An ultrasonic-assisted preparation of resveratrol-protein nanoparticle intestinal targeting delivery system and a preparation method thereof

Abstract

The present application relates to a kind of ultrasound-assisted preparation resveratrol-protein nanoparticle intestinal targeting delivery system and its preparation method, belong to functional food / medical delivery system technical field.The preparation method of resveratrol-protein nanoparticle intestinal targeting delivery system of the present application, including the following steps: S1: resveratrol is combined with ultrasound treatment hydration protein sufficiently, solid-liquid separation, obtain the protein nanoparticle of resveratrol loading;S2: resveratrol-protein nanoparticle of loading, acetylated dis-starch phosphate and sodium alginate are mixed sufficiently, obtain mixed solution;S3: calcium chloride is mixed with mixed solution, crosslinking reaction, obtain the resveratrol-protein nanoparticle intestinal targeting delivery system of the present application.Protect resveratrol from gastrointestinal environment destruction, improve its stability, realize intestinal targeting and amylase responsive release, improve resveratrol bioavailability, have wide application prospect in functional food and medical field.

Description

Technical Field

[0001] This invention relates to the field of functional food / pharmaceutical delivery systems, and more particularly to an ultrasound-assisted resveratrol-protein nanoparticle intestinal targeted delivery system and its preparation method. Background Technology

[0002] Resveratrol, as a natural active polyphenol, has various biological activities such as anti-oxidation, anti-inflammation, and cardiovascular protection. However, it suffers from poor stability, low water solubility, low bioavailability, and easy degradation in the gastrointestinal tract, which limits its application in the food and pharmaceutical fields.

[0003] Currently, protein nanoparticles have attracted widespread attention as delivery carriers for active ingredients. However, traditional methods for preparing protein nanoparticles have low loading efficiency for hydrophobic active ingredients and struggle to achieve specific targeted release of these ingredients into the gut. Existing targeted delivery systems often employ single embedding materials or simple physical mixing methods, resulting in insufficient targeting and low release efficiency. Therefore, developing a delivery system capable of efficiently loading active ingredients and achieving precise targeted release into the gut is of great significance.

[0004] Chinese patent CN119385288A discloses a preparation method and application of a lactoferrin-sodium alginate hydrogel system, but further improvements are still needed to enhance the encapsulation and targeting effects of the active ingredients. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an ultrasound-assisted resveratrol-protein nanoparticle intestinal targeted delivery system and its preparation method.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for preparing a resveratrol-protein nanoparticle intestinal targeted delivery system, characterized by comprising the following steps:

[0008] S1: Resveratrol is fully combined with ultrasonically treated hydrated protein, and solid-liquid separation is performed to obtain protein nanoparticles loaded with resveratrol.

[0009] S2: Thoroughly mix the protein nanoparticles loaded with resveratrol from step S1, acetylated distarch phosphate, and sodium alginate to obtain a mixed solution;

[0010] S3: Mix calcium chloride with the mixed solution from step S2 and perform a cross-linking reaction to obtain the resveratrol-protein nanoparticle intestinal targeted delivery system.

[0011] This invention provides a delivery system for efficiently preparing resveratrol-loaded protein nanoparticles using ultrasound-assisted technology and achieving intestinal targeting and amylase-responsive release via a composite gel network, and provides a preparation method thereof, in order to solve the problems of low active ingredient loading efficiency, poor targeting, and low bioavailability in the prior art, and improve the bioavailability of resveratrol.

[0012] Furthermore, in step S1, the power of the ultrasound is 300-600W. Preferably, it is 300W.

[0013] Furthermore, in step S1, the ultrasound time is 15-25 minutes, preferably 20 minutes.

[0014] Further, in step S1, the ratio of protein to water used in the ultrasonic treatment of the hydrated protein is 1g:(15-20)mL. Preferably, it is 1g:16.7mL.

[0015] Further, in step S1, the protein is first stirred with water for 2-3 hours, and then placed at 4°C for hydration for 20-24 hours to obtain hydrated protein.

[0016] Preferably, in step S1, the protein is first stirred with water for 2 hours, and then hydrated at 4°C for 24 hours.

[0017] Furthermore, in step S1, the resveratrol ethanol solution is fully combined with the ultrasonically treated hydrated protein.

