Artificial bear gall powder preparation as well as preparation method and application thereof

Abstract

The invention discloses an artificial bear gall powder preparation as well as a preparation method and application thereof, the artificial bear gall powder preparation has a remarkable pH-dependent release characteristic, the release rate in an acid environment is high, the release rate in a neutral environment is low, the unique pH-dependent release mode can effectively avoid premature sudden release of the preparation in a non-target area, and the release rate of the artificial bear gall powder preparation is high. The preparation can be released more efficiently in an acidic microenvironment, the drug loading capacity of the preparation is high, the use efficiency is high, and compared with like products, the preparation cost is greatly reduced; and the preparation process is simple, high in operability and suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of artificial bear gall powder, in particular to an artificial bear gall powder preparation, a preparation method and application thereof. BACKGROUND

[0002] As an important traditional Chinese medicine compound preparation, artificial bear gall powder is widely used in the treatment of various hepatobiliary system diseases in clinical practice, such as primary biliary cholangitis (PBC), cholesterosis of gallbladder, and various inflammation-related diseases including bile reflux gastritis and non-alcoholic steatohepatitis (NASH), and plays a variety of pharmacological roles such as promoting bile secretion, regulating cholesterol metabolism, protecting liver, and anti-inflammatory. The main active ingredients include tauroursodeoxycholic acid, ursodeoxycholic acid, taurine chenodeoxycholic acid, chenodeoxycholic acid, cholic acid and other bile acids and other auxiliary ingredients. However, the clinical efficacy of artificial bear gall powder is often limited by the response defects of its traditional dosage form to the complex pH environment of the gastrointestinal tract. The ordinary tablets or capsules on the market will produce uncontrolled "burst release" due to rapid dissolution after entering the small intestine, and it is difficult to maintain long-acting concentration at the specific lesion site. As an improvement, although the enteric-coated preparation can successfully "bypass" the gastric acid, its release mechanism is still a simple "pH switch", that is, it will be released in full after reaching a certain pH threshold, lacking the ability of precise identification and "on-demand release" to pathological microenvironments (such as the acidic environment of inflammatory areas). Therefore, the existing technical solutions have not achieved the ideal mode of stable and slow release of drugs in the physiological environment and accelerated release in the acidic environment of the lesion, which leaves a clear technical gap and clinical demand for the development of a new type of intelligent pH-responsive delivery system. SUMMARY

[0003] In order to solve the technical problems existing in the prior art, the present application provides an artificial bear gall powder preparation, a preparation method and application thereof.

[0004] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows: A preparation method of an artificial bear gall powder preparation, comprising the following steps: Step S1, preparing zinc oxide nanoparticles Step S1.1, dissolving a zinc salt precursor in deionized water, the molar ratio of zinc salt to water being 1:300-1000, and stirring to obtain a zinc salt precursor solution with a concentration of 0.056-0.185 mol / L; Step S1.2, dissolving an alkaline compound in deionized water, the molar ratio of alkaline compound to water being 1:50-100, and stirring to obtain an alkaline solution with a concentration of 0.56-1.11 mol / L; Step S1.3, drop the basic solution into the zinc salt precursor solution, the molar ratio of zinc salt precursor to basic compound is 1:2, react at 50~80℃ for 1~4 hours to generate zinc oxide precipitate; Step S1.4, after centrifugation of the zinc oxide precipitate at a speed of 3000~4000 rpm for 10~20 min, water washing, and drying at a temperature of 60~70℃ for 2~6h, the zinc oxide nanoparticles are obtained; Step S2, preparation of artificial bear gall powder preparation Step S2.1, disperse the zinc oxide nanoparticles prepared in step S1.4 and artificial bear gall powder in anhydrous ethanol at a mass ratio of 1~3:1, then place in a water bath shaker, oscillate at a vibration speed of 180~200 rpm and a water bath temperature of 30~35℃ for 24 hours in the dark to obtain a drug-loaded suspension; Step S2.2, centrifuge the drug-loaded suspension at a speed of 3000~3500 r / pm for 10~20 min, then dry at 30~40℃ for 1~3 hours to obtain the artificial bear gall powder preparation.

