Preparation method, product and application of fisetin nanoparticles

Abstract

According to the fisetin nanoparticle preparation method, the fisetin nanoparticle product and the application, the synergistic effect of amino acid catalysis and a microwave or ultrasonic energy field is matched with mixing self-assembly and subsequent spray drying forming under the acidic condition, on one hand, the traditional Maillard reaction period is greatly shortened, and energy consumption is reduced; on one hand, the whole technological process of wall material preparation and active ingredient loading is simplified, the large-scale production requirement is met, on the other hand, the fisetin encapsulation effect and the nanoparticle structure stability are improved, the defects that fisetin is poor in water solubility and low in bioavailability are overcome, meanwhile, it is guaranteed that the nanoparticle product quality is uniform, the technology efficiency and the product performance superiority are considered, and the method is suitable for industrial production. The core pain points of low efficiency, poor drug loading effect and insufficient product stability of the existing traditional process are effectively overcome.

Description

Technical Field

[0001] This application relates to the field of targeted delivery of bioactive factors, and in particular to a method for preparing fisetin nanoparticles, products and applications. Background Technology

[0002] Fisetin is a natural flavonoid compound with various biological activities, including antioxidant, anti-inflammatory, and neuroprotective effects. However, its inherent characteristics, such as extremely poor water solubility and low bioavailability, severely limit its practical application and large-scale promotion. Current nanodelivery carrier technologies for poorly soluble active ingredients generally suffer from problems such as cumbersome preparation processes, high costs, poor drug encapsulation effects, and insufficient product stability, failing to meet the needs of industrial production and practical applications.

[0003] Protein-polysaccharide Maillard reaction products are commonly used hydrophobic active substance encapsulation wall materials in existing technologies. Most existing technologies employ a conventional process combining freeze-drying and prolonged dry heat to prepare these wall materials, followed by simple physical mixing to load the active ingredients. This traditional process has significant technical drawbacks: First, the core Maillard reaction cycle is extremely long, energy-intensive, and inefficient, making large-scale continuous production difficult. Second, the active ingredient loading method is passive, lacking sufficient encapsulation driving force, resulting in low drug loading and encapsulation efficiency, and the carrier structure is loose, making drug leakage likely. Third, the carrier has a single function, lacks targeted modification and optimization, and has limited application adaptability. Fourth, prolonged high-temperature treatment can easily lead to over-reaction, poor product uniformity, and difficulty in controlling quality stability.

[0004] At present, there is no efficient, controllable and industrially compatible complete solution to address the shortcomings of the existing technologies. The industry urgently needs a simple process, excellent encapsulation effect and high stability for the preparation of fenestrone nanocarriers to break through the existing technological bottlenecks. Summary of the Invention

[0005] This application provides a method for preparing fisetin nanoparticles, products, and applications. Through the synergistic effect of amino acid catalysis and microwave / ultrasonic energy field, combined with mixing self-assembly under acidic conditions and subsequent spray drying, the Maillard reaction efficiency is effectively improved, the reaction cycle is shortened, the water solubility and encapsulation efficiency of fisetin are improved, the structural stability and product uniformity of nanoparticles are enhanced, and the process flow is simplified, making it suitable for industrial production.

[0006] In a first aspect, embodiments of this application provide a method for preparing fisetin nanoparticles, the method comprising: using whey protein and oligosaccharides as reactants and amino acids as Maillard reaction catalysts, applying microwave or ultrasonic treatment to carry out a Maillard reaction to prepare a glycosylated wall material; dissolving fisetin and the glycosylated wall material separately, adjusting the wall material solution to acidity, mixing the fisetin solution with the acidic wall material solution to prepare a fisetin-loaded nanoparticle solution; adding functional additives to the nanoparticle solution, and then spray drying to obtain dried fisetin nanoparticles.

[0007] In some embodiments, the amino acid is an acidic amino acid or a basic amino acid, wherein the acidic amino acid includes aspartic acid and glutamic acid, and the basic amino acid includes lysine, arginine, and histidine.

[0008] In some embodiments, the preparation of glycosylated wall materials by using whey protein and oligosaccharides as reactants and amino acids as Maillard reaction catalysts, and applying microwave or ultrasonic treatment, includes: spray drying a mixed aqueous solution of whey protein, oligosaccharides, and amino acids to obtain a powder; subjecting the powder to microwave treatment at 300W to 800W power for 2min to 15min, and then incubating it at 60℃ to 80℃ for 2h to 8h to complete the Maillard reaction and obtain powdered glycosylated wall materials.

[0009] In some embodiments, the preparation of glycosylated wall materials by using whey protein and oligosaccharides as reactants and amino acids as Maillard reaction catalysts, and applying microwave or ultrasonic treatment, includes: dissolving whey protein, oligosaccharides, and amino acids in an aqueous solution with a pH of 8.0–9.0; and applying ultrasonic treatment with a power of 200W–600W under heating conditions of 60°C–85°C to carry out a Maillard reaction for 0.5 hours–3 hours to obtain a liquid glycosylated wall material.

