Traditional Chinese medicine monomer self-assembled nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses traditional Chinese medicine monomer self-assembled nanoparticles as well as a preparation method and application thereof, and relates to the technical field of pharmaceutical preparations. According to the prepared traditional Chinese medicine monomer self-assembled nanoparticles, namely ginsenoside Rg1-oleanolic acid self-assembled nanoparticles, the self-assembled structural characteristics of ginsenoside Rg1 are ingeniously utilized, the superficial area of the medicine can be effectively increased, the solubility of oleanolic acid can be improved, and therefore the bioavailability of oleanolic acid is effectively improved, and the anti-inflammatory effect of the oleanolic acid is further enhanced. And compared with a physical mixed medicine with the same dosage, the effect is more remarkable. Besides, the preparation method is simple and easy to implement, not only has the advantages of nanoscale drugs, but also realizes self-delivery of pure drugs without auxiliary materials, and greatly improves the delivery efficiency of the drugs.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical preparations, and in particular to a traditional Chinese medicine monomer self-assembled nanoparticle and a preparation method and application thereof. Background Art

[0002] Shenling Baizhu San is a classic Chinese medicine compound. Studies have shown that it can treat a variety of gastrointestinal diseases, including antibiotic diarrhea, weaning stress diarrhea, inflammatory bowel disease, etc. Analysis of the active ingredients of Shenling Baizhu San found that the main active ingredients of Shenling Baizhu San are concentrated in its n-butanol extract, and the n-butanol extract of Shenling Baizhu San contains a variety of ginsenosides, glycyrrhizic acid and oleanolic acid.

[0003] As an important branch of nanotechnology, self-assembly technology has attracted much attention in recent years due to its wide application prospects. In the process of self-assembly, molecules, nanomaterials and other substances spontaneously organize or aggregate into a stable structure with a certain regular geometric appearance under the interaction based on non-covalent bonds, such as nanotubes, nanofibers, nanoparticles and vesicles. In the processing and application of traditional Chinese medicine, the self-assembly phenomenon also plays an important role. The root cause is that traditional Chinese medicine contains rich active ingredients, such as carbohydrates, proteins, flavonoids, terpenes, alkaloids, organic acids, metal ions, etc. These ingredients have the characteristics of self-assembly and can form stable nanostructures through interaction under appropriate conditions. More importantly, the discovery of self-assembled nanoparticles of traditional Chinese medicine provides a new solution to the problems of low bioavailability and short half-life of traditional Chinese medicine in the body.

[0004] Ginsenosides, as the vital active ingredients in ginseng, have multiple therapeutic effects such as antioxidant, anti-inflammatory, vasodilation, anti-allergic, and anti-diabetic. It is particularly worth mentioning that ginsenoside Rg1 (Ginsenoside Rg1, referred to as Rg1), as a member of the tetracyclic triterpenoid derivatives, has attracted much attention for its unique biological activity. Rg1 can quickly relieve fatigue, improve learning and memory, and delay aging. It has the effect of stimulating the central nervous system and inhibiting platelet aggregation. Ginsenoside Rg1 molecules also have good water solubility. They self-assemble in aqueous solution to form spherical and worm-like micelles. Their hydrophobic core can load drugs with poor water solubility, thereby achieving a solubilization effect.

[0005] Oleanolic acid (OA) is a nano-sized pentacyclic triterpenoid compound that exists in a variety of plants as free bodies and glycosides. As a natural compound with multiple biological activities, OA's main functions include immunomodulation and anti-inflammation, antibacterial and anti-infection, liver protection and enzyme reduction, blood sugar regulation, anti-tumor, antioxidant and free radical scavenging. In addition, it may also have other health benefits such as improving sleep and anti-fatigue. Oleanolic acid exhibits important biological activities and health benefits in many aspects, but its stability is poor and it is easily affected by environmental factors; its water solubility is also poor, which to a certain extent limits its absorption and utilization in the body.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a traditional Chinese medicine monomer self-assembled nanoparticles and a preparation method and application thereof.

[0008] The traditional Chinese medicine monomer self-assembled nanoparticles prepared by the present invention, namely, ginsenoside Rg1-oleanolic acid self-assembled nanoparticles, cleverly utilize the self-assembly structural characteristics of ginsenoside Rg1 to improve the water solubility of oleanolic acid. The nanoparticles can increase the surface area of ​​the drug, improve the solubility of the drug, improve its bioavailability, and further enhance its anti-inflammatory effect.

