Method for improving solubility and stability of betulin

Abstract

The invention discloses a method for improving the solubility and stability of betulin, which is characterized in that oleanane terpenoid substance co-assembly and protein-polysaccharide composite matrix encapsulation are utilized to form a betulin loading system with a nanoscale core and a micron-scale shell, so that the solubility and stability of betulin are remarkably improved, and the controlled release of betulin in intestinal tracts is realized. The particle size of the compound formed by assembling the betulin and the oleanane terpenes is 100-120nm, and the compound is uniform in distribution, good in water solubility and high in thermal stability, pH stability and salt stability. After protein-polysaccharide loading is utilized, the water solubility and stability of the assembled compound are further improved, and the compound has the performance of controlled release of betulin in intestinal tracts, also has a synergistic anti-oxidation effect, and is beneficial to improving the bioavailability of the compound.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine, functional food and cosmetics, and in particular to a method for improving the water solubility and stability of betulin. Technical Background

[0002] Betulin is a lupinane-type pentacyclic triterpenoid compound widely found in plants of the genus *Betulia*, such as birch bark and leaves, pomegranate bark and leaves, persimmon calyx, basil, and jujube seed (Grymel et al., 2022). Pharmacological studies have shown that betulin possesses various pharmacological effects, including antiviral, antioxidant, hepatoprotective, anti-inflammatory, and lipid-lowering properties, and is widely used in food additives, pharmaceuticals, and cosmetics (Buko et al., 2018). Studies have shown that betulin has stronger antioxidant properties than vitamin C at low concentrations; however, betulin has low molecular polarity and extremely low solubility in water (approximately 0.08 μg / mL), resulting in low bioavailability and severely limiting its application (Orekhov et al., 2023). Oleanane-type pentacyclic triterpenoids are another type of naturally occurring active compound widely found in plants used for both food and medicine, mainly including oleanolic acid, glycyrrhizic acid, and glycyrrhetinic acid. Numerous studies have found that oleanane-type compounds possess antioxidant, anti-inflammatory, antibacterial, and hepatoprotective biological activities (Xiao et al., 2024). However, oleanane-type molecules also have the disadvantage of low water solubility in applications.

[0003] To overcome the limitations of betulin in application, researchers prepared an assembled complex of rhein and betulin, which significantly improved water solubility and also revealed a synergistic effect in tumor treatment (Wang et al., 2021). Some researchers used rosin acid and oleanolic acid as carriers to prepare nanoparticles loaded with proanthocyanidins via antisolvent precipitation, significantly improving the thermal stability of proanthocyanidins and achieving sustained release (Han et al., 2025). Additionally, some researchers prepared betulin-glycyrrhetinic acid and betulin-oleanolic acid assembled complexes via emulsification evaporation (CN 111632032 A), but this technique is complex, requiring the addition of surfactants and other substances that are difficult to completely remove later. To date, there are few reports on techniques for preparing betulin-oleanane-type molecularly assembled complexes using antisolvent methods based on self-assembly technology to improve their water solubility.

[0004] Although nano-assembled complexes possess excellent water solubility and physiological activity, their stability in aqueous solutions is not high due to their low surface charge, making them susceptible to aggregation caused by factors such as salt, pH, and heat. To address these issues, natural macromolecules are often used for loading to improve the stability of their dispersions. Furthermore, macromolecular loading can protect active molecules from degradation in the acidic environment of the stomach, enabling controlled release in the intestine. Milk proteins, including casein and whey protein, are the main proteins in milk. Milk proteins are characterized by high nutritional value, good biocompatibility, and biodegradability (Meng et al., 2025). Studies have shown that loading curcumin and epigallocatechin gallate using whey protein dual emulsions achieves highly efficient encapsulation of both, with encapsulation efficiencies of 98% and 89.7%, respectively (Cui et al., 2023). In addition, milk proteins are often combined with polysaccharides for loading active molecules. For example, curcumin-loaded nanoparticles made from milk protein and hyaluronic acid exhibit good storage stability and controlled release capability, improving the antioxidant activity and light and heat stability of curcumin (Zhong et al., 2023). However, no reports have been found on the technology of using milk protein and polysaccharide composite matrices to load betulin and oleanane-type substances to assemble complexes.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] This invention preferentially utilizes oleanane-type natural compounds, employing antisolvent technology under ultrasonic drive to assemble them with betulin to form nanoparticles. The structure, solubility, and stability of the nanoparticles are systematically characterized. Furthermore, a protein-polysaccharide composite matrix is ​​used to load the nanoassemblies, preparing microcapsule complexes. The complex structure is characterized, and the complex stability is evaluated. This invention overcomes the application limitations of pentacyclic triterpenes such as betulin and provides functional base materials for functional foods, clinical drugs, and cosmetics.