[0010] In some embodiments, the fisetone is pre-dissolved in an ethanol solution, and the glycosylated wall material is dissolved in pure water.

[0011] In some embodiments, the pH value of the acidic wall material solution is 4.0 to 5.0; the mixing process is carried out under high-speed shear conditions, and the fisetin ethanol solution is rapidly injected into the acidic wall material solution to obtain a fisetin-loaded nanoparticle solution.

[0012] In some embodiments, the functional additive is selected from at least one of phospholipids, PEG, PVP, Tween, and cyclodextrin, and the amount of the functional additive added is 0% to 15% of the total mass of the nanoparticle solution; the spray drying process parameters are an inlet air temperature of 130°C to 200°C and an outlet air temperature of 60°C to 95°C.

[0013] In some embodiments, the amount of amino acids added is 1% to 20% of the whey protein content, and the fisetin nanoparticles obtained have a fisetin loading of 1% to 20%.

[0014] Secondly, embodiments of this application provide fisetin nanoparticles prepared by the method described in the first aspect.

[0015] Thirdly, embodiments of this application provide the application of fisetin nanoparticles as described in the second aspect in functional foods, biomedicines, and cosmetics.

[0016] This application has the following technical advantages: 1) Using amino acids as Maillard reaction catalysts, combined with the synergistic effect of microwave or ultrasonic energy fields, the activation energy of the reaction is effectively reduced, breaking the drawback of long reaction cycles in traditional processes, significantly shortening the preparation time of wall materials, reducing production energy consumption, and avoiding excessive protein denaturation and excessive browning of the reaction, thus ensuring the stability of the basic properties of the wall materials. 2) Adjusting the wall material solution to an acidic environment achieves mixed loading, strengthens the binding force between the wall material and fisetin, replaces the traditional passive mixing method, forms dense nanoparticles, reduces drug leakage, effectively improves fisetin encapsulation efficiency, and prolongs drug loading stability. 3) By utilizing the emulsifying, dispersing and inclusion properties of glycosylated wall materials, the defect of extremely poor water solubility of fisetin itself can be effectively improved, thereby enhancing its water dispersibility and dissolution performance, laying the foundation for improving bioavailability in the future. 4) The nanoparticles are cured and dried in one step by spray drying, which simplifies the subsequent molding process, ensures the uniformity of nanoparticle product quality, and the overall process is simple and coherent, taking into account both process operability and adaptability to large-scale production. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 This is a schematic flowchart illustrating a method for preparing fisetin nanoparticles provided in an embodiment of this application. Detailed Implementation

[0019] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0020] Fisetin is a natural flavonoid compound with various biological activities, including antioxidant, anti-inflammatory, and neuroprotective effects. However, its inherent characteristics, such as extremely poor water solubility and low bioavailability, severely limit its practical application and large-scale promotion. Current nanodelivery carrier technologies for poorly soluble active ingredients generally suffer from problems such as cumbersome preparation processes, high costs, poor drug encapsulation effects, and insufficient product stability, failing to meet the needs of industrial production and practical applications.

[0021] Protein-polysaccharide Maillard reaction products are commonly used hydrophobic active substance encapsulation wall materials in existing technologies. Most existing technologies employ a conventional process combining freeze-drying and prolonged dry heat to prepare these wall materials, followed by simple physical mixing to load the active ingredients. This traditional process has significant technical drawbacks: First, the core Maillard reaction cycle is extremely long, energy-intensive, and inefficient, making large-scale continuous production difficult. Second, the active ingredient loading method is passive, lacking sufficient encapsulation driving force, resulting in low drug loading and encapsulation efficiency, and the carrier structure is loose, making drug leakage likely. Third, the carrier has a single function, lacks targeted modification and optimization, and has limited application adaptability. Fourth, prolonged high-temperature treatment can easily lead to over-reaction, poor product uniformity, and difficulty in controlling quality stability.

[0022] At present, there is no efficient, controllable and industrially compatible complete solution to address the shortcomings of the existing technologies. The industry urgently needs a simple process, excellent encapsulation effect and high stability for the preparation of fenestrone nanocarriers to break through the existing technological bottlenecks.

[0023] To address the aforementioned technical challenges, this application provides a method for preparing fisetin nanoparticles. Through the synergistic effect of amino acid catalysis and microwave / ultrasonic energy fields, combined with acidic conditions for mixing and self-assembly, and subsequent spray drying, this method significantly shortens the traditional Maillard reaction cycle, reduces energy consumption, simplifies the overall process flow for wall material preparation and active ingredient loading, and adapts to the needs of large-scale production. Furthermore, it improves the fisetin encapsulation effect and nanoparticle structural stability, overcoming the shortcomings of poor water solubility and low bioavailability of fisetin, while ensuring uniform nanoparticle product quality. This method balances process efficiency and product performance, effectively overcoming the core pain points of existing traditional processes such as low efficiency, poor drug loading, and insufficient product stability.

[0024] The following describes in detail, with reference to specific embodiments, a method for preparing phenoxy ketone nanoparticles, the product, and its application provided in this application.

