Preparation method and application of corn protein particles selectively embedding ginsenoside Rd

Abstract

The present application relates to the field of active substance embedding delivery technology, and particularly relates to a preparation method and application of corn protein particles for selectively embedding ginsenoside Rd. The preparation method comprises the following steps: (1) dissolving zein in an ethanol solution; (2) adding total ginsenoside to the zein solution; (3) preparing casein buffer, and quickly adding the casein buffer to the mixture; (4) removing ethanol by using a rotary evaporation method; (5) filtering the nanoparticle dispersion; and (6) freeze-drying. The prior art needs to use high-purity ginsenoside Rd monomers as embedding raw materials, and the direct embedding of crude extracts has no selectivity, and the product components are mixed and the quality is uncontrollable. The present application uses the specific binding ability of zein to ginsenoside Rd to selectively enrich ginsenoside Rd from a mixture containing multiple saponins, and the proportion of Rd in the embedded total saponins can reach more than 90%. The overall preparation cost is significantly reduced, and the production is suitable for large-scale industrialization.

Description

Technical Field

[0001] This invention relates to the field of active substance encapsulation and delivery technology, specifically to a method for preparing corn protein particles with selective encapsulation of ginsenoside Rd and its application. Background Technology

[0002] Ginsenoside Rd is one of the important active ingredients in medicinal plants such as ginseng, Panax notoginseng, and American ginseng, belonging to the protopanaxadiol type of saponin monomers. Numerous studies have shown that ginsenoside Rd possesses excellent hepatoprotective, anti-inflammatory, antioxidant, anti-apoptotic, and neuroprotective activities. Especially in alcoholic liver injury models, ginsenoside Rd exerts significant acute hepatoprotective effects through mechanisms such as regulating oxidative stress, inhibiting inflammatory signaling pathways (such as NF-κB), and reducing mitochondrial damage. Therefore, ginsenoside Rd is considered an ideal candidate active ingredient for developing oral hepatoprotective formulations.

[0003] However, ginsenoside Rd faces two major bottlenecks in actual oral delivery: firstly, its water and lipid solubility are both unsatisfactory, resulting in poor stability in the gastrointestinal environment and low oral bioavailability; secondly, the extraction and purification of high-purity ginsenoside Rd monomers is complex and costly, directly limiting the industrialization prospects of using it as a raw material to prepare functional formulations. To address these challenges, researchers have attempted to use nano-encapsulation technology to improve the stability and absorption efficiency of ginsenoside Rd, while simultaneously hoping to achieve efficient encapsulation using lower-cost raw materials.

[0004] Among numerous nanocarriers, zein (commonly known as corn protein) has become a research hotspot for the oral delivery of hydrophobic or amphiphilic bioactive substances due to its excellent self-assembly properties, hydrophobic interaction capabilities, and biodegradability. Corn protein can self-assemble into nanoparticles via antisolvent precipitation, effectively encapsulating various bioactive molecules. Existing literature reports that encapsulating ginsenoside Rd using corn protein nanoparticles can improve its stability and has shown certain hepatoprotective effects in animal experiments.

[0005] However, there are still key technical problems in the existing technology that have not been solved, mainly in the following two aspects: First, there is the issue of raw material cost. Existing encapsulation studies almost exclusively use high-purity ginsenoside Rd monomers as encapsulation raw materials. Because ginsenoside Rd is present in low concentrations in plant materials and coexists with various structurally similar saponins (such as ginsenosides Rb1, Rb2, Rc, Rg1, Re, etc.), obtaining high-purity Rd requires multi-step column chromatography or preparative high-performance liquid chromatography, resulting in extremely high monomer prices. If lower-cost crude ginsenoside extracts (total saponins) are used directly for encapsulation, traditional methods lack selectivity for different saponin monomers, leading to the non-specific encapsulation of multiple saponins from the crude extract. This results in a complex final product composition, unclear active ingredients, and difficulty in quality control. Therefore, how to achieve selective encapsulation of ginsenoside Rd while reducing raw material costs remains a challenge that current technology has not yet solved.

