05); while the effect of the total flavonoids group of jujube seed in Example 10 was more significant ( P <0.01). This indicates that total flavonoids from jujube seed have some significance in improving anxiety-like behaviors.

[0143] Sleep experiment: 30 minutes after the last gavage administration, rats in each group were intraperitoneally injected with 29.3 mg / kg sodium pentobarbital. The sleep latency and sleep duration (from the disappearance to the recovery of the righting reflex) were recorded. The results are shown in Table 17.

[0144] Table 17: Sleep Experiment in CUMS Depressed Rats ( (n = 10)

[0145]

[0146] Note: Compared with the blank group, ## P <0.01, ### P <0.001; compared with the model group, *** P <0.01, ** P <0.01, * P <0.05.

[0147] Compared with the control group, the sleep latency and sleep duration of rats in the model group were significantly increased, and the difference was statistically significant. P <0.01; P <0.001, the model is established; compared with the model group, the positive drug group showed a significant reduction in sleep latency and sleep duration, and the difference was statistically significant. P <0.05; P <0.05); In Examples 5-12, the total flavonoids in jujube seed significantly reduced sleep latency and sleep duration compared to the model group. P <0.05); while the effect of the total flavonoids group of jujube seed in Example 10 was more significant ( P <0.01; P <0.001). This indicates that total flavonoids from jujube seed have some significance in improving insomnia.

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 invention.

Claims

1. A method for preparing a nanoemulsion of total flavonoids from jujube seed, wherein the nanoemulsion has sedative-hypnotic, antidepressant, and anti-anxiety effects, characterized in that: Includes the following steps: (1) Extraction of jujube seed fat oil: The jujube seed medicinal material is pulverized into powder of 10-65 mesh. The jujube seed powder is placed in a supercritical CO2 extraction vessel. Petroleum ether is used as an entrainer. Petroleum ether is added according to the material-liquid ratio of petroleum ether to jujube seed 1:0.05g / mL. The jujube seed fat oil is extracted by supercritical CO2 extraction. The CO2 flow rate is controlled at 25-35L / h, the extraction pressure is 20-40Mpa, the extraction temperature is 40-50℃, and the extraction is carried out for 3-5h. The fat oil is collected for later use. The residual petroleum ether in the defatted jujube seed powder is evaporated and stored for later use. (2) Enzymatic hydrolysis of jujube kernel powder: Distilled water of 2-3 times the weight of jujube kernel powder was added to the defatted jujube kernel powder in step (1), and a compound enzyme with a final concentration of 0.3-0.4 mg / ml was added to enzymatically hydrolyze and destroy the cell wall of jujube kernel. The enzymatic hydrolysis temperature was controlled at 40-60℃, the pH of the enzymatic hydrolysate was 3-5, and the enzymatic hydrolysis was carried out for 1.5-2.5 h. The compound enzyme was a compound enzyme composed of cellulase, pectinase, papain, β-glucanase and hemicellulase mixed in a mass ratio of 2:1:3:1:

1. (3) Fermented Jujube Seed Hydrolysate: After complete enzymatic hydrolysis, the Jujube Seed Hydrolysate is pasteurized at 65-95℃ for 10-30 min. After natural cooling, lactic acid bacteria are added to the Jujube Seed Hydrolysate at a mass percentage of 3%-5%. The mixture is shaken at 27-32℃, 80-140 r / min for 5-10 min. After mixing evenly, fermentation is carried out. The fermentation temperature is controlled at 26-42℃ and the fermentation time is 48-72 h. After fermentation, Jujube Seed Fermentation Broth is obtained. (4) Extraction of total flavonoids from jujube seeds: Add anhydrous ethanol to the fully fermented jujube seed fermentation broth at a material-to-liquid ratio of 15-25:1, adjust its mass concentration to 70%, and extract it in an ultrasonic-microwave synergistic extractor. Control the extraction temperature at 55-85℃, ultrasonic power at 300-500W, and microwave power at 500-600W. Extract for 8-12 minutes. Repeat the extraction 3-4 times. Combine the extract filtrates and concentrate under reduced pressure until there is no alcohol odor. The concentration of the concentrate is 0.9-1.2 g / mL based on the amount of raw medicinal material. Extract the concentrate with ethyl acetate 4-5 times and combine the extracts. Concentrate the ethyl acetate extract under reduced pressure and then evaporate it to dryness in a water bath to obtain the extract. The extract yield is 3.5%-4.0%. The total flavonoid content in the extract is considered to be 10.0%-12.0%, and the flavonoid aglycone content is 70%-95% of the total mass of the total flavonoids. This indicates that the total flavonoids in jujube seeds have been completely extracted. (5) Purification of total flavonoids: The obtained extract was dissolved in hot water and purified using HPD-100 macroporous resin. First, impurities were removed with 5-8 column volumes of water, and then gradient elution was performed using different concentrations of ethanol. The concentration of the loading solution was controlled at 7-10 mg / mL, the loading volume was 3.5-6.5 BV, and the pH was 4.30-5.