[0025] Please see Figure 1 , Figure 1 This is a schematic flowchart illustrating a method for preparing fisetin nanoparticles provided in an embodiment of this application, as shown below. Figure 1 As shown, the method includes steps S101-S103: Step S101: Using whey protein and oligosaccharides as reaction raw materials and amino acids as Maillard reaction catalysts, the Maillard reaction is carried out under microwave or ultrasonic treatment to prepare glycosylated wall materials.

[0026] In this step, amino acids are used as catalysts for the Maillard reaction, combined with the synergistic effect of microwave or ultrasonic physical energy fields: the activation energy of the reaction is reduced through a dual mechanism of amino acid bifunctional catalysis and enhanced mass transfer via physical energy fields. The amino group (-NH2) of the amino acid preferentially binds to the carbonyl group (-C=O) of the oligosaccharide as a nucleophilic site, rapidly forming the Schiff base intermediate, bypassing the high-energy barrier of traditional reactions. Simultaneously, microwave or ultrasonic energy fields enhance reaction kinetics through molecular-level interactions: microwaves induce high-frequency dipole rotation of polar molecules, achieving uniform heating within the system and increasing the frequency of molecular collisions; ultrasound tears apart the molecular hydration layer through cavitation effects, reducing mass transfer resistance. The two work synergistically to achieve a "low-temperature rapid reaction," avoiding high-temperature denaturation and excessive browning of proteins. Furthermore, microwave / ultrasound not only accelerates the reaction but also alters the protein conformation, exposing more hydrophobic sites and enhancing the interaction with fisetin.

[0027] In step S102, the fisetin and the glycosylated wall material are dissolved separately, the wall material solution is adjusted to acidity, and the fisetin solution is mixed with the acidic wall material solution to prepare a fisetin-loaded nanoparticle solution.

[0028] In this process, the wall material solution is in an acidic environment, which neutralizes the charge near the isoelectric point (pI≈4.6) of whey protein. The unfolding of the protein molecule leads to the extensive exposure of hydrophobic domains (such as hydrophobic amino acid side chains). Simultaneously, fisetin, as a hydrophobic flavonoid, experiences a sharp decrease in solubility in the acidic aqueous phase, precipitating out as nanoscale crystal nuclei. Through hydrophobic interactions (van der Waals forces, hydrophobic association) and electrostatic adsorption (π-π stacking of the protein's hydrophobic areas and the fisetin aromatic ring), the two spontaneously form core-shell structured nanoparticles, replacing the traditional weak binding mode of passive mixing.

[0029] Step S103: Add functional additives to the nanoparticle solution, and then spray dry to form dried fisetin nanoparticles.

[0030] Among them, functional additives modify the surface of nanoparticles through non-covalent interactions (such as the inclusion effect of cyclodextrin and the membrane fusion effect of phospholipids), construct a hydration membrane barrier, and improve dispersion stability; spray drying removes solvents in milliseconds through the instantaneous vaporization effect of high-temperature airflow, so that the colloidal structure of nanoparticles can be quickly solidified, avoiding agglomeration and drug leakage during the drying process, and achieving "forming-stabilization" in one step.

[0031] In some embodiments, the amino acid is an acidic amino acid or a basic amino acid, wherein the acidic amino acid includes aspartic acid and glutamic acid, and the basic amino acid includes lysine, arginine, and histidine.

[0032] Among them, the acidic amino acids are selected from aspartic acid and glutamic acid, and the basic amino acids are selected from lysine, arginine, and histidine. All of these amino acids contain bifunctional groups of amino and carboxyl groups, making them highly efficient catalysts for the Maillard reaction. They are well-suited to the subsequent energy field synergistic reaction system, ensuring both reaction acceleration and stable control of the reaction product properties. This avoids problems such as wall material performance degradation and excessive browning caused by inappropriate amino acid selection, further optimizing the overall quality of the glycosylated wall material and providing stable and high-quality wall material raw materials for subsequent nanoparticle preparation. Furthermore, amino acids not only act as reaction catalysts but can also alter protein conformation under the influence of an energy field, exposing more hydrophobic sites and further enhancing the interaction with fisetin, achieving high drug loading and high encapsulation.

[0033] Furthermore, the differences in the side chain structures of the amino acids used can directly affect the types and colors of Maillard reaction products. Among them, basic amino acids can directionally promote the formation of melanoidins during the reaction process, and melanoidins can significantly optimize the emulsification and encapsulation properties of the wall material, laying the foundation for the efficient loading of fisetin in the future.

[0034] Furthermore, the promoting effect of amino acids is more significant in an environment of pH 8-9.

[0035] In some embodiments, the preparation of glycosylated wall materials by using whey protein and oligosaccharides as reactants and amino acids as Maillard reaction catalysts, and applying microwave or ultrasonic treatment, includes: spray drying a mixed aqueous solution of whey protein, oligosaccharides, and amino acids to obtain a powder; subjecting the powder to microwave treatment at 300W to 800W power for 2min to 15min, and then incubating it at 60℃ to 80℃ for 2h to 8h to complete the Maillard reaction and obtain powdered glycosylated wall materials.