[0018] Furthermore, in step S1, the concentration of the resveratrol ethanol solution is 5–8 mg / mL, preferably 6 mg / mL.

[0019] Furthermore, in step S1, the volume concentration of ethanol used in the resveratrol ethanol solution is 50-70%, preferably 70%.

[0020] Furthermore, in step S1, resveratrol and ultrasonically treated hydrated protein are stirred and mixed for 3-5 hours to fully combine, preferably 5 hours.

[0021] Further, in step S1, the mass ratio of resveratrol to the protein used in the ultrasonic treatment of hydrated protein is 1:(10-15). Preferably, it is 1:10.

[0022] Furthermore, in step S1, the solid-liquid separation is centrifugation.

[0023] Furthermore, in step S1, the centrifugation is performed at 6000–8000 r / min for 10–20 min. Preferably, it is performed at 8000 r / min for 20 min.

[0024] Furthermore, in step S1, after solid-liquid separation, the solid is washed 2 to 3 times with ethanol, the volume concentration of which is 30% to 50%, preferably 50%, and preferably 3 times.

[0025] Furthermore, in step S1, after washing with ethanol, the material is freeze-dried to obtain protein nanoparticles loaded with resveratrol.

[0026] Furthermore, in step S1, the protein used in the ultrasonic treatment of the hydrated protein includes soy protein isolate.

[0027] Further, in step S2, the mass ratio of resveratrol-loaded protein nanoparticles to acetylated distarch phosphate is 1:(1-2). Preferably, it is 1:2.

[0028] Further, in step S2, the mass ratio of resveratrol-loaded protein nanoparticles to sodium alginate is 1:(0.3-0.5). Preferably, it is 1:0.5.

[0029] Further, in step S2, the resveratrol-loaded protein nanoparticles, acetylated distarch phosphate, and sodium alginate from step S1 are fully dispersed in water, wherein the ratio of the resveratrol-loaded protein nanoparticles to water is 1 g: (40-50) mL. Preferably, it is 1 g: 50 mL.

[0030] Furthermore, in step S2, the thorough dispersion includes stirring for 0.5 to 1 hour, preferably 1 hour.

[0031] Furthermore, in step S2, after thorough dispersion, the mixture is placed in a water bath at 70-80°C for 10-15 minutes, preferably at 80°C for 15 minutes.

[0032] Further, in step S3, the mass ratio of resveratrol-loaded protein nanoparticles to calcium chloride is 1:(1.5-3). Preferably, it is 1:3.

[0033] Furthermore, in step S3, the calcium chloride aqueous solution is mixed with the mixed solution from step S2.

[0034] Furthermore, in step S3, the mass concentration of the calcium chloride aqueous solution is 3-6%, preferably 6%.

[0035] Secondly, the present invention provides a resveratrol-protein nanoparticle intestinal targeted delivery system, which is prepared by the aforementioned preparation method.

[0036] Thirdly, the present invention provides the application of the resveratrol-protein nanoparticle intestinal targeted delivery system in the preparation of antioxidants.

[0037] Fourthly, the present invention provides an antioxidant containing the resveratrol-protein nanoparticle intestinal targeted delivery system.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] (1) The present invention uses ultrasound-assisted treatment of soybean protein isolate solution. By utilizing the cavitation effect and mechanical action of ultrasound, the structure of protein molecules can be changed, increasing the interaction sites with resveratrol, thereby significantly improving the loading efficiency of resveratrol.

[0040] (2) This invention constructs a cross-linked gel network using acetylated distarch phosphate, sodium alginate, and calcium chloride to effectively encapsulate resveratrol-loaded protein nanoparticles. Sodium alginate is pH-sensitive, stable in the acidic environment of the stomach, and does not readily release the drug, but dissolves in the neutral or weakly alkaline environment of the intestine. Acetylated distarch phosphate can be degraded by amylases in the intestine. The synergistic effect of these two components, combined with the cross-linking effect of calcium chloride, enables the delivery system to achieve targeted release in the intestine, reducing premature release of resveratrol in the stomach and increasing its effective concentration in the intestine.

[0041] (3) The raw materials used in this invention are all food-grade materials, which are highly safe and the preparation process is simple and easy to implement, with mild operating conditions, making it suitable for industrial production.

