Epigallocatechin gallate starch nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses epigallocatechin gallate starch nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. According to the method, high-proportion branched-chain cassava starch is used as a raw material, ultrasonic microwave assistance is adopted, in the nanometer starch alcohol precipitation process, epigallocatechin gallate is synchronously packaged to form nanometer-level epigallocatechin gallate starch nanometer particles, and embedding of epigallocatechin gallate is completed. The embedding rate of the epigallocatechin gallate starch nanoparticles can reach 50.20%, the drug loading capacity can reach 311mg / g, the drug slow release performance is obvious in the process of simulating gastrointestinal digestion, the sustained release time can be 20 hours, the drug release capacity can reach 72.33% and 90.10% respectively, and the preparation process is simple, easy to operate, low in preparation cost and suitable for large-scale production and application.

Description

Technical Field

[0001] The invention belongs to the technical field of drug sustained release, and particularly relates to epigallocatechin gallate starch nanoparticles and a preparation method and application thereof. Background Art

[0002] Epigallocatechin gallate (EGCG) is a natural polyhydroxycatechin compound with excellent physiological and pharmacological effects. EGCG is the most potent active ingredient in tea polyphenols, and its unique structure endows it with multiple biological functions. Due to its unique stereochemical structure, EGCG exhibits remarkable antioxidant, anticancer, and antitumor effects. However, EGCG is extremely unstable, and its pharmacological effects are easily affected by dosage, in vivo release, and environmental factors. To fully explore the medicinal value of EGCG, new formulation technologies are needed. Encapsulation and sustained-release loading have become research directions for EGCG. The biocompatibility of natural materials has made it a research hotspot for drug carriers.

[0003] As a natural polymer material, starch has been widely used as a drug carrier due to its good biocompatibility. Due to the limitations of the properties of native starch itself, most starch is used as a drug carrier by physical adsorption of drugs in aqueous phase. It is limited by its own swelling properties in organic solvents, and the drug loading effect is not ideal. There are also problems such as long adsorption cycle and complex operation. With the rise of nanotechnology, nano starch (Nanometer starch, SNPs) has broad development prospects as a drug carrier that effectively combines with drugs and effectively releases and absorbs them in the human body. The article "Preparation and Drug Loading Performance of Cassava Starch Nanoparticles" studied the adsorption performance of cassava starch nanoparticles on curcumin. In this method, the adsorption and drug loading process needs to be divided into two steps: first, synthesizing drug-loaded nano starch particles, and then continuing to adsorb the drug. The steps are complex and the cycle is long. The adsorption time is also long, and the drug loading time takes 25 minutes. In addition, the drug adsorption amount is low, with the maximum adsorption amount being only 81.14 mg / g.

[0004] In the field of nano-starch-drug conjugation, patent publication number CN 113577307 A discloses a method for preparing kaempferol starch nanoparticles and their application. This method forms nanoscale kaempferol starch nanoparticles through hydrogen bonding between starch molecules and kaempferol and the hydrophobic cavities of amylose, thereby encapsulating kaempferol. In this technique, kaempferol is dissolved in 30-60% anhydrous ethanol. The resulting kaempferol alcohol solution serves as the non-solvent phase and is introduced into a starch emulsion. The volume ratio of the starch emulsion to the kaempferol alcohol solution is 1:0.4-1.5. In this process, ethanol is used as the non-solvent phase, and the large proportion of the starch emulsion in the solvent phase easily causes starch gelation or gel formation, which can easily lead to a loss in the yield of the drug-loaded nanostarch. Furthermore, the resulting drug-loaded nanostarch has uneven morphology and size, with severe agglomeration and gelation. This also results in low drug loading, with the drug loading capacity for kaempferol being only 3.78 mg / g.