[0036] Among them, the energy field treatment and power need to be coordinated with the amino acid induction system to avoid local overheating that could lead to protein aggregation or excessive Maillard browning. Treatment at 300W~800W power for 2 to 15 minutes can ensure that the reaction starts without charring.

[0037] In some embodiments, the preparation of glycosylated wall materials by using whey protein and oligosaccharides as reactants and amino acids as Maillard reaction catalysts, and applying microwave or ultrasonic treatment, includes: dissolving whey protein, oligosaccharides, and amino acids in an aqueous solution with a pH of 8.0–9.0; and applying ultrasonic treatment with a power of 200W–600W under heating conditions of 60°C–85°C to carry out a Maillard reaction for 0.5 hours–3 hours to obtain a liquid glycosylated wall material.

[0038] In some embodiments, the fisetone is pre-dissolved in an ethanol solution, and the glycosylated wall material is dissolved in pure water.

[0039] In some embodiments, the pH value of the acidic wall material solution is 4.0 to 5.0; the mixing process is carried out under high-speed shear conditions, and the fisetin ethanol solution is rapidly injected into the acidic wall material solution to obtain a fisetin-loaded nanoparticle solution.

[0040] Within the pH range of 4.0–5.0, proteins approach their isoelectric point, exposing hydrophobic regions. When a fisetin ethanol solution is rapidly injected into the pure water system of the acidic glycosylated wall material, a solvent displacement effect occurs instantaneously, causing fisetin to become instantaneously supersaturated in the aqueous phase and rapidly precipitate. This instantaneous precipitation characteristic, combined with the physical capability of high-speed shearing, enables fisetin molecules to rapidly combine with wall material molecules that are already near their isoelectric point and have fully exposed hydrophobic regions through hydrophobic interactions, electrostatic adsorption, and π-π stacking, forming stable core-shell structured nanoparticles. Compared to directly adding solid fisetin to the liquid phase system, the solvent combination in this embodiment significantly improves the encapsulation efficiency and the structural regularity of the nanoparticles. Deviations from the pH range of 4.0–5.0 will result in a decrease in encapsulation efficiency of >20%.

[0041] In some embodiments, the functional additive is selected from at least one of phospholipids, PEG, PVP, Tween, and cyclodextrin, and the amount of the functional additive added is 0% to 15% of the total mass of the nanoparticle solution; the spray drying process parameters are an inlet air temperature of 130°C to 200°C and an outlet air temperature of 60°C to 95°C.

[0042] Phospholipids can improve the affinity of nanoparticles to the membrane and enhance oral absorption efficiency; PEG, PVP, and Tween, as polymeric surfactants and emulsifiers, can optimize the surface potential of nanoparticles, prevent particle aggregation, and improve water dispersion stability and storage stability; cyclodextrin can further improve the solubility of fisetin through inclusion complexation and enhance drug loading stability. The above adjuvants can be used alone or in combination, taking into account both functional compatibility and process versatility.

[0043] The addition amount is limited to 0% to 15% of the total mass of the nanoparticle solution. The lower limit of 0% covers the basic scheme without adding additives, which is suitable for minimal process scenarios. 15% is the upper limit threshold. Adding an amount below this range can avoid problems such as abnormal increase in nanoparticle size, interference with the wall material embedding effect, and increased cost caused by excessive additives. At the same time, it can ensure that the additives play a stable and modifying role, balancing process costs and product performance.

[0044] In step S103, the spray drying parameters are limited to an inlet air temperature of 130℃~200℃ and an outlet air temperature of 60℃~95℃, which represents the optimal parameter range for synergistic matching. If the inlet air temperature is below 130℃, the solvent vaporization rate is too slow, and the nanoparticles cannot solidify quickly, easily leading to adhesion and agglomeration. If the temperature is above 200℃, the instantaneous temperature is too high, easily causing the destruction of fisetin activity and denaturation and inactivation of the protein wall material. The outlet air temperature, as a core process control indicator, needs to be compatible with the inlet air temperature. A temperature range of 60℃~95℃ ensures sufficient drying of the system and meets the moisture content requirements, while avoiding excessive damage to the nanoparticle structure and active ingredients from residual heat. This ensures that the final dried powder has good flowability, intact nanostructure, and stable drug loading performance, making it suitable for large-scale continuous production.

[0045] In some embodiments, the amount of amino acids added is 1% to 20% of the whey protein content, and the fisetin nanoparticles obtained have a fisetin loading of 1% to 20%.

[0046] The amount of amino acids added is set at 1%–20% of the whey protein content. If the addition is too low (below 1%), the amino acids are insufficient to effectively catalyze the Maillard reaction, failing to significantly reduce the activation energy and offering limited improvement to the reaction rate and wall material performance. If the addition is too high (above 20%), it will lead to an excess of small molecules in the system, interfering with the normal cross-linking between proteins and sugars, potentially causing a decrease in the emulsificability of the wall material, poor product uniformity, and increased costs. Controlling it at 1%–20% ensures that the amino acids fully play their role in catalyzing and regulating the reaction pathway while maintaining system stability, resulting in a glycosylated wall material with optimal overall performance.

