Pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles as well as preparation and application thereof

Abstract

The invention discloses pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles as well as preparation and application thereof, and relates to the technical field of pharmaceutical preparations. Inspired by compatibility and solubilization of traditional Chinese medicines, the pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles (AB4-OA NPs) are prepared by taking natural active components, namely micromolecular pulsatilla saponin B4 and oleanolic acid, as raw materials; the oleanolic acid has the effects of promoting dissolution and resisting inflammation, the water solubility of the oleanolic acid is improved, the bioavailability is improved, and the anti-inflammatory effect of the oleanolic acid is enhanced. Experimental results show that the AB4-OA NPs can effectively improve the dissolution rate of the oleanolic acid, so that the bioavailability of the oleanolic acid is effectively improved, meanwhile, the AB4-OA NPs has a better anti-inflammatory effect, and the effect is better than that of a single drug with the same dosage. In addition, the synthetic preparation method is simple and convenient, has the advantages of nano-scale drugs, realizes auxiliary-material-free pure drug self-delivery, and improves the delivery efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and more specifically to self-assembled nanoparticles of pulsatilla saponin B4-oleanolic acid and preparation and application thereof. Background Art

[0002] With the rise of nanotechnology, self-assembly has become one of the hot topics that has received widespread attention in recent years. Self-assembly is the process by which molecules form nanostructures such as nanotubes, nanofibers, particles and vesicles through non-covalent bonds. More and more studies have shown that nano-shaped products are commonly found in Chinese herbal medicines during the process of harvesting, processing, decoction, preparation and entering the body. The main reason is that the active ingredients of Chinese herbal medicines, such as sugars, proteins, flavonoids, terpenes, alkaloids, organic acids, metal ions, etc., have self-assembly characteristics and can associate with different components based on interaction forces to improve the solubility of poorly soluble drugs, enhance pharmacological activity, and reduce adverse reactions. The discovery of self-assembled nanoparticles of Chinese herbal medicines is of great significance for improving the problems of poor in vivo stability, low bioavailability, and short half-life of Chinese herbal medicines.

[0003] Pulsatilla scutellaria is a traditional Chinese medicine, first recorded in Shennong's Herbal Classic. The medicinal part is the dried root of the Ranunculaceae plant Pulsatilla scutellaria. It is bitter and cold in nature, and enters the stomach and large intestine meridians. It has the effects of clearing away heat and detoxification, cooling blood and stopping dysentery, drying dampness and killing insects. It is mainly used in the treatment of amoebic dysentery, acute and chronic inflammation, peptic ulcers, etc. Pulsatilla scutellaria saponins are pentacyclic triterpenoid components with biological activity in Pulsatilla scutellaria, which have multiple pharmacological effects such as anti-schistosomiasis, anti-inflammatory, antibacterial, vascular activation, and anti-tumor. Pulsatilla scutellaria saponins B4 (Anemoside B4, AB4) is an effective saponin among them, with abundant content, low price, easy to be converted into clinical application drugs, and also has anti-tumor, anti-inflammatory, antiviral, kidney damage protection, analgesia, immunomodulation and other activities. Pulsatilla scutellaria saponins B4 has good water solubility due to its unique amphiphilic structure, and can self-assemble in water to form particles with a hydrophobic core, and its hydrophobic core can load drugs with poor water solubility, thereby achieving the effect of solubilization.

[0004] Oleanolic acid (OA) is a pentacyclic triterpenoid compound and a commonly used hepatoprotective and anti-inflammatory drug in clinical practice. It is rich in plants such as Aralia, Prunella Vulgaris, Green Leaf Gall, Ligustrum lucidum, Clove, Jujube, Plum, Achyranthes bidentata, and Cornus officinalis. It mainly exists in the form of free or combined glycosides. Oleanolic acid has a variety of biological activities: protecting the liver, protecting the kidneys, reducing inflammation, lowering blood lipids and blood sugar, etc. Nowadays, oleanolic acid is mostly used clinically in: prevention of liver damage caused by anti-tuberculosis drugs, treatment of chronic toxic hepatitis, acute viral hepatitis, improving patients' immune function, and as an anti-cancer adjuvant drug. However, oleanolic acid is relatively lipophilic, has poor solubility, and low oral bioavailability, which limits the full play of its pharmacological effects.

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

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a kind of Pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles and the preparation and application thereof.

[0007] The pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles prepared by the invention have the functions of promoting dissolution and anti-inflammatory, improving the water solubility of oleanolic acid, improving the bioavailability and enhancing the anti-inflammatory effect thereof.