[0005] Further, in step S1, the zinc salt precursor is zinc nitrate hexahydrate, zinc acetate or zinc chloride.

[0006] Further, in step S1, the basic compound is sodium hydroxide or potassium hydroxide.

[0007] An artificial bear gall powder preparation prepared by the above method.

[0008] Further, the 24h release rate of the artificial bear gall powder preparation in a phosphate buffer solution with a pH value of 7.4 is <40%, and the 24h release rate in a phosphate buffer solution with a pH value of 5.5 is >60%.

[0009] Further, the drug loading of the preparation is 10.2~14.8%.

[0010] Further, the preparation is a composite nanoparticle powder, and the physical structure is that the artificial bear gall powder is loaded on the surface of the zinc oxide nanoparticles by physical adsorption.

[0011] Further, the preparation comprises artificial bear gall powder and zinc oxide nanoparticles, wherein the artificial bear gall powder comprises the following components: Tauroursodeoxycholic acid 40~55%; Tauroursodeoxycholic acid 15~30%; Ursodeoxycholic acid 2~5%; Chenodeoxycholic acid 1~3%; Other bile acids and trace components 5~10%.

[0012] The application of the artificial bear gall powder preparation in primary biliary cholangitis (PBC), cholesterosis, bile reflux gastritis and non-alcoholic steatohepatitis (NASH).

[0013] Compared with the prior art, the application has the following beneficial effects: The artificial bear gall powder preparation provided by the application has a significant pH-dependent release characteristic, i.e., a high release rate in an acidic environment and a low release rate in a neutral environment. The drug loading capacity of the preparation can reach 10-15%, which improves the use efficiency of the drug and reduces the cost. The preparation process is simple, easy to operate and suitable for industrial production. According to literature reports, zinc oxide has good anti-inflammatory ability and forms a synergistic therapeutic effect with artificial bear gall powder, which can significantly improve the bioavailability of the drug. BRIEF DESCRIPTION OF DRAWINGS

[0014] The disclosure of the application will be described with reference to the accompanying drawings. It should be understood that the drawings are only for illustrative purposes, and are not intended to limit the scope of protection of the application. In the drawings, the same reference numerals are used to refer to the same parts. Among them: Figure 1 is an XRD pattern of ZnO nanoparticles prepared in Examples 1, 2 and 3; Figure 2 is an SEM image of ZnO nanoparticles prepared in Example 1; Figure 3 is an SEM image of ZnO nanoparticles prepared in Example 2; Figure 4 is an SEM image of ZnO nanoparticles prepared in Example 3; Figure 5 is an optical photograph of the artificial bear gall powder preparation prepared in Examples 1-3. DETAILED DESCRIPTION

[0015] It is easy to understand that, according to the technical solution of the application, those skilled in the art can propose various structures and implementation manners that can be replaced with each other without changing the essential spirit of the application. Therefore, the following detailed description and the drawings are only exemplary descriptions of the technical solution of the application, and should not be regarded as the whole or as a limitation or restriction on the technical solution of the application.

[0016] The technical solution of the application will be described in detail below with reference to the accompanying drawings. The description of the exemplary embodiments is only for illustrative purposes, and is not intended to limit or restrict the application or its application or use.

[0017] Example 1 Step 1, Preparation of ZnO nanoparticles Step 1.1, Preparation of solution A1: 2.975 g of zinc nitrate hexahydrate Zn(NO3)2·6H2O was dissolved in 90.1 g of deionized water (molar ratio of zinc salt to water was 1:500), and stirred to obtain solution A1 with a concentration of 0.111 mol / L. Step 1.2, Preparation of solution B1: 0.8 g of sodium hydroxide NaOH was dissolved in 36.03 g of deionized water (molar ratio of base to water was 1:100), and stirred to obtain solution B1 with a concentration of 0.56 mol / L. Step 1.3, Reaction: Solution B1 was added dropwise to solution A1, and stirred at 50°C for 2 hours to generate white precipitate C1, wherein the molar ratio of zinc nitrate hexahydrate Zn(NO3)2·6H2O to sodium hydroxide NaOH was 1:2.