[0005] There are no existing methods for preparing epigallocatechin gallate starch nanoparticles through simultaneous nanoencapsulation. To fully explore the medicinal value of EGCG, the present invention has developed a method for preparing drug-loaded starch nanoparticles through simultaneous nanoencapsulation of EGCG, which has both high drug loading efficiency and significant sustained-release properties. Summary of the Invention

[0006] To address the above problems, the present invention provides epigallocatechin gallate starch nanoparticles, a preparation method, and applications thereof. By using high-proportion amylopectin starch as raw material, methanol as a non-solvent phase, and adopting ultrasonic microwave assistance, highly drug-loaded starch nanoparticles with good morphology and uniform size can be prepared in a short time. During the nano-alcohol precipitation process, EGCG is simultaneously encapsulated to form nano-scale epigallocatechin gallate starch nanoparticles, thereby completing the embedding of EGCG.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for preparing epigallocatechin gallate starch nanoparticles comprises the following steps:

[0009] (1) Starch pregelatinization: Add water to cassava starch to prepare a starch solution, stir and gelatinize to obtain a gelatinized starch solution;

[0010] (2) Preparation of starch milk: The gelatinized starch solution is ultrasonically treated to obtain starch milk;

[0011] (3) Synchronous nano-encapsulation of epigallocatechin gallate: Epigallocatechin gallate is dissolved in anhydrous methanol, and the obtained epigallocatechin gallate alcohol solution is used as the non-solvent phase. At a constant temperature of 65-70°C, starch milk is quickly passed into the epigallocatechin gallate alcohol solution under ultrasonic conditions to obtain epigallocatechin gallate starch nanosuspension. After centrifugation and removal of the supernatant, the precipitate is dispersed and washed with anhydrous methanol, and then freeze-dried to obtain epigallocatechin gallate starch nanoparticles.

[0012] Furthermore, in step (3), the mass concentration of the epigallocatechin gallate alcohol solution is 1 to 5 mg / mL.

[0013] Furthermore, in step (3), the volume ratio of the starch milk to the epigallocatechin gallate alcohol solution is 1:8-10.

[0014] Furthermore, in step (3), the starch milk is quickly passed into the epigallocatechin gallate alcohol solution under ultrasonic conditions of 200 to 300 W, and ultrasonicated for 5 to 10 minutes.

[0015] Furthermore, in step (3), the centrifugal speed is 2500-3000 r / min, and the time is 2-4 min; and the freeze-drying is vacuum freeze-drying at -40--50°C.

[0016] Furthermore, in step (2), the ultrasonic treatment is specifically as follows: placing the gelatinized starch solution under an ultrasonic microwave combined instrument at 400-500W for 15-20 minutes, and the microwave temperature is controlled at 60-70°C.

[0017] Furthermore, in step (1), the mass concentration of the starch solution is 1 to 3%.

[0018] Furthermore, in step (1), the gelatinization temperature is 85-100° C., and the time is 40-50 minutes.

[0019] Epigallocatechin gallate starch nanoparticles prepared by the preparation method described above.

[0020] The invention relates to an application of the epigallocatechin gallate starch nanoparticles as described above in preparing an anti-tumor drug.

[0021] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0022] 1. Unlike kaempferol in chemical structure and biological activity, the compound EGCG in the method of the present invention has more phenolic hydroxyl active sites in its structure, especially gallate groups, which provide it with stronger antioxidant activity. In terms of biological activity, its active sites can form strong interactions with starch molecules, which is conducive to the formation of stable inclusion complexes, thereby improving the medicinal value of EGCG and the bioavailability of starch.

[0023] 2. Unlike traditional adsorption drug-loading methods, the present invention uses simultaneous nano-encapsulation technology to prepare EGCG-SNPs particles, which can obtain highly drug-loaded starch nanoparticles in a very short time, simplifying the operation steps and achieving the effect of time and labor saving. The present invention uses simple physical encapsulation to isolate EGCG from the outside air, effectively solving the problems of EGCG instability and the susceptibility of its efficacy to dose and internal environmental factors, and fully realizing the medicinal value of EGCG.