[0007] The objective of this invention is achieved through the following technical solution: A method for improving the solubility and stability of betulin includes the following steps and process conditions: (1) Dissolve betulin and oleanane molecules together in anhydrous ethanol at a mass ratio of 8:2 to 2:8 to prepare a binary mixed solution with a concentration of 1 to 10 mg / mL. (2) Place the binary mixed solution in a jacketed reactor with a 25-35℃ circulating water bath, and add it to 15-25 times its volume of deionized water at a rate of 1-3 mL / min with ultrasonic assistance. The ultrasonic frequency is 20 kHz, the amplitude is 20-40%, and the pulse mode is 2-4s on and 2-4s off. After the addition is complete, continue ultrasonication for 6-8 min to obtain the assembled complex solution of betulin and oleanane type molecules. (3) The complex solution was placed in a membrane filter with a molecular weight cutoff of 1000~3000 Da for filtration, and the retentate was collected after being concentrated 3~6 times. (4) Dissolve the protein (natural edible water-soluble protein such as milk protein) and polysaccharide (edible water-soluble polysaccharide) in water respectively to prepare a 2% solution. Then mix the protein and polysaccharide solution evenly in a volume ratio of 1:2 to 2:1. Then mix it with the retentate in step (3) in a volume ratio of 1:1 to 2:1. Place it in a jacketed reactor with a 25-35℃ circulating water bath for ultrasonic treatment. The frequency is 20kHz, the amplitude is 20-40%, the pulse mode is 2-4s on and 2-4s off, and the ultrasonic time is 6-8min to obtain a mixture. (5) Spray dry the mixture in step (4) with an inlet temperature of 130~150℃, an atomization pressure of 0.05~0.1MPa, a needle-impact interval of 1s, a needle-impact execution time of 1s, and a material flow rate of 200~300mL / h to obtain the protein-polysaccharide loaded betulinic acid assembly complex powder.

[0008] The present invention has the following advantages and effects compared with the prior art: (1) Based on the assembly ability of pentacyclic triterpenoid molecules, this invention selects oleanane-type molecules and, through extensive experimental exploration, develops an assembly process for them with betulin in the antisolvent process. An assembly complex of betulin-oleanane-type molecules is prepared under ultrasonic drive, providing a technical basis for the assembly of betulin and oleanane-type molecules via the antisolvent method. The ultrasonic assistance in this invention increases the collision frequency of active molecules, promotes conformational deformation of molecules, and thus improves assembly efficiency. The assembled mixture is effectively filtered through a membrane to remove unassembled free molecules, while simultaneously achieving efficient concentration of the assembled complex. This technology is simple, easy to implement, mild, environmentally friendly, and suitable for large-scale development.

[0009] (2) The main molecule of this invention, lupinane-type betulin, possesses excellent antioxidant and anti-inflammatory activities and is a natural active molecule that is both food and medicine. The preferred guest molecule is oleanane-type pentacyclic triterpenoid, which is also a natural active molecule that is both food and medicine and has outstanding biological activity. The above molecules can be used as dietary supplements, as drugs, and as the main anti-inflammatory and antioxidant ingredients in cosmetics, with a very wide range of applications. The two molecules form nanoparticles through self-assembly technology, which not only effectively improves water solubility but also has a synergistic effect, significantly improving biological activity and bioavailability, and giving it a wider range of applications.

