Silibinin freeze-dried powder and preparation method thereof
By using gradient cooling and vacuum freeze-drying technology, adjusting the mass ratio of active ingredients to solubilizers, and processing in stages, the problems of low encapsulation rate and poor stability of silymarin freeze-dried powder were solved, achieving high encapsulation rate, rapid reconstitution, and improved safety.
Patent Information
- Application Number
- CN202511412604.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-29
- Publication Date
- 2025-11-18
AI Technical Summary
Existing silymarin lyophilized powder has low encapsulation efficiency, easily damaged structure, slow reconstitution rate, and poor stability. Furthermore, the organic solvents in traditional formulations can easily cause adverse reactions.
The gradient cooling method and vacuum freeze-drying technology were adopted. The mass ratio of active ingredient to solubilizer was adjusted to 1:4~16, and the freeze-drying process was carried out in three stages, including equilibration at 4 ℃, cooling to -50 ℃ and holding, vacuum sublimation and vacuum heating treatment, combined with nitrogen protection.
It improves the encapsulation efficiency and reconstitution rate of silymarin, enhances the stability of the lyophilized powder, reduces the risk of adverse reactions, and meets the clinical needs for intravenous administration.
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Figure CN120960156A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the pharmaceutical field, and in particular to a lysmarin freeze-dried powder and its preparation method. Background Technology
[0002] Silymarin has significant antioxidant, anti-inflammatory, anti-fibrotic, and hepatoprotective effects, and is widely used clinically to treat alcoholic liver disease, non-alcoholic fatty liver disease, drug-induced liver injury, and viral hepatitis.
[0003] Currently, commercially available silymarin formulations are mainly in oral dosage forms (such as capsules and tablets), with injectable formulations being scarce. Traditional silymarin injections require the use of organic solvents (such as ethanol and propylene glycol) for solubilization, but these excipients can easily cause adverse reactions such as hemolysis and allergies, and the safety of long-term administration is questionable. In addition, silymarin is easily degraded in a liquid environment, and conventional aqueous injections cannot guarantee stability during storage.
[0004] Preparing silymarin into lyophilized powder avoids the degradation risks associated with liquid formulations, allows for immediate clinical use, reduces reliance on organic solvents, and improves medication safety. However, traditional silymarin lyophilized powders have low encapsulation rates of the active ingredient, and the inclusion complex structure is easily destroyed during lyophilization, resulting in slow reconstitution rates and poor stability. Summary of the Invention
[0005] In view of the above-mentioned shortcomings in the prior art, the purpose of this application is to provide a lyophilized powder of silymarin with high encapsulation efficiency, high stability and rapid reconstitution.
[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, embodiments of this application provide a method for preparing silymarin freeze-dried powder, comprising: The solubilizer, cosolvent, and metal chelating agent are dissolved in water for injection, and the pH is adjusted to 4-9 using a pH adjuster to obtain the first solution. The active ingredient is added to the first solution, stirred to dissolve, diluted to a final volume, and filtered to obtain the second solution; the active ingredient is silymarin and / or its pharmaceutically acceptable salt; the mass ratio of the active ingredient to the solubilizer is 1:4~16; The second solution was equilibrated at 4 °C for 30 min, then cooled to -50 °C at a rate of 1 °C / min and held for 2-5 h to obtain the pre-frozen product. The pre-frozen product was placed in a vacuum device with a vacuum degree of 0.01~0.5 mbar and a temperature of -35 ℃~-20 ℃ for 10~20 h to allow the free water in the pre-frozen product to sublimate, thus obtaining the first dried product. The vacuum level of the vacuum device was maintained at 0.01~0.5 mbar, and the temperature was increased to 10 ℃~30 ℃ at a heating rate of 0.1~0.3 ℃ / min and maintained for 4~8 h. Nitrogen purging was then performed to obtain silymarin freeze-dried powder.
[0007] Secondly, embodiments of this application provide a silymarin freeze-dried powder, which is prepared by the silymarin freeze-dried powder preparation method of the first aspect.
[0008] Compared with the prior art, the technical solution provided in this application has the following beneficial effects: Firstly, by scientifically and rationally adjusting the mass ratio of active ingredient to solubilizer to 1:4~16, the encapsulation rate of active ingredient can be improved; secondly, by adopting a gradient cooling method, the freeze-drying process is carried out in three stages. In the first stage, the second solution is placed at 4°C for equilibration for 30 min, and then cooled to -50°C at a cooling rate of 1°C / min, and maintained for 2~5 h to obtain a pre-frozen product; in the second stage, the pre-frozen product is placed in a vacuum device with a vacuum degree of 0.01~0.5 mbar and a temperature of -35°C~-20°C for 10~20 h to allow the free water in the pre-frozen product to sublimate, obtaining a first dried product; in the third stage, the vacuum degree of the vacuum device is maintained at 0.01~0.5 mbar, and the temperature is increased to 10°C~30°C at a heating rate of 0.1~0.3°C / min, and maintained for 4~8 h. Nitrogen purging is used to obtain silymarin freeze-dried powder, which not only avoids the destruction of the inclusion complex structure during freeze-drying, but also results in a fast reconstitution rate and good stability of the freeze-dried powder, which is beneficial to improving the safety of medication. Attached Figure Description
[0009] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0010] Figure 1 This is the HPLC chromatogram of the lyophilized silymarin powder prepared in Example 3 of this application; Figure 2 These are photographs of the reconstituted solution of silymarin freeze-dried powder prepared in Examples 1-3 of this application, taken 12 hours after reconstitution. Detailed Implementation
[0011] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are only for explaining this application, but the implementation of this application is not limited thereto.