[0009] The structural formula of the ginsenoside Rg1 is:

[0010]

[0011] The structural formula of the oleanolic acid is:

[0012]

[0013] The purpose of the present invention is achieved through the following technical solutions:

[0014] A method for preparing traditional Chinese medicine monomer self-assembled nanoparticles comprises the following steps:

[0015] (1) mixing ginsenoside Rg1 and water to obtain a ginsenoside Rg1 solution;

[0016] (2) mixing oleanolic acid and anhydrous ethanol to obtain an oleanolic acid solution;

[0017] (3) adding the oleanolic acid solution dropwise into the ginsenoside Rg1 solution to obtain a mixed solution;

[0018] (4) The mixed solution is stirred and then ultrasonicated to obtain a ginsenoside Rg1-oleanolic acid self-assembled nanoparticle solution, i.e., a Chinese medicine monomer self-assembled nanoparticle solution.

[0019] Preferably, in step (2), the concentration of the oleanolic acid solution is 1±0.2 mg / mL; further 1 mg / mL;

[0020] Preferably, in step (3), in the mixed solution, the mass ratio of ginsenoside Rg1 to oleanolic acid is (10±0.3):1; further 10:1.

[0021] Preferably, in step (3), the final concentration of ginsenoside Rg1 in the mixed solution is 1±0.2 mg / mL; further 1 mg / mL;

[0022] Preferably, in step (4), the stirring condition is 500±100 rpm at 25±3°C for 60±5 min; further 500 rpm at 25°C for 60 min;

[0023] Preferably, in step (4), the ultrasonic condition is ultrasonic at 25±3°C for 10 to 15 min; further ultrasonic at 25°C for 15 min.

[0024] Furthermore, the preparation method of the present invention may further include the step of drying the ginsenoside Rg1-oleanolic acid self-assembled nanoparticle solution after step (4).

[0025] Preferably, the drying is freeze-drying.

[0026] A kind of traditional Chinese medicine monomer self-assembled nanoparticles is prepared by the above preparation method.

[0027] Application of the traditional Chinese medicine monomer self-assembled nanoparticles in the preparation of anti-inflammatory drugs;

[0028] Application of the traditional Chinese medicine monomer self-assembled nanoparticles in the preparation of drugs for treating colitis;

[0029] Application of the traditional Chinese medicine monomer self-assembled nanoparticles in preparing medicine for treating acute colitis.

[0030] Preferably, the colitis or acute colitis is DSS-induced colitis or acute colitis.

[0031] Compared with the prior art, the present invention has the following advantages and effects:

[0032] (1) The present invention is inspired by the solubilization and synergism of traditional Chinese medicine compatibility, and uses the natural active ingredients, small molecule ginsenoside Rg1 and oleanolic acid, as basic raw materials to prepare ginsenoside Rg1-oleanolic acid self-assembled nanoparticles. Therefore, the present invention designs a preparation method of nanoparticles based on ginsenoside Rg1 and oleanolic acid as raw materials and its anti-inflammatory application.

[0033] (2) The present invention designs a preparation method of ginsenoside Rg1-oleanolic acid self-assembled nanoparticles and its anti-inflammatory application, wherein ginsenoside Rg1-oleanolic acid self-assembled nanoparticles (Rg1-OANPs) can effectively increase the surface area of ​​the drug, improve the solubility of oleanolic acid, thereby effectively improving the bioavailability of oleanolic acid, and at the same time have a good anti-inflammatory effect and compared with the same dose of physically mixed drugs, its effect is more significant. In addition, the preparation method is simple and easy, not only has the advantages of nano-scale drugs, but also realizes the self-delivery of pure drugs without excipients, greatly improving the delivery efficiency of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the appearance result of the self-assembled nanoparticle solution at different oleanolic acid concentrations.

[0035] Figure 2 This is the dynamic light scattering result of ginsenoside Rg1-oleanolic acid self-assembled nanoparticles (Rg1-OA NPs).

[0036] Figure 3 This is the SEM electron micrograph of ginsenoside Rg1-oleanolic acid self-assembled nanoparticles (Rg1-OANPs).

[0037] Figure 4 This is the SEM electron microscope image of oleanolic acid (OA).

[0038] Figure 5 This is the SEM electron microscope image of ginsenoside Rg1.

[0039] Figure 6 This is the UV characterization result of ginsenoside Rg1-oleanolic acid self-assembled nanoparticles (Rg1-OANPs).