[0006] Second, there is the issue of freeze-drying stability. Freeze-drying is a common post-processing method for protein nanocarriers to extend the storage time of nanoparticles and facilitate transportation. However, our previous research revealed that when ginsenoside Rd is encapsulated in corn protein nanoparticles, the strong interaction between Rd and corn protein induces a more ordered arrangement of the corn protein molecular chains, significantly increasing the crystallinity of the nanoparticles. High crystallinity makes the particles more prone to irreversible aggregation during freeze-drying, resulting in a sharp decrease in redispersibility, a significant increase in particle size, and even the formation of large particle precipitates. This freeze-drying instability severely weakens the storage stability, reconstitution effect, and in vivo absorption efficiency of the formulation, making corn protein-Rd nanoparticles difficult to apply practically. Although crystallization behavior can be adjusted by adding other excipients, arbitrarily adding excipients often significantly reduces the encapsulation rate of ginsenoside Rd, leading to the loss of active ingredients.

[0007] Currently, no technical solutions have been reported that simultaneously address both of the aforementioned issues. On the one hand, there is a lack of methods for preparing corn protein particles that can selectively enrich Rd from crude ginsenoside extracts while maintaining high encapsulation efficiency; on the other hand, there is also a lack of strategies to effectively reduce Rd-induced crystallinity and improve freeze-drying stability without sacrificing Rd encapsulation efficiency. Therefore, developing a low-cost, highly selective, and freeze-drying-stable ginsenoside Rd nano-encapsulation system is of great significance for promoting its practical application in functional foods or pharmaceutical preparations, such as those for alcoholic acute liver injury.

[0008] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the inventors studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0009] This invention relates to the field of active substance encapsulation and delivery technology, specifically to a method for preparing corn protein particles with selective encapsulation of ginsenoside Rd and its application.

[0010] To address the issues of the high cost of ginsenoside Rd monomers, the excessive cost of directly using ginsenoside Rd monomers for encapsulation, and the reduced freeze-drying stability due to the interaction between ginsenoside Rd and corn protein increasing the crystallinity of corn protein particles, this invention utilizes the selective binding ability of corn protein to ginsenoside Rd. It selects and purchases lower-cost crude ginsenoside extracts for encapsulation, preparing particles with selectively encapsulated ginsenoside Rd. Simultaneously, casein is used to reduce the crystallinity of corn protein, improving the freeze-drying stability of the corn protein particles without significantly affecting the encapsulation rate of ginsenoside Rd.

[0011] To address the aforementioned technical problems, one objective of this invention is to provide a method for rapidly separating ginsenoside Rd from total ginsenosides, comprising the following steps: (1) Dissolve zein in an ethanol solution to obtain a zein solution; (2) Add total ginsenosides to the zein solution and wait for it to dissolve completely to obtain a mixture; (3) Prepare casein buffer solution. Quickly add casein buffer solution to the above mixture and stir to form nanoparticle dispersion. (4) Ethanol in the nanoparticle dispersion was removed by rotary evaporation; (5) The nanoparticle dispersion was filtered with a filter membrane with a pore size of 100~200 kDa to retain the corn protein particles encapsulating ginsenoside Rd, remove unbound free saponins and large particle impurities, and obtain purified corn protein particles. (6) The purified nanoparticle dispersion was pre-frozen and then freeze-dried.

[0012] According to a preferred embodiment, in step (1), the volume fraction of the ethanol solution is 60%~80%. Preferably, in step (1), the volume fraction of the ethanol solution is 70%. Alternatively, the volume fraction of the ethanol solution is 60%. Or, the volume fraction of the ethanol solution is 80%.

[0013] According to a preferred embodiment, in step (2), the mass ratio of zein to total ginsenosides is 0.5~2:1. Preferably, in step (2), the mass ratio of zein to total ginsenosides is 1:1. The mass ratio of zein to total ginsenosides is 0.5:1. The mass ratio of zein to total ginsenosides is 2:1.