[0012] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this application pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; the amounts of experimental reagents used are, unless otherwise specified, the amounts used in conventional experimental operations; and the experimental methods used are, unless otherwise specified, conventional methods.
[0013] In a first aspect, embodiments of this application provide a method for preparing silymarin freeze-dried powder, comprising: S1. Dissolve the solubilizer, co-solvent, and metal chelating agent in water for injection, and adjust the pH to 4-9 using a pH adjuster to obtain the first solution.
[0014] S2. Add the active ingredient to the first solution, stir to dissolve, make up to volume, and filter to obtain the second solution; the active ingredient is silymarin and / or its pharmaceutically acceptable salt; the mass ratio of the active ingredient to the solubilizer is 1:4~16.
[0015] When the mass ratio of active ingredient to solubilizer is too low, the encapsulation efficiency of the active ingredient is low; when the mass ratio is too high, it leads to waste of raw materials and increases the viscosity of the solution. Through extensive experimental research, the inventors of this application have discovered that the optimal balance between encapsulation efficiency and economic efficiency is achieved when the mass ratio of active ingredient to solubilizer is 1:4 to 16.
[0016] S3. The second solution is placed at a temperature of 4 ℃ for 30 min to equilibrate, and then cooled to -50 ℃ at a cooling rate of 1 ℃ / min and held for 2~5 h to obtain the pre-frozen product.
[0017] S4. Place the pre-frozen product in a vacuum device with a vacuum degree of 0.01~0.5 mbar and a temperature of -35 ℃~-20 ℃ for 10~20 h to allow the free water in the pre-frozen product to sublimate and obtain the first dried product.
[0018] S5. Maintain the vacuum level of the vacuum device at 0.01~0.5 mbar, heat to 10 ℃~30 ℃ at a heating rate of 0.1~0.3 ℃ / min, maintain for 4~8 h, and then purge with nitrogen to obtain silymarin freeze-dried powder.
[0019] The technical solution provided in this application embodiment, on the one hand, can improve the encapsulation rate and solubility of the active ingredient by scientifically and reasonably adjusting the mass ratio of the active ingredient to the solubilizer to 1:4~16. On the other hand, a gradient cooling method was adopted, and the freeze-drying process was carried out in three stages. In the first stage, the second solution was placed at 4 ℃ for 30 min to equilibrate, and then cooled to -50 ℃ at a cooling rate of 1 ℃ / min and held for 2~5 h to obtain a pre-frozen product. In the second stage, the pre-frozen product was placed in a vacuum device with a vacuum degree of 0.01~0.5 mbar and a temperature of -35 ℃~-20 ℃ for 10~20 h to allow the free water in the pre-frozen product to sublimate and obtain the first dried product. In the third stage, the vacuum degree of the vacuum device was maintained at 0.01~0.5 mbar, and the temperature was increased to 10 ℃~30 ℃ at a heating rate of 0.1~0.3 ℃ / min and held for 4~8 h. Nitrogen purging was then performed to obtain silymarin freeze-dried powder. This method not only avoids the destruction of the inclusion complex structure during freeze-drying, but also produces a freeze-dried powder with a fast reconstitution rate and good stability, which is beneficial to reducing adverse reactions, improving drug safety, and meeting the needs of clinical intravenous administration.
[0020] In some embodiments, in step S1, the solubilizer and co-solvent are dissolved in water for injection, and the pH is adjusted to 7-8 using a pH adjuster to obtain a first solution.
[0021] In some embodiments, the mass ratio of the active ingredient to the solubilizer is 1:4 to 8. For example, the mass ratio of the active ingredient to the solubilizer may be 1:4, 1:5, 1:6, 1:7, or 1:8, etc.
[0022] In some embodiments, the solubilizer is at least one of hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, or sodium sulfobutyl-β-cyclodextrin.
[0023] Preferably, the solubilizer is sodium sulfobutyl-β-cyclodextrin.
[0024] The technical solution provided in this application uses cyclodextrin and its derivatives as solubilizers. Through their hydrophobic cavities, they encapsulate the active ingredients, which can significantly improve the water solubility and stability of the active ingredients. In some embodiments, the active ingredient is at least one of silybin, silybin meglumine, or silybin disuccinate disodium salt.
