A preparation method of an apigenin oral nano-preparation

Abstract

This invention discloses a method for preparing an oral nanoparticle formulation of apigenin, belonging to the field of traditional Chinese medicine preparation technology. The method uses a mixture of soybean protein and xylan as the aqueous phase and an apigenin ethanol solution as the organic phase. Nanoparticles are prepared by antisolvent precipitation, followed by adsorption of tannic acid to obtain the final product. The nanoparticles prepared by this invention have a particle size of less than 600 nm and a high encapsulation efficiency. Utilizing the synergistic effect of soybean protein, xylan, and tannic acid, apigenin is protected from gastric acid degradation, achieving targeted intestinal delivery and significantly improving its oral bioavailability. This method can be used to prepare drugs for treating intestinal diseases such as inflammatory bowel disease, irritable bowel syndrome, or colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a method for preparing a apigenin preparation. Background Technology

[0002] Apogenin is a natural flavonoid compound widely found in various vegetables, fruits, and traditional Chinese medicines, such as celery, parsley, and chamomile. Numerous studies have demonstrated that apigenin possesses significant anti-inflammatory, antioxidant, antibacterial, and immunomodulatory activities. In recent years, increasing research has confirmed the potential application value of apigenin in the prevention and treatment of intestinal diseases such as inflammatory bowel disease (including ulcerative colitis and Crohn's disease), irritable bowel syndrome, and colorectal cancer. Studies have shown that apigenin can directly bind to and activate adenosine monophosphate-activated protein kinase (AMPK), enhancing antioxidant capacity and inhibiting the NF-κB signaling pathway, thus reducing oxidative stress and inflammatory damage. Furthermore, it can directly bind to the pyridine-domain-containing NOD-like receptor protein 3 (NLRP3) inflammasome, inhibiting its assembly and activation, thereby reducing the maturation and release of interleukin-1β (IL-1β). This repairs the intestinal mucosal barrier and improves pathological symptoms such as weight loss and colonic shortening in mice with ulcerative colitis. (Natural Dietary Flavonoid Apigenin Mitigates Ulcerative Colitisvia Modulating the AMPK / NF-κB / NLRP3 Signaling Axis[J]. Journal of Agricultural and Food Chemistry , 2025, 74: 983-997).

[0003] However, the efficacy of apigenin in actual oral applications is severely limited. Apigenin is a hydrophobic flavonoid compound with extremely low solubility in water, making it difficult to effectively dissolve and disperse in gastrointestinal fluids. Its absolute oral bioavailability is low, and under strongly acidic conditions (gastric pH 1.0–3.0), structural degradation or transformation may occur, further reducing the drug concentration reaching the effective site in the intestine.

[0004] Currently, soybean protein is a readily available, inexpensive, biodegradable, and biocompatible natural plant protein with good emulsifying and gel-forming abilities. Xylan, a type of natural hemicellulose, possesses excellent intestinal-targeted delivery characteristics and can be specifically degraded by colonic flora. Tannic acid, a natural polyphenol compound with multiple ortho- and posterior phenolic hydroxyl groups, can form complexes with proteins or polysaccharides through hydrogen bonding and hydrophobic interactions, providing protection under acidic conditions. Currently, there are no reports on the use of a nano-delivery system combining soybean protein, xylan, and tannic acid for the intestinal-targeted delivery of apigenin and its application in the treatment of intestinal diseases. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for preparing an oral nano-formulation of apigenin. This nano-formulation can simultaneously utilize soybean protein and xylan as the core nanocarriers to carry apigenin, with an outer layer adsorbing tannins to resist gastric acid degradation, thus solving the problem of oral delivery of apigenin.

[0006] This invention provides a method for preparing an oral nano-formulation of apigenin. The nano-formulation is prepared by the following method: soybean protein is dissolved in an alkaline aqueous solution, and the pH is adjusted to neutral after dissolution; xylan is dissolved in water and mixed with the soybean protein solution as an aqueous phase; apigenin is dissolved in an organic solvent as an organic phase; the aqueous phase is added dropwise to the organic phase under stirring conditions, and the organic solvent is evaporated to obtain the oral nano-formulation of apigenin.

[0007] In the preparation method of the nano-formulation, the organic solvent is ethanol.