[0010] (3) The preferred loading matrix consists of natural edible proteins and polysaccharides. By cleverly utilizing their charged or hydrophobic groups, they statically bind to the pentacyclic triterpenoid assembly complex through electrostatic and hydrophobic interactions. Spray drying technology effectively encapsulates the active molecules within the microcapsules, significantly improving the dispersibility and stability of the aqueous solution. This ensures that the nano-assembly complex maintains its structural integrity during subsequent processing or use, maximizing its activity, while also integrating well with other matrices in the system, resulting in a uniform texture. The loading matrix is ​​not only safe and edible with good biocompatibility but also possesses high nutritional value and poses no safety risks. Furthermore, the assembly process involves no exogenous chemical reagents, making it green and sustainable.

[0011] (4) The nanocomposite prepared by the self-assembly technology of this invention has a particle size between 100 and 120 nm, uniform particle size, and high thermal stability, pH stability, and salt stability in aqueous solution. After loading with proteins and polysaccharides, it forms a complete micron-sized microcapsule, which releases the complete nanocomposite upon resolution in water, thus maintaining the structural stability of the assembled product. Nanoparticles can penetrate biological membranes more effectively, promote the transport and absorption of active molecules in vivo, and further improve bioavailability. Attached Figure Description

[0012] Figure 1 This is a scanning electron microscope image (magnification: 30000×) of betulin-oleanane type molecular self-assembled nanoparticles after membrane filtration in an embodiment of the present invention.

[0013] Figure 2 This is a scanning electron microscope (SEM) image (magnification: 10000×) of the spray-dried product of the betulin-oleanane type molecular self-assembled complex encapsulated with protein-polysaccharide in an embodiment of the present invention.

[0014] Figure 3 This is a scanning electron microscope (SEM) image (magnification: 10000×) of the spray-dried product of the betulinol-oleanane type molecular self-assembled complex encapsulated with protein-polysaccharide in an embodiment of the present invention after resolution in water.

[0015] Figure 4 The particle size, polydispersity index (PDI), and zeta potential of the betulin-oleanane type molecular self-assembled complex and the protein-polysaccharide-encapsulated betulin-oleanane type molecular self-assembled complex spray-dried products in the embodiments of the present invention are shown.

[0016] Figure 5 The images show the infrared and XRD patterns of the betulin-oleanane type molecular self-assembled complex and the protein-polysaccharide-encapsulated betulin-oleanane type molecular self-assembled complex spray-dried products in the embodiments of the present invention.

[0017] Figure 6The solution appearance and solubility of the betulin-oleanane type molecular self-assembly complex and the protein-polysaccharide-encapsulated betulin-oleanane type molecular self-assembly complex in the embodiments of the present invention are shown.

[0018] Figure 7 The water contact angles of the betulin-oleanane type molecular self-assembly complex and the protein-polysaccharide-encapsulated betulin-oleanane type molecular self-assembly complex powders in the embodiments of the present invention are shown.

[0019] Figure 8 The particle size and PDI of the betulin-oleanane type molecular self-assembled complex and the protein-polysaccharide-encapsulated betulin-oleanane type molecular self-assembled complex aqueous solution in the embodiments of the present invention are obtained by heating in a water bath at 60°C, 70°C and 80°C for 1 hour, respectively.

[0020] Figure 9 The particle size and PDI of the betulin-oleanane type molecular self-assembled complex and the protein-polysaccharide-encapsulated betulin-oleanane type molecular self-assembled complex in aqueous solutions at pH 5, 7, and 9 are shown in the embodiments of the present invention.

[0021] Figure 10 The particle size and PDI of the betulin-oleanane type molecular self-assembled complex and the protein-polysaccharide-encapsulated betulin-oleanane type molecular self-assembled complex in 50mM, 100mM, 250mM, 500mM and 1000mM NaCl aqueous solutions are shown in the embodiments of the present invention.

[0022] Figure 11 The images show the in vitro simulated digestion and release curves of the betulin-oleanane type molecular self-assembly complex and the protein-polysaccharide-encapsulated betulin-oleanane type molecular self-assembly complex in the embodiments of the present invention.

[0023] Figure 12 The free radical scavenging activities of the betulin-oleanane type molecular self-assembly complex and the protein-polysaccharide-encapsulated betulin-oleanane type molecular self-assembly complex aqueous solution in the embodiments of the present invention are DPPH and ABTS free radical scavenging activities.