[0025] Preferably, the active ingredient is silymarin meglumine.
[0026] In some embodiments, the cosolvent is meglumine.
[0027] In some embodiments, the pH adjuster is at least one of sodium hydroxide, potassium hydroxide, meglumine, hydrochloric acid, sodium carbonate, or potassium carbonate.
[0028] In some embodiments, the metal chelating agent is at least one of ethylenediaminetetraacetic acid tetrasodium (EDTA-4Na), ethylenediaminetetraacetic acid disodium (EDTA-2Na), and ethylenediaminetetraacetic acid (EDTA).
[0029] In some embodiments, the active ingredient is added to a first solution, stirred to dissolve, diluted to a fixed volume, and filtered to obtain a second solution, comprising: The active ingredient is added to the first solution, stirred to dissolve, and then diluted to a final volume to obtain the third solution. Add needle-grade activated carbon to the third solution for adsorption for 30-45 min, then perform decarbonization treatment using a 1 µm titanium rod, coarse filtration using a 0.45 µm filter cartridge, and sterilization filtration using a 0.22 µm filter cartridge to obtain the second solution.
[0030] Secondly, embodiments of this application provide a silymarin freeze-dried powder, which is prepared by the silymarin freeze-dried powder preparation method of the first aspect.
[0031] This application has undergone multiple experiments, and some of the test results are presented here for reference to further describe the invention in detail. The following is a detailed description in conjunction with specific embodiments.
[0032] Example 1 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 1: Table 1
[0033] The preparation steps of the lyophilized milk thistle powder in this embodiment are as follows: Sodium sulfonyl-β-cyclodextrin, meglumine, and disodium edetate (EDTA-2Na) were dissolved in water for injection, and the pH was adjusted to 7.5 using sodium hydroxide or hydrochloric acid to obtain the first solution. Silymarin meglumine was added to the first solution, stirred to dissolve, and the volume was brought to 35 mL. Activated carbon for injection was added, and adsorption was performed for 30 minutes. The solution was then decarbonized using a 1 µm titanium rod, coarsely filtered using a 0.45 µm filter, and sterilized using a 0.22 µm filter to obtain the second solution. In the first stage of lyophilization, the second solution was equilibrated at 4 °C for 30 min, then cooled to -50 °C at a rate of 1 °C / min and maintained for 4 h to obtain the pre-frozen product. In the second stage of lyophilization, the pre-frozen product was placed in the chamber of a vacuum apparatus, and the vacuum pump was started to reduce the chamber pressure to 0.01 mbar. The partition temperature was maintained at -30 °C for 15 h to allow the free water in the pre-frozen product to sublimate, yielding the first dried product. In the third stage of freeze drying, the vacuum degree of the vacuum device was maintained at 0.01 mbar, the temperature was increased to 25°C at a heating rate of 0.2°C / min, and maintained for 6 h. Nitrogen was then used to fill the container and press it into a stopper to obtain silymarin freeze-dried powder.
[0034] Example 2 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 2: Table 2
[0035] The preparation steps of the lyophilized milk thistle powder in this embodiment are as follows: Sodium sulfonyl-β-cyclodextrin, meglumine, and disodium edetate (EDTA-2Na) were dissolved in water for injection, and the pH was adjusted to 7.0 using sodium hydroxide or hydrochloric acid to obtain the first solution. Silymarin meglumine was added to the first solution, stirred to dissolve, and the volume was brought to 35 mL. Activated carbon for injection was added, and adsorption was performed for 45 minutes. The solution was then decarbonized using a 1 µm titanium rod, coarsely filtered using a 0.45 µm filter, and sterilized using a 0.22 µm filter to obtain the second solution. In the first stage of lyophilization, the second solution was equilibrated at 4 °C for 30 min, then cooled to -50 °C at a rate of 1 °C / min and maintained for 5 h to obtain the pre-frozen product. In the second stage of lyophilization, the pre-frozen product was placed in the chamber of a vacuum apparatus, and the vacuum pump was started to reduce the chamber pressure to 0.2 mbar. The partition temperature was maintained at -35 °C for 12 h to allow the free water in the pre-frozen product to sublimate, yielding the first dried product. In the third stage of freeze drying, the vacuum degree of the vacuum device was maintained at 0.2 mbar, and the temperature was increased to 20°C at a heating rate of 0.3°C / min and maintained for 8 h. Nitrogen was then used to fill the container and press it into a stopper to obtain silymarin freeze-dried powder.