[0042] (4) This delivery system can protect resveratrol from the damage of the gastrointestinal environment and improve its stability. At the same time, it can achieve intestinal targeting and amylase-responsive release, effectively improving the bioavailability of resveratrol. It has broad application prospects in the fields of functional foods and medicines. Attached Figure Description

[0043] Figure 1 The particle size distribution of resveratrol-loaded soy protein isolate nanoparticles is shown in the images of unultrasonicated and sonicated at different power levels.

[0044] Figure 2 This is a graph showing the encapsulation efficiency of resveratrol.

[0045] Figure 3 This is a graph showing the retention rate of resveratrol under heat treatment.

[0046] Figure 4 The graph shows the retention rate of resveratrol under ultraviolet light treatment.

[0047] Figure 5The graph shows the in vitro release rate of resveratrol. A represents the in vitro release rate of resveratrol in Examples 1-4 and Comparative Examples 1-4 in simulated gastric juice and amylase-containing simulated intestinal juice environments; B represents the in vitro release rate of resveratrol in Example 1 after treatment in a simulated gastric juice environment in simulated intestinal juice environments with or without amylase; C represents the in vitro release rate of resveratrol in Examples 1 and Comparative Examples 5-7 in simulated gastric juice and amylase-containing simulated intestinal juice environments.

[0048] Figure 6 This is a graph showing the DPPH free radical scavenging rate. Detailed Implementation

[0049] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0050] Example 1

[0051] 1. Preparation of resveratrol-loaded soy protein isolate nanoparticles

[0052] (1) Accurately weigh 3g of soy protein isolate, disperse it in 50mL of deionized water, stir with a magnetic stirrer for 2h at room temperature to fully disperse the soy protein isolate, and place it in a 4℃ refrigerator for 24h to hydrate and obtain a soy protein isolate solution.

[0053] (2) The soy protein isolate solution was placed in an ultrasonic instrument and ultrasonically treated for 20 minutes at 300W under ice bath conditions to obtain the ultrasonically treated soy protein isolate solution. During the ultrasonication process, the ice bath was maintained to prevent the protein structure from being damaged by excessively high temperature.

[0054] (3) Weigh 300 mg of resveratrol and dissolve it in 50 mL of 70% ethanol. Stir until completely dissolved to obtain a resveratrol solution.

[0055] (4) Slowly add the resveratrol solution to the soy protein isolate solution after ultrasonic treatment, and continue to stir and mix at room temperature for 5 hours to allow the resveratrol to fully combine with the protein, and obtain mixed solution 1.

[0056] (5) Place the mixed solution 1 after step (4) into a centrifuge and centrifuge at 8000 r / min for 20 min to precipitate the protein nanoparticles loaded with resveratrol and collect the precipitate after centrifugation.

[0057] (6) The precipitate was washed three times with 50% anhydrous ethanol to remove unbound resveratrol and other impurities, and then freeze-dried to obtain resveratrol-loaded soy protein isolate nanoparticles.

[0058] 2. Preparation of an intestinal-targeted cross-linking gel-supported system

[0059] (1) Weigh 1g of the resveratrol-loaded soy protein isolate nanoparticles, 1g of acetylated distarch phosphate, and 0.5g of sodium alginate obtained in step 1, disperse them together in 50mL of deionized water, stir with a magnetic stirrer for 1h to make them fully mixed, place them in a constant temperature water bath at 80℃ for 15min, then take them out and let them stand, and let them cool naturally to room temperature to obtain mixed solution 2.

[0060] (2) Weigh 1.5g of calcium chloride, add deionized water to dissolve it, and prepare a calcium chloride solution with a mass concentration of 3%.

[0061] (3) Slowly pour the mixed solution 2 into the calcium chloride solution while stirring to carry out the cross-linking reaction and form a gel, thus obtaining the resveratrol-protein nanoparticle intestinal targeted delivery system.

[0062] Example 2

[0063] The only difference from Example 1 is that the ultrasonic power is 600W in step 1, while the other operating steps and conditions are the same.

[0064] Example 3

[0065] The only difference from Example 1 is that in step 2, the amount of calcium chloride used is 3.0g, which is dissolved in deionized water to prepare a calcium chloride solution with a mass concentration of 6%. All other operation steps and conditions are the same.

[0066] Example 4

[0067] The only difference from Example 1 is that the amount of acetylated distarch phosphate used in step 2 is 2g, while the other operation steps and conditions are the same.