[0047] The fisetin drug loading is set at 1%–20%, with 1% as the lower limit of effective loading, meeting the application scenarios requiring low dosage and high stability. 20% is the upper limit of the system's loading capacity; within this range, the nanoparticle structure remains dense and intact, with stable encapsulation efficiency, preventing problems such as excessive free fisetin, particle aggregation, and drug leakage due to drug overload. This wide-range drug loading design allows the product to adapt to the dosage requirements of different application scenarios such as functional foods, health products, and cosmetics, enhancing the technology's versatility and industrialization value.

[0048] In some embodiments, the fisetin nanoparticles have a particle size ≤500nm, a polydispersity index ≤0.3, a solubility ≥0.3mg / ml, and an encapsulation efficiency ≥40%.

[0049] Among them, the particle size of the nanoparticles is controlled within 500 nm. This particle size range can significantly improve the dispersibility, dissolution rate and mucosal permeability of the formulation in vivo, which is beneficial to improving the bioavailability of fisetin. At the same time, the smaller particle size can avoid particle aggregation and sedimentation, ensure the uniformity and stability of the formulation system, and meet the basic requirements of oral or delivery systems for nanocarriers.

[0050] The polydispersity index is used to characterize the uniformity of nanoparticle size distribution. Controlling it to 0.3 or below indicates a narrow nanoparticle size distribution and good system uniformity, which avoids problems such as local aggregation, encapsulation efficiency fluctuations, and inconsistent in vivo behavior caused by uneven particle size, thus improving batch-to-batch stability and reliability.

[0051] Feselon itself has extremely low water solubility, approximately 0.03 mg / mL. Through nanoparticle encapsulation and wall material solubilization, the solubility of the system can be increased to over 0.3 mg / mL. This can significantly improve the dissolution and dispersion of feselon in the aqueous phase, solving problems such as poor absorption and low utilization caused by its poor solubility, and providing a foundation for subsequent applications.

[0052] Encapsulation efficiency reflects the proportion of active ingredient that is effectively encapsulated. An encapsulation efficiency higher than 40% indicates that most of the fisetin is stably embedded inside the nanoparticles, which can reduce problems such as leakage, oxidation, and poor stability caused by free drug, ensuring drug loading efficiency and formulation quality, and achieving efficient loading and stable delivery.

[0053] The technical solutions proposed in this application will be described in more detail below with specific embodiments, but this application is not limited to these embodiments.

[0054] Example 1: Preparation of wall materials and pH-driven self-assembly of fisetin nanoparticles via amino acid-induced microwave-dry heat coupling method. A method for preparing wall materials based on amino acid-induced microwave-dry heat coupling and pH-driven self-assembly of 10% fisetone nanoparticles comprises the following components by weight percentage: Fesetone 2.0 g 8.0 g whey protein isolate Lysine 1.0 g 7.0 g of galacto-oligosaccharides 2.0g phospholipids The preparation process is as follows: (1) Preparation of glycosylated wall material by amino acid-induced-microwave-dry heat coupling method: whey protein isolate (hereinafter referred to as WPI), galactooligosaccharide (hereinafter referred to as GOS) and lysine were dissolved in PBS buffer (pH 7.0), stirred for 2 h for hydration, homogenized and sprayed at an inlet air temperature of 160℃ and an outlet air temperature of >60℃ to obtain WPI-GOS mixed powder. The mixed powder was then spread evenly in a container and placed in a microwave reactor and treated at 600W power for 10 min. The powder was then immediately transferred to a blower dryer and incubated at 60℃ for 6 h to complete the Maillard reaction. After the reaction was completed, a light yellow wall material powder was obtained and set aside for use. (2) pH-driven self-assembly of loaded fisetin: Dissolve 2.0g fisetin in 300ml anhydrous ethanol and stir until dissolved. Take 16g of all the wall material powder from (1) and disperse it in 150g of purified water. Stir to redissolve for 1h. Adjust the pH to 4.5 with 0.5M HCl solution. Under high-speed shearing of 10000rpm, add the fisetin ethanol solution to the acidic wall material solution. After observing that the system becomes a light yellow dispersion, continue shearing for 20min. Then, remove the ethanol from the dispersion under reduced pressure at 60℃ to obtain a nano suspension for later use. (3) Spray drying molding: 2.0g of phospholipid was added under high-speed shearing at 10000rpm and sheared for 10min to obtain a uniform nanoparticle suspension modified with phospholipid. Then, the nanoparticle suspension was spray dried at an inlet air temperature of 160℃, an outlet air temperature of ≥60℃, a feed rate of 5mL / min, and an atomization pressure of 0.3MPa to obtain dried yellow fisetin nanoparticle powder.