[0018] Step 1.4, Post-treatment: The white precipitate C1 was centrifuged at 3500 rpm for 10 min, washed with water for 3 times, and dried at 60°C for 4 hours to obtain ZnO nanoparticles, as shown in Figure 2 .

[0019] The X-ray diffraction pattern of the prepared material is shown in Figure 1 , and the phase retrieval result indicates that the material preparation is successful; the SEM image, as shown in Figure 2 , shows that the nanoparticles are irregular agglomerates with rough and porous surfaces, which is consistent with the typical characteristics of physical agglomeration of nanoparticles, and is conducive to the adsorption and loading of artificial bear bile powder drugs.

[0020] Step 2, Preparation of artificial bear bile powder preparation Step 2.1, Drug loading step: 200 mg of ZnO nanoparticles and 100 mg of artificial bear bile powder were dissolved in 20 mL of anhydrous ethanol, and oscillated in the dark at a vibration speed of 180 rpm for 24 hours at a water bath temperature of 30°C to obtain drug-loaded suspension D1. Step 2.2, Post-treatment: The drug-loaded suspension D1 was centrifuged at a speed of 3500 rpm for 10 min, and then dried at 40°C for 1 hour to obtain artificial bear bile powder preparation E1.

[0021] Example 2 Step 1, Preparation of ZnO nanoparticles Step 1.1, Preparation of solution A2: 2.195 g of zinc acetate Zn(CH3COO)2·2H2O was dissolved in 54.1 g of deionized water (molar ratio of zinc salt to water was 1:300), and stirred to obtain solution A2 with a concentration of 0.185 mol / L.

[0022] Step 1.2, Preparation of solution B2: 1.12 g of potassium hydroxide KOH was dissolved in 40 mL of deionized water (molar ratio of base to water was 1:80), and stirred to obtain a solution with a concentration of 0.694 mol / L.

[0023] Step 1.3, Reaction: Solution B2 was added dropwise to solution A2, and stirred at 70°C for 1 hour to generate white precipitate C2, wherein the molar ratio of zinc acetate Zn(CH3COO)2·2H2O to potassium hydroxide KOH was 1:2.

[0024] Step 1.4, Post-treatment: White precipitate C2 was centrifuged at a speed of 4000 rpm for 15 min, washed with water for 3 times, and then dried at 70°C for 4 hours to obtain ZnO nanoparticles.

[0025] The X-ray diffraction pattern of the prepared material is shown in Figure 1 , and the phase retrieval result indicates that the material preparation is successful; as shown in Figure 3 , the SEM image shows that the nanoparticles are irregular agglomerates with rough and porous surfaces, which is consistent with the typical characteristics of physical agglomeration of nanoparticles, and is beneficial for the adsorption and loading of artificial bear gall powder drugs.

[0026] Step 2, Preparation of artificial bear gall powder preparation Step 2.1, Drug loading step: 100 mg of ZnO nanoparticles and 100 mg of artificial bear gall powder were dissolved in 20 mL of anhydrous ethanol, and oscillated at a speed of 200 rpm in the dark at 35°C for 24 hours to obtain a drug-loaded suspension D2.

[0027] Step 2.2, Post-treatment: The drug-loaded suspension D2 was centrifuged at a speed of 4000 rpm for 15 min, and then dried at 35°C for 2 hours to obtain artificial bear gall powder preparation E2.

[0028] Example 3 Step 1, Preparation of ZnO nanoparticles Step 1.1, Preparation of solution A3: 1.36 g of zinc chloride ZnCl2 was dissolved in 180.2 mL of deionized water (molar ratio of zinc salt to water was 1:1000), and stirred to obtain a solution with a concentration of 0.056 mol / L.