[0036] Example 3 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 3: Table 3
[0037] The preparation steps of the lyophilized milk thistle powder in this embodiment are as follows: Sodium sulfonyl-β-cyclodextrin, meglumine, and disodium edetate (EDTA-2Na) were dissolved in water for injection, and the pH was adjusted to 7.8 using sodium hydroxide or hydrochloric acid to obtain the first solution. Silymarin meglumine was added to the first solution, stirred to dissolve, and the volume was brought to 35 mL. Activated carbon for injection was added, and adsorption was performed for 40 minutes. The solution was then decarbonized using a 1 µm titanium rod, coarsely filtered using a 0.45 µm filter, and sterilized using a 0.22 µm filter to obtain the second solution. In the first stage of lyophilization, the second solution was equilibrated at 4 °C for 30 min, then cooled to -50 °C at a rate of 1 °C / min and maintained for 3 h to obtain the pre-frozen product. In the second stage of lyophilization, the pre-frozen product was placed in the chamber of a vacuum apparatus, and the vacuum pump was started to reduce the chamber pressure to 0.1 mbar. The partition temperature was maintained at -25 °C for 20 h to allow the free water in the pre-frozen product to sublimate, yielding the first dried product. In the third stage of freeze drying, the vacuum level of the vacuum device was maintained at 0.1 mbar, and the temperature was increased to 30°C at a heating rate of 0.3°C / min and maintained for 4 h. Nitrogen was then used to fill the container and press it into a stopper to obtain silymarin freeze-dried powder.
[0038] The lyophilized silymarin powder prepared in Example 3 was analyzed by HPLC, and the HPLC chromatogram is shown below. Figure 1 As shown.
[0039] Example 4 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 4: Table 4
[0040] The preparation steps of the lyophilized milk thistle powder in this embodiment are as follows: Sodium sulfonyl-β-cyclodextrin, meglumine, and disodium edetate were dissolved in water for injection, and the pH was adjusted to 7.0 using sodium hydroxide or hydrochloric acid to obtain the first solution. Silymarin meglumine was added to the first solution, stirred to dissolve, and the volume was brought to 35 mL. Activated carbon for injection was added, and adsorption was performed for 30 minutes. The solution was then decarbonized using a 1 µm titanium rod, coarsely filtered using a 0.45 µm filter, and sterilized using a 0.22 µm filter to obtain the second solution. In the first stage of lyophilization, the second solution was equilibrated at 4 °C for 30 min, then cooled to -50 °C at a rate of 1 °C / min and maintained for 5 h to obtain the pre-frozen product. In the second stage of lyophilization, the pre-frozen product was placed in the chamber of a vacuum apparatus, and the vacuum pump was started to reduce the chamber pressure to 0.3 mbar. The partition temperature was maintained at -35 °C for 18 h to allow the free water in the pre-frozen product to sublimate, yielding the first dried product. In the third stage of freeze drying, the vacuum level of the vacuum device was maintained at 0.05 mbar, and the temperature was increased to 15 ℃ at a heating rate of 0.1 ℃ / min and maintained for 7 h. Nitrogen was then used to fill the container and press it into a stopper to obtain silymarin freeze-dried powder.
[0041] Example 5 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 5: Table 5
[0042] The preparation steps of the lyophilized milk thistle powder in this embodiment are as follows: Sodium sulfonyl-β-cyclodextrin, meglumine, and disodium edetate were dissolved in water for injection, and the pH was adjusted to 8.0 using sodium hydroxide or hydrochloric acid to obtain the first solution. Silymarin meglumine was added to the first solution, stirred to dissolve, and the volume was brought to 35 mL. Activated carbon for injection was added, and adsorption was performed for 45 minutes. The solution was then decarbonized using a 1 µm titanium rod, coarsely filtered using a 0.45 µm filter, and sterilized using a 0.22 µm filter to obtain the second solution. In the first stage of lyophilization, the second solution was equilibrated at 4 °C for 30 min, then cooled to -50 °C at a rate of 1 °C / min and held for 2 h to obtain the pre-frozen product. In the second stage of lyophilization, the pre-frozen product was placed in the chamber of a vacuum apparatus, and the vacuum pump was started to reduce the chamber pressure to 0.5 mbar. The partition temperature was maintained at -20 °C for 20 h to allow the free water in the pre-frozen product to sublimate, yielding the first dried product. In the third stage of freeze drying, the vacuum level of the vacuum device was maintained at 0.2 mbar, and the temperature was increased to 25 ℃ at a heating rate of 0.2 ℃ / min and maintained for 6 h. Nitrogen was then used to fill the container and press it into a stopper to obtain silymarin freeze-dried powder.
[0043] Example 6 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 6: Table 6
[0044] The preparation steps of the lyophilized milk thistle powder in this embodiment are as follows: Sodium sulfonyl-β-cyclodextrin, meglumine, and disodium edetate were dissolved in water for injection, and the pH was adjusted to 7.5 using sodium hydroxide or hydrochloric acid to obtain the first solution. Disodium silymarin dihexasuccinate was added to the first solution, stirred to dissolve, and the volume was brought to 35 mL. Activated carbon for injection was added, and adsorption was performed for 45 minutes. The solution was then decarbonized using a 1 µm titanium rod, coarsely filtered using a 0.45 µm filter, and sterilized using a 0.22 µm filter to obtain the second solution. In the first stage of lyophilization, the second solution was equilibrated at 4 °C for 30 min, then cooled to -50 °C at a rate of 1 °C / min and maintained for 4 h to obtain the pre-frozen product. In the second stage of lyophilization, the pre-frozen product was placed in the chamber of a vacuum apparatus, and the vacuum pump was started to reduce the chamber pressure to 0.1 mbar. The partition temperature was maintained at -30 °C for 16 h to allow the free water in the pre-frozen product to sublimate, yielding the first dried product. In the third stage of freeze drying, the vacuum level of the vacuum device was maintained at 0.1 mbar, and the temperature was increased to 20°C at a heating rate of 0.3°C / min and maintained for 8 h. Nitrogen was then used to fill the container and press it into a stopper to obtain silymarin freeze-dried powder.