[0024] Figure 13 The hydroxyl radical scavenging activity and reducing power of the betulin-oleanane type molecular self-assembly complex and the protein-polysaccharide-encapsulated betulin-oleanane type molecular self-assembly complex aqueous solution in the embodiments of the present invention are shown. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise stated, the raw materials used in the embodiments of the present invention are conventionally purchased raw materials, and the testing methods are conventional testing methods. Example 1

[0026] (1) Weigh 80 mg of betulin and 20 mg of glycyrrhetinic acid respectively, dissolve them in 100 mL of anhydrous ethanol, and prepare a binary mixed solution; (2) The binary mixed solution was placed in a jacketed reactor with a 25°C circulating water bath and added to 2500 mL of deionized water at a rate of 3 mL / min under ultrasonic assistance. The ultrasonic frequency was 20 kHz, the amplitude was 20%, and the pulse mode was 4 s on and 4 s off. After the addition was completed, the ultrasonic treatment was continued for 8 min under the same conditions to obtain the betulinol-glycyrrhetinic acid assembled complex solution. (3) Place the complex solution in a membrane separation device, select a spiral wound membrane with a molecular weight cutoff of 1000 Da for ultrafiltration, and concentrate it by 3 to obtain the assembled complex retentate. (4) Dissolve whey protein and sodium hyaluronate separately in water to prepare a 2% solution, and then mix them evenly in a volume ratio of 1:2. Take 200 mL of the retentate from step (3), mix it with 100 mL of whey protein-sodium hyaluronate mixture, and place it in a jacketed reactor with a 25°C circulating water bath for ultrasonic treatment. The frequency is 20 kHz, the amplitude is 20%, the pulse mode is 4 seconds on and 4 seconds off, and the ultrasonic time is 8 minutes to obtain the mixture. (5) Spray dry the mixture in step (4) with an inlet temperature of 130°C, an atomization pressure of 0.05 MPa, a needle-impact interval of 1 s, a needle-impact execution time of 1 s, and a material flow rate of 200 mL / h to obtain whey protein-sodium hyaluronate-betulin-glycyrrhetinic acid complex powder. Example 2

[0027] (1) Weigh 20 mg of betulin and 80 mg of glycyrrhetinic acid respectively, dissolve them in 10 mL of anhydrous ethanol, and prepare a binary mixed solution; (2) Place the binary mixed solution in a jacketed reactor with a 35°C circulating water bath, and add 150 mL of deionized water at a rate of 1 mL / min under ultrasonic assistance. The ultrasonic frequency is 20 kHz, the amplitude is 40%, and the pulse mode is 2 s on and 2 s off. After the addition is complete, continue ultrasonic treatment under the same conditions for 6 min to obtain the betulinol-glycyrrhetinic acid assembled complex solution. (3) Place the complex solution in an ultrafiltration tube with a molecular weight cutoff of 3000 Da and centrifuge at 6000 g for 30 min to obtain the nanocomposite retentate; (4) Dissolve whey protein and sodium hyaluronate separately in water to prepare a 2% solution, and then mix them evenly in a volume ratio of 2:1. Take 50 mL of the retentate from step (3), mix it with 100 mL of whey protein-sodium hyaluronate mixture, and place it in a jacketed reactor with a 35°C circulating water bath for ultrasonic treatment. The frequency is 20 kHz, the amplitude is 40%, the pulse mode is 2 seconds on and 2 seconds off, and the ultrasonic time is 6 min to obtain the mixture. (5) Spray dry the mixture in step (4) with an inlet temperature of 150°C, an atomization pressure of 0.1 MPa, a needle-impact interval of 1 s, a needle-impact execution time of 1 s, and a material flow rate of 300 mL / h to obtain whey protein-sodium hyaluronate-betulin-glycyrrhetinic acid complex powder. Example 3