[0014] According to a preferred embodiment, in step (2), the mass fraction of ginsenoside Rd in the total ginsenosides is not less than 5%. Preferably, in step (2), the mass fraction of ginsenoside Rd in the total ginsenosides is 5%~10%.

[0015] According to a preferred embodiment, in step (3), the volume of casein buffer added is 2 to 6 times the volume of corn protein solution. Preferably, the volume of casein buffer added is 2 times the volume of corn protein solution. Preferably, the volume of casein buffer added is 3 times the volume of corn protein solution. Preferably, the volume of casein buffer added is 4 times the volume of corn protein solution. Preferably, the volume of casein buffer added is 5 times the volume of corn protein solution. Preferably, the volume of casein buffer added is 6 times the volume of corn protein solution.

[0016] According to a preferred embodiment, casein is stirred overnight in a dilute alkaline solution until completely dissolved, and then diluted with 10 mM pH 8.6 phosphate buffer to a final concentration of 5 mg / mL.

[0017] According to a preferred embodiment, in step (3), the volume ratio of the mixture to water is 1:3 to 6. Preferably, the volume ratio of the mixture to water is 1:3. The volume ratio of the mixture to water is 1:4. The volume ratio of the mixture to water is 1:5. The volume ratio of the mixture to water is 1:6.

[0018] According to a preferred embodiment, in step (4), the rotary evaporation temperature is 40~50°C. Preferably, the rotary evaporation temperature is 46°C.

[0019] According to a preferred embodiment, in step (5), the pore size of the filter membrane is 100 kDa. The pore size of the filter membrane is 150 kDa. The pore size of the filter membrane is 200 kDa.

[0020] According to a preferred embodiment, in step (6), the pre-freezing temperature is -60~-80℃, and the vacuum degree of freeze drying is less than 20 Pa.

[0021] One of the objectives of this invention is to provide corn protein particles that selectively encapsulate ginsenoside Rd, the corn protein particles being prepared based on the above-described method for preparing corn protein particles with selective encapsulation of ginsenoside Rd.

[0022] One of the objectives of this invention is to provide the application of the corn protein particles with selectively encapsulated ginsenoside Rd in the preparation of oral functional foods, health products or hepatoprotective drugs for the prevention, relief or treatment of alcoholic acute liver injury.

[0023] One objective of this invention is to provide a method for improving the freeze-drying stability of ginsenoside Rd-supported zein nanoparticles. Specifically, casein is compounded into the zein-encapsulated ginsenoside Rd system, with a casein to zein mass ratio of 0.1-2:1. Preferably, casein coating the particle surface causes the nanoparticle's zeta potential to change from positive to negative. Casein coating the particle surface reduces Rd-induced zein crystallinity, ensuring that the particle size ratio before and after freeze-drying is no greater than 1.15.

[0024] The present invention has the following beneficial effects: 1. Significantly reduce raw material preparation costs and achieve low-cost industrial production. Existing technologies require the use of high-purity ginsenoside Rd monomers as encapsulation raw materials. Direct encapsulation of crude extracts lacks selectivity, resulting in mixed product components and uncontrollable quality. This invention utilizes the specific binding ability of zein to ginsenoside Rd, allowing the direct use of inexpensive crude ginsenoside extracts as raw materials. It selectively enriches ginsenoside Rd from mixtures containing multiple saponins, achieving an Rd content of over 90% in the total encapsulated saponins, with clearly defined and controllable active ingredients. It eliminates the expensive steps and consumable costs of multi-step column chromatography and liquid-phase purification of Rd monomers, significantly reducing overall preparation costs and making it suitable for large-scale industrial production.