[0040] Figure 7 This is the infrared characterization result of ginsenoside Rg1-oleanolic acid self-assembled nanoparticles (Rg1-OANPs).

[0041] Figure 8 These are the results of the long-term storage stability experiment of ginsenoside Rg1-oleanolic acid self-assembled nanoparticles (Rg1-OA NPs); A: particle size results; B: PDI results.

[0042] Fig. 9The figure is the effect of ginsenoside Rg1-oleanolic acid self-assembled nanoparticles (Rg1-OANPs) on DSS-induced acute colitis in mice; A: body weight of mice in each group at each stage, g; B: DAI score of each group; C: body weight of each group on Day14, g; D: DAI score of each group on Day14; E: colon length of each group, cm; F: spleen index of each group; GJ: mRNA expression levels of TNF-α, IL-1β, IL-6, and iNOS in serum of each group; Note: Compared with Control, *P<0.05, **P<0.01, ***P<0.001; Compared with DSS group, #P<0.05, ##P<0.01, ###P<0.001. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the raw materials used in the examples of the present invention are conventionally purchased raw materials.

[0044] The ginsenoside Rg1 used in the examples has a CAS number of 22427-39-0; ginsenoside Rf has a CAS number of 52286-58-5; ginsenoside Re has a CAS number of 52286-59-6; ginsenoside Rg3 has a CAS number of 14197-60-5; matrine has a CAS number of 519-02-8; and oleanolic acid has a CAS number of 508-02-1.

[0045] Example 1

[0046] 1. Self-assembled nanoparticles prepared from different Chinese medicine monomers and oleanolic acid

[0047] Different Chinese medicine monomers (ginsenoside Rg1, ginsenoside Rf, ginsenoside Re, ginsenoside Rg3 or matrine) were mixed with water to obtain a Chinese medicine monomer solution; oleanolic acid was mixed with anhydrous ethanol to obtain an oleanolic acid solution with a mass concentration of 1 mg / mL; the oleanolic acid solution was added dropwise to the ginsenoside Rg1 solution to obtain a mixed solution, wherein the final concentration of the Chinese medicine monomer in the mixed solution was 1 mg / mL, and the mass ratio of the Chinese medicine monomer to oleanolic acid was 10:1; the mixed solution was first stirred at 25°C and 500 rpm for 60 min, and then ultrasonicated at 25°C for 15 min to obtain a Chinese medicine monomer-oleanolic acid self-assembled nanoparticle solution, which were respectively recorded as ginsenoside Rg1-oleanolic acid self-assembled nanoparticles (Rg1-OA NPs) solution, ginsenoside Rf-oleanolic acid self-assembled nanoparticles (Rf-OA NPs) solution, ginsenoside Re-oleanolic acid self-assembled nanoparticles (Re-OA NPs) solution, ginsenoside Rg3-oleanolic acid self-assembled nanoparticles (Rg3-OA NPs) solution or matrine-oleanolic acid self-assembled nanoparticles (matrine-OANPs) solution. The obtained Chinese medicine monomer-oleanolic acid self-assembled nanoparticle solution was placed in the sample cell of the dynamic light scattering instrument to test its particle size and PDI. The results are shown in Table 1. Among the five different Chinese medicine monomer-oleanolic acid self-assembled nanoparticles, the particle size and PDI of ginsenoside Rg1-oleanolic acid self-assembled nanoparticles (Rg1-OA NPs) are the smallest, so Rg1-OANPs are finally selected.

[0048] Table 1 Particle size and PDI results of different Chinese medicine monomer-oleanolic acid self-assembled nanoparticles

[0049] drug Particle size Average particle size PDI Rg1-OANPs 214.4~247.8nm 232.64±11.25nm 0.330±0.044 Rf-OANPs 1220.0~1497.0nm 1394.00±121.33nm 0.913±0.099 Re-OANPs 617.9~823.2nm 722.25±85.22nm 0.966±0.069 Rg3-OANPs 762.9~834.3nm 799.26±30.24nm 0.302±0.054 Matrine-OA NPs 317.2~346.5nm 332.60±14.71nm 0.577±0.039

[0050] 2. Single Factor Test

[0051] Single factor experiments were conducted on factors such as oleanolic acid concentration, volume ratio, stirring temperature, stirring speed, stirring time, and ultrasonic time to determine the effects of each factor on the particle size and PDI of ginsenoside Rg1-oleanolic acid self-assembled nanoparticles.

