A nano preparation for treating colitis and its preparation method and application

Abstract

The present invention belongs to the field of pharmaceutical preparations, and relates to a nano preparation for treating colitis, and a preparation method and application thereof. The components of the nano preparation include: piperine, glycyrrhizic acid, a stabilizer, a colon targeting material, and a pore-forming agent. The particle size of the nano preparation is 100-300 nm, the encapsulation rate of piperine and glycyrrhizic acid is 80.5-99.5%, and the drug loading is 20.5-60.5%. The nano preparation can achieve colon-specific release, increase the synchronous release of piperine and glycyrrhizic acid in the colon, synergistically enhance the anti-colitis effect, and significantly improve the anti-inflammatory effect compared with a single drug and a common nano preparation. In addition, the nano preparation can be used as an intermediate to prepare oral granules, capsules, tablets, etc., for clinical effective treatment of colitis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and more particularly relates to a nano preparation for treating colitis, a preparation method thereof and an application thereof. Background Art

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease whose incidence is rapidly increasing with improvements in modern living standards. Clinical symptoms include rectal bleeding, diarrhea, abdominal pain, and weight loss, which severely impact patients' quality of life and increase their risk of cancer. Current clinical treatments for colitis primarily include biologic therapies such as 5-aminosalicylic acid (5-ASA), glucocorticoids, immunosuppressants, and infliximab, but these are associated with high costs, strong drug resistance, and severe side effects.

[0003] Piperine (PIP) is the primary active compound in pepper and belongs to the cinnamic alkaloid class. Piperine exhibits anti-inflammatory, immunomodulatory, antioxidant, and antitumor effects, but it suffers from low solubility, relatively slow drug release, and poor colon targeting. Glycyrrhizic acid (GA), the primary active ingredient in the root extract of the legume licorice plant, is composed of two hydrophilic glucuronic acid molecules and one hydrophobic glycyrrhetinic acid molecule. It exhibits multiple biological activities, including anti-inflammatory, hepatoprotective, immunomodulatory, antiviral, and anticancer activities. However, glycyrrhizic acid also suffers from the drawbacks of being easily degraded in the gastrointestinal tract and having poor colon targeting. Therefore, the efficacy of either drug alone in the treatment of colitis is limited.

[0004] Combination therapy offers new hope for the treatment of colitis, but only by increasing and synchronizing the release of multiple drugs in the colon can the synergistic therapeutic effect be achieved. Nanoparticle formulations offer a promising technological solution to this problem.

[0005] Nanoformulation technology is a new nanotechnology that can improve the bioavailability of poorly soluble drugs. Due to its advantages such as large specific surface area, improved solubility and easy modification, it has been widely used in the development and research of poorly soluble drug formulations. However, simple nanoformulations have small particle size and large surface area, which can easily lead to premature release and degradation of drugs, reducing the efficacy of the drugs and causing adverse side effects. Therefore, how to construct dual-drug nanoformulations and prevent their leakage and degradation in the gastrointestinal tract, target the colon and release two drugs synchronously to synergistically enhance the anti-colitis effect. This is a problem that those skilled in the art urgently need to solve. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of the prior art, the present invention provides a colon-targeted nanoformulation loaded with piperine and glycyrrhizic acid. Based on the passive colon-targeting mechanism of the nanoformulation, a suitable stabilizer is selected, and the types and dosages of the colon-targeting material and pore-forming agent are optimized. The nanoformulation is constructed using colon-targeting materials. The stabilizer and glycyrrhizic acid are used to construct a hydrophobic core structure encapsulating piperine, while the colon-targeting material is embedded in the outer shell, with the pore-forming agent bound to the surface. This nanoformulation avoids degradation and absorption in the gastrointestinal tract, increases the simultaneous release of piperine and glycyrrhizic acid in the colon, and achieves a synergistically enhanced anti-colitis efficacy.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A nanoformulation for treating colitis, characterized in that the components of the nanoformulation include: piperine, glycyrrhizic acid, a stabilizer, a colon targeting material, and a pore-forming agent; the ratio of the piperine, glycyrrhizic acid, and stabilizer is 2:0.5-4:0.5-4; the colon targeting material accounts for 10-15% of the mass of the nanoformulation; and the pore-forming agent accounts for 1-12% of the colon targeting material.

[0009] Preferably, the particle size of the nanoformulation is 100 to 300 nm, and the zeta potential is -40 mV to -20 mV; the encapsulation efficiency of the piperine and glycyrrhizic acid is 80.5 to 99.5%, and the drug loading is 20.5 to 60.5%.

[0010] The beneficial effects of the above technical solution are: the nano-preparation can co-encapsulate piperine and glycyrrhizic acid to achieve synchronous release in the colon, and has the characteristics of high drug loading capacity, uniform particle size and stability.

[0011] Preferably, the nanoformulation can achieve colon-specific release; in the SGF (0-2h), SIF (3-5h), and SCF (6-24h) transfer release media, the in vitro release of piperine and glycyrrhizic acid achieves synchronous release, and the cumulative release of piperine and glycyrrhizic acid within 2h is both <20%; the cumulative release of piperine and glycyrrhizic acid within 5h is both <35%; and the cumulative release of piperine and glycyrrhizic acid within 24h is both 60-100%.