[0025] 2. The problem of freeze-drying agglomeration was solved with almost no loss of ginsenoside Rd encapsulation efficiency, resulting in excellent freeze-drying reconstitution stability. Ginsenoside Rd interacts strongly with corn protein, increasing the protein molecule's order and particle crystallinity. Traditional casein-free systems exhibit irreversible aggregation after freeze-drying, with the reconstituted particle size increasing dramatically from 70 nm to over 200 nm, easily leading to precipitation and a significant decrease in the storage and usability of the formulation. This invention uses a compound of food-grade milk casein coated onto the surface of nanoparticles: (1) DSC and XRD characterization confirmed that casein can destroy the highly ordered crystalline structure of corn protein induced by Rd, reduce the overall crystallinity of particles, and inhibit particle aggregation in the freeze-drying process from the root. (2) When the optimal ratio of casein to corn protein is 0.5:1, the Rd loading rate is still maintained at about 5.9%, which is close to the 6.2% encapsulation level of the system without casein. The retention rate of active substances is high and there is no obvious loss of active ingredients. (3) The particle size is stably maintained at 50~80 nm before and after freeze-drying, and the freeze-dried / fresh particle size ratio is as low as 1.0~1.1. There is no obvious large particle precipitation after reconstitution. Microscopic electron microscopy shows that the particles are spherical and uniformly dispersed, which greatly improves the storage, transportation and reconstitution performance of freeze-dried formulations.

[0026] 3. The surface charge of nanoparticles is controllable, improving the stability of colloidal dispersion. Corn protein-Rd nanoparticles without casein have a strong positive charge (+51 mV) on their surface, making the system prone to electrostatic aggregation. After adding an appropriate proportion of casein, the zeta potential of the particles changes to a strong negative charge of -30 to -60 mV, which enhances the electrostatic repulsion between particles and significantly improves the colloidal stability of the fresh dispersion when stored at room temperature, reducing the risk of particle aggregation during preparation and storage.

[0027] 4. The small particle size of the finished product is beneficial to improving oral bioavailability. The nanoparticles prepared by this invention maintain an ultra-small particle size of 50-80 nm after both fresh preparation and freeze-drying and reconstitution. Compared with the large particles after traditional freeze-drying and agglomeration, the smaller particle size can improve the permeability of the gastrointestinal mucosa, improve the inherent defects of ginsenoside Rd such as poor water solubility, poor oral absorption, and low bioavailability, and improve in vivo delivery efficiency.

[0028] 5. The entire process is green and safe, and the operation is simple and easy to scale up. The entire preparation process uses only an ethanol-water system and food-grade zein and casein, without any toxic or harmful organic solvents. The steps are as follows: protein dissolution, saponin co-dissolution, casein antisolvent self-assembly, rotary evaporation to remove alcohol, membrane filtration purification, and freeze drying. The process is continuous and controllable, the reaction conditions are mild, the equipment is conventional and readily available, and there is no need for complex and precise purification equipment, making it easy to scale up for industrial production.

[0029] 6. The freeze-dried product retains complete biological activity and has a better liver-protective effect than existing formulations. Animal models of acute alcoholic liver injury confirmed that oral administration of the freeze-dried and reconstituted nanoparticles of this invention can significantly reduce serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in mice, alleviating alcohol-induced hepatocellular damage. At the same Rd dosage, its hepatoprotective effect is significantly better than that of a simple mixture of crude ginsenoside extract and blank protein particles, and also better than that of the positive control drug silymarin. This demonstrates that the selective encapsulation system not only improves physicochemical stability but also enhances the in vivo hepatoprotective efficacy of Rd, making it suitable for the development of hepatoprotective functional foods, health products, and oral hepatoprotective drugs.

[0030] 7. One process solves two major industry pain points simultaneously, expanding application scenarios. Existing technologies can only address one specific issue: raw material cost or freeze-drying stability. No integrated solution simultaneously addresses both selective Rd enrichment and freeze-drying aggregation inhibition. This invention's single preparation process achieves two core advantages: low-cost selective encapsulation and high freeze-drying reconstitution stability. It can be used directly as a functional oral dry powder formulation or further processed into tablets, capsules, oral liquids, and other dosage forms, possessing significant practical application value in the prevention and intervention of alcoholic liver injury. Attached Figure Description