[0052] 2.1 Effect of oleanolic acid concentration on particle size and PDI

[0053] Ginsenoside Rg1 was mixed with water to obtain ginsenoside Rg1 solution; oleanolic acid was mixed with anhydrous ethanol to obtain oleanolic acid solution with a mass concentration of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL; the same volume of oleanolic acid solution was added dropwise to the ginsenoside Rg1 solution to obtain a mixed solution, wherein the final concentration of ginsenoside Rg1 in the mixed solution was 1 mg / mL, and the mass ratio of ginsenoside Rg1 to oleanolic acid was 10:1, 10:2, 10:3, 10:4, and 10:5; the mixed solution was stirred at 25°C and 500 rpm for 60 min, and then ultrasonicated at 25°C for 15 min to obtain ginsenoside Rg1-oleanolic acid self-assembled nanoparticle solution. The effect of oleanolic acid concentration on particle size and PDI was investigated.

[0054] The results are shown in Table 2. The particle size and PDI of the prepared Rg1-OANPs are similar at different oleanolic acid concentrations. Figure 1 As shown, it can be clearly observed from the appearance of the solution that only the nanoparticle solution with an oleanolic acid concentration of 1 mg / ml is the clearest and most transparent, without any flocculation or aggregation. Therefore, the final oleanolic acid concentration of 1 mg / mL was selected, that is, the mass ratio of ginsenoside Rg1 to oleanolic acid was 10:1.

[0055] Table 2 Particle size and PDI results of self-assembled nanoparticles at different oleanolic acid concentrations

[0056] Oleanolic acid concentration Particle size Average particle size PDI 1mg / mL 214.4~247.8nm 232.64±11.25nm 0.330±0.044 2mg / mL 243.4~259.2nm 251.92±5.97nm 0.447±0.027 3mg / mL 210.5~257.2nm 227.70±17.77nm 0.383±0.084 4mg / mL 207.2~222.0nm 213.48±6.36nm 0.464±0.080 5mg / mL 230.1~256.0nm 243.30±10.41nm 0.403±0.034

[0057] 2.2 Effect of volume ratio on particle size and PDI

[0058] Ginsenoside Rg1 was mixed with water to obtain a ginsenoside Rg1 solution with a mass concentration of Oleanolic acid was mixed with anhydrous ethanol to obtain oleanolic acid solution with a mass concentration of 1 mg / mL, 0.5 mg / mL, Oleanolic acid solution was added dropwise to ginsenoside Rg1 solution to obtain a mixed solution, wherein the final concentration of ginsenoside Rg1 in the mixed solution was 1 mg / mL, the final concentration of oleanolic acid was 0.1 mg / mL, and the mass ratio of ginsenoside Rg1 to oleanolic acid was 10:1; the mixed solution was first stirred at 500 rpm for 60 min at 25°C, and then ultrasonicated at 25°C for 15 min to obtain ginsenoside Rg1-oleanolic acid self-assembled nanoparticle solution. The effect of volume ratio on particle size and PDI was investigated.

[0059] The results are shown in Table 3. Among the different volume ratios of aqueous phase to organic phase, the particle size and PDI of Rg1-OANPs prepared when the volume ratio was 9:1 were the smallest, so the volume ratio of aqueous phase to organic phase was finally selected as 9:1.

[0060] Table 3 Particle size and PDI results of self-assembled nanoparticles at different volume ratios of aqueous phase to organic phase

[0061] Volume ratio of aqueous phase to organic phase Particle size Average particle size PDI 9：1 214.4~247.8nm 232.64±11.25nm 0.330±0.044 8：2 335.1~353.4nm 347.10±7.14nm 0.712±0.026 7：3 445.4~463.2nm 458.28±8.61nm 0.396±0.038 6：4 893.1~979.8nm 934.4±43.50nm 0.745±0.044

[0062] 2.3 Effect of stirring temperature on particle size and PDI

[0063] Ginsenoside Rg1 was mixed with water to obtain ginsenoside Rg1 solution; oleanolic acid was mixed with anhydrous ethanol to obtain oleanolic acid solution with a mass concentration of 1 mg / mL; oleanolic acid solution was added dropwise to ginsenoside Rg1 solution to obtain a mixed solution, wherein the final concentration of ginsenoside Rg1 in the mixed solution was 1 mg / mL, and the mass ratio of ginsenoside Rg1 to oleanolic acid was 10:1; the mixed solution was first stirred at 500 rpm for 60 min at different temperatures (25°C, 37°C or 60°C), and then ultrasonicated at 25°C for 15 min to obtain ginsenoside Rg1-oleanolic acid self-assembled nanoparticle solution. The effect of stirring temperature on particle size and PDI was investigated.