[0012] Preferably, the nanoformulation is a dual-drug nanoformulation loaded with piperine and glycyrrhizic acid, which has a colon-targeted synergistically enhanced anti-colitis effect.

[0013] Preferably, the stabilizer is selected from one or two of TPGS, povidone K30, soy lecithin, and bovine serum albumin; the colon targeting material is selected from one of Eudragit L100, Eudragit L100-55, Eudragit S100, and HPMCAS-MF; and the porogen is selected from one of HPC, Tween 80, and Span 80.

[0014] The beneficial effect of the above technical solution is that the stabilizer can form a stable nano preparation with piperine and glycyrrhizic acid.

[0015] Preferably, the stabilizer is TPGS; the colon targeting material is Eudragit S100; and the porogen is Tween 80.

[0016] The beneficial effects of the above technical solution are as follows: TPGS is a polymer containing vitamin E lipophilic groups and polyethylene glycol hydrophilic long chains. During the preparation process, TPGS and glycyrrhizic acid are combined through intermolecular hydrophobic interactions and hydrogen bonds, providing a hydrophobic cavity structure for piperine, laying the foundation for the formation of a stable nanoformulation and a high drug loading capacity.

[0017] Eudragit S100 is a pH-dependent amphiphilic enteric polymer. Its ester functional groups interact hydrophobically with hydrophobic molecules, embedding it on the surface of the nanoformulation to prevent degradation and absorption in the stomach and small intestine. Tween 80, which has a relatively high number of polyoxyethylene groups, binds to the hydrophilic sites of Eudragit S100, enhancing drug release in the colon.

[0018] Preferably, the nanoformulation is a colon-targeted nanoformulation co-loaded with piperine and glycyrrhizic acid, and the nanoformulation achieves colon-targeted delivery of piperine and glycyrrhizic acid to synergistically regulate the polarization of macrophages to exert immune anti-inflammatory effects and enhance the anti-colitis effect.

[0019] The present invention also provides a method for preparing the above-mentioned nanoformulation for treating colitis, wherein the preparation method adopts a combination of one or more of a media grinding method, a high-pressure homogenization method, and an emulsification-solvent evaporation method.

[0020] The beneficial effect of the above technical solution is that nanoformulations that meet the requirements can be prepared by adopting a variety of different methods, and a variety of methods can be used in combination to achieve high efficiency in the preparation of nanoformulations.

[0021] Preferably, the specific steps of the emulsification-solvent evaporation method are as follows:

[0022] (1) Add glycyrrhizic acid, stabilizer and porogen into water and stir evenly with magnetic stirring to form an aqueous phase;

[0023] (2) dissolving piperine and colon targeting material in methanol and fully dissolving them by ultrasonication to form an organic phase;

[0024] (3) Under ultrasound, the organic phase is added dropwise to the aqueous phase for ultrasound;

[0025] (4) Magnetic stirring is performed at room temperature to evaporate methanol and obtain a nanoformulation.

[0026] Preferably, the ultrasound in step (3) is ice bath ultrasound, the ultrasound power is 65%, and the ultrasound time is 10 minutes; the ultrasound conditions are working for 3 seconds and intervals of 2 seconds.

[0027] The beneficial effect of the above technical solution is that piperine and the colon targeting material are uniformly dispersed in the aqueous solution, and the obtained nano preparation has the characteristics of uniformity and stability.

[0028] The present invention also provides the use of the nano preparation in preparing medicine for treating colitis.

[0029] The present invention also provides an evaluation of the anti-colitis effect of the above-mentioned nanoformulation, wherein the anti-colitis effect includes mouse body weight, disease index, colon length, spleen weight, colon tissue characteristics or repair of intestinal mucosal damage.

[0030] The beneficial effects of the above technical solution are: the nanoformulation effectively treats colitis, the nanoformulation achieves a synergistic enhancement of the anti-colitis effect of piperine and glycyrrhizic acid, the anti-inflammatory effect of the combined drug is significantly better than that of a single drug, and the colon-targeted nanoformulation is better than ordinary nanoformulation.

[0031] The present invention also provides a mechanism of action of the above-mentioned nanoformulation against colitis, which includes the ratio of M1 macrophages to M2 macrophages, and the expression of mammalian rapamycin protein (mTOR) and hypoxia-inducible factor-1α (HIF-1α) protein.

[0032] The beneficial effects of the above technical solution are: the above nanoformulation can exert anti-inflammatory effects through immune regulation, among which piperine can act on mTOR to increase the polarization of M2 macrophages; glycyrrhizic acid can act on HIF-1α to reduce the polarization of M1 macrophages. The two drugs synergistically regulate macrophages for the treatment of colitis.