[0031] Figure 1The encapsulation of saponin monomers in total ginsenosides by nanoparticles formed to increase the amount of casein added (represented by liquid phase diagram). Figure 2 To determine the loading efficiency of total ginsenosides and ginsenoside Rd at different mass ratios of added casein to zein, and the proportion of ginsenoside Rd in the loaded total ginsenosides; Figure 3 To investigate the effects of increasing the amount of casein added on the particle's (A) zeta potential, (B) particle size, (C) particle size of the lyophilized particles after redispersement, and (D) the ratio of the average particle size after lyophilization to that before lyophilization. Figure 4 Transmission electron microscopy (TEM) images of saponin-loaded nanoparticles after freeze-drying and redispersion. The casein-coated nanoparticles contain 0.5 times the amount of corn gluten. Figure 5 DSC spectra of ginsenoside Rd (A) unloaded and (B) loaded at different mass ratios of added casein to zein. Figure 6 XRD patterns of ginsenoside Rd (A) unloaded and (B) loaded at different mass ratios of added casein to zein. Figure 7 To investigate the protective activity of ginsenoside-loaded particles against alcoholic acute liver injury. Detailed Implementation

[0032] In the description of this invention, terminology is used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly defined.

[0033] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; materials, reagents, or instruments whose manufacturers are not specified are all commercially available; conditions not specified in the examples are performed according to conventional conditions or conditions recommended by the manufacturer. Furthermore, this invention does not limit the source of the raw materials used; unless otherwise specified, all raw materials used in this invention are commercially available products commonly found in this technical field. Zein was purchased from Beijing Solarbio Science & Technology Co., Ltd., with a purity ≥95%. Total ginsenosides were purchased from Shaanxi Jinkangtai Biotechnology Co., Ltd. Unless otherwise specified, all solvents used were of analytical grade. Polyethersulfone filter membranes were purchased from Merck. Casein (purity ≥90%) was purchased from Sigma-Aldrich. Anhydrous ethanol, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium hydroxide, etc., were all of analytical grade. A dynamic light scattering particle size analyzer (DLS, Malvern Zetasizer Nano ZS) was used to determine particle size and zeta potential. A transmission electron microscope (TEM, JEOL JEM-2100F) was used to observe particle morphology. Ultrafiltration tubes (100 kDa molecular weight cutoff) were used to separate unreintegrated casein and unloaded ginsenosides. A Christ Alpha 1-2 LDplus freeze dryer was used.

[0034] Example 1: Preparation of casein-corn protein nanoparticles selectively encapsulating ginsenoside Rd This embodiment provides nanoparticles based on corn protein and casein, which can selectively encapsulate ginsenoside Rd from crude ginsenoside extract and have good freeze-drying stability.

[0035] 1) Preparation of zein solution: Weigh 1.0 g of zein powder and dissolve it in 100 mL of 70% (v / v) ethanol-water solution. Stir at room temperature until completely dissolved to obtain a zein solution with a concentration of 10 mg / mL.

[0036] (2) Preparation of ginsenoside crude extract solution: Weigh 0.2 g of ginsenoside crude extract powder and add it to the above zein solution. Continue stirring until completely dissolved to obtain a zein-ginsenoside crude extract mixture. The mass ratio of ginsenoside crude extract to zein is 1:5.

[0037] (3) Preparation of casein solution: Weigh 0.5 g of casein powder, disperse it in 50 mL of deionized water, adjust the pH to 8.6 with 0.1 M NaOH solution, and stir overnight at 4°C until completely dissolved. Then dilute with 10 mM pH 8.6 phosphate buffer to a final concentration of 5 mg / mL to obtain the casein solution. In this example, the mass ratio of casein to corn protein is 0.5:1.

[0038] (4) Formation of nanoparticles: Under vigorous stirring (800 rpm), 4 times the volume (400 mL) of the above casein solution was quickly added to 100 mL of corn protein-ginsenoside crude extract mixture, and stirring was continued for 30 minutes to obtain a milky white nanoparticle solid dispersion.

[0039] (5) Removal of ethanol: The above dispersion was transferred to a rotary evaporator and evaporated at 46°C and 0.08 MPa for 30 minutes to remove ethanol from the system.