10. The concentration of ethanol used for gradient elution was 10%-60%. The elution volume of each gradient was 4-5 BV, and the elution rate was 3.5-4.5 BV·h. -1 After elution, the ethanol from each elution gradient is recovered, and the mixture is concentrated and dried under reduced pressure in a water bath at 70-80℃ to obtain total flavonoid powder from jujube seed. The yield of total flavonoid fraction from jujube seed is 0.6%-1.0%, and the purity of total flavonoids from jujube seed is calculated to be 75%-90%, with flavonoid aglycone content accounting for 70%-95% of the total flavonoid mass. (6) Preparation of nanoemulsion by self-emulsification method: According to the volume ratio of emulsifier polyoxyethylene octylphenol ether-10 (OP-10) to co-emulsifier anhydrous ethanol of 2-3:1, anhydrous ethanol is added to OP-10 and stirred at 60-120 r / min for 20-30 min at room temperature until a uniform emulsifier / co-emulsifier system is formed. The jujube seed fatty oil obtained in step (1) is mixed with ethyl oleate at a volume ratio of 1:1-2 to form the oil phase; Slowly add the oil phase to the emulsifier / co-emulsifier system and stir at 60-120 r / min for 20-30 min; add the purified total flavonoid powder of jujube seed in step (5) to the mixture and add purified water dropwise while stirring at 60-120 r / min at room temperature until the solution is clear and transparent, which is the total flavonoid nanoemulsion of jujube seed; wherein: the mass percentage of total flavonoid powder of jujube seed to emulsifier / co-emulsifier system, oil phase and water is 1:8-10:1-2:18-20.

2. The method for preparing a total flavonoid nanoemulsion from jujube seed according to claim 1, characterized in that: In step (1), the jujube seed medicinal material is placed in a pulverizer and pulverized at 1500-2000r / min for 1-3s.

3. The method for preparing a total flavonoid nanoemulsion from jujube seed according to claim 1, characterized in that: The extract in step (4) is concentrated under reduced pressure to obtain a concentrated solution. The working pressure for the reduced pressure concentration is 0.07-0.09 MPa, and the water bath temperature is 70-80℃. The ratio of the concentrated solution to ethyl acetate is 1:2-3 for extraction.

4. The method for preparing a total flavonoid nanoemulsion from jujube seed according to claim 1, characterized in that: In steps (4) and (5), rutin was used as a reference standard, and the total flavonoid content in the extract was determined by spectrophotometry. Daphne flavonoid, genistein, quercetin, kaempferol and apigenin were used as reference standards, and the total amount of total flavonoid aglycones in the above-mentioned jujube seed was determined by HPLC-DAD chromatography, and the jujube seed flavonoid aglycone content was calculated.

5. The method for preparing a total flavonoid nanoemulsion from jujube seed according to claim 1, characterized in that: The total flavonoid nanoemulsion of jujube seed described in step (6) has a particle size of 20-30 nm, a polydispersity index (PDI) of 0.1-0.3, and an encapsulation efficiency of ≥97%.

6. A method for preparing a soft capsule of total flavonoids from jujube seed using the nanoemulsion of total flavonoids from jujube seed prepared by the method of claim 1, characterized in that: The specific preparation method is as follows: 20-25g of gelatin is added to 20mL of water, the temperature is controlled at 15℃-20℃, and after stirring to accelerate expansion, it is heated to 75℃-80℃ to melt. 1-1.5g of polyethylene glycol and 8-10mL of glycerin are added and mixed well. The mixture is degassed under reduced pressure until the melt is transparent and free of bubbles. The prepared total flavonoid nanoemulsion solution and gelatin solution are pressed into soft capsules using a rotary molding machine. The surface oil layer is washed off with anhydrous ethanol, and the capsules are dried in a ventilated environment at 24℃-27℃ to obtain the total flavonoid nanoemulsion soft capsules of jujube seed. The ratio of the total flavonoid nanoemulsion solution of jujube seed to the gelatin solution is 1:1-1.

5.

7. The preparation method according to claim 6, characterized in that: The working pressure for degassing is 0.06-0.08 MPa.

8. A nanoemulsion soft capsule containing total flavonoids from jujube seed prepared according to claim 6.

9. The use of the total flavonoid nanoemulsion soft capsules of jujube seed as described in claim 8 in the preparation of medicines for improving insomnia, depression and anxiety.

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