[0033] It can be seen from the above technical solutions that, compared with the prior art, the present invention provides a nanoformulation for treating colitis, and its preparation method and application, which have the following beneficial effects:

[0034] (1) The nanoformulation of the present invention is a colon-targeted nanoformulation that co-loads two drugs, which can improve the water solubility of piperine and prevent piperine and glycyrrhizic acid from being degraded by the gastrointestinal tract after oral administration, thereby increasing the drug content and retention time in the colon, thereby better exerting the anti-colitis effect.

[0035] (2) The glycyrrhizic acid in the nanoformulation of the present invention not only enhances the anti-inflammatory effect but also increases the stability of the nanoformulation. On the one hand, glycyrrhizic acid and the stabilizer are bound by intermolecular hydrophobic interactions and hydrogen bonds, and the nanoformulation formed by the colon-targeting material and the pore-forming agent is embedded on the surface. On the other hand, glycyrrhizic acid and piperine synergistically exert anti-colitis effects. Both drugs have high encapsulation efficiency and drug loading, that is, the encapsulation efficiency of piperine and glycyrrhizic acid is 80.5-99.5%, and the drug loading is 20.5-60.5%.

[0036] (3) The nanoformulation of the present invention can achieve the simultaneous release of piperine and glycyrrhizic acid in the colon. Under the condition of colonic fluid (SCF) pH>7, the cumulative release of piperine and glycyrrhizic acid from the nanoformulation is 2-5 times higher than that of the coarse suspension group. In addition, after oral administration of different drugs to mice, the content of piperine and glycyrrhizic acid in the nanoformulation in the colon is 2-6 times higher than that of the coarse suspension group.

[0037] (4) The nanoformulation of the present invention can regulate the body's immunity, effectively alleviate the symptoms of colitis in mice and repair the barrier damage of the intestinal tract. Compared with single drugs and ordinary nanoformulations, it improves the anti-colitis effect.

[0038] (5) The nanoformulation of the present invention can simultaneously regulate the M1 and M2 phenotypes of macrophages to exert an immunomodulatory effect. Piperine can act on mammalian rapamycin protein (mTOR) to increase the polarization of M2 macrophages; glycyrrhizic acid can act on hypoxia-inducible factor (HIF-1α) to reduce the polarization of M1 macrophages. The two drugs synergistically regulate the balance of macrophages and enhance the anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0040] Figure 1 This is a schematic structural diagram of a nanoformulation for treating colitis according to the present invention.

[0041] Figure 2 Characterization of the different substances in the present invention. Figures a and b show transmission electron microscopy (TEM) patterns, c show powder X-ray diffraction patterns (XRD), d show differential scanning calorimetry (DSC), and e show Fourier transform infrared (FT-IR) spectra (PIP represents the piperine API, PM represents a physical mixture (PIP, GA, TPGS, and Eudragit S100), NCs represent the nanoformulation prepared in Comparative Example 1, and ENCs represent the nanoformulation prepared in Example 5).

[0042] Figure 3 The in vitro release curve of a nanoformulation for treating colitis of the present invention is shown in Figure 1, where a represents the in vitro release in piperine and b represents the in vitro release in glycyrrhizic acid (SGF, SIF, and SCF simulate gastric juice, small intestinal fluid, and colonic fluid, respectively).

[0043] Figure 4 This is an investigation of the colon targeting of a nanoparticle formulation for treating colitis according to the present invention, wherein a represents the distribution in the body and b represents the colon targeting.

[0044] Figure 5 This is an in vivo evaluation of the anti-inflammatory effect of a nanoformulation for treating colitis according to the present invention. Wherein, a represents the mouse modeling and dosing period, b represents the mouse weight, c represents the disease index, d represents colon and spleen radiographs, e represents the colon length, f represents the spleen weight, g represents colon hematoxylin and eosin staining, h represents the expression of inflammatory factors in colon tissue, and i represents the immunohistochemistry of zona occludin (ZO-1) and occludin in colon tissue.

[0045] Figure 6 This is a study of the in vivo anti-inflammatory mechanism of a nanoformulation for treating colitis of the present invention. A and B are flow cytometric measurements of CD206 and CD11c expression in spleen cells, and C is immunohistochemical analysis of Arg1, iNOS, mTOR, and HIF-1α in colon tissue (CD206 is a surface receptor for M2 macrophages, CD11c is a surface receptor for M1 macrophages, iNOS is nitric oxide synthase, Arg1 is arginine 1, mTOR is mammalian protein of rapamycin, and HIF-1α is hypoxia-inducible factor-1α).

[0046] Figure 7 The present invention is used to evaluate the biosafety of a nano-preparation for treating colitis, and hematoxylin and eosin staining of the mouse heart, liver, spleen, lung and kidney is performed. DETAILED DESCRIPTION

[0047] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0048] Example 1

[0049] A nanoformulation for treating colitis, comprising components of piperine, glycyrrhizic acid, a stabilizer colon targeting material, and a pore-forming agent; the mass ratio of piperine, glycyrrhizic acid, and stabilizer is 2:1:1, the colon targeting material accounts for 12.5% ​​of the mass of the nanoformulation, and the pore-forming agent accounts for 10% of the mass of the colon targeting material.