[0040] (6) Purification: The nanoparticle dispersion after removing ethanol was filtered through a polyethersulfone membrane with a pore size of 100 kDa to remove unloaded ginsenosides and any large particulate impurities that may form. The filtrate was collected to obtain the purified nanoparticle dispersion.

[0041] (7) Freeze-drying: The above nanoparticle dispersion was pre-frozen at -80℃ for 12 hours, and then transferred to a freeze dryer and dried at a vacuum degree <10 Pa and -50℃ for 48 hours to obtain freeze-dried powdered casein-corn protein nanoparticles selectively encapsulating ginsenoside Rd.

[0042] Example 2: Effect of different casein ratios on nanoparticle properties To optimize the amount of casein added, following the method in Example 1, only the mass ratio of casein to corn protein was changed, and the following experimental groups were set up: 0:1 (i.e., no casein added), 0.25:1, 0.5:1, and 1:1. The amount of corn protein was fixed at 1.0 g, and the amount of crude ginsenoside extract was fixed at 0.2 g. Nanoparticles were prepared for each group, and the following indicators were measured: (1) Determination of loading rate: Free saponins were separated by ultrafiltration. 5 mL of nanoparticle dispersion was placed in a 10 kDa ultrafiltration centrifuge tube and centrifuged at 8000 g for 30 minutes. The filtrate was collected for the determination of free ginsenosides. ① The total ginsenoside content was determined by colorimetric analysis using the sulfuric acid-phenol method, followed by UV absorbance measurement at 490 nm. A standard curve was plotted using ginsenoside Re as a standard to calculate the total saponin content. ② The ginsenoside Rd content was determined by high performance liquid chromatography: chromatographic column C18 (4.6×250 mm, 5 μm); mobile phase acetonitrile-water (gradient elution: 0-30 min, 20% acetonitrile → 40% acetonitrile); flow rate 1.0 mL / min; detection wavelength 203 nm; column temperature 30℃. The amount of embedded saponins in the filter bottom (retained portion) was calculated by subtracting the free amount from the total added amount. Loading rate (%) = (mass of encapsulated saponins / mass of protein particles) × 100%.

[0043] Experimental results are as follows Figure 2As shown (A is the loading efficiency of total ginsenosides and Rd, and B is the proportion of Rd in the loaded total ginsenosides).

[0044] The results showed that without the addition of casein (mass ratio 0:1), the average loading rate of ginsenoside Rd was 6.2%, the total saponin loading rate was approximately 6.2%, and Rd accounted for 100% of the total encapsulated saponins. As the casein ratio increased to 0.5:1, the Rd loading rate slightly decreased to 5.9%, while the total saponin loading rate increased to 8.3%. When the casein ratio further increased to 1:1, the loading rates of both Rd and total saponins continued to decrease.

[0045] (2) Particle size and zeta potential: Freshly prepared and lyophilized nanoparticles were diluted with deionized water to a suitable concentration (0.02 mg / mL) and measured using a nanoparticle size and zeta potential analyzer.

[0046] The results are as follows Figure 3 As shown: without the addition of casein, the average particle size of fresh particles was 68 nm, and the zeta potential was +51 mV; after lyophilization and redispersion, the particle size increased sharply to over 100 nm. Figure 3 C), when ginsenoside Rd was encapsulated, the increase in particle size after freeze-drying was more significant, increasing from 70 nm to over 200 nm. When the casein ratio increased to 0.5:1, the Zeta potential of the unencapsulated ginsenoside Rd particles changed from positive to -51 mV ( Figure 3 A) indicates that casein was successfully coated on the particle surface; the average particle size of fresh particles was 68 nm, and the particle size remained basically unchanged after freeze-drying and redispersing. Figure 3 The particle size ratio (after lyophilization / before lyophilization) of B and C is 1.1, and 1.0 when saponin is encapsulated (particle sizes before and after lyophilization are 62 nm and 64 nm, respectively), which demonstrates excellent lyophilized redispersibility after the addition of casein. Considering the loading rate of ginsenoside Rd, 0.5:1 is the preferred ratio.