[0008] The structural formula of the pulsatilla saponin B4 is:

[0009]

[0010] The structural formula of the oleanolic acid is:

[0011]

[0012] The purpose of the present invention is achieved through the following technical solutions:

[0013] A kind of pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles, wherein the active ingredients thereof are composed of pulsatilla saponin B4 and oleanolic acid;

[0014] Preferably, the mass ratio of pulsatilla saponin B4 to oleanolic acid is 10-15:1; further 10:1.

[0015] A method for preparing pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles, comprising the following steps:

[0016] (1) mixing Pulsatilla saponin B4 and water to obtain a Pulsatilla saponin B4 solution;

[0017] (2) mixing oleanolic acid and anhydrous ethanol to obtain an oleanolic acid solution;

[0018] (3) adding the oleanolic acid solution dropwise to the pulsatilla saponin B4 solution, stirring to obtain a mixed solution;

[0019] (4) The mixed solution is dialyzed and dried to obtain scutellaria saponin B4-oleanolic acid self-assembled nanoparticles.

[0020] Preferably, in step (2), the concentration of the oleanolic acid solution is 0.5 to 10 mg / mL; further 0.5 to 5 mg / mL; further 1±0.5 mg / mL;

[0021] Preferably, in step (3), in the mixed solution, the mass ratio of pulsatilla saponin B4 to oleanolic acid is 10-15:1; further 10:1.

[0022] Preferably, in step (3), in the mixed solution, the final concentration of pulsatilla saponin B4 is 0.5 to 10 mg / mL; further 0.5 to 5 mg / mL; further 1±0.5 mg / mL;

[0023] Preferably, in step (3), the stirring condition is 1000±100 rpm at 25±1°C for 60±5 min; further 1000 rpm at 25°C for 60 min;

[0024] Preferably, in step (4), the molecular weight cut-off used for dialysis is 3000-3500 Da; further 3500 Da.

[0025] Preferably, in step (4), the dialysis condition is dialysis with deionized water for 12±3 h; further, 12 h.

[0026] Preferably, in step (4), the drying is freeze-drying.

[0027] Application of the pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles in the preparation of anti-inflammatory drugs.

[0028] Use of the pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles in preparing drugs for treating and / or preventing enteritis;

[0029] Furthermore, the pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles are used in the preparation of drugs for treating and / or preventing Escherichia coli infection.

[0030] Preferably, the Escherichia coli is pathogenic Escherichia coli; further includes enterotoxigenic Escherichia coli; further includes Escherichia coli (E. coli) ETEC K88.

[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 of traditional Chinese medicine compatibility, and uses the natural active ingredient small molecule scutellaria saponin B4 and oleanolic acid as raw materials to prepare scutellaria saponin B4-oleanolic acid self-assembled nanoparticles. Therefore, the present invention designs a preparation method of nanoparticles based on scutellaria saponin B4 and oleanolic acid as raw materials and its anti-inflammatory application.

[0033] (2) The present invention designs a preparation method of AB4-OA self-assembled nanoparticles and its anti-inflammatory application, wherein AB4-OA self-assembled nanoparticles can effectively 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 are better than the effect of a single drug at the same dose. In addition, the synthetic preparation method is simple and has the advantages of nano-scale drugs, while achieving self-delivery of pure drugs without excipients, thereby improving the delivery efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the assembly model of the pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles of the present invention.

[0035] Figure 2 This is a graph showing the particle size and PDI results of scutellaria saponin B4-oleanolic acid self-assembled nanoparticles at different mass ratios.

[0036] Figure 3 This is the dynamic light scattering result of scutellaria saponin B4-oleanolic acid self-assembled nanoparticles at a mass ratio of 10:1.

[0037] Figure 4 This is the SEM electron microscope image of the self-assembled nanoparticles of Pulsatilla saponin B4-oleanolic acid at a mass ratio of 10:1.

[0038] Figure 5 This is the SEM electron microscope image of oleanolic acid (OA).

[0039] Figure 6 This is the SEM electron microscope image of Pulsatilla saponin B4.

[0040] Figure 7 This is the infrared characterization result of self-assembled nanoparticles of scutellaria baicalensis saponin B4-oleanolic acid at a mass ratio of 10:1.

[0041] Figure 8 This is the dissolution test result of Pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles at a mass ratio of 10:1.