[0008] Preferably, the nano-formulation is prepared by the following method: soybean protein is dissolved in an alkaline aqueous solution with a concentration of 0.5-5 mg / mL, and the pH is adjusted to neutral after dissolution; xylan is dissolved in water with a concentration of 0.5-10 mg / mL, and mixed with the soybean protein solution as the aqueous phase; apigenin is dissolved in an organic solvent as the organic phase with a concentration of 0.01-1 mg / mL, and the aqueous phase is added dropwise to the organic phase under stirring conditions, wherein the volume ratio of the organic solvent to water is 2:1; the organic solvent is evaporated to obtain the oral apigenin nano-formulation.

[0009] In the preparation method, after evaporating the organic solvent, tannic acid is added under stirring conditions. The mass ratio of tannic acid to soybean protein is 0.2-2:1, thus obtaining an oral nano-formulation of apigenin protected by tannic acid. Tannic acid can be firmly adsorbed to the soybean protein / xylan system, protecting the soybean protein from being decomposed by gastric acid.

[0010] The apigenin oral nanoparticle formulation has a particle size of less than 600 nm.

[0011] In the preparation method of the nano-formulation, soybean protein is dissolved in an aqueous solution with a pH of 8-14.

[0012] In the preparation method of the nano-formulation, the aqueous phase is added dropwise to the organic phase under stirring conditions, and the stirring speed is 200 rpm-1000 rpm.

[0013] Compared with the prior art, the preparation method of apigenin nanoparticles provided by the present invention has a uniform and stable particle size and a high drug encapsulation rate. Attached Figure Description

[0014] Figure 1 Scanning electron microscope image of the formulation in Example 1; Figure 2 Scanning electron microscope image of the formulation in Example 5; Figure 3 Example 5: Particle size and PDI changes of the formulation at different NaCl concentrations; Figure 4 Example 5: In vitro release curves of the formulation in different release media. Detailed Implementation

[0015] The present invention will be further illustrated below through embodiments. It should be understood that the embodiments of the present invention are merely provided for illustrative purposes and are not intended to limit the invention. Therefore, any simple modifications to the present invention based on the method of the present invention are within the scope of protection claimed by the present invention.

[0016] Example 1 5 mg of soy protein was dissolved in 10 mL of pH 12.5 aqueous solution, and the pH was adjusted to neutral after dissolution; 5 mg of xylan was dissolved in 10 mL of water, and the mixture was combined with the soy protein solution to form the aqueous phase; 0.4 mg of apigenin was dissolved in 40 mL of ethanol to form the organic phase. The aqueous phase was added dropwise to the organic phase under magnetic stirring at 200 rpm, and the organic solvent was removed by rotary evaporation to obtain the oral nano-formulation of apigenin.

[0017] Example 2 50 mg of soy protein was dissolved in 10 mL of pH 8 aqueous solution, and the pH was adjusted to neutral after dissolution. 50 mg of xylan was dissolved in 10 mL of water, and the solution was mixed with the soy protein solution to form the aqueous phase. 40 mg of apigenin was dissolved in 40 mL of ethanol to form the organic phase. The aqueous phase was added dropwise to the organic phase under magnetic stirring at 500 rpm, and the organic solvent was removed by rotary evaporation to obtain the oral nano-formulation of apigenin.

[0018] Example 3 25 mg of soy protein was dissolved in 10 mL of pH 14 aqueous solution, and the pH was adjusted to neutral after dissolution; 100 mg of xylan was dissolved in 10 mL of water, and the solution was mixed with the soy protein solution to form the aqueous phase; 5 mg of apigenin was dissolved in 40 mL of ethanol to form the organic phase. The aqueous phase was added dropwise to the organic phase under magnetic stirring at 1000 rpm, and the organic solvent was removed by rotary evaporation to obtain the oral nano-formulation of apigenin.

[0019] Example 4 5 mg of soy protein was dissolved in 10 mL of pH 12.5 aqueous solution, and the pH was adjusted to neutral after dissolution; 20 mg of xylan was dissolved in 2 mL of water, and the solution was mixed with the soy protein solution to form the aqueous phase; 1 mg of apigenin was dissolved in 24 mL of ethanol to form the organic phase. The aqueous phase was added dropwise to the organic phase under magnetic stirring at 600 rpm, the organic solvent was removed by rotary evaporation, 1 mg of tannic acid was added, and magnetic stirring was continued for 5 h to obtain the oral nano-formulation of apigenin.