[0047] (3) Transmission electron microscopy observation: Take freeze-dried and redispersed nanoparticles (casein ratio 0.5:1), drop them onto a copper grid, stain with 2% phosphotungstic acid, dry them and observe them under TEM.

[0048] The results are as follows Figure 4 As shown: the casein-coated nanoparticles are spherical, uniform in size (200 nm), well dispersed, and show no obvious aggregation; while the control sample without casein (not shown in the figure) shows a large number of aggregates.

[0049] Example 3: Effects of casein on crystallinity and freeze-drying stability (DSC and XRD analysis) Nanoparticles unloaded (without ginsenosides) and loaded with ginsenoside Rd (Rd monomer was used instead of crude extract to investigate the interaction between Rd and corn protein separately) were prepared according to different casein ratios in Example 2. After freeze-drying, DSC and XRD tests were performed.

[0050] DSC determination: Weigh 5 mg of lyophilized powder, seal it in an aluminum crucible, and heat it from 20°C to 200°C at a rate of 10°C / min under a nitrogen atmosphere.

[0051] The results are as follows Figure 5 As shown: Particles loaded with Rd ( Figure 5 B) When the proportion of corn protein is high, an endothermic peak of 100℃±3℃ appears, which corresponds to the dissociation of the ordered structure (crystalline region) of corn protein; after the addition of casein, the endothermic peak shifts to a lower temperature (85℃±3℃) and the peak area decreases, indicating that casein reduces the stability of the ordered structure induced by Rd.

[0052] XRD measurement: Cu-Kα rays (λ=0.154 nm) were used, with a tube voltage of 40 kV, a tube current of 40 mA, a scanning range of 2θ=5-50°, and a step size of 0.02°.

[0053] The results are as follows Figure 6 As shown: After loading Rd, corn protein exhibits distinct diffraction peaks at 2θ≈20° and 30°. Figure 6 B), crystallinity increases; as the proportion of casein increases, the intensity of the diffraction peak gradually weakens, indicating that casein effectively inhibits Rd-induced crystallization.

[0054] The above results indicate that the addition of casein significantly reduces the risk of freeze-drying agglomeration in the Rd / corn protein system due to increased crystallinity, thereby improving freeze-drying stability.

[0055] Example 4: Evaluation of protective activity against alcoholic acute liver injury Animal model establishment: Male C57BL / 6J mice (6-8 weeks old, weighing 20-22 g) were randomly divided into 5 groups (n=8 per group): normal control group, alcohol model group, positive control group (silymarin, 200 mg / kg), casein-corn protein encapsulation group prepared in Example 1 (hereinafter referred to as "encapsulation group", 50 mg / kg based on Rd content), and simple mixture group of unencapsulated ginsenoside crude extract and blank nanoparticles (hereinafter referred to as "simple mixture group", same dosage). After 3 days of acclimatization, except for the normal control group, the other groups of mice were given Lieber-DeCarli alcoholic liquid diet (containing 5% v / v alcohol) for free drinking for 10 consecutive days to induce acute alcoholic liver injury. The normal control group was given an isocaloric control liquid diet. Every morning, the drug administration group was orally administered the corresponding drug (dissolved in 0.5% sodium carboxymethyl cellulose), while the model group and normal control group were given an equal volume of solvent.

[0056] Indicator detection: After fasting for 12 hours following the last gavage, blood was collected from the orbital rim under anesthesia, and serum was separated. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured using a fully automated biochemical analyzer.

[0057] Result: As Figure 7 As shown (using AST as a representative), the AST activity in the alcohol model group significantly increased to 94 U / L (compared to approximately 18 U / L in the normal group). The AST activity in the simple mixture group was 65 U / L, and in the positive control group it was 56 U / L. However, the AST activity in the encapsulated group decreased to 39 U / L, significantly lower than that in the simple mixture group and the positive control group (p<0.05). The ALT change trend was consistent. These results indicate that the casein-corn protein nanoparticles selectively encapsulated with ginsenoside Rd prepared in this invention, after lyophilization and reconstitution, can still significantly alleviate alcohol-induced acute liver injury, and its protective effect is superior to that of the unencapsulated simple mixture and the positive control drug silymarin.