[0042] Fig. 9 The figure is the effect of scutellaria saponin B4-oleanolic acid self-assembled nanoparticles on the secretion of inflammatory factors by LPS-stimulated macrophages; where, AF: the NO content in the cell supernatant and the mRNA expression levels of IL-10, IL-1β, TNF-α, iNOS, and COX-2 in the cells, respectively; Note: groups marked with different letters have significant differences (P<0.05); groups marked with the same letters have no significant differences (P>0.05).

[0043] Fig.10The effect of scutellaria saponin B4-oleanolic acid self-assembled nanoparticles on Escherichia coli disease in mice; A: body weight of mice in each group at each stage; B: mortality rate of each group; C: spleen index of each group; D: liver index of each group; E: diarrhea score of each group; FH: Escherichia coli content in feces, spleen and liver of each group; Note: groups or groups at the same stage with different letters have significant differences (P<0.05); no difference or the same letters indicate no significant difference (P>0.05). DETAILED DESCRIPTION

[0044] 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.

[0045] The assembly model of the pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles of the present invention is shown in Figure 1 .

[0046] In the examples, Pulsatilla saponin B4 (98%) was purchased from Chengdu Kangbang Biotechnology Co., Ltd.; oleanolic acid (98%) was purchased from Beijing Solaibao Technology Co., Ltd.

[0047] The scutellaria saponin B4 used in the examples has a CAS number of 129741-57-7 and oleanolic acid has a CAS number of 508-02-1.

[0048] Example 1 Preparation of Pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles (AB4-OA NPs)

[0049] Mix scutellaria baicalensis saponin B4 with water to obtain scutellaria baicalensis saponin B4 solution; mix oleanolic acid with anhydrous ethanol to obtain oleanolic acid solution with mass concentrations of 1 mg / mL, 2 mg / mL and 5 mg / mL; add the same volume of oleanolic acid solution dropwise into the scutellaria baicalensis saponin B4 solution, stir at 1000 rpm for 60 min at 25°C to obtain a mixed solution, wherein the final concentration of scutellaria baicalensis saponin B4 in the mixed solution is 1 mg / mL, and the mass ratio of scutellaria baicalensis saponin B4 to oleanolic acid is 10:1, 5:1 or 2:1; load the mixed solution into a dialysis bag with a molecular weight cutoff of 3500Da, dialyze with deionized water for 12 hours, and freeze-dry to obtain scutellaria baicalensis saponin B4-oleanolic acid self-assembled nanoparticles, recorded as AB4-OA NPs.

[0050] Example 2 Dynamic light scattering experiment of AB4-OA NPs

[0051] The AB4-OANPs powders of different mass ratios prepared in Example 1 were uniformly dispersed in water to prepare a 1 mg / mL mother solution, and then placed in a dynamic light scattering instrument sample cell to test the particle size, PDI, and Zeta potential. The results are as follows: Figure 2 As shown in the figure, the particle size of AB4-OANPs is 200.53 nm, and the PDI is 0.31. The particle sizes of the mass ratios of 2:1 and 5:1 are larger than those of the mass ratio of 10:1, so the mass ratio of 10:1 is selected for subsequent experiments. Figure 3 As shown, the average particle size of AB4-OA NPs with a mass ratio of 10:1 is 200.53±1.84nm, the PDI is 0.31±0.03, and the potential is -22.16±0.33mV. The above results show that the particles formed by the freeze-dried and re-dissolved AB4-OANPs with a mass ratio of 10:1 are nanometer-sized, relatively stable, not easy to form precipitation, and relatively uniform in size, maintaining good stability.

[0052] Example 3 Scanning electron microscopy observation of AB4-OANPs

[0053] In this experiment, the AB4-OANPs prepared in Example 1 with a mass ratio of 10:1 were observed by scanning electron microscopy. AB4-OA NPs, AB4 and OA powders were uniformly dispersed on the conductive glue, and then sprayed with gold after drying. The morphology and particle size were observed by SEM. Figures 4 to 6 As shown, it is shown that AB4-OANPs with a mass ratio of 10:1 formed spherical particles, whose morphology is completely different from that of AB4 and OA monomers.