[0068] Comparative Example 1

[0069] The only difference from Example 1 is that ultrasonic treatment was not performed in step 1; all other operating steps and conditions are the same.

[0070] Comparative Example 2

[0071] The only difference from Example 1 is that acetylated distarch phosphate was not added in step 2; all other operating steps and conditions are the same.

[0072] Comparative Example 3

[0073] The only difference from Example 1 is that calcium chloride was not added in step 2; all other operating steps and conditions are the same.

[0074] Comparative Example 4

[0075] Only step 1 was performed, i.e., preparing resveratrol-loaded soy protein isolate nanoparticles, without step 2.

[0076] Comparative Example 5

[0077] The only difference from Example 1 is that in step 2, acetylated distarch phosphate is replaced with phosphate starch; all other operating steps and conditions are the same.

[0078] Comparative Example 6

[0079] The only difference from Example 1 is that in step 2, acetylated distarch phosphate is replaced with hydroxypropyl distarch phosphate; all other operating steps and conditions are the same.

[0080] Comparative Example 7

[0081] The only difference from Example 1 is that in step 2, acetylated distarch phosphate is replaced with acetylated distarch adipate; all other operating steps and conditions are the same.

[0082] Example 1

[0083] To further verify the effect of ultrasonic-assisted treatment on the particle size of resveratrol-loaded soy protein isolate nanoparticles in this invention, the particle size of the resveratrol-loaded soy protein isolate nanoparticles prepared in Example 1, Example 2 and Comparative Example 1 (SPI) was measured.

[0084] The particle size of the three samples was determined using a dynamic light scattering particle size analyzer (DLS). Each sample was measured three times, and the average value and standard deviation were recorded.

[0085] like Figure 1 As shown, the resveratrol-loaded soy protein isolate nanoparticles without ultrasonic treatment have a larger particle size, approximately 300 nm; while the particle size of the resveratrol-loaded soy protein isolate nanoparticles treated with 300 W ultrasonic treatment is significantly reduced, and the particle size of the resveratrol-loaded soy protein isolate nanoparticles treated with 600 W ultrasonic treatment is between the two.

[0086] Ultrasonic treatment can influence the molecular structure of soy protein isolate (SPI) through cavitation and mechanical action. Under ultrasound, the aggregated state of protein molecules is disrupted, forming smaller particles and thus reducing the particle size of nanoparticles. At 300W ultrasound treatment, the cavitation and mechanical action resulted in sufficient deaggregation of protein molecules, significantly reducing the nanoparticle size. While increasing the ultrasound power to 600W further affects the protein molecules, the excessive energy may cause over-aggregation or denaturation of some protein molecules, leading to a slight increase in particle size compared to the 300W treatment, but still smaller than the untreated SPI nanoparticles. These experimental results demonstrate that ultrasound-assisted treatment can effectively control the particle size of resveratrol-loaded SPI nanoparticles. Appropriate ultrasound power helps prepare smaller, more uniform nanoparticles. Smaller nanoparticle sizes increase specific surface area, improve resveratrol loading efficiency and stability, and also exhibit better dispersibility and biocompatibility in vivo, further highlighting the advantages of the ultrasound-assisted preparation method of this invention in constructing a resveratrol-protein nanoparticle intestinal targeted delivery system.

[0087] Example 2

[0088] To further explore the performance advantages of the resveratrol-protein nanoparticle intestinal targeted delivery system prepared in this invention, the encapsulation efficiency of each embodiment and comparative example was tested.

[0089] Anhydrous ethanol was mixed with the resveratrol-protein nanoparticle intestinal targeted delivery system at a volume ratio of 10:1. The mixture was sonicated for 60 min at room temperature and centrifuged at 8000 r / min for 10 min. The resulting precipitate was extracted twice more with anhydrous ethanol, and all supernatants were collected and combined. The absorbance of the supernatant was measured at 306 nm using a UV spectrophotometer. The resveratrol content was determined according to a pre-prepared standard curve, and the encapsulation efficiency (EE) was calculated.

[0090] EE (%) = Mc / Mt × 100%

[0091] Where Mc is the mass of the bioactive substance measured, and Mt is the total mass of the bioactive substance.