[0029] Step 1.2, Preparation of solution B3: 0.8 g of sodium hydroxide was dissolved in 18 mL of deionized water (molar ratio of base to water was 1:50), and stirred to obtain a solution with a concentration of 1.11 mol / L.

[0030] Step 1.3, Reaction: Solution B3 was added dropwise to solution A3, and stirred at 80°C for 4 hours to generate white precipitate C3, wherein the molar ratio of zinc chloride ZnCl2 to ammonia was 1:2.

[0031] Step 1.4, post-treatment: centrifuge the white precipitate C3 at 3000 rpm for 20 min, wash with water for 3 times, and dry at 70℃ for 4 hours to obtain ZnO nanoparticles.

[0032] The X-ray diffraction pattern of the prepared material is shown in Figure 1, and the phase retrieval result indicates that the material preparation is successful. Figure 1 Figure 4 The SEM image shown in Figure 2 indicates that the nanoparticles are irregular agglomerates with rough and porous surfaces, which is a typical characteristic of physical agglomeration of nanoparticles and is beneficial for the adsorption and loading of artificial bear gall powder drugs.

[0033] Step 2, preparation of artificial bear gall powder preparation Step 2.1, drug loading step: 300 mg of ZnO nanoparticles and 100 mg of artificial bear gall powder were dissolved in 20 mL of anhydrous ethanol, and the mixture was shaken at 35℃ and 200 rpm for 24 hours in the dark to obtain a drug-loaded suspension D3.

[0034] Step 2.2, post-treatment: centrifuge the obtained drug-loaded suspension D3 at 3000 rpm for 20 min, and then dry at 30℃ for 3 hours to obtain artificial bear gall powder preparation E3.

[0035] Effect Example 1 The artificial bear gall powder preparation E1 prepared in Example 1 was tested for two contents: ① drug loading amount ② pH response effect.

[0036] ① Drug loading amount determination method: (a) Determine the initial drug concentration of artificial bear gall powder (pure powder) Take the initial sample of artificial bear gall powder (pure powder) solution, dilute it, and then determine the concentration of active ingredient chenodeoxycholic acid C initial by liquid chromatography.

[0037] Calculate the initial total drug amount: m initial = C initial ×V initial In the formula, m initial is the initial total drug amount, and V initial is the initial drug volume. (b) Determine the free drug concentration of artificial bear gall powder (pure powder) In Example 1, Step 2.2, after centrifugation, the supernatant was collected, and the remaining drug concentration C free was determined by liquid chromatography. 1) Calculate the amount of unloaded drug: m free = C free ×V 上清液 In the formula, m free ​V is the amount of drug loaded 上清液 V is the volume of supernatant; 2) Calculate the actual drug loading: 21) Drug loading: m loaded = m initial -m free In the formula, m loaded is the amount of drug loaded; 22) Drug loading (%): Drug loading = m loaded / (m ZnO + m loaded ) x 100% In the formula, m ZnO is the amount of ZnO nanoparticles.

[0038] According to the above method, the drug loading of artificial bear gall powder preparation E1 is measured to be 12.5%.

[0039] ② pH response effect test The pH response release experiment of artificial bear gall powder preparation E1 is carried out, specifically: The artificial bear gall powder preparation E1 is dispersed in PBS buffer with pH 7.4 and pH 5.5, and placed in a 37°C constant temperature shaker at a speed of 100 rpm for dissolution experiment. The concentration of artificial bear gall powder preparation E1 is determined by sampling at regular intervals, and the following results are obtained:

[0040] At the same time, the artificial bear gall powder (pure powder) used in step 2.1 of Example 1 is subjected to experimental control analysis.