[0054] Example 4 Infrared characterization experiment of AB4-OA NPs

[0055] In this experiment, AB4, OA and AB4-OA NPs prepared in a mass ratio of 10:1 were characterized by infrared spectroscopy. 2 mg of AB4, OA and AB4-OA NPs, as well as 2 mg of AB4-OA (AB4 and OA were mixed in a mass ratio of 10:1) were weighed, 200 mg of infrared dried potassium bromide powder was added, mixed and pressed into potassium bromide tablets, and detected in an infrared spectrometer with a resolution of 4 cm -1 , signal-to-noise ratio 50000:1, scan 64 times, record the spectrum. Figure 7 As shown, the C=O peak of OA is located at 1691 cm -1 The -OH peak is located at 3458.97 cm -1No new characteristic peaks appeared in AB4-OANPs, indicating that the formed nanoparticles did not destroy the skeleton structure of the monomer, and no chemical bond was formed between OA and AB4. However, the stretching vibration peak intensity of the carbonyl group of OA was significantly weakened, and only the characteristic peak of AB4 was retained, indicating that AB4 successfully encapsulated OA.

[0056] Example 5 Dissolution Determination of AB4-OA NPs

[0057] The aqueous solution was used as the dissolution medium, the speed was 100 rpm, and the temperature was 37°C. 1 mg of oleanolic acid and lyophilized powder of scutellariae saponin B4-oleanolic acid self-assembled nanoparticles (containing 1 mg of oleanolic acid) were accurately weighed and placed on the medium. 500 μL of samples were taken at 5, 10, 20, 40, 60, 90, and 120 min, respectively, filtered through a 0.45 μm filter membrane, and an equal amount of medium was added. 20 μL of the filtrate was taken and injected into the liquid chromatograph to determine the content of oleanolic acid and draw the dissolution curve. The results are as follows: Figure 8 As shown, the concentration of oleanolic acid monomer that can be dissolved in water is very low, while the cumulative dissolution of the nanoparticles within 2 hours reaches 76%.

[0058] Example 6 Anti-inflammatory activity experiment

[0059] Grouping: normal control group (CON), model group (LPS), AB4 group, OA group, AB4-OA group, AB4-OA NPs group, each group with 3 replicate wells, Note: AB4-OA NPs refers to AB4-OA NPs with a mass ratio of 10:1 prepared in Example 1, and AB4-OA is obtained by mixing AB4 and OA at a mass ratio of 10:1.

[0060] Macrophages RAW264.7 (mouse peritoneal macrophages) were inoculated in 12-well plates, with 1*10 5 cells; the normal control group (CON) was added with an equal volume of basal medium; the remaining groups were added with medium containing drugs (final concentration of 500 μg / mL) or an equal volume of basal medium, incubated for 2 h, and then LPS (lipopolysaccharide) with a final concentration of 1 μg / mL was added and cultured for 24 h. The cell pellets were collected, total RNA was extracted, reverse transcribed to synthesize cDNA, and RT-PCR was performed with the designed and synthesized primers. -ΔΔCT The data were analyzed by relative quantitative analysis, and the NO content in the cell supernatant and the mRNA expression levels of IL-1β, TNF-α, IL-10, iNOS, and COX-2 in the cells were determined, with the GADPH gene as the internal reference gene; the results are shown in Fig. 9: After LPS treatment, macrophages can induce significantly increased expression levels of pro-inflammatory factors such as NO, IL-1β, TNF-α, iNOS, and COX-2, and significantly reduced the expression level of the anti-inflammatory factor IL-10. AB4-OANPs treatment can significantly improve the expression levels of these factors. At the same time, compared with the AB4 and OA single-drug groups and the AB4-OA physical mixture group, the AB4-OA NPs treatment group had better effect, indicating that the synthetic AB4-OANPs can enhance the anti-inflammatory effects of the two monomers.

[0061] Example 7 Effect on Escherichia coli Disease in Mice

[0062] Experimental subjects: 5-week-old C57 female mice;

[0063] Experimental groups: After one week of adaptation, 84 C57 mice were randomly divided into 6 groups, namely blank control group (Control), model group (ETEC), nanoparticle group (AB4-OANPS 50 mg / kg), physical mixing group (AB4-OA 50 mg / kg), anemonin B4 group (AB4 50 mg / kg), and oleanolic acid group (OA 50 mg / kg); Note: AB4-OA NPs refers to AB4-OANPs with a mass ratio of 10:1 prepared in Example 1; AB4-OA refers to AB4 and OA mixed in a mass ratio of 10:1.