[0092] like Figure 2As shown, in terms of encapsulation efficiency, the encapsulation efficiency of Examples 1-4 was significantly higher than that of Comparative Examples 1-4. Specifically, the encapsulation efficiency of Example 2 (600W ultrasound) was lower than that of Example 1 (300W ultrasound). Examples 3 (increased calcium chloride content) and 4 (increased acetylated distarch phosphate content) also showed different trends in encapsulation efficiency compared to Example 1. Comparative Example 1, without ultrasound treatment, had a low encapsulation efficiency; Comparative Example 2, without acetylated distarch phosphate, Comparative Example 3, without calcium chloride, and Comparative Example 4, without a cross-linked gel loading system, all had low encapsulation efficiency. The results indicate that ultrasound treatment can alter the molecular structure of soy protein isolate, increasing the binding sites with resveratrol, thereby improving the encapsulation efficiency. For example, the 300W ultrasound treatment in Example 1 resulted in a more significant change in protein structure, promoting resveratrol encapsulation and increasing the encapsulation efficiency. The cross-linked gel network constructed from acetylated distarch phosphate, sodium alginate, and calcium chloride effectively encapsulated and protected the nanoparticles. Example 3 increased the amount of calcium chloride, and Example 4 increased the amount of acetylated distarch phosphate, altering the gel network structure and affecting the encapsulation ability of resveratrol. The comparative examples, lacking ultrasonic treatment, key raw materials, or cross-linking gel construction steps, failed to form an effective encapsulation structure, resulting in poor resveratrol encapsulation. This invention, through ultrasonic-assisted treatment combined with specific raw materials and ratios, can significantly improve the encapsulation rate of the resveratrol-protein nanoparticle intestinal targeted delivery system, powerfully demonstrating the superiority of this invention in constructing a highly efficient resveratrol intestinal targeted delivery system.

[0093] Example 3

[0094] The thermal stability of the resveratrol-protein nanoparticle intestinal targeted delivery system prepared in each example and comparative example was tested.

[0095] The retention rate of resveratrol was determined after heat treatment at 90℃ for 120 min. Immediately after heat treatment, the samples were placed in an ice-water bath to detect the remaining resveratrol content. The percentage of resveratrol remaining in the sample relative to the initial amount was defined as the retention rate, which characterizes thermal stability. Each sample was measured in triplicate, and the average value and standard deviation were recorded.

[0096] like Figure 3As shown, the thermal stability of Examples 1-4 is significantly better than that of Comparative Examples 1-4. Examples 1-4 exhibit higher resveratrol retention rates during heating, with Examples 3 and 4 showing relatively higher retention rates. Comparative Examples 1-4 show lower retention rates, especially Comparative Example 4, which has a significantly lower retention rate than the other groups. In this invention, ultrasound-assisted treatment and a specific combination of raw materials (a cross-linked gel network constructed from acetylated distarch phosphate, sodium alginate, and calcium chloride) play a crucial role in improving the thermal stability of the resveratrol-protein nanoparticle intestinal targeted delivery system. Ultrasound treatment alters the structure of soy protein isolate, allowing it to more tightly encapsulate resveratrol and reducing resveratrol loss under heat. The cross-linked gel network provides a stable microenvironment for resveratrol, effectively preventing damage from external factors during heating. Increasing the amount of calcium chloride in Example 3 and increasing the amount of acetylated distarch phosphate in Example 4 further optimized the gel network structure, enhanced temperature tolerance, and enabled better retention of resveratrol at high temperatures. The comparative examples, lacking ultrasonic treatment, key raw materials, or a fully constructed cross-linked gel network, failed to provide effective protection for resveratrol, leading to its easy degradation or escape upon heating and resulting in poor thermal stability. This invention, through ultrasonic-assisted treatment and a rational raw material formulation, significantly improves the thermal stability of the resveratrol-protein nanoparticle intestinal targeted delivery system, ensuring the stability of resveratrol under certain temperature conditions and providing strong assurance for the stability of this delivery system in practical applications.

[0097] Example of effect 4

[0098] The stability of the resveratrol-protein nanoparticle intestinal targeted delivery system prepared in each example and comparative example was tested under ultraviolet light irradiation.

[0099] The sample in a 5 mL beaker was irradiated with ultraviolet light at a wavelength of 365 nm for 120 min. Immediately after irradiation, the sample was taken, and the retention rate of resveratrol in the sample was calculated. The percentage of resveratrol remaining relative to the initial amount was defined as the retention rate, which measures ultraviolet stability. Each sample was measured in triplicate, and the average value and standard deviation were recorded.