[0024] 3. In the traditional method, ethanol is used as the non-solvent phase, which easily causes the loss of drug-loaded nano starch production, and the obtained drug-loaded nano starch has uneven morphology and size, and serious agglomeration and gelation. However, the present invention uses methanol as the non-solvent phase to obtain smaller and more uniform drug-loaded starch nanoparticles, which have lower viscosity and higher polarity, contribute to the diffusion of EGCG during the drug loading process, effectively promote the dispersion and refinement of drug-loaded starch nanoparticles, reduce the solubility of starch molecules in the solvent phase, increase the drug loading and dispersion stability, and improve the synthesis yield. In the process of achieving simultaneous nanoencapsulation, methanol has a weak competition for the active sites of EGCG and starch molecules, which is more conducive to embedding EGCG in starch, obtaining a higher drug loading in the drug-loaded nanoparticles, and can better control the formation of specific crystal forms of drug-loaded nano starch, which is beneficial to the precise control and release of the drug delivery system and improves the biocompatibility and degradability of drug-loaded nanoparticles.

[0025] 4. The order and ratio of solvent and non-solvent phase addition affect the physicochemical properties of drug-loaded starch nanoparticles. Increasing the proportion of the non-solvent phase can easily cause starch gelatinization or gel formation. Therefore, during the simultaneous nanoencapsulation of EGCG, the present invention introduces starch emulsion into the EGCG alcohol solution, controlling the volume ratio of the two to 1:8-10. By adjusting the order and ratio of solvent and non-solvent phase addition, the resulting drug-loaded starch nanoparticles have a better morphology and uniform size, while also increasing the yield of drug-loaded starch nanoparticles, simplifying the operating steps, and saving raw materials.

[0026] 5. Since the ratio of carrier and drug in the drug loading process is the most important factor affecting the drug loading effect, the present invention achieves a high drug loading effect by adjusting the ratio of EGCG to starch milk, controlling the mass concentration of the EGCG alcohol solution to 1-5 mg / mL and the starch milk concentration to 1-3%. After obtaining short-chain starch molecules by ultrasonic microwave assistance, more EGCG molecules are dissolved in the methanol solution and delivered to the various binding sites of the starch molecules by diffusion, which can increase the drug loading capacity of the drug-loaded nanostarch while increasing the synthesis yield, thereby improving the utilization rate of the starch raw material and reducing the loss of the original drug.

[0027] 6. The present invention combines the stability of EGCG with the structural state of starch molecules to control the temperature of EGCG simultaneous nano-encapsulation to 65-70°C. At this temperature, the starch molecules are in a stretched state, and the starch hydroxyl groups have more binding sites for EGCG. When the temperature cools, the starch molecules coil and form a helical structure. At this time, the nano-alcohol precipitation can embed EGCG within the drug-loaded nano starch particles, achieving successful loading of EGCG simultaneous nano-encapsulation.

[0028] 7. The EGCG-SNPs particles prepared by the present invention have an embedding rate of up to 50.20% and a drug loading capacity of up to 311 mg / g. The drug sustained-release performance is obvious in the simulated gastrointestinal digestion process, and the drug release can be sustained for 20 hours, with the drug release amount reaching 72.33% and 90.10%, respectively. In addition, the preparation process is simple, easy to operate, and has low preparation cost. The drug loading time during the preparation process is short (only 5 to 10 minutes), which is suitable for large-scale production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The embedding rate and drug loading capacity of starch milk with the same concentration for different EGCG alcohol concentrations.

[0030] Figure 2 The encapsulation efficiency and drug loading of different starch concentrations with the same EGCG alcohol concentration.

[0031] Figure 3 This is the SEM electron microscope image of the EGCG-SNPs particles in Example 1.

[0032] Figure 4 This is the sustained release effect of EGCG-SNPs particles in Example 1 under a simulated gastric digestion environment.

[0033] Figure 5 This is the sustained release effect of EGCG-SNPs particles in Example 1 under a simulated pancreatic digestion environment. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below through examples. These examples are only used to illustrate the present invention and do not limit the scope of protection of the present invention.