[0041] The experimental method used is: 30 mg of artificial bear gall powder (pure powder) is weighed and placed in containers containing 40 mL of pH 7.4 PBS buffer and pH 5.5 PBS buffer, respectively, and placed in a 37°C constant temperature shaker at a speed of 100 rpm for dissolution experiment. The concentration of artificial bear gall powder (pure powder) is determined by sampling at regular intervals, and the following results are obtained:

[0042] Comparing the experimental results of artificial bear gall powder preparation E1 and artificial bear gall powder (pure powder), it can be seen that: Artificial bear bile powder (pure powder) exhibited extremely rapid and uncontrolled release behavior in buffer solutions at pH 7.4 and pH 5.5. At pH 7.4, dissolution of the artificial bear bile powder (pure powder) was essentially complete within half an hour; at pH 5.5, the dissolution rate was slightly slower, but still approached complete dissolution within 2 hours. Subsequent time points (4-24 hours) showed almost no increase in drug dissolution, further confirming its rapid and complete release characteristics.

[0043] The artificial bear bile powder formulation E1 prepared in this embodiment exhibited extremely low release behavior in a buffer solution at pH 7.4, with the dissolution rate remaining controllable after 24 hours. However, the dissolution rate in a buffer solution at pH 5.5 was 2 to 3 times higher than that at pH 7.4 at the same time point, demonstrating superior pH responsiveness.

[0044] In summary, artificial bear bile powder (pure powder) exhibits uncontrollable release behavior, while the artificial bear bile powder formulation prepared by this invention can demonstrate continuous and controllable sustained-release characteristics. It plays a key role in regulating the release behavior of artificial bear bile powder, avoiding excessively high initial blood drug concentrations, and prolonging the duration of drug action, and has significant technical advantages.

[0045] Example 2 The artificial bear bile powder preparation E2 prepared in Example 2 was tested in two aspects: ① drug loading ② pH response effect.

[0046] ① Method for determining drug loading: (a) Determination of the initial drug concentration of artificial bear bile powder (pure powder) An initial sample of artificial bear bile powder (pure powder) solution was taken, diluted, and the concentration of the active ingredient ursodeoxycholic acid (C) was determined by liquid chromatography. initial .

[0047] Calculate the initial total amount of medicine: m initial = C initial ×V initial In the formula, m initial V represents the initial total drug dosage. initial This represents the initial drug volume; (b) Determination of free drug concentration in artificial bear bile powder (pure powder) In step 2.2 of Example 2, after centrifugation, the supernatant was collected, and the concentration of the remaining drug C was determined by liquid chromatography. free ; 1) Calculate the amount of unloaded drug: m free = C free ×V 上清液 In the formula, mfree V is the volume of the supernatant; 上清液 V is the volume of the supernatant; 2) Calculate the actual drug loading: 21) Drug loading: m loaded = m initial -m free In the formula, m loaded is the drug loading; 23) Drug loading (%): Drug loading = m loaded / (m ZnO + m loaded ) x 100% In the formula, m ZnO is the drug loading of ZnO nanoparticles.

[0048] According to the above method, the drug loading of artificial bear gall powder preparation E2 was measured to be 14.8%.

[0049] ②pH response effect test The pH response release experiment of artificial bear gall powder preparation E2 was carried out, specifically: The artificial bear gall powder preparation E2 was dispersed in PBS buffer with pH 7.4 and pH 5.5, and placed in a 37°C constant temperature shaker at a speed of 100 rpm for dissolution experiment. The concentration of artificial bear gall powder preparation E2 was determined by sampling at regular intervals, and the following results were obtained:

[0050] Effect Example 3 The two contents of artificial bear gall powder preparation E2 prepared in Example 3 were tested: ① drug loading ② pH response effect.

[0051] ① The drug loading of artificial bear gall powder preparation E3 was measured to be 10.2%.

[0052] (②pH response effect test The pH response release experiment of artificial bear gall powder preparation E3 was carried out, specifically: The artificial bear gall powder preparation E3 was dispersed in PBS buffer with pH 7.4 and pH 5.5, and placed in a 37°C constant temperature shaker at a speed of 100 rpm for dissolution experiment. The concentration of artificial bear gall powder preparation E2 was determined by sampling at regular intervals, and the following results were obtained:

[0053] It should be noted that the artificial bear gall powder preparations prepared in Examples 1-3, such as Figure 5As shown, it is shown that the artificial bear gall powder preparation presents irregular micron-sized aggregates with a size in the range of 10-100 microns, which is a composite nanoparticle powder, and the particle surface is rough, which is consistent with the characteristics of physical adsorption loading. This porous, loose aggregate structure formed by the agglomeration of nanoparticles provides favorable conditions for efficient loading and subsequent controlled release of drugs.