[0100] like Figure 4As shown, the retention rates of resveratrol in Examples 1-4 under UV irradiation were significantly higher than those in Comparative Examples 1-4. Examples 3 and 4 showed relatively higher retention rates, while Examples 1 and 2 also exhibited good stability. The retention rates of the comparative examples were generally lower, with Comparative Example 4 showing a significantly lower retention rate than the other groups. Results analysis: Ultrasonic-assisted treatment and the specific composite gel network structure are crucial for improving the UV stability of resveratrol in this invention. Ultrasonic treatment alters the structure of soy protein isolate, making it more tightly bound to resveratrol, and the resulting nanoparticles can, to some extent, block the damage of internal resveratrol by UV light. The cross-linked gel network constructed from acetylated distarch phosphate, sodium alginate, and calcium chloride acts as a barrier, effectively reducing the intensity of UV irradiation on the resveratrol within the nanoparticles and decreasing its photodegradation. Increasing the amount of calcium chloride in Example 3 and increasing the amount of acetylated distarch phosphate in Example 4 further optimized the gel network, enhancing its resistance to UV light and allowing for better retention of resveratrol under UV irradiation. The comparative examples, lacking ultrasonic treatment, key raw materials, or a complete cross-linked gel network construction, could not provide sufficient UV protection for resveratrol, leading to its easy degradation and low retention rate under UV irradiation. This invention, through ultrasound assistance and a rational raw material formulation, can significantly improve the stability of the resveratrol-protein nanoparticle intestinal targeted delivery system under UV light, providing strong assurance for the stability of this delivery system in practical applications, especially in scenarios potentially affected by UV light.

[0101] Example 5

[0102] The in vitro release characteristics of the resveratrol-protein nanoparticle intestinal targeted delivery systems prepared in each example and comparative example were tested.

[0103] Release experiment in simulated gastric juice environment: Take an appropriate amount of the sample to be tested and place it in simulated gastric juice (pH=1.2, containing pepsin). Shake at 37℃ and 100r / min. Take samples at time intervals (0.5h, 1h, 1.5h and 2h). After centrifugation and filtration pretreatment, determine the content of resveratrol and calculate the cumulative release rate.

[0104] Simulated intestinal fluid environment release experiment: Simulated intestinal fluid containing amylase: The test samples were placed in simulated intestinal fluid (pH=7.4, containing amylase) and shaken at 37℃ and 100 r / min. Samples were taken at regular intervals (2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, and 6h). After centrifugation and filtration pretreatment, the resveratrol content was determined, and the cumulative release rate was calculated. This step was used to investigate the release of 11 groups of samples (Examples 1-4 and Comparative Examples 1-7) in the intestinal environment and under the action of amylase.

[0105] Taking the sample from Example 1 as an example, after release in a simulated gastric fluid environment, release was performed in a simulated intestinal fluid environment (with or without amylase). The resveratrol content was measured, and the cumulative release rate was calculated. This step was used to compare the release differences in Example 1 with and without amylase, and to explore the enzyme-targeted release characteristics.

[0106] Each sample was measured in parallel three times, and the average value and standard deviation were recorded.

[0107] like Figure 5 As shown in Figure A, in a simulated gastric fluid environment, the release rates of all samples were low within the first 2 hours, with the release rates of Examples 1-4 generally lower than those of Comparative Examples 1-4. In a simulated intestinal fluid environment containing amylase, the resveratrol release rates of Examples 1-4 gradually increased over time. Differences in release curves existed between the examples due to variations in factors such as ultrasonic power and raw material dosage. Figure 5 As shown in Figure B, the release of resveratrol in Example 1 was examined separately in simulated intestinal fluid containing and without amylase. It was found that in simulated intestinal fluid containing amylase, the release rate of resveratrol was faster and the final release rate was higher; while in simulated intestinal fluid without amylase, the release rate was relatively slower and the final release rate was lower. In a simulated gastric fluid environment, the samples prepared in Examples 1-4 of this invention exhibited stable gel structures under acidic conditions due to the pH sensitivity of sodium alginate, effectively inhibiting premature release of resveratrol and resulting in a lower release rate than the comparative group. In a simulated intestinal fluid environment containing amylase, the composite gel network of Examples 1-4 (constructed from acetylated distarch phosphate, sodium alginate, and calcium chloride) played a crucial role. On one hand, sodium alginate gradually dissolved in a neutral environment; on the other hand, acetylated distarch phosphate degraded under the action of amylase. This dual action promoted rapid release of resveratrol, with a release rate higher than the comparative group. The comparative group, lacking key raw materials or structures, could not achieve effective targeted release. Comparing the release of acetylated distarch phosphate in simulated intestinal fluid containing and without amylase in Example 1 fully demonstrates the responsiveness of acetylated distarch phosphate to amylase. In the presence of amylase, the degradation of acetylated distarch phosphate accelerates gel network disruption and promotes resveratrol release, proving that the delivery system of this invention has excellent enzyme-targeted release characteristics. The resveratrol-protein nanoparticle intestinal targeted delivery system prepared in this invention can effectively achieve intestinal targeting and amylase-responsive release, reduce premature release of resveratrol in the stomach, and increase its effective concentration in the intestine, showing promising application prospects.