[0050] The stabilizer is TPGS, the colon targeting material is Eudragit S100, and the pore-forming agent is Tween 80.

[0051] A method for preparing a nanoformulation for treating colitis, wherein the method adopts an emulsification-solvent evaporation method and specifically comprises the following steps:

[0052] (1) Weigh 12.5 mg of glycyrrhizic acid, 12.5 mg of TPGS, and 0.625 mg of Tween 80 and add them to 10 ml of water. Stir magnetically to form an aqueous phase.

[0053] (2) Weigh 25 mg of piperine and 6.25 mg of Eudragit S100 and dissolve them in 1 ml of methanol. Ultrasonicate to fully dissolve them to form an organic phase.

[0054] (3) Under ultrasound, the organic phase is added dropwise to the aqueous phase for ultrasound;

[0055] (4) By volatilizing methanol under magnetic stirring at room temperature, a nanoformulation for treating colitis can be obtained.

[0056] The ultrasonic treatment in step 3) is ice bath ultrasonic treatment, with an ultrasonic power of 65% and a duration of 10 min; the ultrasonic treatment conditions are 3 s on and 2 s off.

[0057] Example 2

[0058] A nanoformulation for treating colitis, comprising components of piperine, glycyrrhizic acid, a stabilizer, a colon targeting material, and a pore-forming agent; the mass ratio of the piperine, glycyrrhizic acid, and stabilizer is 2:1:1; the colon targeting material accounts for 12.5% ​​of the mass of the nanoformulation; and the pore-forming agent accounts for 10% of the mass of the colon targeting material.

[0059] The stabilizer is TPGS, the colon targeting material is Eudragit S100, and the porogen is Span 80.

[0060] A method for preparing a nanoformulation for treating colitis, wherein the method adopts an emulsification-solvent evaporation method and specifically comprises the following steps:

[0061] (1) Weigh 12.5 mg of glycyrrhizic acid, 12.5 mg of TPGS, and 0.625 mg of Span 80 and add them to 10 ml of water. Stir magnetically to form an aqueous phase.

[0062] (2) Weigh 25 mg of piperine and 6.25 mg of Eudragit S100 and dissolve them in 1 ml of methanol. Ultrasonicate to fully dissolve them to form an organic phase.

[0063] (3) Under ultrasound, the organic phase is added dropwise to the aqueous phase for ultrasound;

[0064] (4) By volatilizing methanol under magnetic stirring at room temperature, a nanoformulation for treating colitis can be obtained.

[0065] The ultrasonic treatment in step 3) is ice bath ultrasonic treatment, with an ultrasonic power of 65% and a duration of 10 min; the ultrasonic treatment conditions are 3 s on and 2 s off.

[0066] Example 3

[0067] A nanoformulation for treating colitis, comprising components of piperine, glycyrrhizic acid, a stabilizer, a colon targeting material, and a pore-forming agent; the mass ratio of the piperine, glycyrrhizic acid, and stabilizer is 2:1:1; the colon targeting material accounts for 12.5% ​​of the mass of the nanoformulation; and the pore-forming agent accounts for 10% of the mass of the colon targeting material.

[0068] The stabilizer is TPGS, the colon targeting material is Eudragit S100, and the porogen is HPC.

[0069] A method for preparing a nanoformulation for treating colitis, wherein the method adopts an emulsification-solvent evaporation method and specifically comprises the following steps:

[0070] (1) Weigh 12.5 mg of glycyrrhizic acid, 12.5 mg of TPGS, and 0.625 mg of HPC and add them to 10 ml of water. Stir magnetically to form an aqueous phase.

[0071] (2) Weigh 25 mg of piperine and 6.25 mg of Eudragit S100 and dissolve them in 1 ml of methanol. Ultrasonicate to fully dissolve them to form an organic phase.

[0072] (3) Under ultrasound, the organic phase is added dropwise to the aqueous phase for ultrasound;

[0073] (4) By volatilizing methanol under magnetic stirring at room temperature, a nanoformulation for treating colitis can be obtained.

[0074] The ultrasonic treatment in step 3) is ice bath ultrasonic treatment, with an ultrasonic power of 65% and a duration of 10 min; the ultrasonic treatment conditions are 3 s on and 2 s off.

[0075] Example 4

[0076] A nanoformulation for treating colitis, comprising components of piperine, glycyrrhizic acid, a stabilizer, a colon targeting material, and a pore-forming agent; the mass ratio of the piperine, glycyrrhizic acid, and stabilizer is 2:1:1; the colon targeting material accounts for 12.5% ​​of the mass of the nanoformulation; and the pore-forming agent accounts for 8% of the mass of the colon targeting material.

[0077] The stabilizer is TPGS, the colon targeting material is Eudragit S100, and the pore-forming agent is Tween 80.

[0078] A method for preparing a nanoformulation for treating colitis, wherein the method adopts an emulsification-solvent evaporation method and specifically comprises the following steps:

[0079] (1) Weigh 12.5 mg of glycyrrhizic acid, 12.5 mg of TPGS, and 0.5 mg of Tween 80 and add them to 10 ml of water. Stir magnetically to form an aqueous phase.