[0028] (1) Weigh 50 mg of betulin and 50 mg of oleanolic acid respectively, dissolve them in 50 mL of anhydrous ethanol, and prepare a binary mixed solution; (2) Place the binary mixed solution in a jacketed reactor with a 30°C circulating water bath, and add 100 mL of deionized water at a rate of 2 mL / min under ultrasonic assistance. The ultrasonic frequency is 20 kHz, the amplitude is 30%, and the pulse mode is 3 seconds on and 3 seconds off. After the addition is complete, continue ultrasonic treatment under the same conditions for 7 minutes to obtain the betulinol-oleanolic acid assembled complex solution. (3) Place the complex solution in an ultrafiltration tube with a molecular weight cutoff of 2000 Da and centrifuge at 5000 g for 40 min to obtain the nanocomposite retentate; (4) Dissolve casein micelles and sodium alginate in water to prepare a 2% solution, and then mix them evenly in a volume ratio of 1:1. Take 50 mL of the retentate from step (3), mix it with 75 mL of casein micelle-sodium alginate mixture, and place it in a jacketed reactor with a 30°C circulating water bath for ultrasonic treatment. The frequency is 20 kHz, the amplitude is 30%, the pulse mode is 3 seconds on and 3 seconds off, and the ultrasonic time is 7 minutes to obtain the mixture. (5) Spray dry the mixture in step (4) with an inlet temperature of 140°C, an atomization pressure of 0.07 MPa, a needle-impact interval of 1 s, a needle-impact execution time of 1 s, and a material flow rate of 250 mL / h to obtain casein micelles-sodium alginate-betulin-oleanolic acid complex powder. Example 4

[0029] (1) Weigh 40 mg of betulin and 60 mg of glycyrrhetinic acid respectively, dissolve them in 100 mL of anhydrous ethanol, and prepare a binary mixed solution; (2) Place the binary mixed solution in a jacketed reactor with a 30°C circulating water bath, and add 2000 mL of deionized water at a rate of 3 mL / min under ultrasonic assistance. The ultrasonic frequency is 20 kHz, the amplitude is 20%, and the pulse mode is 4 s on and 4 s off. After the addition is complete, continue ultrasonic treatment under the same conditions for 8 min to obtain the betulinol-glycyrrhetinic acid assembled complex solution. (3) Place the complex solution in a membrane separation device, select a spiral wound membrane with a molecular weight cutoff of 2000 Da for ultrafiltration, and concentrate it by 4 to obtain the nanocomposite retentate. (4) Dissolve sodium caseinate and sodium hyaluronate in water to prepare a 2% solution, and then mix them evenly in a volume ratio of 1:1. Take 200 mL of the retentate from step (3), mix it with 100 mL of sodium caseinate-sodium hyaluronate mixture, and place it in a jacketed reactor with a 30°C circulating water bath for ultrasonic treatment. The frequency is 20 kHz, the amplitude is 20%, the pulse mode is 4 seconds on and 4 seconds off, and the ultrasonic time is 8 minutes to obtain the mixture. (5) Spray dry the mixture in step (4) with an inlet temperature of 130°C, an atomization pressure of 0.1 MPa, a needle-impact interval of 1 s, a needle-impact execution time of 1 s, and a material flow rate of 300 mL / h to obtain betulinol-glycyrrhetinic acid complex powder supported on sodium caseinate-sodium hyaluronate. Example 5

[0030] (1) Weigh 70 mg of betulin and 30 mg of glycyrrhetinic acid respectively, dissolve them in 20 mL of anhydrous ethanol, and prepare a binary mixed solution; (2) Place the binary mixed solution in a jacketed reactor with a 30°C circulating water bath, and add 500 mL of deionized water at a rate of 2.5 mL / min under ultrasonic assistance. The ultrasonic frequency is 20 kHz, the amplitude is 40%, and the pulse mode is 2 s on and 2 s off. After the addition is complete, continue ultrasonic treatment under the same conditions for 7 min to obtain the betulinol-glycyrrhetinic acid assembled complex solution. (3) Place the complex solution in an ultrafiltration tube with a molecular weight cutoff of 3000 Da and centrifuge at 6000 g for 30 min to obtain the nanocomposite retentate; (4) Dissolve whey protein and sodium alginate in water to prepare a 2% solution, and then mix them evenly in a volume ratio of 2:1. Take 100mL of the retentate in step (3), mix it with 100mL of whey protein-sodium alginate mixture, and place it in a jacketed reactor with a 30℃ circulating water bath for ultrasonic treatment. The frequency is 20kHz, the amplitude is 30%, the pulse mode is 3s on and 3s off, and the ultrasonic time is 7min to obtain the mixture. (5) Spray dry the mixture in step (4) with an inlet temperature of 150°C, an atomization pressure of 0.08 MPa, a needle-impact interval of 1 s, a needle-impact execution time of 1 s, and a material flow rate of 280 mL / h to obtain whey protein-sodium alginate-betulin-glycyrrhetinic acid complex powder.