[0020] Example 5 5 mg of soy protein was dissolved in 10 mL of pH=12 aqueous solution, and the pH was adjusted to neutral after dissolution; 20 mg of xylan was dissolved in 2 mL of water, and mixed with the soy protein solution as the aqueous phase; 1 mg of apigenin was dissolved in 24 mL of ethanol as the organic phase. The aqueous phase was added dropwise to the organic phase under magnetic stirring at 600 rpm, the organic solvent was removed by rotary evaporation, 10 mg of tannic acid was added, and magnetic stirring was continued for 1 h to obtain the oral nano-formulation of apigenin.

[0021] Comparative Example 1 5 mg of human serum albumin was dissolved in 10 mL of pH 12.5 aqueous solution, and the pH was adjusted to neutral after dissolution; 5 mg of xylan was dissolved in 10 mL of water, and the solution was mixed with the human serum albumin solution to form the aqueous phase; 0.4 mg of apigenin was dissolved in 40 mL of ethanol to form the organic phase. The aqueous phase was added dropwise to the organic phase under magnetic stirring at 200 rpm, and the organic solvent was removed by rotary evaporation.

[0022] Comparative Example 2 5 mg of soy protein was dissolved in 10 mL of pH 12.5 aqueous solution, and the pH was adjusted to neutral after dissolution to form the aqueous phase. 0.4 mg of apigenin was dissolved in 40 mL of ethanol to form the organic phase. The aqueous phase was added dropwise to the organic phase under magnetic stirring at 200 rpm, and the organic solvent was removed by rotary evaporation.

[0023] Comparative Example 3 100 mg of soy protein was dissolved in 10 mL of pH 12.5 aqueous solution, and the pH was adjusted to neutral to form the aqueous phase; 5 mg of sucrose was dissolved in 10 mL of water, and the solution was mixed with the soy protein solution to form the aqueous phase; 40 mg of apigenin was dissolved in 40 mL of ethanol to form the organic phase. The aqueous phase was added dropwise to the organic phase under magnetic stirring at 200 rpm, and the organic solvent was removed by rotary evaporation.

[0024] Comparative Example 4 50 mg of soy protein was dissolved in 10 mL of pH 7.0 aqueous solution; 50 mg of xylan was dissolved in 10 mL of water, and the mixture was combined with the soy protein solution to form the aqueous phase; 40 mg of apigenin was dissolved in 40 mL of dichloromethane to form the organic phase. The aqueous phase was added dropwise to the organic phase under magnetic stirring at 500 rpm, and the organic solvent was removed by rotary evaporation.

[0025] Comparative Example 5 50 mg of soy protein was dissolved in 10 mL of pH 8 aqueous solution, and the pH was adjusted to neutral after dissolution; 50 mg of xylan was dissolved in 2 mL of water, and mixed with the soy protein solution as the aqueous phase; 40 mg of paclitaxel was dissolved in 40 mL of ethanol as the organic phase. The aqueous phase was added dropwise to the organic phase under magnetic stirring at 500 rpm, and the organic solvent was removed by rotary evaporation.

[0026] Comparative Example 6 5 mg of soy protein was dissolved in 10 mL of pH 12.5 aqueous solution, and the pH was adjusted to neutral after dissolution; 20 mg of xylan was dissolved in 2 mL of water, and mixed with the soy protein solution as the aqueous phase; 1 mg of apigenin was dissolved in 24 mL of ethanol as the organic phase. The aqueous phase was added dropwise to the organic phase under magnetic stirring at 600 rpm, the organic solvent was removed by rotary evaporation, 1 mg of dopamine was added, and magnetic stirring was continued for 5 h.

[0027] Verification of Examples 1. The solutions obtained in Examples 1-5 and Comparative Examples 1-6 were filtered using a 0.45-micron microporous membrane. The particle size and polydispersity index of the resulting liquids were monitored using a laser particle size analyzer.

[0028] Table 1. Particle size and polydispersity index (PDI) of each embodiment The results are shown in Figure 1. The nanoparticles prepared in the embodiments of the present invention have a normal distribution, a particle size of less than 600 nm, a PDI of less than 0.3, and uniform particle size.