[0064] The results are shown in Table 4. Among the different stirring temperatures, the particle size and PDI of Rg1-OANPs prepared at a stirring temperature of 25°C are the smallest, so 25°C is finally selected as the stirring temperature.

[0065] Table 4 Particle size and PDI results of self-assembled nanoparticles at different stirring temperatures

[0066] Stirring temperature Particle size Average particle size PDI 25℃ 214.4~247.8nm 232.64±11.25nm 0.330±0.044 37℃ 327.3~396.6nm 354.44±28.27nm 0.761±0.051 60℃ 227.8~284.0nm 257.67±33.13nm 0.588±0.042

[0067] 2.4 Effect of stirring speed on particle size and PDI

[0068] Ginsenoside Rg1 was mixed with water to obtain ginsenoside Rg1 solution; oleanolic acid was mixed with anhydrous ethanol to obtain oleanolic acid solution with a mass concentration of 1 mg / mL; oleanolic acid solution was added dropwise to ginsenoside Rg1 solution to obtain a mixed solution, wherein the final concentration of ginsenoside Rg1 in the mixed solution was 1 mg / mL, and the mass ratio of ginsenoside Rg1 to oleanolic acid was 10:1; the mixed solution was first stirred at different speeds (500 rpm, 1000 rpm or 1500 rpm) at 25°C for 60 min, and then ultrasonicated at 25°C for 15 min to obtain ginsenoside Rg1-oleanolic acid self-assembled nanoparticle solution. The effect of stirring speed on particle size and PDI was investigated.

[0069] The results are shown in Table 5. Under different stirring speeds, the particle size and PDI of Rg1-OANPs prepared at a stirring speed of 500 rpm are the smallest, so 500 rpm is finally selected as the stirring speed.

[0070] Table 5 Particle size and PDI results of self-assembled nanoparticles at different stirring speeds

[0071] Stirring speed Particle size Average particle size PDI 500rpm 214.4~247.8nm 232.64±11.25nm 0.330±0.044 1000rpm 397.5~469.1nm 424.13±39.16nm 0.672±0.051 1500rpm 374.4~438.2nm 401.13±33.13nm 0.480±0.094

[0072] 2.5 Effect of stirring time on particle size and PDI

[0073] Ginsenoside Rg1 was mixed with water to obtain ginsenoside Rg1 solution; oleanolic acid was mixed with anhydrous ethanol to obtain oleanolic acid solution with a mass concentration of 1 mg / mL; oleanolic acid solution was added dropwise to ginsenoside Rg1 solution to obtain a mixed solution, wherein the final concentration of ginsenoside Rg1 in the mixed solution was 1 mg / mL, and the mass ratio of ginsenoside Rg1 to oleanolic acid was 10:1; the mixed solution was first stirred at 500 rpm at 25°C for different times (15min, 30min, 60min, 120min), and then ultrasonicated at 25°C for 15min to obtain ginsenoside Rg1-oleanolic acid self-assembled nanoparticle solution. The effect of stirring time on particle size and PDI was investigated.

[0074] The results are shown in Table 6. Under different stirring times, the particle size and PDI of Rg1-OANPs prepared when the stirring time is 60 min are the smallest, so 60 min is finally selected as the stirring time.

[0075] Table 6 Particle size and PDI results of self-assembled nanoparticles at different stirring times

[0076] Mixing time Particle size Average particle size PDI 15min 713.2~987.8nm 893.03±3.41nm 1.000±0.000 30min 363.1~472.1nm 416.13±56.73nm 0.765±0.076 60min 214.4~247.8nm 232.64±11.25nm 0.330±0.044 120min 353.5~359.2nm 358.24±3.51nm 0.435±0.019

[0077] 2.6 Effect of ultrasonic time on particle size and PDI

[0078] Ginsenoside Rg1 was mixed with water to obtain a ginsenoside Rg1 solution with a mass concentration of 1 mg / mL; oleanolic acid was mixed with anhydrous ethanol to obtain an oleanolic acid solution with a mass concentration of 1 mg / mL; the oleanolic acid solution was added dropwise to the ginsenoside Rg1 solution to obtain a mixed solution, wherein the final concentration of ginsenoside Rg1 in the mixed solution was 1 mg / mL, and the mass ratio of ginsenoside Rg1 to oleanolic acid was 10:1; the mixed solution was first stirred at 500 rpm at 25°C for 60 min, and then ultrasonicated at 25°C for different time (0 min, 15 min or 30 min) to obtain a ginsenoside Rg1-oleanolic acid self-assembled nanoparticle solution. The effect of ultrasonic time on particle size and PDI was investigated.