[0031] The structures and properties of the assembled complexes and protein-polysaccharide-encapsulated complexes prepared in Examples 1-5 were characterized, and the results are as follows: The microstructure of the assembled complexes prepared in step (3) of Examples 1-5 was observed by scanning electron microscopy, as shown in the attached figure. Figure 1 As shown in the figure, betulin, glycyrrhetinic acid, and oleanolic acid all self-assembled to form spherical nanoparticles with uniform particle size. Figure 2 It can be seen that after encapsulation with protein-polysaccharide, the spray-dried complex consists of micron-sized capsule-shaped particles with surface depressions, a typical morphology of macromolecular spray-dried products. (From the attached...) Figure 3 As can be seen, after the spray-dried composite is dissolved, the assembled composite is released and remains as spherical nanoparticles in water. The spray-drying process did not damage its structure, thus achieving encapsulation. (See attached...) Figure 4 It can be seen that the particle size of the assembled complex prepared in step (3) is between 100 and 120 nm, the PDI is less than 0.23, and the potential is between -8 and -12 mV. This indicates that the assembled complex consists of nanoscale particles with small particle size, uniform particle size distribution, and a negative surface charge. After resolution of the protein-polysaccharide-encapsulated assembled complex, the particle size of the system increases due to the macromolecular characteristics of the protein and polysaccharide, but remains at the nanoscale. Simultaneously, the negative charge on the surface of the complex system increases due to the negative charge of the protein and polysaccharide, thus enhancing its stability. (From the attached...) Figure 5 It can be seen that after assembly, the intensity of the infrared and XRD characteristic peaks of betulin and oleanane molecules decreased or disappeared, confirming the occurrence of self-assembly. After encapsulation with protein-polysaccharide, the intensity of the infrared and XRD characteristic peaks of the assembled complex decreased or disappeared, indicating that the nano-assemblies were encapsulated inside the macromolecules, forming a core-shell structured complex.

[0032] From the appendix Figure 6 It can be seen that the free betulin before assembly is basically insoluble in water, while the aqueous solution of the assembled complex prepared in step (3) is uniformly milky white with a solubility of over 75%; after encapsulation with protein and polysaccharide, the solubility of the complex is as high as over 90%. Figure 7 It can be seen that the free monomeric pentacyclic triterpenoid molecule is hydrophobic, with a water contact angle of approximately 140º; the water contact angle of the assembled complex prepared in step (3) is significantly reduced, exhibiting hydrophilicity, and it still maintains good hydrophilicity after protein-polysaccharide encapsulation. Therefore, the betulin complex prepared by the technology of this invention has excellent water solubility.

[0033] The stability of the assembly complex prepared in step (3) and the protein-polysaccharide-encapsulated assembly complex prepared in step (5) was evaluated, and the results are shown in the appendix. Figure 8 It can be seen that the particle size of the assembled complex aqueous solution prepared in step (3) increased significantly after heat treatment at different temperatures, indicating slight aggregation or dissociation between particles, but they are still nanoscale particles with good dispersibility; the particle size of the complex aqueous solution encapsulated with protein-polysaccharide did not change much after heat treatment. (From the attached...) Figure 9 It can be seen that the assembled complex prepared in step (3) has high stability at pH 5 and 7, but it partially dissociates and increases in particle size at pH 9; after protein-polysaccharide encapsulation, the particle size of the complex does not change much in the pH range of 5-9. Figure 10 It can be seen that the assembled complex has poor salt stability, with a significant increase in particle size in sodium chloride solutions of different concentrations. However, after encapsulation with protein-polysaccharide, its salt stability is significantly improved, and the particle size change is small, remaining at the nanoscale. In summary, the betulinol complex prepared by the technology of this invention exhibits extremely high thermal stability, pH stability, and salt stability.