[0045] Example 7 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 7: Table 7
[0046] The preparation steps of the lyophilized milk thistle powder in this embodiment are the same as those in Example 3.
[0047] Example 8 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 8: Table 8
[0048] The preparation steps of the lyophilized milk thistle powder in this embodiment are as follows: Sodium sulfonyl-β-cyclodextrin, meglumine, and disodium edetate were dissolved in water for injection, and the pH was adjusted to 7.8 using sodium hydroxide or hydrochloric acid to obtain the first solution. Silymarin meglumine was added to the first solution, stirred to dissolve, and the volume was brought to 35 mL. Activated carbon for injection was added, and adsorption was performed for 30 minutes. The solution was then decarbonized using a 1 µm titanium rod, coarsely filtered using a 0.45 µm filter, and sterilized using a 0.22 µm filter to obtain the second solution. In the first stage of lyophilization, the second solution was equilibrated at 4 °C for 30 min, then cooled to -50 °C at a rate of 1 °C / min and maintained for 5 h to obtain the pre-frozen product. In the second stage of lyophilization, the pre-frozen product was placed in the chamber of a vacuum apparatus, and the vacuum pump was started to reduce the chamber pressure to 0.03 mbar. The partition temperature was maintained at -30 °C for 12 h to allow the free water in the pre-frozen product to sublimate, yielding the first dried product. In the third stage of freeze drying, the vacuum level of the vacuum device was maintained at 0.033 mbar, and the temperature was increased to 20 ℃ at a heating rate of 0.2 ℃ / min and maintained for 8 h. Nitrogen was then used to fill the container and press it into a stopper to obtain silymarin freeze-dried powder.
[0049] Example 9 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 9: Table 9
[0050] The preparation steps of the lyophilized milk thistle powder in this embodiment are as follows: Sodium sulfonyl-β-cyclodextrin, meglumine, and disodium edetate were dissolved in water for injection, and the pH was adjusted to 8.0 using sodium hydroxide or hydrochloric acid to obtain the first solution. Silymarin meglumine was added to the first solution, stirred to dissolve, and the volume was brought to 35 mL. Activated carbon for injection was added, and adsorption was performed for 30 minutes. The solution was then decarbonized using a 1 µm titanium rod, coarsely filtered using a 0.45 µm filter, and sterilized using a 0.22 µm filter to obtain the second solution. In the first stage of lyophilization, the second solution was equilibrated at 4 °C for 30 min, then cooled to -50 °C at a rate of 1 °C / min and maintained for 3 h to obtain the pre-frozen product. In the second stage of lyophilization, the pre-frozen product was placed in the chamber of a vacuum apparatus, and the vacuum pump was started to reduce the chamber pressure to 0.02 mbar. The partition temperature was maintained at -35 °C for 15 h to allow the free water in the pre-frozen product to sublimate, yielding the first dried product. In the third stage of freeze drying, the vacuum level of the vacuum device was maintained at 0.02 mbar, and the temperature was increased to 20 ℃ at a heating rate of 0.2 ℃ / min and maintained for 7 h. Nitrogen was then used to fill the container and press it into a stopper to obtain silymarin freeze-dried powder.
[0051] Example 10 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 10: Table 10
[0052] The preparation steps of the lyophilized milk thistle powder in this embodiment are the same as those in Example 3.
[0053] Example 11 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 11: Table 11
[0054] The preparation steps of the lyophilized milk thistle powder in this embodiment are the same as those in Example 3.
[0055] Example 12 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 12: Table 12
[0056] The preparation steps of the lyophilized milk thistle powder in this embodiment are the same as those in Example 3.
[0057] Example 13 The formulation of the lyophilized milk thistle powder in this embodiment is shown in Table 13: Table 13
[0058] The preparation steps of the lyophilized milk thistle powder in this embodiment are the same as those in Example 3.
[0059] The encapsulation efficiency and related substances (total impurities) of the lysozyme lyophilized powders prepared in Examples 1-13 of this application were tested, and the test results are shown in Table 14.