[0080] (2) Weigh 25 mg of piperine and 6.25 mg of Eudragit S100 and dissolve them in 1 ml of methanol. Ultrasonicate to fully dissolve them to form an organic phase.

[0081] (3) Under ultrasound, the organic phase is added dropwise to the aqueous phase for ultrasound;

[0082] (4) By volatilizing methanol under magnetic stirring at room temperature, a nanoformulation for treating colitis can be obtained.

[0083] The ultrasonic treatment in step 3) is ice bath ultrasonic treatment, with an ultrasonic power of 65% and a duration of 10 min; the ultrasonic treatment conditions are 3 s on and 2 s off.

[0084] Example 5

[0085] A nanoformulation for treating colitis, comprising components of piperine, glycyrrhizic acid, a stabilizer, a colon targeting material, and a pore-forming agent; the mass ratio of the piperine, glycyrrhizic acid, and stabilizer is 2:1:1; the colon targeting material accounts for 12.5% ​​of the mass of the nanoformulation; and the pore-forming agent accounts for 4% of the mass of the colon targeting material.

[0086] The stabilizer is TPGS, the colon targeting material is Eudragit S100, and the pore-forming agent is Tween 80.

[0087] A method for preparing a nanoformulation for treating colitis, wherein the method adopts an emulsification-solvent evaporation method and specifically comprises the following steps:

[0088] (1) Weigh 12.5 mg of glycyrrhizic acid, 12.5 mg of TPGS, and 0.25 mg of Tween 80 and add them to 10 ml of water. Stir magnetically to form an aqueous phase.

[0089] (2) Weigh 25 mg of piperine and 6.25 mg of Eudragit S100 and dissolve them in 1 ml of methanol. Ultrasonicate to fully dissolve them to form an organic phase.

[0090] (3) Under ultrasound, the organic phase is added dropwise to the aqueous phase for ultrasound;

[0091] (4) By volatilizing methanol under magnetic stirring at room temperature, a nanoformulation for treating colitis can be obtained.

[0092] The ultrasonic treatment in step 3) is ice bath ultrasonic treatment, with an ultrasonic power of 65% and a duration of 10 min; the ultrasonic treatment conditions are 3 s on and 2 s off.

[0093] Comparative Example 1

[0094] The difference from Example 5 is that no porogen is added in step (1) and no colon targeting material is added in step (2).

[0095] Effect verification

[0096] 1. Schematic diagram of a nanoparticle preparation for treating colitis

[0097] Figure 1 In the example, PIP stands for piperine, GA stands for glycyrrhizic acid, TPGS stands for vitamin E polyethylene glycol succinate, and Eudragit S100 is a 1:2 copolymer of methacrylic acid and methyl methacrylate. The nanoformulation prepared in Example 5 is composed of TPGS and glycyrrhizic acid bound by intermolecular hydrophobic interactions and hydrogen bonds, providing a hydrophobic cavity for piperine. Eudragit S100 is a pH-dependent amphiphilic enteric polymer whose ester functional groups interact hydrophobically with hydrophobic molecules and are embedded on the surface of the nanoformulation, preventing degradation and absorption in the stomach and small intestine. Tween 80 has a relatively large number of polyoxyethylene groups, a hydrophilic group. A small amount of Tween 80 binds to the hydrophilic sites of Eudragit S100, enhancing drug release in the colon.

[0098] 2. Morphological observation of a nanoformulation for the treatment of colitis

[0099] A crude suspension of piperine and the nanoformulations prepared in Example 5 and Comparative Example 1 were dripped onto a copper grid and negatively stained with phosphotungstic acid. The morphological characteristics of the nanocrystals were observed and photographed using a transmission electron microscope. After freeze-dried powders of the nanoformulations prepared in Example 5 and Comparative Example 1 were placed, appropriate amounts of the piperine API and freeze-dried powders of the nanoformulations prepared in Example 5 and Comparative Example 1 were placed on a sample table and sprayed with gold. Observation and photography were performed using a Hitachi S-3400 scanning electron microscope. The results are shown in Figure 2. Figure 2 As shown in a and b.

[0100] from Figure 2As can be seen from a and b, the piperine particles are larger and more agglomerated, while the corresponding nanoformulations are evenly dispersed and have uniform columnar particles.