[0079] The results are shown in Table 7. Under different ultrasonic times, the particle size and PDI of Rg1-OANPs prepared when the ultrasonic time is 15 min and 30 min are not much different. However, heat will continue to be released during the ultrasonic process, and the temperature increase is not conducive to maintaining the stability of Rg1-OANPs. Therefore, 15 min was finally selected as the ultrasonic time.

[0080] Table 7 Particle size and PDI results of self-assembled nanoparticles under different ultrasonic times

[0081] Ultrasound time Particle size Average particle size PDI 0min 312.1~381.8nm 338.34±3.41nm 0.066±0.058 15min 214.4~247.8nm 232.64±11.25nm 0.330±0.044 30min 214.9~224.0nm 221.7±3.84nm 0.365±0.030

[0082] 3. Dynamic light scattering experiment of Rg1-OA NPs

[0083] The Rg1-OA NPs solution prepared in step 1 of Example 1 was placed in a dynamic light scattering instrument sample cell to test its particle size, PDI, and Zeta potential. The results are as follows: Figure 2 As shown, the particle size of Rg1-OA NPs is 232.64±11.25nm, the PDI is 0.330±0.044, and the potential is -2.58±0.34mV. The above results show that the Rg1-OA NPs solution is evenly dispersed, the nanoparticles are nanoscale, and the size is uniform.

[0084] 4. Scanning electron microscopy observation of Rg1-OA NPs

[0085] In this experiment, the Rg1-OA NPs solution prepared in step 1 of Example 1 was observed by scanning electron microscopy. Rg1 was dissolved in water at a concentration of 1 mg / mL; OA was dissolved in water at a concentration of 1 mg / mL; Rg1-OA NPs refers to the Rg1-OA NPs solution prepared in step 1 of Example 1; the above solutions were dropped on tin foil, which was placed in a drying oven for drying, and then the tin foil was fixed on the conductive glue, sprayed with gold, and its morphology and particle size were observed using SEM. The results are shown in Figure 2. Figure 3 to Figure 5 As shown, it was shown that Rg1-OANPs formed a large number of spherical and worm-like micelles in aqueous solution, and their morphologies were completely different from those of Rg1 and OA monomers.

[0086] 5. UV characterization experiment of Rg1-OA NPs

[0087] In this experiment, Rg1, OA and the Rg1-OA NPs solution prepared in step 1 of Example 1 were characterized by UV. Rg1 was dissolved in water at a concentration of 1 mg / mL; OA was dissolved in anhydrous ethanol at a concentration of 1 mg / mL; Rg1-OA NPs refers to the Rg1-OA NPs solution prepared in step 1 of Example 1; Rg1-OA refers to the mixed solution of Rg1 and OA prepared in step 1 of Example 1; 2 mL of each solution was placed in a cuvette and tested in a UV spectrophotometer. The results are shown in Figure 6 As shown, the characteristic peak of OA appears at 210 nm, and the characteristic peak of Rg1 appears at 200 nm; while Rg1-OA NPs have no obvious characteristic peak at 210 nm, indicating that Rg1 may encapsulate OA.

[0088] 6. Infrared characterization experiment of Rg1-OA NPs

[0089] In this experiment, Rg1, OA and Rg1-OA NPs powder obtained by freeze-drying the Rg1-OA NPs solution prepared in step 1 of Example 1 were characterized by infrared. 2 mg of Rg1, OA and Rg1-OANPs were weighed, 200 mg of infrared dried potassium bromide powder was added, mixed and pressed into potassium bromide tablets, and placed in an infrared spectrometer for detection. The resolution of the spectrometer was 4 cm -1 , signal-to-noise ratio 50000:1, scan 64 times, record the spectrum. Figure 7 As shown, 3470.34cm -1 The characteristic absorption peak of -OH belonging to OA is 1696.87 cm -1 It is the characteristic absorption peak of C=O, 1645.38cm -1 The C=O absorption characteristic peak of Rg1. Rg1-OA NPs retain certain OA characteristic peaks, but the peak shape and peak position have changed to a certain extent. The C=O characteristic peak of OA moves to 1690.96 cm -1 、1644.64cm -1 , no new characteristic peaks appeared, indicating that the formed nanoparticles did not destroy the skeleton structure of the monomer, and no chemical bond was formed between OA and Rg1, indicating that OA was successfully encapsulated by Rg1 rather than a single physical mixture, which was consistent with the UV results.