[0108] In addition, such as Figure 5As shown in Figure C, based on the analysis of release kinetics, Comparative Example 5 had the fastest initial release rate, with resveratrol released rapidly; Comparative Example 6 had the second fastest release rate, with an initial release rate slightly lower than Comparative Example 5, and the release rate gradually approaching that of Comparative Example 6; Example 1 had a relatively slow release process, with a final release rate lower than Comparative Examples 5 and 6, exhibiting the characteristic of "slow release in the early stage, accelerated release in the later stage, but low overall release level"; Comparative Example 7 had the slowest release rate, with a release rate significantly lower than other groups, extremely slow release in the early stage, and although there was an increase in the later stage, the magnitude was limited. The strong hydrophilicity of the phosphate group causes it to rapidly absorb water and swell in gastric juice, disrupting the binding of the starch network to the carrier, leading to the release of resveratrol, resulting in rapid release in the early stage and a relatively high overall release rate. Hydroxypropyl distarch phosphate combines the hydrophilicity of the hydroxypropyl group with the flexibility of the molecular chain, so the swelling is slightly slower, but as time goes on, the release accelerates after the network disintegrates, with the overall release rate falling between Comparative Example 5 and Examples 1 and 7. Due to the strong resistance of the hydroxypropyl ether bond to enzymatic degradation, the enzyme-targeted degradation efficiency is insufficient. Acetylated distarch phosphate, through the hydrophobicity of the acetyl groups, hinders rapid water penetration, delaying carrier swelling and network disruption, thus slowing resveratrol release and preventing premature large-scale release in the stomach. The phosphate groups are gradually hydrolyzed by amylase in simulated intestinal fluid, promoting carrier dissolution and increasing the release rate; however, due to the initial network density, the final release rate is still lower than that of comparative examples 5 and 6. Adipic acid strengthens the cross-linking of starch molecules through "bridging," forming a network structure with smaller pores and greater rigidity. During hydration, carrier swelling and disintegration are extremely difficult, and resveratrol is spatially confined and strongly bound, resulting in extremely slow initial release and limited release in the later stages. In summary, the bifunctional modification of acetylated distarch phosphate can better achieve the synergy of intestinal targeted retention and enzyme-targeted degradation, thus exhibiting release performance more suited to the design requirements in in vitro release experiments.

[0109] Example 6

[0110] The antioxidant properties of the resveratrol-protein nanoparticle intestinal targeted delivery system prepared in each example and comparative example were tested, and the DPPH free radical scavenging rate was performed.

[0111] Anhydrous ethanol was mixed with the resveratrol-protein nanoparticle intestinal targeted delivery system at a volume ratio of 10:1. The mixture was sonicated at room temperature for 60 min, centrifuged at 8000 rpm for 10 min, and the resulting precipitate was extracted twice more. All supernatants were collected and combined as the sample solution. The liquid sample was analyzed using a DPPH free radical scavenging assay kit. Each sample was measured in triplicate, and the average value and standard deviation were recorded.