[0101] 2. Characterization of the physicochemical properties of a nanoformulation for the treatment of colitis

[0102] The piperine API, the physical mixture (the mixture of piperine, glycyrrhizic acid, TPGS, and Eudragit S100 as described in Example 5), and the freeze-dried powder of the nanoformulation prepared in Example 5 and Comparative Example 1 were placed on a sample table, with a sample size of 5-10 mg. The glass slide was gently compacted and powder X-ray diffraction analysis (XRD) was performed within a diffraction angle (2θ) range of 0-100° with a scanning step size of 0.02. The piperine API, the physical mixture (the mixture of piperine, glycyrrhizic acid, TPGS, and Eudragit S100 as described in Example 5), and the freeze-dried powder of the nanoformulation prepared in Example 5 and Comparative Example 1 were placed in an aluminum crucible in a sample size of 2-5 mg. The heating rate was 10°C / min, and the temperature range was 25°C to 300°C. The test was performed under dynamic nitrogen conditions at a flow rate of 50 mL / min and differential scanning calorimetry (DSC) was used for analysis. The piperine API, the physical mixture (a mixture of piperine, glycyrrhizic acid, TPGS, and Eudragit S100 as in Example 5), and the freeze-dried powder of the nanoformulation prepared in Example 5 and Comparative Example 1 were placed on a sample table and a mortar, respectively. Dry potassium bromide was added, the mixture was fully ground, tableted, and sampled. The sample was analyzed by Fourier transform infrared spectroscopy (FTIR). The results are shown in FIG. Figure 2 As shown in c~d.

[0103] from Figure 2 As can be seen from Figures c to d, the crystalline form of piperine in the nanoformulation prepared in Example 5 disappears, indicating that piperine is dispersed in the nanoformulation and has an amorphous structure. The stabilizer TPGS and glycyrrhizic acid combine through intermolecular hydrophobic interactions and hydrogen bonds, providing a hydrophobic cavity structure for piperine, thereby achieving a stable nanoformulation.

[0104] 3. In vitro release assay of a nanoformulation for the treatment of colitis

[0105] The release rate of the piperine crude suspension, glycyrrhizic acid crude suspension, and the nanoformulations prepared in Example 5 and Comparative Example 1 was determined as follows: the in vitro drug release characteristics were detected using the dialysis bag method. The experimental operation is as follows: 0.5 mL of the piperine crude suspension, glycyrrhizic acid crude suspension, and the nanoformulations prepared in Example 5 and Comparative Example 1 were respectively taken and placed in a dialysis bag (MW = 8000-14000 Da), the two ends were tied tightly, and immersed in a centrifuge tube containing 40 mL of buffer solution, and released in the transfer medium gastric juice SGF (0-2h), small intestinal fluid SIF (3-5h), and colonic fluid SCF (6-24h). The centrifuge tube was placed in a water bath constant temperature oscillation chamber with a temperature of 37°C and a speed of 100 rpm. According to the set time, 2 ml of the drug-containing medium was taken at a fixed point, and an equal amount of the medium was added to the centrifuge tube. After filtering through a 0.22 μm filter membrane, it was detected by high performance liquid chromatography, and the results are shown as follows. Figure 3 shown.

[0106] from Figure 3 It can be seen that the nanoformulation prepared in Comparative Example 1 has a cumulative release of piperine and glycyrrhizic acid of 35.42% and 36.34% within 2 hours in the SGF, SIF, and SCF transfer release media, a cumulative release of piperine and glycyrrhizic acid of 59.18% and 76.73% within 5 hours, and a cumulative release of piperine and glycyrrhizic acid of 96.55% and 95.89% within 24 hours in the SGF, SIF, and SCF transfer release media; the nanoformulation prepared in Example 5 has a cumulative release of piperine and glycyrrhizic acid of 6.53% and 19.03% within 2 hours in the SGF, SIF, and SCF transfer release media, a cumulative release of piperine and glycyrrhizic acid of 13.68% and 32.93% within 5 hours, and a cumulative release of piperine and glycyrrhizic acid of 70.53% and 91.10% within 24 hours. Compared with the nanoformulation prepared in Comparative Example 1, the nanoformulation prepared in Example 5 can reduce the release of piperine and glycyrrhizic acid in gastric juice and intestinal juice, increase the release in colonic juice, and achieve synchronous release of piperine and glycyrrhizic acid in the colon; in the colonic juice, the cumulative release amounts of piperine and glycyrrhizic acid in the nanoformulation prepared in Example 5 are 1.5 times and 3 times that of the nanoformulation prepared in Comparative Example 1, respectively, and the cumulative release amounts of piperine and glycyrrhizic acid in the nanoformulation prepared in Example 5 are 4.4 times and 2.5 times that of the crude suspension of piperine and glycyrrhizic acid, respectively.

[0107] 4. In vivo colon targeting study of a nanoformulation for the treatment of colitis

[0108] Colitis mouse model: Female C57BL / 6J mice were fed 3% (w / v) DSS in water for 7 consecutive days to induce colitis. Body weight and disease index (DAI) scores were measured daily. The disease index (DAI) was scored as follows: weight loss (0, 0; 1, 1%, 1%-5%, 6%-10%, 11%-20%, 4, >20%); stool consistency (0, normal; 1, soft but still formed; 2, soft but not formed; 3, very wet and sticking to the cage wall; 4, diarrhea); and anal bleeding (0, no bleeding; 1, minimal bleeding; 2, small bleeding; 3, visible blood flow in the stool; 4, heavy bleeding).

[0109] Preparation of DiRNCs nanoformulation: The operation method is the same as that of Comparative Example 1, except that the cell membrane near-infrared fluorescent probe DiR is used instead of piperine.