[0054] The above-mentioned artificial bear gall powder preparation can be widely used in primary biliary cholangitis PBC, cholesterosis of gallbladder, bile reflux gastritis and non-alcoholic fatty liver disease NASH.

[0055] The technical scope of the present application is not limited only to the content in the above description, and those skilled in the art can make various modifications and modifications to the above embodiments without departing from the technical idea of the present application, and these modifications and modifications should all belong to the protection scope of the present application.

Claims

1. A method for preparing an artificial bear gall powder preparation, characterized by, The method comprises the following steps: Step S1, preparing zinc oxide nanoparticles Step S1.1, dissolving zinc salt precursor in deionized water, the molar ratio of zinc salt to water is 1:300-1000, stirring to obtain a zinc salt precursor solution with a concentration of 0.056-0.185 mol / L; Step S1.2, dissolving an alkaline compound in deionized water, the molar ratio of the alkaline compound to water is 1:50-100, stirring to obtain an alkaline solution with a concentration of 0.56-1.11 mol / L; Step S1.3, adding the alkaline solution to the zinc salt precursor solution, the molar ratio of zinc salt precursor to alkaline compound is 1:2, reacting at 50-80°C for 1-4 hours to generate zinc oxide precipitate; Step S1.4, centrifuging the zinc oxide precipitate at a speed of 3000-4000 rpm for 10-20 min, then washing with water, and drying at a temperature of 60-70°C for 2-6 hours to obtain zinc oxide nanoparticles; Step S2, preparing artificial bear gall powder preparation Step S2.1, dispersing the zinc oxide nanoparticles prepared in step S1.4 and artificial bear gall powder in anhydrous ethanol at a mass ratio of 1-3:1, then placing in a water bath shaker, oscillating at a vibration speed of 180-200 rpm and a water bath temperature of 30-35°C for 24 hours in the dark to obtain a drug-loaded suspension; Step S2.2, centrifuging the drug-loaded suspension at a speed of 3000-3500 r / pm for 10-20 min, then drying at 30-40°C for 1-3 hours to obtain artificial bear gall powder preparation.

2. The method for preparing the artificial bear bile powder formulation according to claim 1, characterized in that, In the step S1, the zinc salt precursor is zinc nitrate hexahydrate, zinc acetate or zinc chloride.

3. The method for preparing the artificial bear bile powder formulation according to claim 1, characterized in that, In the step S1, the alkaline compound is sodium hydroxide or potassium hydroxide.

4. An artificial bear gall powder preparation prepared by the method of any one of claims 1-3.

5. The artificial bear gall powder preparation according to claim 4, characterized in that, The 24h release rate of the artificial bear gall powder preparation in a phosphate buffer solution with a pH value of 7.4 is <40%, and the 24h release rate in a phosphate buffer solution with a pH value of 5.5 is >60%.

6. The artificial bear gall powder preparation according to claim 4, characterized in that, The drug loading amount of the preparation is 10.2-14.8%.

7. The artificial bear gall powder preparation according to claim 4, characterized in that, The preparation is a composite nanoparticle powder, and the physical structure is that the artificial bear gall powder is loaded on the surface of the zinc oxide nanoparticles by physical adsorption.

8. The artificial bear gall powder preparation according to claim 4, characterized in that, The artificial bear gall powder preparation comprises the following components: Tauroursodeoxycholic acid 40-55%; Tauroursodeoxycholic acid 40-55%; Tauroursodeoxycholic acid 40-55%; Tauroursodeoxycholic acid 40-55%; Other bile acids and trace components 5-10%.

9. The artificial bear gall powder preparation according to any one of claims 1-4 is used in primary biliary cholangitis PBC, cholesterosis, bile reflux gastritis and non-alcoholic steatohepatitis NASH.

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