[0060] Encapsulation efficiency test method: Take a certain volume of the test solution and add it to an ultrafiltration centrifuge tube. Centrifuge at 12,000 rpm for about 20 minutes. Free drug amount: The breakthrough liquid (filtrate) contains unencapsulated free silymarin. Take the filtrate and determine the concentration using a concentration method to calculate the free drug amount. Total drug amount: Take another equal volume of the test solution (without centrifugation), dilute it directly, and determine the total drug amount. Encapsulation efficiency = (Total drug amount - Free drug amount) / Total drug amount * 100%. The encapsulation efficiency of this product should be greater than 80.0%.
[0061] The method for detecting related substances (total impurities) is as follows: using octadecylsilane-bonded silica gel as the packing material, methanol-water-glacial acetic acid as the mobile phase, and HPLC at a detection wavelength of 288 nm, the impurities are detected. The sum of the peak areas of all impurities in the test solution shall not exceed 1.2 times (3.0%) the peak area of the main peak in the control solution.
[0062] Table 14
[0063] As shown in Table 14, the lysozyme lyophilized powder prepared by the preparation methods provided in Examples 1-13 of this application has a high encapsulation rate, which can reach more than 92%, and the content of related substances is less than 2%, with good stability, which is beneficial to improving the safety of medication.
[0064] The comparison results between Examples 3 and Examples 10-13 show that, compared with using sodium sulfobutyl-β-cyclodextrin as a single solubilizer, using at least two of hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, or sodium sulfobutyl-β-cyclodextrin in combination can reduce the amount of solubilizer used, which is beneficial to reducing the cost of excipients, while ensuring the encapsulation rate and stability of the active ingredients.
[0065] The lyophilized silymarin powder prepared in Examples 1-13 of this application was reconstituted using 0.9% sodium chloride injection, and the stability of the reconstituted solution was examined at 0h, 6h, and 12h, respectively. The test results are shown in Table 15.
[0066] Content detection method: Using octadecylsilane-bonded silica gel as the packing material and methanol-water-glacial acetic acid as the mobile phase, the detection wavelength is 288 nm, and the method is HPLC. The content of silymarin in this product should be 90.0%~110.0% of the labeled amount.
[0067] Table 15
[0068] As shown in Table 15, the reconstituted solutions of silymarin lyophilized powder prepared by the preparation methods provided in Examples 1 to 13 of this application have good stability. They are all clear, pale yellow liquids with no precipitation or sedimentation. The pH value remains basically unchanged within 12 hours. After 12 hours of reconstitution, the content meets the requirements and is stable without significant decrease.
[0069] Figure 2 These are photographs of the reconstituted solution of silymarin freeze-dried powder prepared in Examples 1-3 of this application, taken 12 hours after reconstitution.
[0070] The silymarin lyophilized powders prepared in Examples 1-13 of this application were subjected to a reconstitution test using 0.9% sodium chloride injection, and the reconstitution time (the time required for solid silymarin lyophilized powder to completely dissolve in 0.9% sodium chloride injection) of each group of test samples was recorded. The test results are shown in Table 16.
[0071] Table 16
[0072] As shown in Table 16, the lysozyme lyophilized powder prepared by the preparation methods provided in Examples 1-13 of this application has high reconstitution efficiency and a reconstitution time of less than 1 min.
[0073] Comparative Example 1 The formulation of the lyophilized milk thistle powder for this comparative example is shown in Table 17: Table 17
[0074] The preparation steps of the lyophilized milk thistle powder in this comparative example are the same as those in Example 3.
[0075] Comparative Example 2 The formulation of the lyophilized milk thistle powder for this comparative example is shown in Table 18: Table 18
[0076] The preparation steps of the lyophilized milk thistle powder in this comparative example are the same as those in Example 3.
[0077] Comparative Example 3 The formulation of the lyophilized milk thistle powder for this comparative example is shown in Table 19: Table 19
[0078] The preparation steps of the lyophilized milk thistle powder in this comparative example are the same as those in Example 3.
[0079] Comparative Example 4 The formulation of the lyophilized milk thistle powder in this comparative example is the same as that in Example 3.
[0080] The preparation steps of the lyophilized milk thistle powder in this comparative example are as follows: Sodium sulfonyl-β-cyclodextrin and meglumine were dissolved in water for injection, and the pH was adjusted to 7.8 using sodium hydroxide or hydrochloric acid to obtain the first solution. Silymarin meglumine was added to the first solution, stirred to dissolve, and the volume was brought to 35 mL. Activated carbon for injection was added, and adsorption was performed for 40 minutes. The solution was then decarbonized using a 1 µm titanium rod, coarsely filtered using a 0.45 µm filter, and sterilized using a 0.22 µm filter to obtain the second solution. In the first stage of lyophilization, the second solution was cooled to -50 °C at a rate of 1 °C / min and maintained for 3 hours to obtain a pre-frozen product. In the second stage of lyophilization, the pre-frozen product was placed in the chamber of a vacuum apparatus, and the vacuum pump was started to reduce the chamber pressure to 0.1 mbar. The partition temperature was maintained at -25 °C for 20 hours to allow the free water in the pre-frozen product to sublimate, yielding the first dried product. In the third stage of freeze drying, the vacuum level of the vacuum device was maintained at 0.1 mbar, and the temperature was increased to 30 ℃ at a heating rate of 0.3 ℃ / min and maintained for 4 h. Nitrogen was then used to fill the container and press it into a stopper to obtain silymarin freeze-dried powder.