[0035] 1. Experiment on the effect of different epigallocatechin gallate (EGCG) alcohol concentrations on drug loading

[0036] In order to study the effect of different EGCG alcohol concentrations on drug loading, 8 different EGCG alcohol concentrations (0.11, 0.17, 0.39, 1, 2, 3, 4, and 5 mg / mL) were set for the experiment. The specific experimental process is as follows:

[0037] Take cassava starch, add water to prepare a starch solution with a concentration of 3%, place it in a constant temperature magnetic stirrer at 90°C, stir for 50 minutes, and then place the obtained gelatinized starch solution in an ultrasonic microwave combined instrument at 500W for 20 minutes to reduce the viscosity. The microwave controls the temperature at 70°C to obtain a low-viscosity starch milk. EGCG is dissolved in anhydrous methanol to prepare EGCG alcohol solutions of different concentrations and use it as a non-solvent. Under a constant temperature of 70°C, the starch milk is quickly passed into EGCG alcohol solutions of different concentrations under 200W ultrasonic conditions. The volume ratio of starch milk to EGCG alcohol solution is 1:9. At the same time, ultrasonication is carried out for 10 minutes to obtain EGCG starch nanoparticles (denoted as EGCG-SNPs) suspensions with different EGCG alcohol concentrations.

[0038] EGCG-SNPs suspensions with different EGCG alcohol concentrations were centrifuged at 3000 rpm for 3 minutes in a high-speed refrigerated centrifuge, and the supernatant was collected. The supernatant was fixed to a certain volume, and the absorbance A was measured using a UV spectrophotometer to obtain the mass concentration of EGCG. The embedding efficiency and drug loading were then calculated. The embedding efficiency and drug loading were calculated using the following formula:

[0039] Embedding rate (%) = (m 总 -m 游离 ) / m 总 ×100 (1)

[0040] Drug loading (mg / g) = (m 总 -m 游离 ) / m 复合物 (2)

[0041] Where: m 总 is the total mass of EGCG added to the EGCG-SNPs suspension, mg; m 游离 is the mass of free EGCG remaining in the supernatant, mg; m 复合物 is the mass of EGCG-SNPs, g.

[0042] The experimental results are as follows Figure 1 shown. Figure 1Results showed that as EGCG concentration increased, the SNPs' entrapment efficiency and drug loading of EGCG showed little effect at low concentrations, but initially increased and then slowed down at higher concentrations. At an EGCG concentration of 1 mg / mL, the SNPs achieved a maximum EGCG entrapment efficiency of 50.02% during sedimentation. The drug loading slowly increased with increasing EGCG concentration, reaching a maximum of 273.2 mg / g at 5 mg / mL. During SNP sedimentation, short-chain starch molecules moved from the water solvent phase into the methanol nonsolvent phase. Hydroxyl groups between the short-chain starch molecules reassociate with the numerous phenolic hydroxyl groups within the EGCG structure, forming hydrogen bonds and forming an EGCG-starch inclusion complex. At a certain EGCG molecular weight, the EGCG molecular weight facilitates partial penetration into the SNP structure through solvent-driven diffusion. The hydroxyl groups on the EGCG molecules more readily interact with those on the short-chain starch molecules, forming hydrogen bonds and, as the starch molecules settle, are entrapped within the SNPs.

[0043] While lower EGCG concentrations showed little change in diffusion, higher EGCG concentrations resulted in competition for binding sites between EGCG and starch, causing more EGCG to dissolve in methanol and resulting in a decreased encapsulation efficiency. As EGCG concentration increased, the drug loading capacity of EGCG-SNPs gradually reached a relatively balanced value. During this process, EGCG concentration significantly influenced both the encapsulation efficiency and drug loading capacity. Therefore, based on the above analysis, a more suitable EGCG concentration of 1-5 mg / mL achieved optimal drug loading.