[0055] Example 2: Preparation of wall materials and pH-driven self-assembly of fisetin nanoparticles using an amino acid-induced-ultrasound-hygrothermal coupling method. A method for preparing wall materials based on amino acid-induced-ultrasound-hygrothermal coupling and pH-driven self-assembly of 10% fisetone nanoparticles comprises the following components by weight percentage: Fesetone 2.0 g 8.0 g whey protein isolate Lysine 1.0 g 7.0 g of galacto-oligosaccharides 2.0g phospholipids The preparation process is as follows: (1) Preparation of glycosylated wall material by amino acid-induced-ultrasound-wet heat coupling method: WPI, GOS and lysine were dissolved in purified water and hydrated by stirring for 2 hours. Then the pH was adjusted to 8.0. The solution was placed in a constant temperature water bath at 75℃ and sonicated with an ultrasonic power of 400W for 2 hours. After the reaction was completed, the solution color turned light brown and a uniform WPI-GOS glycosylated wall material liquid was obtained. It was cooled to room temperature and ready for use. (2) pH-driven self-assembly of loaded fisetin: Dissolve 2.0 g fisetin in 300 ml of anhydrous ethanol and stir until dissolved. Take the wall material solution from (1) and adjust the pH to 4.5 with 0.5 M HCl solution. Under high-speed shearing of 10000 rpm, add the fisetin ethanol solution to the acidic wall material solution. After observing that the system becomes a pale yellow dispersion, continue shearing for 20 min. Then, remove the ethanol from the dispersion by rotary evaporation under reduced pressure at 60 °C to obtain a nano-suspension for later use. (3) Spray drying molding: 2.0g of phospholipid was added under high-speed shearing at 10000rpm and sheared for 10min to obtain a uniform nanoparticle suspension modified with phospholipid. Then, the nanoparticle suspension was spray dried at an inlet air temperature of 160℃, an outlet air temperature of ≥60℃, a feed rate of 5mL / min, and an atomization pressure of 0.3MPa to obtain dried yellow fisetin nanoparticle powder.

[0056] Example 3: Process Comparison Experiment - Sample Preparation Using Traditional Dry Heat Method: The traditional dry heat method for preparing 10% fisetone nanoparticles comprises the following components by weight percentage: Fesetone 2.0 g Whey protein isolate 9.0 g 9.0 g of galacto-oligosaccharides The preparation process is as follows: (1) Preparation of wall material: Referring to the example in CN113855811A, WPI and GOS were dissolved in PBS buffer and lyophilized for the first time. Then, the mixture was reacted at 60°C under humidity provided by saturated magnesium nitrate for 24-48 hours. The mixture was washed three times with 75% ethanol and then lyophilized a second time to obtain purified wall material for later use.

[0057] (2) Fesetone loading: Dissolve 2.0g of fesetone in 300ml of anhydrous ethanol and stir until dissolved. Then dissolve 18g of purified wall material in 160g of purified water. Add the fesetone ethanol solution dropwise to the wall material aqueous solution and stir at room temperature (25℃) for 12h. (3) Freeze-drying molding: After removing alcohol by rotary evaporation, freeze-dry for 48 hours to obtain the finished product of fisetin nanoparticles.

[0058] Example 4: Comparison of Fesetone Loading Mechanisms - pH-Driven Self-Assembly vs. Simple Mixing: Group A (pH-driven self-assembly): Same formulation and preparation process as in Example 1.

[0059] Control group B (simple mixing without adjusting the pH of the wall material, maintaining pH 7.0): 2.0 g of fisetin was dissolved in 300 ml of anhydrous ethanol and stirred until dissolved, then set aside. 16 g of the wall material powder was dispersed in 150 g of purified water and stirred to reconstitute for 1 h. Then, under high-speed shearing at 10000 rpm, the fisetin ethanol solution was added to the wall material solution. After observing the appearance of a yellow flocculated state, shearing was continued for 20 min. The dispersion was then subjected to reduced pressure rotary evaporation at 60 °C to remove the ethanol, resulting in a nano-suspension. 2.0 g of phospholipid was then added under high-speed shearing at 10000 rpm and sheared for 10 min to obtain a phospholipid-modified suspension. The suspension was then spray-dried at an inlet air temperature of 160 °C, an outlet air temperature ≥60 °C, a feed rate of 5 mL / min, and an atomization pressure of 0.3 MPa to obtain dried yellow fisetin nanoparticle powder.

[0060] Example 5: Preparation of wall materials and pH-driven self-assembly of fisetin nanoparticles using an amino acid-induced microwave-dry heat coupling method (increasing loading to 20%): A method for preparing wall materials based on amino acid-induced microwave-dry heat coupling and pH-driven self-assembly of 20% fisetone nanoparticles comprises the following components by weight percentage: Fesedon 4.0 g Whey protein isolate 7.0 g Lysine 1.0 g 6.0 g of galacto-oligosaccharides 2.0g phospholipids The preparation process is as follows: (1) Preparation of glycosylated wall material by amino acid-induced-microwave-dry heat coupling method: WPI, GOS and lysine were dissolved in PBS buffer (pH 7.0), stirred for 2 h for hydration, homogenized and sprayed at an inlet air temperature of 160℃ and an outlet air temperature of >60℃ to obtain WPI-GOS mixed powder. The mixed powder was then spread evenly in a container and placed in a microwave reactor and treated at 600W power for 10 min. The powder was then immediately transferred to a blower dryer and incubated at 60℃ for 6 h to complete the Maillard reaction. After the reaction was completed, a light yellow wall material powder was obtained and set aside for use. (2) pH-driven self-assembly of loaded fisetin: Dissolve 4.0g fisetin in 600ml anhydrous ethanol and stir until dissolved. Take 16g of all the wall material powder from (1) and disperse it in 150g purified water. Stir to redissolve for 1h. Adjust the pH to 4.5 with 0.1M HCl solution. Under high-speed shearing of 10000rpm, add the fisetin ethanol solution to the acidic wall material solution. Observe that the system becomes a light yellow dispersion and continue shearing for 20min. Then remove the ethanol from the dispersion under reduced pressure at 60℃ to obtain a nano suspension for later use. (3) Spray drying molding: 2.0g of phospholipid was added under high-speed shearing at 10000rpm and sheared for 10min to obtain a uniform nanoparticle suspension modified with phospholipid. Then, the nanoparticle suspension was spray dried at an inlet air temperature of 160℃, an outlet air temperature of ≥60℃, a feed rate of 5mL / min, and an atomization pressure of 0.3MPa to obtain dried yellow fisetin nanoparticle powder.