[0081] Comparative Example 5 The formulation of the lyophilized milk thistle powder in this comparative example is the same as that in Example 3.
[0082] The preparation steps of the lyophilized milk thistle powder in this comparative example are as follows: Sodium sulfonyl-β-cyclodextrin and meglumine were dissolved in water for injection, and the pH was adjusted to 7.8 using sodium hydroxide or hydrochloric acid to obtain the first solution. Silymarin meglumine was added to the first solution, stirred to dissolve, and the volume was brought to 35 mL. Activated carbon for injection was added, and adsorption was performed for 40 minutes. The solution was then decarbonized using a 1 µm titanium rod, coarsely filtered using a 0.45 µm filter, and sterilized using a 0.22 µm filter to obtain the second solution. In the first stage of lyophilization, the second solution was placed in the chamber of a vacuum apparatus. The vacuum pump was started, and the chamber pressure was reduced to 0.1 mbar. The partition temperature was maintained at -25°C for 20 h to allow the free water in the pre-frozen product to sublimate, yielding the first dried product. In the second stage of lyophilization, the vacuum apparatus was maintained at 0.1 mbar, and the temperature was increased to 30°C at a rate of 0.3°C / min and maintained for 4 h. Nitrogen was then used to fill the chamber and press the stopper to obtain silymarin lyophilized powder.
[0083] Comparative Example 6 The formulation of the lyophilized milk thistle powder in this comparative example is the same as that in Example 3.
[0084] The preparation steps of the lyophilized milk thistle powder in this comparative example are as follows: Sodium sulfonyl-β-cyclodextrin and meglumine were dissolved in water for injection, and the pH was adjusted to 7.8 using sodium hydroxide or hydrochloric acid to obtain the first solution. Silymarin meglumine was added to the first solution, stirred to dissolve, and the volume was brought to 35 mL. Activated carbon for injection was added, and adsorption was performed for 40 minutes. The solution was then decarbonized using a 1 µm titanium rod, coarsely filtered using a 0.45 µm filter, and sterilized using a 0.22 µm filter to obtain the second solution. In the first stage of lyophilization, the second solution was equilibrated at 4 °C for 30 min, then cooled to -50 °C at a rate of 1 °C / min and maintained for 3 h to obtain the pre-frozen product. In the second stage of lyophilization, the pre-frozen product was placed in the chamber of a vacuum apparatus, and the vacuum pump was started to reduce the chamber pressure to 0.1 mbar. The partition temperature was maintained at -25 °C for 20 h to allow the free water in the pre-frozen product to sublimate, yielding the first dried product. In the third stage of freeze drying, the vacuum device was kept at a vacuum level of 0.1 mbar, and the temperature was increased to 50 ℃ at a heating rate of 0.3 ℃ / min and maintained for 4 h. Nitrogen was then used to fill the container and press it into a stopper to obtain silymarin freeze-dried powder.
[0085] The encapsulation efficiency and related substances (total impurities) of the lyophilized milk thistle powder prepared in Comparative Examples 1 to 6 were tested according to the above experimental method. The test results are shown in Table 20.
[0086] Table 20
[0087] As can be seen from Tables 14 and 19, the comparison results between Comparison 1-3 and Example 3 show that when the mass ratio of active ingredient to solubilizer is 2:1 or 1:1-2, the encapsulation efficiency of the active ingredient is relatively low.
[0088] The comparison results between Comparative Examples 4-5 and Example 3 show that placing the second solution at a temperature of 4 °C for 30 min in the first stage of freeze drying, and then cooling it to -50 °C at a cooling rate of 1 °C / min and maintaining it for 3 h, is beneficial to improve the encapsulation rate of the active ingredients.
[0089] The comparison results between Comparative Example 6 and Example 3 show that in the third stage of freeze drying, maintaining the vacuum degree of the vacuum device at 0.1 mbar, heating to 30 ℃ at a heating rate of 0.3 ℃ / min, and maintaining it for 4 h can reduce the content of related substances (total impurities), which is beneficial to improving the stability of the formulation and the safety of medication.
[0090] The lyophilized silymarin powder prepared in Comparative Examples 1-4 was reconstituted using 0.9% sodium chloride injection, and the stability of the reconstituted solution was examined at 0h, 6h, and 12h. The test results are shown in Table 21.
[0091] Table 21
[0092] As can be seen from Tables 15 and 21, the comparison results between Comparison 1-3 and Example 3 show that when the mass ratio of active ingredient to solubilizer is 2:1 or 1:1-2, the stability of the reconstituted solution of the formulation is poor.