[0110] Preparation of DiRENCs nanoformulation: The operation method is the same as that of Example 5, except that the cell membrane near-infrared fluorescent probe DiR is used instead of piperine.

[0111] A colitis mouse model was established as described above, with 3 mice per group fasting for 12 hours with free access to water. The mice were weighed and gavage-administered with DiR, DiRNCs, and DiRENCs (DiR at 0.5 mg / kg). Gastrointestinal tissues were removed 2, 6, 12, and 24 hours after mice were sacrificed, and images were acquired using an IVIS spectral imaging system. Colonic tissue drug content was further assayed by gavage with a coarse suspension of piperine, a coarse suspension of glycyrrhizic acid, a coarse suspension of piperine and glycyrrhizic acid, and the nanoformulations prepared in Example 5 and Comparative Example 1 (piperine at 10 mg / kg and glycyrrhizic acid at 5 mg / kg). Six hours after administration, mice were sacrificed, colonic tissue was excised, homogenized with phosphate buffer (1:5), and centrifuged at 12,000 rpm for 10 minutes. The supernatant was removed, and methanol was added to precipitate protein. The supernatant was removed by repeated centrifugation, dried, and concentrated with a nitrogen blower. The supernatant was redissolved with 200 μL of methanol and centrifuged at 12,000 rpm for 20 min. The drug concentration in the colon was determined by high performance liquid chromatography. The results were as follows: Figure 4 shown.

[0112] from Figure 4It can be seen that the fluorescence in the gastrointestinal tract reflects the distribution of DiR. At 2 hours, DiR fluorescence is distributed in the stomach and small intestine, while the fluorescence intensity of DiRENCs is lower, which may be due to the coating of the enteric film material. At 6 hours, the fluorescence of free DiR and DiRNCs shifts to the small intestine, and the fluorescence of DiRENCs is distributed in the colon and small intestine. After 12 and 24 hours, the fluorescence of free DiR and DiRNCs almost disappears, while the fluorescence of DiRENCs remains in the colon. Compared with DiRNCs, DiRENCs can target the colon and increase the retention time. Quantitative analysis further confirmed that the content of piperine and glycyrrhizic acid in the inflamed colon of the nanoformulation prepared in Example 5 was 4.3 times and 5.1 times that of the piperine crude suspension and glycyrrhizic acid crude suspension, respectively; the content of piperine and glycyrrhizic acid in the inflamed colon of the nanoformulation prepared in Example 5 was 3 times that of Comparative Example 1.

[0113] 5. Evaluation of the anti-colitis effect of a nanoformulation for treating colitis in vivo.

[0114] (1) Establish a colitis model as described above;

[0115] (2) Experimental Grouping Eighteen C57BL / 6J mice were randomly divided into the following six groups, with six mice in each group. The groups were as follows:

[0116] 1) Blank control group: healthy mice were given only normal saline by gavage;

[0117] 2) Model group: Model mice were only given normal saline by gavage;

[0118] 3) Administration group: piperine coarse suspension, piperine and glycyrrhizic acid coarse suspension, and the nanoformulation prepared in Example 5 and Comparative Example 1 (administered dose of piperine: 10 mg / kg, glycyrrhizic acid: 5 mg / kg)

[0119] (3) Dosage regimen: The mice were dosed according to the experimental groups starting from the third day of modeling, once a day for 7 consecutive days. Starting from the first day, the weight and drinking water of the animals were recorded every day, and the weight, blood in stool, and fecal morphology of the mice were observed. On the 11th day, the mice were killed and the colon, spleen and other tissues were removed. They were photographed, measured and weighed, and the pathological characteristics of the colon tissue, the expression of inflammatory factors and the repair of mucosal damage were measured. The results are as follows: Figure 5 shown.

[0120] from Figure 5It can be seen that compared with other groups, the nanoformulation prepared in Example 5 alleviated the symptoms of colitis such as weight loss, increased disease index, shortened colon length, and increased spleen weight in mice; significantly improved pathological features such as submucosal edema, infiltration of a large number of inflammatory cells, and disappearance of crypts in colon tissue; increased the expression of anti-inflammatory factors (IL-10, TGF-β1) and reduced the expression of pro-inflammatory factors (IL-17A, IL-1β); at the same time, it also increased the expression of intestinal epithelial cell tight junction proteins. Therefore, the prepared nanoformulation can play a synergistically enhanced anti-colitis effect, and the significant effect is better than the nanoformulation prepared in Comparative Example 1.

[0121] 6. Study on the anti-colitis mechanism of a nanoparticle preparation for treating colitis in vivo

[0122] After the above-mentioned treatment, the spleens of the mice were removed, and spleen cells were extracted and stained with antibodies (F4 / 80, CD11c, CD206) for 30 minutes. The cells were then rinsed with 1× Permeabilization Buffer and resuspended. 300 μl of Flow Cytometry Staining Buffer was added to resuspend the cells, and the proportion of M cells was detected by flow cytometry. Immunohistochemistry was used to detect the expression of markers related to M1 and M2 macrophages (nitric oxide synthase (iNOS) and arginine 1 (Arg1)) and metabolic pathway-related proteins (mammalian protein of rapamycin (mTOR) and hypoxia-inducible factor-1α (HIF-1α)). The results are shown in Figure 2. Figure 6 shown.