[0064] The mice were gavaged for 7 days according to the above groups, once a day, 200 μL each time, and the drug concentration was 5 mg / mL; the blank group and the model group were gavaged with the same volume of normal saline. After gavage on the 7th day, the blank group was intraperitoneally injected with 0.4 mL of normal saline, and the other groups were intraperitoneally injected with 0.4 mL of E. coli ETEC K88 (1×10 8 CFU / mL). During the experiment, the general behavior of the mice was observed and recorded every day, and diarrhea and death were recorded. On the third day after modeling, the diarrhea rate and body weight of each group were calculated, the liver and spleen were separated, weighed and recorded, and the organ index was calculated [organ index (g / kg) = organ fresh weight (g) / live weight (kg)]. The results are shown in Fig.10 After intraperitoneal injection of E. coli ETEC K88, mice showed symptoms such as mental depression, loss of appetite, and yellow loose stools. Fig.10As shown in A, B, and E in the figure, compared with the ETEC group, the diarrhea score of the AB4-OANPS group (diarrhea scoring criteria: dry and hard: 0; relatively loose: 1; loose: 2; loose stools: 3; watery stools: 4) was significantly reduced, and it was able to significantly alleviate the weight loss of mice caused by E. coli, while the mixed group and the single drug group had no significant effect; at the same time, the survival rate of mice in the ETEC group was reduced to 64%, while the survival rate of mice in the AB4-OA NPS group recovered to 93%, which was better than the mixed group and the single drug group. Fig.10 As shown in Figures C and D, compared with the ETEC group, the spleen index and liver index of the AB4-OANPS group were significantly lower than those of the ETEC group, and the effect was better than that of the mixed group and the single drug group. Fig.10 As can be seen from F, G, and H, compared with the ETEC group, the E. coli content in the liver, spleen, and feces of the AB4-OANPS group was reduced, and the effect was also better than that of the mixed group and the single drug group. This shows that AB4-OANPS has a certain preventive effect on E. coli disease in mice.

[0065] In summary, the comprehensive performance of Pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles in anti-inflammatory and mouse Escherichia coli disease is better than that of single drugs and physical mixtures, and has better dissolution than single drug groups, effectively improving the release of oleanolic acid. The ingredients of the nanoparticles are all from natural plants, without adding antibiotics, and are not easy to develop drug resistance. They will not expose a large number of livestock and poultry or even humans to the risk of infection caused by drug-resistant strains, and will not bring hidden dangers to food safety. Therefore, the nanoparticles are expected to provide new ideas for antibiotic-free, green and healthy breeding models.

[0066] 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.

[0067] 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 self-assembled nanoparticle of Pulsatilla saponin B4-oleanolic acid, characterized in that: Its active ingredients are composed of pulsatilla saponin B4 and oleanolic acid.

2. The pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles according to claim 1, characterized in that: The mass ratio of the pulsatilla saponin B4 to oleanolic acid is 10-15:

1.

3. The method for preparing the self-assembled nanoparticles of Pulsatilla saponin B4-oleanolic acid according to claim 1 or 2, characterized in that: The steps include: (1) mixing Pulsatilla saponin B4 and water to obtain a Pulsatilla saponin B4 solution; (2) mixing oleanolic acid and anhydrous ethanol to obtain an oleanolic acid solution; (3) adding the oleanolic acid solution dropwise into the pulsatilla saponin B4 solution, stirring to obtain a mixed solution; (4) The mixed solution is dialyzed and dried to obtain scutellaria saponin B4-oleanolic acid self-assembled nanoparticles.

4. The preparation method according to claim 3, characterized in that: In step (2), the concentration of the oleanolic acid solution is 0.5 to 10 mg / mL; further 0.5 to 5 mg / mL; further 1±0.5 mg / mL.

5. The preparation method according to claim 3, characterized in that: In step (3), in the mixed solution, the final concentration of pulsatilla saponin B4 is 0.5-10 mg / mL; further 0.5-5 mg / mL; further 1±0.5 mg / mL.

6. The preparation method according to claim 3, characterized in that: In step (3), the stirring condition is 1000±100 rpm at 25±1°C for 60±5 min; In step (4), the molecular weight cut-off used for dialysis is 3000-3500Da; In step (4), the dialysis condition is to use deionized water for 12±3h; In step (4), the drying is freeze-drying.

7. Use of the pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles according to claim 1 or 2 in the preparation of anti-inflammatory drugs.

8. Use of the Pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles according to claim 1 or 2 in the preparation of drugs for treating and / or preventing enteritis.

9. Use of the Pulsatilla saponin B4-oleanolic acid self-assembled nanoparticles according to claim 1 or 2 in the preparation of a medicament for treating and / or preventing Escherichia coli infection.

10. The use according to claim 9, characterized in that: The Escherichia coli is pathogenic Escherichia coli; further includes enterotoxigenic Escherichia coli; and further includes Escherichia coli (E. coli) ETEC K88.

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