[0044] 2. Experiment on the effect of different starch concentrations on drug loading

[0045] In order to study the effect of different starch concentrations on drug loading, four different starch concentrations (0.5, 1, 3, and 5%) were set for the experiment. The specific experimental process is as follows:

[0046] Take cassava starch, add water to prepare starch solutions of different concentrations, place them in a constant temperature magnetic stirrer at 90°C, stir for 50 minutes, and then place the obtained gelatinized starch solution in an ultrasonic microwave combined instrument at 500W for 20 minutes to reduce the viscosity. The microwave controls the temperature at 70°C to obtain starch milk of different concentrations with low viscosity. EGCG is dissolved in anhydrous methanol to prepare an EGCG alcohol solution with a concentration of 1 mg / mL and used as a non-solvent. At a constant temperature of 70°C, starch milks of different concentrations are quickly passed into the EGCG alcohol solution under 200W ultrasonic conditions. The volume ratio of starch milk to EGCG alcohol solution is 1:9. At the same time, ultrasonication is carried out for 10 minutes to obtain EGCG starch nanoparticles (denoted as EGCG-SNPs) suspensions with different starch concentrations.

[0047] EGCG-SNP suspensions with varying starch concentrations were centrifuged at 3000 rpm for 3 minutes in a high-speed refrigerated centrifuge. The supernatant was then taken. The supernatant was fixed to a desired volume and the absorbance (A) was measured using a UV spectrophotometer to determine the EGCG mass concentration. The encapsulation efficiency and drug loading were then calculated using the same methods as in Experiment 1 above.

[0048] The experimental results are as follows Figure 2 shown. Figure 2 The results showed that the concentration of starch milk had a small effect on the embedding efficiency of SNPs, but a large effect on the drug loading capacity of SNPs. During the sedimentation process, although the drug loading capacity was high at 0.5% low concentration starch, the yield of EGCG-SNPs prepared was not ideal. In order to obtain a higher yield of EGCG-SNPs, it was necessary to increase the concentration of starch milk. The ratio of the original drug to the carrier in the sedimentation system largely determines the drug loading effect of the drug delivery system. Figure 1 The effect of the initial EGCG concentration on the EGCG-SNPs embedding rate and drug loading capacity. Choosing a starch milk concentration of 1% and an initial EGCG concentration of 1 mg / mL can not only obtain a higher EGCG-SNPs yield, but also achieve a better drug loading effect, and reduce the loss and waste of the original drug EGCG in this process, which is a better choice.

[0049] The EGCG loading in EGCG-SNPs decreased with increasing starch emulsion concentration. This is because the EGCG content in the starch nanoparticles decreased with sedimentation of higher starch emulsion concentrations in the sedimentation system, reducing the EGCG content in the EGCG-SNPs. However, higher starch emulsion concentrations allowed more short-chain starch molecules to bind to EGCG molecules, resulting in more EGCG being embedded within the starch nanoparticles as they settled. The embedding efficiency increased to 50.20% at a starch emulsion volume fraction of 1%, while further increases in starch emulsion concentration did not significantly change the embedding efficiency. In this scenario, a competitive relationship exists between starch molecules, the nonsolvent phase, and EGCG, resulting in some EGCG remaining in the methanol. However, the starch molecular chains lack sufficient affinity to displace EGCG from the methanol, resulting in a negligible increase in the embedding efficiency. Therefore, based on the above analysis, a starch concentration of 1-3% is optimal for optimal drug delivery.

[0050] Example 1

[0051] A method for preparing epigallocatechin gallate starch nanoparticles comprises the following steps:

[0052] (1) Starch pregelatinization: Cassava starch was prepared by adding water to a 3% starch solution, which was placed in a constant temperature magnetic stirrer at 90°C and stirred for 50 min to obtain a gelatinized starch solution;

[0053] (2) Preparation of starch milk: The gelatinized starch solution was ultrasonically treated at 500W for 20 minutes under an ultrasonic microwave combination instrument to reduce the viscosity. The microwave temperature was controlled at 70°C to obtain a low-viscosity starch milk.