[0058] Comparative Example 1: Using ginsenoside Rd monomer instead of crude extract Following the method of Example 1, but replacing the crude ginsenoside extract with an equimolar amount (based on Rd) of high-purity ginsenoside Rd monomer (purity ≥98%), and setting the mass ratio of casein to corn protein to 0.5:1, nanoparticles were prepared.

[0059] The results showed that the loading rate of Rd monomer reached 6.5%, and the redispersed particle size after freeze-drying was 65 nm, similar to that of Example 1. However, raw material cost calculations showed that the raw material cost of using ginsenoside Rd monomer was approximately 15 times that of using crude extract (calculated based on Rd content). Therefore, despite good encapsulation performance, the cost was too high, which is not conducive to industrialization.

[0060] Comparative Example 2: No casein added The method of Example 1 was followed, but in step (3), an equal volume of 10 mM pH 8.6 phosphate buffer was used instead of the casein solution, i.e., no casein was added, while the other steps were the same. The nanoparticles prepared had an average particle size of 70 nm before lyophilization and an Rd loading rate of 6.2%; however, after lyophilization, severe aggregation occurred, and the redispersed particle size reached 205 nm, with obvious precipitation visible after redispersement. DSC and XRD showed high crystallinity. This comparative example illustrates that without the addition of casein, Rd-induced crystallization leads to extremely poor lyophilization stability, which cannot meet the requirements of the formulation.

[0061] It should be noted that the specific embodiments described above are exemplary, and those skilled in the art can devise various solutions inspired by the disclosure of this invention, all of which fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification is illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. A method for preparing corn protein particles with selectively encapsulated ginsenoside Rd, characterized in that, Includes the following steps: (1) Dissolve zein in an ethanol solution to obtain a zein solution; (2) Add ginsenosides to the zein solution and obtain a mixture after complete dissolution; (3) Prepare casein buffer solution. Quickly add casein buffer solution to the above mixture and stir to form nanoparticle dispersion. (4) The ethanol in the nanoparticle dispersion is removed by rotary evaporation; (5) The nanoparticle dispersion was filtered with a filter membrane with a pore size of 100~200 kDa to retain the corn protein particles encapsulating ginsenoside Rd, remove unbound free saponins and large particulate impurities, and obtain purified corn protein particles. (6) The purified nanoparticle dispersion was pre-frozen and then freeze-dried.

2. The method according to claim 1, characterized in that, In step (2), the mass fraction of ginsenoside Rd in total ginsenosides is not less than 5%.

3. The method according to claim 2, characterized in that, In step (3), the volume of the casein buffer added is 2 to 6 times the volume of the corn protein solution.

4. The method according to claim 1, characterized in that, In step (2), the mass ratio of zein to total ginsenosides is 0.5 to 2:

1.

5. The method according to claim 4, characterized in that, In step (2) and step (3), the volume ratio of the mixture to water is 1:3~6.

6. The method according to claim 1, characterized in that, In step (4), the temperature of rotary evaporation is 40~50℃.

7. The method according to claim 1, characterized in that, In step (6), the pre-freezing temperature is -60~-80℃, and the vacuum degree of freeze drying is less than 20 Pa.

8. A corn protein particle selectively encapsulating ginsenoside Rd, characterized in that, The corn protein particles were prepared using the method for selectively encapsulating ginsenoside Rd as described in any one of claims 1-7.

9. The use of the corn protein particles selectively encapsulating ginsenoside Rd as described in claim 8 in the preparation of oral functional foods, health products, or hepatoprotective drugs for the prevention, relief, or treatment of alcoholic acute liver injury.

10. A method for improving the freeze-drying stability of ginsenoside Rd-supported zein nanoparticles, characterized in that, In a system where ginsenoside Rd is encapsulated in zein, casein is compounded, with a casein to zein mass ratio of 0.1 to 2:1.

Images