[0061] Example 6: Preparation of wall materials and pH-driven self-assembly of fisetin nanoparticles using an amino acid-induced microwave-dry heat coupling method – further enhancement of solubility using cyclodextrin: A method for preparing wall materials based on amino acid-induced microwave-dry heat coupling and pH-driven self-assembly of 10% fisetone nanoparticles comprises the following components by weight percentage: Fesetone 2.0 g 8.0 g whey protein isolate Lysine 1.0 g 7.0 g of galacto-oligosaccharides Cyclodextrin 2.0g The preparation process is as follows: (1) Preparation of glycosylated wall material by amino acid-induced-microwave-dry heat coupling method: WPI, GOS and lysine were dissolved in PBS buffer (pH 7.0), stirred for 2 h for hydration, homogenized and sprayed at an inlet air temperature of 160℃ and an outlet air temperature of >60℃ to obtain WPI-GOS mixed powder. The mixed powder was then spread evenly in a container and placed in a microwave reactor and treated at 600W power for 10 min. The powder was then immediately transferred to a blower dryer and incubated at 60℃ for 6 h to complete the Maillard reaction. After the reaction was completed, a light yellow wall material powder was obtained and set aside for use. (2) pH-driven self-assembly of loaded fisetin: Dissolve 2.0g fisetin in 300ml anhydrous ethanol and stir until dissolved. Take 16g of all the wall material powder from (1) and disperse it in 150g of purified water. Stir to redissolve for 1h. Adjust the pH to 4.5 with 0.5M HCl solution. Under high-speed shearing of 10000rpm, add the fisetin ethanol solution to the acidic wall material solution. After observing that the system becomes a light yellow dispersion, continue shearing for 20min. Then, remove the ethanol from the dispersion under reduced pressure at 60℃ to obtain a nano suspension for later use. (3) Spray drying molding: 2.0 g of cyclodextrin was added under high-speed shearing at 10000 rpm and sheared for 10 min to obtain a uniform nanoparticle suspension. Then, the nanoparticle suspension was spray dried at an inlet air temperature of 160℃, an outlet air temperature of ≥60℃, a feeding rate of 5 mL / min, and an atomization pressure of 0.3 MPa to obtain dried light yellow fisetin nanoparticle powder.

[0062] Example 7: Performance Evaluation of Fesetone Nanoparticles The results of solubility, encapsulation efficiency, and particle size analysis of the samples from Examples 1 to 6 are shown in Table 1. Table 1. Results of Fissone nanoparticle loading, solubility, encapsulation efficiency, polydispersity index, and particle size analysis.

[0063] From the above embodiments 1-6 and Table 1, the following significant advantages of the present invention can be observed: (1) Significantly improved process efficiency: The Maillard reaction technology induced by amino acids and assisted by microwave and ultrasound shortens the reaction time of the traditional dry heat method, which takes several days or even longer, to less than 8 hours, increasing the reaction efficiency by more than 75%, reducing energy consumption, saving costs, and facilitating continuous and large-scale production.

[0064] (2) Significant optimization of wall material performance and encapsulation effect: The introduction of amino acids not only accelerates the reaction but also promotes the formation of melanoidin-like polymers. Melanoidin-like polymers are excellent natural emulsifiers and antioxidant polymers, which enable the prepared glycosylated wall material to have stronger interfacial stability and fundamentally improve the encapsulation rate of nanoparticles. As can be seen from Examples 1, 2 and 3 (comparative examples) and Group B (comparative example) in Example 4 of this application, the combination of the "amino acid-induced rapid method for preparing wall materials" and "pH-driven self-assembly" in this application can achieve an encapsulation rate of more than 45% in both dry heat and wet heat methods, which is a significant improvement compared to the encapsulation rate of about 20% in the traditional method. The saturated solubility also increases from about 0.2 mg / ml in the traditional method to about 0.4 mg / ml, which is a significant improvement in solubility. This proves the strong synergistic effect between the two modules.