[0093] The comparison results between Comparative Examples 4-5 and Example 3 show that placing the second solution at 4 °C for equilibration for 30 min in the first stage of freeze drying, and then cooling it to -50 °C at a cooling rate of 1 °C / min and maintaining it for 3 h, is beneficial to improve the stability of the reconstituted solution of the formulation.
[0094] The silymarin lyophilized powders prepared in Comparative Examples 1, 4-6 were reconstituted using 0.9% sodium chloride injection. The reconstitution time (the time required for solid silymarin lyophilized powder to completely dissolve in 0.9% sodium chloride injection) of each group of test samples was recorded. The test results are shown in Table 22.
[0095] Table 22
[0096] As can be seen from Tables 16 and 22, the comparison results between Comparison 1 and Example 3 show that when the mass ratio of active ingredient to solubilizer is 2:1, the reconstitution time of the formulation is greater than 5 min, and the reconstitution efficiency is low.
[0097] The comparison results between Comparative Examples 4-6 and Example 3 show that in the first stage of freeze drying, the second solution is first placed at a temperature of 4 °C for equilibration for 30 min, and then cooled to -50 °C at a cooling rate of 1 °C / min and held for 3 h. The reconstitution time of the formulation is greater than 3 min, and the reconstitution efficiency is low.
[0098] The comparison results between Comparative Example 6 and Example 3 show that in the third stage of freeze drying, the vacuum degree of the vacuum device is maintained at 0.1 mbar, the temperature is increased to 30 ℃ at a heating rate of 0.3 ℃ / min, and maintained for 4 h. The reconstitution time of the formulation is greater than 2 min, which is inferior to the reconstitution efficiency of the formulation in Example 3.
[0099] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be included within the protection scope of this application.
Claims
1. A method for preparing silymarin freeze-dried powder, characterized in that, include: The solubilizer, cosolvent, and metal chelating agent are dissolved in water for injection, and the pH is adjusted to 4-9 using a pH adjuster to obtain the first solution. The active ingredient is added to the first solution, stirred to dissolve, diluted to a final volume, and filtered to obtain the second solution; the active ingredient is silymarin and / or its pharmaceutically acceptable salt. The mass ratio of the active ingredient to the solubilizer is 1:4~16; The second solution was placed at 4 °C for equilibration for 30 min, then cooled to -50 °C at a rate of 1 °C / min and held for 2-5 h to obtain the pre-frozen product. The pre-frozen product is placed in a vacuum device with a vacuum degree of 0.01~0.5 mbar and a temperature of -35 ℃~-20 ℃ for 10~20 h to allow the free water in the pre-frozen product to sublimate, thus obtaining the first dried product. The vacuum degree of the vacuum device is maintained at 0.01~0.5 mbar, and the temperature is increased to 10 ℃~30 ℃ at a heating rate of 0.1~0.3 ℃ / min and maintained for 4~8 h. Nitrogen purging is then performed to obtain silymarin freeze-dried powder.
2. The method for preparing silymarin freeze-dried powder according to claim 1, characterized in that, The mass ratio of the active ingredient to the solubilizer is 1:4~8.
3. The method for preparing silymarin freeze-dried powder according to claim 1, characterized in that, The solubilizer is at least one of hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, or sodium sulfobutyl-β-cyclodextrin.
4. The method for preparing silymarin freeze-dried powder according to claim 3, characterized in that, The solubilizer is sodium sulfobutyl-β-cyclodextrin.
5. The method for preparing silymarin freeze-dried powder according to claim 1, characterized in that, The active ingredient is at least one of silybin, silybin meglumine, or silybin disuccinate disodium salt.
6. The method for preparing silymarin freeze-dried powder according to claim 5, characterized in that, The active ingredient is silymarin meglumine.
7. The method for preparing silymarin freeze-dried powder according to claim 1, characterized in that, The co-solvent is meglumine.
8. The method for preparing silymarin freeze-dried powder according to claim 1, characterized in that, The pH adjuster is at least one of sodium hydroxide, potassium hydroxide, meglumine, hydrochloric acid, sodium carbonate, or potassium carbonate. The metal chelating agent is at least one of ethylenediaminetetraacetic acid tetrasodium, ethylenediaminetetraacetic acid disodium, and ethylenediaminetetraacetic acid.
9. The method for preparing silymarin freeze-dried powder according to claim 1, characterized in that, The active ingredient is added to the first solution, stirred to dissolve, diluted to a fixed volume, and filtered to obtain a second solution, comprising: The active ingredient is added to the first solution, stirred to dissolve, and then diluted to a final volume to obtain the third solution; Add needle-grade activated carbon to the third solution for adsorption for 30-45 min, then perform decarbonization treatment using a 1 µm titanium rod, coarse filtration treatment using a 0.45 µm filter element, and sterilization filtration treatment using a 0.22 µm filter element to obtain the second solution.
10. A lyophilized milk thistle powder, characterized in that, The silymarin freeze-dried powder is prepared by the method for preparing silymarin freeze-dried powder according to any one of claims 1 to 9.