[0054] (3) Synchronous nanoencapsulation of epigallocatechin gallate: EGCG was dissolved in anhydrous methanol to prepare an EGCG alcohol solution with a mass concentration of 1 mg / mL and used as a non-solvent. At a constant temperature of 70°C, starch milk was quickly passed into the EGCG alcohol solution under 200W ultrasonic conditions. The volume ratio of starch milk to EGCG alcohol solution was 1:9. Ultrasonication was performed for 10 minutes to obtain an EGCG starch nanosuspension. The suspension was then centrifuged at 3000 r / min for 3 minutes. The supernatant was removed and the precipitate was dispersed and washed three times with anhydrous methanol. The suspension was then freeze-dried at -50°C in a vacuum oven to a constant weight to obtain epigallocatechin gallate starch nanoparticles.

[0055] Example 2

[0056] A method for preparing epigallocatechin gallate starch nanoparticles comprises the following steps:

[0057] (1) Starch pregelatinization: Take cassava starch, add water to prepare a starch solution with a concentration of 1%, place it in a constant temperature magnetic stirrer at 85°C, and stir for 45 minutes to obtain a gelatinized starch solution;

[0058] (2) Preparation of starch milk: The gelatinized starch solution was ultrasonically treated at 400W for 15 minutes under an ultrasonic microwave combination instrument to reduce the viscosity. The microwave temperature was controlled at 60°C to obtain a low-viscosity starch milk.

[0059] (3) Synchronous nanoencapsulation of epigallocatechin gallate: EGCG was dissolved in anhydrous methanol to prepare an EGCG alcohol solution with a mass concentration of 3 mg / mL and used as a non-solvent. At a constant temperature of 65°C, starch milk was quickly passed into the EGCG alcohol solution under 200W ultrasonic conditions. The volume ratio of starch milk to EGCG alcohol solution was 1:8. Ultrasonication was performed for 5 minutes to obtain an EGCG starch nanosuspension. The suspension was centrifuged at 2500 r / min for 4 minutes, the supernatant was removed, and the precipitate was dispersed and washed three times with anhydrous methanol. Then, it was freeze-dried at -40°C in a vacuum oven to a constant weight to obtain epigallocatechin gallate starch nanoparticles.

[0060] Example 3

[0061] A method for preparing epigallocatechin gallate starch nanoparticles comprises the following steps:

[0062] (1) Starch pregelatinization: Take cassava starch, add water to prepare a starch solution with a concentration of 2%, place it in a constant temperature magnetic stirrer at 100°C, and stir for 40 minutes to obtain a gelatinized starch solution;

[0063] (2) Preparation of starch milk: The gelatinized starch solution was ultrasonically treated at 500W for 15 minutes under an ultrasonic microwave combination instrument to reduce the viscosity. The microwave temperature was controlled at 65°C to obtain a low-viscosity starch milk.

[0064] (3) Synchronous nanoencapsulation of epigallocatechin gallate: EGCG was dissolved in anhydrous methanol to prepare an EGCG alcohol solution with a mass concentration of 5 mg / mL and used as a non-solvent. At a constant temperature of 70°C, starch milk was quickly passed into the EGCG alcohol solution under 300W ultrasonic conditions. The volume ratio of starch milk to EGCG alcohol solution was 1:10. The suspension was ultrasonicated for 10 minutes to obtain an EGCG starch nanosuspension. The suspension was centrifuged at 3000 r / min for 2 minutes, the supernatant was removed, and the precipitate was dispersed and washed three times with anhydrous methanol. The suspension was then freeze-dried at -45°C in a vacuum oven to a constant weight to obtain epigallocatechin gallate starch nanoparticles.

[0065] (1) Material characterization and analysis

[0066] Scanning electron microscopy (SEM) was used to characterize the surface morphology of cassava starch, SNPs and EGCG-SNPs in Example 1. Figure 3 . Figure 3 In the figure, Figure (3a) is the SEM image of cassava starch, Figure (3b), Figure (3d) and Figure (3e) are the SEM images of SNPs at different magnifications, and Figure (3c) and Figure (3f) are the SEM images of EGCG-SNPs at different magnifications.