[0112] The DPPH radical scavenging results show that Comparative Example 4 had the lowest scavenging rate, while Comparative Examples 2 and 3 also had lower scavenging rates than the Example. In Examples 1-4 and Comparative Examples 5-7, the groups using acetylated distarch phosphate showed better overall scavenging rates. This phenomenon is attributed to the stability of the encapsulation system, the compatibility of the starch structure, and the preparation process. Firstly, Comparative Example 4 lacked a gel network constructed from cross-linked starch, sodium alginate, and calcium chloride, leading to easy loss and oxidative degradation of resveratrol during centrifugation, washing, and extraction, resulting in the exposure and loss of the active ingredient. Comparative Example 3 lacked calcium chloride, preventing sodium alginate from undergoing ionic cross-linking, resulting in a loose gel structure, poor nanoparticle dispersion, and reduced encapsulation efficiency. In contrast, the Example and complete formulation groups, through the spatial support of acetylated distarch phosphate and the cross-linking reinforcement of sodium alginate and calcium chloride, formed a dense three-dimensional network, effectively trapping nanoparticles and reducing resveratrol loss during processing and extraction. Secondly, the type of starch determines the encapsulation compatibility. Compared to phosphate distarch, hydroxypropyl distarch phosphate, and acetylated distarch adipate, acetylated distarch phosphate, with its acetyl modification providing a hydrophilic-hydrophobic balance, results in a gel pore size that better matches the nanoparticle size after cross-linking, thus improving encapsulation stability. Furthermore, there is an efficiency threshold for ultrasonic power. The clearance rate of Example 1 (300W) is better than that of Example 2 (600W), indicating that 300W ice-bath ultrasonication effectively disrupts the soybean protein isolate aggregates, achieving molecular-level dispersion and better binding of resveratrol. Higher power, however, did not further optimize the encapsulation interface, suggesting that ultrasonic parameters need to be adjusted in conjunction with system characteristics. The low clearance rate in the component deficiency experiments (Comparative Example 2 lacking starch, Comparative Example 3 lacking calcium chloride) further demonstrates the synergistic effect of the system: starch provides mechanical support, and calcium chloride drives dynamic cross-linking. The interfacial interaction between the two and the protein stabilizes the encapsulation structure, ensuring the retention and release of resveratrol's antioxidant activity. In summary, the stable gel encapsulation system significantly enhances the DPPH radical scavenging ability by reducing the loss of active ingredients, adapting to the structural compatibility of starch, and optimizing preparation parameters, providing a scientific basis for the encapsulation design of functional nanoparticles and the regulation of intermolecular interactions and structures.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for the preparation of an intestinal targeted delivery system of resveratrol-protein nanoparticles, characterized by, The method comprises the following steps: S1: mixing resveratrol with ultrasonic-treated hydrated protein, and separating the solid from the liquid to obtain resveratrol-loaded protein nanoparticles; S2: mixing the resveratrol-loaded protein nanoparticles obtained in step S1, acetylated distarch phosphate and sodium alginate to obtain a mixed solution; S3: mixing calcium chloride with the mixed solution obtained in step S2, and performing cross-linking reaction to obtain the resveratrol-protein nanoparticle intestinal-targeted delivery system. The protein is soybean protein isolate. In step S2, the mass ratio of the resveratrol-loaded protein nanoparticles to acetylated distarch phosphate is 1:(1-2).

2. The production method according to claim 1, wherein In step S1, the power of the ultrasonic treatment is 300-600 W.

3. The production method according to claim 1, wherein In step S1, the ratio of the protein to water used in the ultrasonic treatment of the hydrated protein is 1 g:(15-20) mL.

4. The production method according to claim 1, wherein In step S1, the mass ratio of resveratrol to the protein used in the ultrasonic treatment of the hydrated protein is 1:(10-15).

5. The production method according to claim 1, wherein In step S2, the mass ratio of the resveratrol-loaded protein nanoparticles to sodium alginate is 1:(0.3-0.5).

6. The production method according to claim 1, wherein In step S3, the mass ratio of the resveratrol-loaded protein nanoparticles to calcium chloride is 1:(1.5-3).

7. An enteric targeted delivery system of resveratrol-protein nanoparticle characterized in that, The resveratrol-protein nanoparticle intestinal-targeted delivery system is prepared by the method according to any one of claims 1-6.

8. Use of the resveratrol-protein nanoparticle intestinal-targeted delivery system according to claim 7 in the preparation of an antioxidant.

9. An antioxidant agent characterized in that, The antioxidant contains the resveratrol-protein nanoparticle intestinal-targeted delivery system according to claim 7.

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