[0090] 7. Stability experiment of Rg1-OA NPs

[0091] 7.1. Centrifugation and dilution stability test:

[0092] Centrifugal stability: Take an appropriate amount of the Rg1-OA NPs solution prepared in step 1 of Example 1 and place it in a centrifuge tube. Centrifuge it at a low speed of 3000 r / min for 0.5 h; and at a high speed of 10000 r / min for 15 min. After centrifugation, observe whether there is drug precipitation in the solution to judge the stability of the nanoparticle solution. The results show that no drug precipitation is found in the centrifuge tube after centrifugation of the Rg1-OA NPs solution, indicating that low and high speed centrifugation will not affect the stability of the Rg1-OA NPs solution.

[0093] Dilution stability: Take 3 equal parts of the Rg1-OA NPs solution prepared in step 1 of Example 1, add different volumes (100, 50, 10 times) of distilled water to dilute and stir evenly, leave it at room temperature overnight, and observe the effect of the dilution multiple on the physical stability of the nanoparticle solution. After the sample was diluted with different multiples of distilled water, no flocculation, precipitation or phase change of the Rg1-OA NPs solution was observed, indicating that the dilution multiple would not affect its stability. This result shows that the Rg1-OANPs solution has good physical stability and is not prone to phase separation or stratification after dilution.

[0094] 7.2. Long-term storage stability test:

[0095] In this experiment, the Rg1-OA NPs solution prepared in step 1 of Example 1 was subjected to a long-term storage experiment at room temperature to investigate the stability of the sample. An appropriate amount of Rg1-OA NPs solution was stored at -20°C, 4°C, and 25°C, and samples were taken and observed at 1d, 2d, 3d, 5d, and 7d to investigate the stability of the sample during storage. The Rg1-OANPs solution has good stability at 4°C, and can maintain its original transparent state after long-term storage, and no flocculation or agglomeration is found. After being placed at -20°C for 1 day, the solution was taken out for observation, and it was found that the appearance of the solution was slightly turbid, and flocculation and agglomeration occurred; after being placed at 25°C for 3 days, the solution was taken out for observation, and it was found that the appearance of the solution was slightly turbid, and flocculation and agglomeration occurred; the above results are consistent with Figure 8 The measured particle size and PDI results are consistent. The results suggest that the nanoparticle solution should not be placed in a frozen or room temperature environment and should be stored at 4°C.

[0096] Example 2 Effect on DSS-induced acute colitis in mice

[0097] 1. Experimental methods

[0098] Experimental subjects: Male C57 mice aged 7 to 8 weeks;

[0099] Experimental groups: After one week of adaptation, C57 mice were randomly divided into 4 groups, namely, blank control group (Control), model group (DSS), nanoparticle group (Rg1-OA NPs, equivalent to a dose of 20 mg / kg of monomer Rg1 and 2 mg / kg of monomer OA), and physical mixture group (Rg1-OA, a dose of 20 mg / kg of monomer Rg1 and 2 mg / kg of monomer OA); 6 mice in each group. Note: Rg1-OA NPs refers to the Rg1-OANPs solution prepared in step 1 of Example 1; Rg1-OA refers to the mixed solution of Rg1 and OA prepared in step 1 of Example 1.

[0100] The blank control group (Control) was given sterile water to drink freely, and the other three groups were given 3% dextran sulfate sodium (DSS) solution to drink freely, and the model was established for 7 days. After 7 days of modeling, all groups were given sterile water to drink, and were gavaged for 7 days according to the above grouping. The blank control group (Control) and the model group (DSS) were gavaged with an equal volume of normal saline. During the experiment, the body weight of the mice was recorded every day, and 6 mice were randomly selected every other day to record the stool viscosity (0-3 points), fecal occult blood (0-4 points), and weight loss percentage (0-4 points), and the DAI score was calculated (the sum of these three results was divided by 3 to obtain the DAI value). On the 7th day after modeling, the body weight of each group was recorded, the spleen was separated, weighed and recorded, and the organ index was calculated [organ index (g / kg) = organ fresh weight (g) / live weight (kg)]; the colon length (cm) of each group was recorded; the RT-PCR method was used to detect and use 2 -ΔΔCT The data were analyzed by relative quantitative analysis, and the expression levels of inflammatory factors (TNF-α, IL-1β, IL-6 and iNOS) in the serum of each group were determined, and the β-actin gene was used as the internal reference gene.