[0067] Depend on Figure 3 It can be seen that cassava starch is spherical or hemispherical, with a smooth surface and no ripples. The particle size is relatively large and distributed between 10 and 20 μm. Ultrasonic fragmentation has a greater effect on the particle size. SNPs are spherical, with a particle size within 100 nm, and there is a slight agglomeration and adhesion phenomenon between the particles. The main morphology of EGCG-SNPs prepared by one-step nanoencapsulation is nanospheres, with relatively uniform size distribution, ripples on the surface, and good dispersion. The morphology and size of SNPs and EGCG-SNPs prepared by sedimentation method changed significantly. Compared with SNPs, the addition of EGCG reduced the agglomeration of SNPs. The size of EGCG-SNPs was larger than that of SNPs, with a particle size of about 100 nm, but it did not affect the formation of SNPs during the sedimentation process. The prepared drug-loaded nanoparticles had good structural integrity.

[0068] (2) Drug release performance test

[0069] (1) Take 2.00g of sodium chloride and dissolve it in 500mL of water. Adjust the pH to 2.0 with 0.10mol / L hydrochloric acid. Take 0.26g of pepsin and dissolve it in water. Mix the two solutions and dilute them to 1000mL with water as a sustained-release solution. Tween-80 with a mass concentration of 1.00% is used as a surfactant in the release medium. 15mg of EGCG-SNPs in Example 1 is dispersed in 5mL of the same sustained-release solution and placed in a dialysis bag (MD44=500). The dialysis bag is placed in a beaker containing 500mL of the same medium. The entire system is placed in a 37°C constant-temperature water bath for release experiments at a speed of 80r / min.

[0070] At specific time points (0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0, and 25.0 h), 3 mL of dissolution medium was removed from the beaker and an equal amount of fresh sustained-release solution was added to the beaker. The EGCG content in the sample was determined using a UV spectrophotometer, the cumulative release rate was calculated, and a release characteristic curve was plotted. The cumulative release rate of the original drug EGCG in gastric fluid was determined using the same method, and the release characteristic curve of the original drug EGCG was plotted as a control.

[0071] (2) Take 6.80g of potassium dihydrogen phosphate, add 500mL of water to dissolve, adjust the pH value to 6.8 with 0.10mol / L sodium hydroxide solution, take 2.00g of trypsin, add appropriate amount of water to dissolve it, mix the two liquids, and dilute with water to 1000mL as a sustained-release solution. In the release medium, Tween-80 with a mass concentration of 1.00% is used as a surfactant. 15mg of EGCG-SNPs in Example 1 are dispersed in mL of the same sustained-release solution and placed in a dialysis bag (MD44=500), which is placed in a beaker containing 500mL of the same medium. The entire system is placed in a 37°C constant temperature water bath for release experiments at a speed of 80r / min.

[0072] At specific time points (0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0, and 25.0 h), 3 mL of dissolution medium was removed from the beaker and an equal amount of fresh sustained-release solution was added to the beaker. The EGCG content in the sample was determined by UV spectrophotometry, the cumulative release rate was calculated, and the release characteristic curve was plotted. The cumulative release rate of the original drug EGCG in the pancreatic juice environment was determined by the same method, and the release characteristic curve of the original drug EGCG was plotted as a control.

[0073] The calculation formula for the cumulative release rate of drug sustained release is:

[0074] Cumulative release rate (%) = (M t +M c) / M0×LC×100 (3)

[0075] Where: M t is the mass of EGCG in the sample solution at time t, mg; M c is the mass of EGCG in the loss solution, mg; M0 is the mass of EGCG-SNPs, mg; LC is the drug loading capacity of EGCG-SNPs to EGCG, %.

[0076] The release results of EGCG and EGCG-SNPs drugs are as follows Figure 4 and 5 shown.