[0029] 3. The solutions obtained in Examples 1 and 5 were filtered through a 0.45-micrometer microporous membrane, and the resulting liquids were freeze-dried and observed using a scanning electron microscope.

[0030] The results are as follows Figure 1 and 2 The nano-formulation prepared in the embodiments of the present invention shown is approximately spherical with relatively uniform particle size.

[0031] 4. The solutions obtained from Examples 1-5 and Comparative Examples 1-4 and 6 were filtered using a 0.45-micron microporous membrane. 200 μL of the resulting liquid was diluted 5 times with methanol to break the emulsion. The apigenin drug content was detected at 340 nm using a UV spectrophotometer, and the encapsulation rate of apigenin in the nano-formulation was calculated.

[0032] Table 2. Encapsulation efficiency of Examples 1-5 and Comparative Examples 1-4, 6 The results are shown in Table 2. The nano-formulation of apigenin prepared by this invention has a high encapsulation rate.

[0033] 5. The nano-formulation obtained in Example 5 was filtered through a 0.45-micron microporous membrane and placed in NaCl solutions with concentrations of 20, 50, 75, 100, and 150 mmol / L, respectively. The particle size and polydispersity index of the resulting liquids were monitored using a laser particle size analyzer.

[0034] The results are as follows Figure 3 The results showed that the particle size and polydispersity index (PDI) of apigenin oral nanoparticles were relatively stable in 20–150 mmol / L NaCl solutions.

[0035] 6. The nano-formulation obtained in Example 5 was filtered using a 0.45-micron microporous membrane. 1 mL of the nano-formulation liquid was placed in a dialysis bag and then in 40 mL of in vitro release medium. An in vitro release experiment was conducted by shaking in a 37°C water bath. Different release media were prepared to simulate artificial gastric juice, small intestinal juice, and colonic juice. The release order was: artificial gastric juice (0.5 h, 1 h, 2 h), artificial small intestinal juice (3 h, 4 h, 5 h), and artificial colonic juice (6 h, 8 h, 12 h, 24 h). 0.5 mL of sample was taken at each time point, and 0.5 mL of release medium was added accordingly. Ultraviolet absorption was measured at a wavelength of 340 nm. The cumulative release rate was calculated, and a release curve was plotted.

[0036] The results are as follows Figure 4 The results showed that apigenin was relatively stable in a simulated gastric acid environment, with almost no drug release; after 2 hours, the drug was gradually released when placed in simulated small intestinal fluid; after 5 hours, the cumulative drug release rate reached 66.59% when placed in simulated colonic fluid.

[0037] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an oral nano-formulation of apigenin, characterized in that, The preparation method is as follows: Soy protein is dissolved in an alkaline aqueous solution, and the pH is adjusted to neutral after dissolution. Xylan aqueous solution is then added and mixed to form the aqueous phase. Apigenin is dissolved in an organic solvent to form the organic phase. The aqueous phase is added dropwise to the organic phase under stirring conditions, and the organic solvent is removed to obtain the oral nano-formulation of apigenin.

2. The method for preparing apigenin oral nanoparticles according to claim 1, characterized in that, The organic solvent is ethanol.

3. The method for preparing apigenin oral nanoparticles according to claim 1, characterized in that, The soybean protein concentration is 0.5-5 mg / mL after being dissolved in an alkaline aqueous solution. The concentration of the xylan aqueous solution is 0.5-10 mg / mL; In the organic phase, the concentration of apigenin is 0.01-1 mg / mL; The volume ratio of organic solvent to water incorporated into the organic phase and aqueous phase is 2:

1.

4. The method for preparing apigenin oral nanoparticles according to claim 3, characterized in that, The pH of the alkaline aqueous solution is 8-14.

5. The method for preparing apigenin oral nanoparticles according to claim 3, characterized in that, In the preparation method, the aqueous phase is added dropwise to the organic phase under stirring conditions, and the stirring speed is 200 rpm-1000 rpm.

6. The method for preparing apigenin oral nanoparticles according to claim 1, characterized in that, In the preparation method, after the organic solvent is removed, tannic acid is added under stirring conditions. The mass ratio of tannic acid to soybean protein is 0.2-2:1, thus obtaining tannic acid-protected apigenin oral nano-formulation.

7. The method for preparing apigenin oral nanoparticles according to claim 1, characterized in that, The apigenin oral nanoparticle formulation has a particle size of less than 600 nm.

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