[0123] from Figure 6 It can be seen that compared with the saline group, the nanoformulation prepared in Example 5 can restore the number of F4 / 80+CD206+ macrophages from 1.20% to 2.51%, and can reduce the number of F4 / 80+CD11c+ macrophages from 8.01% to 4.28%. At the same time, immunohistochemistry results showed that the nanoformulation reduced the expression of iNOS, a marker of M1 macrophages, and increased the expression of Arg, a marker of M2 macrophages. The above results indicate that the nanoformulation can significantly exert immune anti-inflammatory effects on different subtypes of macrophages. Further study of the related mechanisms affecting macrophages showed that compared with other groups, the nanoformulation significantly increased the expression of mTOR protein and reduced the expression of HIF-1α protein. Therefore, the prepared nanoformulation can simultaneously regulate macrophages of both M1 and M2 phenotypes to exert immune anti-inflammatory effects, wherein it is mainly considered that piperine acts on mTOR to increase the polarization of M2 macrophages, and glycyrrhizic acid acts on HIF-1α to reduce the polarization of M1 macrophages. The two drugs synergistically regulate the balance of macrophages, enhance the anti-inflammatory effect, and are superior to the nanoformulation prepared in Comparative Example 1.

[0124] 7. Biosafety investigation of a nanoparticle preparation for the treatment of colitis

[0125] After the above treatment, the main organs of the mice (heart, liver, spleen, lung, and kidney) were stained with hematoxylin and eosin. Figure 7 shown.

[0126] from Figure 7 It can be seen that the prepared nanoformulation has no obvious toxic side effects on major organs. Based on the above anti-inflammatory effect evaluation, the nanoformulation of the present invention for treating colitis is safe and effective.

[0127] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. Reference can be made to the common and similar parts between the various embodiments. For the solutions disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple, and the relevant parts can be referred to the method section.

[0128] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nano preparation for treating colitis, characterized in that: The nano preparation is composed of the following components: piperine, glycyrrhizic acid, stabilizer TPGS, colon targeting material Eudragit S100, and pore-forming agent Tween 80; the mass ratio of piperine, glycyrrhizic acid and stabilizer is 2:0.5-4:0.5-4; the colon targeting material accounts for 10-15% of the mass of the nano preparation; the pore-forming agent accounts for 1-12% of the mass of the colon targeting material; The nanoformulation can achieve colon-specific release; in the simulated gastrointestinal fluid SGF (0-2h), SIF (3-5h), SCF (6-24h) transfer release medium, the in vitro release of piperine and glycyrrhizic acid of the nanoformulation achieves synchronous release, and the cumulative release of piperine and glycyrrhizic acid within 2h is both less than 20%; the cumulative release of piperine and glycyrrhizic acid within 5h is both less than 35%; and the cumulative release of piperine and glycyrrhizic acid within 24h is both 60-100%.

2. A nano preparation for treating colitis according to claim 1, characterized in that: The particle size of the nano preparation is 100-300nm, and the zeta potential is -40mV--20mV; the encapsulation rates of the piperine and glycyrrhizic acid are both 80.5-99.5%, and the drug loading amounts are both 20.5-60.5%.

3. A nano preparation for treating colitis according to claim 1, characterized in that: The nanoformulation is a colon-targeted nanoformulation co-loaded with piperine and glycyrrhizic acid. The nanoformulation achieves colon-targeted delivery of piperine and glycyrrhizic acid to synergistically regulate the polarization of macrophages to exert immune anti-inflammatory effects and enhance the anti-colitis effect.

4. The method for preparing a nano preparation for treating colitis according to any one of claims 1 to 3, characterized in that: The preparation method adopts one or more of the following methods: medium grinding method, high pressure homogenization method, and emulsification-solvent volatilization method.

5. The method for preparing a nano preparation for treating colitis according to claim 4, characterized in that: The specific steps of the emulsification-solvent evaporation-emission method are as follows: (1) adding glycyrrhizic acid, a stabilizer and a porogen into water, stirring the mixture evenly with a magnetic stirrer to form an aqueous phase; (2) dissolving piperine and colon targeting material in methanol and fully dissolving them by ultrasonication to form an organic phase; (3) under the action of ultrasound, the organic phase is added dropwise to the aqueous phase for ultrasound; (4) Magnetic stirring is performed at room temperature to evaporate methanol and obtain a nanoformulation.

6. The method for preparing a nano preparation for treating colitis according to claim 5, characterized in that: The ultrasound in step (3) is ice bath ultrasound, the ultrasound power is 65%, and the ultrasound time is 10 minutes; the ultrasound conditions are working for 3 seconds and interval for 2 seconds.

7. Use of the nano preparation for treating colitis according to any one of claims 1 to 3 in the preparation of a medicament for treating colitis.

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