[0077] Depend on Figure 4 and Figure 5 It was found that free EGCG was rapidly released in the gastrointestinal fluid digestion simulation, with the cumulative release rate reaching over 90% within 2 hours, indicating that free EGCG can diffuse smoothly through the dialysis membrane. However, the cumulative release rate of EGCG from EGCG-SNPs prepared by nanoencapsulation was only 1.85% (pH = 2) and 8.95% (pH = 6.8) within 2 hours, indicating that SNPs can effectively delay the release of EGCG. EGCG from EGCG-SNPs was slowly released within 5 hours, with the cumulative release rate reaching only 12.40% (pH = 2) and 20.55% (pH = 6.8). This may be due to the enzymatic hydrolysis of starch in the drug-loaded starch nanoparticles under the action of gastrointestinal enzymes. At this time, only EGCG on the surface of the drug-loaded starch nanoparticles diffuses through dialysis, resulting in a slower diffusion.

[0078] Subsequently, EGCG was rapidly released within 5 to 20 hours, with the cumulative release rate reaching 72.33% (pH = 2) and 90.10% (pH = 6.8) in 20 hours. This indicates that SNPs can effectively protect and control the release of EGCG during the simulated digestion process in gastrointestinal fluid. The reason for the different cumulative sustained-release rates of EGCG in gastrointestinal fluid within the same period of time may be that EGCG is less stable at near-neutral pH than in an acidic environment, resulting in an increased cumulative release rate. In summary, SNPs can serve as an effective EGCG nanocarrier, which is beneficial for prolonging drug efficacy and reducing the number of dosing times.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing epigallocatechin gallate starch nanoparticles, characterized in that: The following steps are involved: (1) Starch pre-gelatinization: Add water to cassava starch to prepare a starch solution, and stir to gelatinize to obtain a gelatinized starch solution; (2) Preparation of starch milk: The gelatinized starch solution is subjected to ultrasonic treatment to obtain starch milk; (3) Synchronous nano-encapsulation of epigallocatechin gallate: epigallocatechin gallate is dissolved in anhydrous methanol, and the obtained epigallocatechin gallate alcohol solution is used as a non-solvent phase. At a constant temperature of 65-70° C., starch milk is quickly passed into the epigallocatechin gallate alcohol solution under ultrasonic conditions to obtain an epigallocatechin gallate starch nanosuspension. After centrifugation and removal of the supernatant, the precipitate is dispersed and washed with anhydrous methanol, and then freeze-dried to obtain epigallocatechin gallate starch nanoparticles.

2. The method for preparing epigallocatechin gallate starch nanoparticles according to claim 1, characterized in that: In step (3), the mass concentration of the epigallocatechin gallate alcohol solution is 1 to 5 mg / mL.

3. The method for preparing epigallocatechin gallate starch nanoparticles according to claim 2, characterized in that: In step (3), the volume ratio of the starch milk to the epigallocatechin gallate alcohol solution is 1:8-10.

4. The method for preparing epigallocatechin gallate starch nanoparticles according to claim 3, characterized in that: In step (3), the starch milk is quickly passed into the epigallocatechin gallate alcohol solution under 200-300W ultrasonic conditions, and ultrasonication is performed for 5-10 minutes.

5. The method for preparing epigallocatechin gallate starch nanoparticles according to claim 1, characterized in that: In step (3), the centrifugal speed is 2500-3000 r / min, and the time is 2-4 min; the freeze-drying is vacuum freeze-drying at -40--50°C.

6. The method for preparing epigallocatechin gallate starch nanoparticles according to claim 1, characterized in that: In step (2), the ultrasonic treatment is specifically as follows: placing the gelatinized starch solution under an ultrasonic microwave apparatus at 400-500W for 15-20 minutes, and the microwave temperature is controlled at 60-70°C.

7. The method for preparing epigallocatechin gallate starch nanoparticles according to claim 1, characterized in that: In step (1), the mass concentration of the starch solution is 1 to 3%.

8. The method for preparing epigallocatechin gallate starch nanoparticles according to claim 1, characterized in that: In step (1), the gelatinization temperature is 85-100° C. and the time is 40-50 min.

9. Epigallocatechin gallate starch nanoparticles prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the epigallocatechin gallate starch nanoparticles as claimed in claim 9 for preparing anti-tumor drugs.

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