[0034] The nano-assembled complexes prepared in step (3) of Examples 1-5 and the betulin complex encapsulated with protein-polysaccharides were subjected to simulated gastrointestinal digestion. The results are shown in the appendix. Figure 11 It can be seen that the assembled complex prepared in step (3) released approximately 60% of betulin in gastric juice; after 5 hours of digestion, the cumulative release rate of betulin was approximately 85%. After protein-polysaccharide encapsulation, the release rate of betulin in gastric juice from the complex was significantly reduced, but the cumulative release rate after 5 hours still reached over 80%. Therefore, the betulin encapsulated with protein-polysaccharide prepared using the technology of this invention releases less betulin in gastric juice and exhibits significant intestinal release characteristics.

[0035] The antioxidant properties of the nano-assembled complexes prepared in step (3) of Examples 1-5 and the betulin complex encapsulated with protein-polysaccharides were characterized, and the results are shown in the appendix. Figure 12 and attached Figure 13 As shown in the figure, the DPPH, ABTS, and hydroxyl radical scavenging rates, as well as the reducing power of the nano-assembled composite, are significantly higher than those of the free monomer molecules. Furthermore, the antioxidant activity is further significantly enhanced after encapsulation with protein-polysaccharides. Therefore, the betulin complex prepared using the technology of this invention exhibits good antioxidant activity.

[0036] In summary, after treatment with the technology of this invention, betulin has significantly increased water solubility, significantly improved stability, significantly enhanced antioxidant properties, and exhibits intestinal-targeted release characteristics, making it an extremely high-performance active complex.

[0037] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for improving the solubility and stability of betulin, characterized in that... This includes the following steps and process conditions: (1) Dissolve betulin and guest molecules together in anhydrous ethanol at a mass ratio of 8:2 to 2:8 to prepare a binary mixed solution with a concentration of 1 to 10 mg / mL; (2) Place the mixed solution in step (1) into a jacketed reactor with a circulating water bath at 25~35℃, and slowly add it dropwise to 15~25 times its volume of deionized water under ultrasonic assistance. The ultrasonic frequency is 20kHz, the amplitude is 20~40%, and the pulse mode is 2~4s on and 2~4s off. After the addition is complete, continue ultrasonication for 6~8min to obtain the assembly complex solution of betulin and oleanane type molecules. (3) The complex solution was placed in a membrane filter with a molecular weight cutoff of 1000~3000 Da for filtration, and the retentate was collected after being concentrated 3~6 times. (4) Dissolve the protein and polysaccharide separately in water to prepare a 2% solution. Then mix the protein and polysaccharide solution evenly in a volume ratio of 1:2 to 2:

1. Then mix it with the retentate in step (3) in a volume ratio of 1:1 to 2:

1. Place it in a jacketed reactor with a 25-35℃ circulating water bath and perform ultrasonic treatment. The ultrasonic frequency is 20kHz, the amplitude is 20-40%, and the pulse mode is 2-4s on and 2-4s off. After the addition is complete, continue ultrasonic treatment for 6-8 minutes to obtain the mixture. (5) Spray dry the mixture in step (4) with an inlet temperature of 130~150℃, an atomization pressure of 0.05~0.1MPa, a needle-impact interval of 1s, a needle-impact execution time of 1s, and a material flow rate of 200~300mL / h to obtain the protein-polysaccharide loaded betulinic acid assembly complex powder.

2. The preparation method according to claim 1, characterized in that: The guest molecules in step (1) are oleanolic acid, glycyrrhizic acid, glycyrrhetinic acid and other oleanane-type terpenoids; the proteins in step (4) are edible water-soluble proteins such as milk protein; the polysaccharides are edible water-soluble polysaccharides such as sodium alginate, hyaluronic acid, pectin and other alginates; the dropping rate of the binary mixture in step (2) is 1~3 mL / min.

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