[0101] 2. Experimental results

[0102] Results Fig. 9 ,like Fig. 9 As shown in A, B, C, and D in Figure 3, compared with the DSS group, the DAI score of the Rg1-OA NPs group was significantly reduced, and it could significantly alleviate the weight loss of mice caused by DSS, while the weight of the Rg1-OA group was not significantly different from that of the DSS group. Fig. 9 As shown in Figure E, compared with the DSS group, the colon length of the Rg1-OA NPs group was significantly greater than that of the DSS group, while there was no significant effect in the Rg1-OA group. Fig. 9 As shown in Figure F, the spleen index of the Rg1-OA NPs group was significantly lower than that of the DSS group, and the effect was better than that of the Rg1-OA group. Fig. 9As shown in G, H, I, and J, compared with the DSS group, the inflammatory factors TNF-α, IL-1β, IL-6, and iNOS in the Rg1-OA NPs group were significantly reduced, and the effect was better than that in the Rg1-OA group. The above results show that Rg1-OA NPs have a significant improvement effect on the shortened colon and splenomegaly induced by DSS in mice, can significantly reduce the level of inflammatory factors induced by DSS, and have a significant relief and therapeutic effect on the DSS-induced acute colitis model in mice.

[0103] In summary, ginsenoside Rg1-oleanolic acid self-assembled nanoparticles performed better than physical mixing in relieving acute colitis in mice, and effectively improved the dissolution of oleanolic acid. The components of the nanoparticles are all extracted from traditional Chinese medicine, and no antibiotics are added throughout the process, which effectively avoids the problem of drug resistance and prevents a large number of livestock and poultry and even humans from facing the risk of infection due to drug-resistant strains. At the same time, it also builds a solid line of defense for food safety. In view of this, the nanoparticles are expected to become a powerful force to promote the innovation of antibiotic-free and green and environmentally friendly breeding models.

[0104] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

[0105] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing self-assembled nanoparticles of traditional Chinese medicine monomers, characterized in that: The method comprises the following steps: (1) mixing ginsenoside Rg1 and water to obtain a ginsenoside Rg1 solution; (2) mixing oleanolic acid and anhydrous ethanol to obtain an oleanolic acid solution; (3) adding the oleanolic acid solution dropwise into the ginsenoside Rg1 solution to obtain a mixed solution; (4) The mixed solution is stirred and then ultrasonicated to obtain a ginsenoside Rg1-oleanolic acid self-assembled nanoparticle solution, i.e., a Chinese medicine monomer self-assembled nanoparticle solution.

2. The preparation method according to claim 1, characterized in that: In step (3), in the mixed solution, the mass ratio of ginsenoside Rg1 to oleanolic acid is (10±0.3):

1.

3. The preparation method according to claim 2, characterized in that: In step (3), the final concentration of ginsenoside Rg1 in the mixed solution is 1±0.2 mg / mL.

4. The preparation method according to claim 1, characterized in that: In step (2), the concentration of the oleanolic acid solution is 1±0.2 mg / mL.

5. The preparation method according to claim 1, characterized in that: In step (4), the stirring condition is 500±100 rpm at 25±3°C for 60±5 min; In step (4), the ultrasonic condition is 25±3° C. for 10 to 15 minutes.

6. The preparation method according to any one of claims 1 to 5, characterized in that: After step (4), the method further includes the step of drying the ginsenoside Rg1-oleanolic acid self-assembled nanoparticle solution.

7. A Chinese medicine monomer self-assembled nanoparticle, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the Chinese medicine monomer self-assembled nanoparticles according to claim 7 in the preparation of anti-inflammatory drugs.

9. Use of the Chinese medicine monomer self-assembled nanoparticles according to claim 7 in preparing drugs for treating colitis.

10. The use according to claim 9, characterized in that: Application in the preparation of medicines for treating acute colitis.

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