[0065] (3) Superior product performance: ① Significantly improved solubility and bioavailability potential: The saturated solubility of all nanoparticle samples is significantly higher than that of fisetone raw material (0.03 mg / ml). It can form a clear nano-dispersion of up to 0.4 mg / ml in water, which is about 14 times higher than that of the raw material, laying a solid foundation for significantly improving in vivo bioavailability.

[0066] ② High encapsulation efficiency and wide drug loading range: This invention successfully achieves a wide drug loading range from 1% to 20%. This demonstrates that the wall material has excellent drug encapsulation capabilities, and the encapsulation rate remains stable at over 40%.

[0067] ③ Uniform and controllable nanoparticle size, meeting the requirements for targeted delivery: The particle size D90 of all samples was controlled below 500 nm. This particle size range is beneficial for long-term drug circulation and potential tissue targeting.

[0068] (4) Highly versatile technology: As demonstrated in Examples 1, 2, and 3 (comparative examples) and Group B (comparative example) of Example 4 in this application, the use of the "amino acid-energy field" Maillard reaction shortens the wall material preparation time to 6 hours, and all core indicators show significant improvement: the reaction time is shortened by 75%, the encapsulation efficiency and drug loading are increased by approximately 2.5 times, the solubility is increased by approximately 13 times, and the product homogeneity (PDI) is better. This comprehensive performance improvement proves the substantial progress of this application, significantly reduces energy consumption, and is extremely easy to implement in industrial applications. This method is not only applicable to fisetin, but can also be extended to the construction of nano-encapsulation and targeted delivery systems for other poorly soluble natural active ingredients, possessing good technology transfer potential.

[0069] In summary, the fisetone nanoparticles provided in this application, through innovative wall material design and optimized preparation process, achieve high-quality indicators such as high solubility, high encapsulation efficiency and suitable particle size, while also achieving the two major goals of process simplification and ease of industrialization. This effectively solves the industry pain points of complex processes and difficulty in scale-up in existing technologies, and provides a solution with great market application prospects for the efficient delivery of poorly soluble functional components.

Claims

1. A method for preparing fisetin nanoparticles, characterized in that, The method includes: Using whey protein and oligosaccharides as reactants and amino acids as Maillard reaction catalysts, a Maillard reaction was carried out under microwave or ultrasonic treatment to prepare glycosylated wall materials. Fesetone and the glycosylated wall material were dissolved separately, the wall material solution was adjusted to acidity, and the fesetone solution was mixed with the acidic wall material solution to prepare a fesetone-loaded nanoparticle solution. Functional additives were added to the nanoparticle solution, followed by spray drying to obtain dried fisetin nanoparticles.

2. The preparation method according to claim 1, characterized in that, The amino acids are acidic or basic amino acids. The acidic amino acids include aspartic acid and glutamic acid, and the basic amino acids include lysine, arginine, and histidine.

3. The preparation method according to claim 1, characterized in that, The method involves using whey protein and oligosaccharides as reactants, amino acids as Maillard reaction catalysts, and applying microwave or ultrasonic stimulation to carry out the Maillard reaction, thereby preparing glycosylated wall materials, including: A powder was obtained by spray drying a mixed aqueous solution of whey protein, oligosaccharides, and amino acids. The powder was microwaved at 300W to 800W for 2 to 15 minutes, and then incubated at 60℃ to 80℃ for 2 to 8 hours to complete the Maillard reaction, thus obtaining powdered glycosylated wall material.

4. The preparation method according to claim 1, characterized in that, The method involves using whey protein and oligosaccharides as reactants, amino acids as Maillard reaction catalysts, and applying microwave or ultrasonic stimulation to carry out the Maillard reaction, thereby preparing glycosylated wall materials, including: Dissolve whey protein, oligosaccharides, and amino acids in an aqueous solution with a pH of 8.0–9.0; Under heating conditions of 60℃~85℃, ultrasonic action with a power of 200W~600W is applied to carry out Maillard reaction for 0.5h~3h to obtain liquid glycosylated wall material.

5. The preparation method according to claim 1, characterized in that, The fisetin is pre-dissolved in an ethanol solution, and the glycosylated wall material is dissolved in pure water.

6. The preparation method according to claim 5, characterized in that, The pH value of the acidic wall material solution is 4.0 to 5.0; The mixing process is carried out under high-speed shear conditions, in which a fisetin ethanol solution is rapidly injected into an acidic wall material solution to obtain a fisetin-loaded nanoparticle solution.

7. The preparation method according to claim 1, characterized in that, The functional additive is selected from at least one of phospholipids, PEG, PVP, Tween, and cyclodextrin, and the amount of the functional additive added is 0% to 15% of the total mass of the nanoparticle solution. The spray drying process parameters are: inlet air temperature 130℃~200℃, outlet air temperature 60℃~95℃.

8. The preparation method according to claim 1, characterized in that, The amount of amino acids added is 1% to 20% of the whey protein content, and the fisetin nanoparticles prepared have a fisetin loading of 1% to 20%.

9. Fesetone nanoparticles prepared by the method according to any one of claims 1-8.

10. The application of the fisetone nanoparticles as described in claim 9 in functional foods, biomedicine, and cosmetics.

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