SOD (superoxide dismutase) freeze-drying preparation and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological agents, and relates to an SOD freeze-dried preparation and a preparation method and application thereof. The invention provides an SOD (superoxide dismutase) freeze-drying preparation which is obtained by dissolving raw materials in a proper amount of solvent and then freeze-drying the raw materials, and the raw materials comprise SOD and collagen; the mass ratio of the SOD to the collagen is 1: (0.5-4). The collagen is added into the raw materials of the SOD freeze-drying preparation, so that the loss of the activity of SOD in the freeze-drying process can be well prevented, and meanwhile, the technical problem that the heat stability of the SOD freeze-drying preparation is insufficient is solved.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and relates to a freeze-dried SOD formulation, its preparation method and application. Background Technology

[0002] SOD is an abbreviation for Superoxide Dismutase, an important antioxidant enzyme widely distributed in various organisms such as animals, plants, and microorganisms. SOD possesses unique physiological activity and is the primary substance for scavenging free radicals in the body. The level of SOD in an organism is a direct indicator of aging and death; it has been confirmed that more than 60 diseases are caused by oxygen free radicals. It can combat and block the damage caused to cells by oxygen free radicals, and promptly repair damaged cells, thus reversing the damage caused by free radicals to cells.

[0003] Superoxide dismutase (SOD) is an effective scavenger of superoxide anion free radicals and is currently used in cosmetics and pharmaceuticals, but its effects are not significant because SOD is easily inactivated. Therefore, there is an urgent need to find a modifier to stabilize superoxide dismutase. CN1245831A provides a method for preparing superoxide dismutase that is heat-resistant and does not inactivate. This method involves adding 2-8% of a solid protective agent to the extracted solid powder superoxide dismutase. The solid protective agent can be any one of trehalose, sucrose, lactose, galactose, arabinose, ribose, dextran, polysucrose, vitamin C, vitamin E, lipoic acid, glutathione, and tea polyphenols. The method involves mixing them in the above proportions and then performing freeze-drying. To reduce storage and transportation costs and improve the market competitiveness of products, it is necessary to further develop SOD freeze-dried formulations with better freeze-drying protection and high-temperature stability. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by developing SOD lyophilized formulations with enhanced freeze-drying protection and high-temperature stability.

[0005] On one hand, the present invention relates to a SOD lyophilized formulation, wherein the SOD lyophilized formulation is obtained by lyophilizing raw materials after dissolving them in an appropriate amount of solvent, wherein the raw materials include: SOD and collagen; The ratio of SOD to collagen protein is 1:0.5~4.

[0006] Furthermore, in the SOD lyophilized formulation provided by the present invention, the raw material also includes a lyophilization protectant.

[0007] Furthermore, in the SOD lyophilized formulation provided by the present invention, the raw materials also include a lyophilization protectant, which is lactose and / or mannitol.

[0008] Furthermore, in the SOD lyophilized formulation provided by the present invention, the mass ratio of SOD to the lyophilization protectant is 1:0.1~30.

[0009] Furthermore, in the SOD lyophilized formulation provided by the present invention, the raw materials include: SOD, lactose, mannitol and collagen; The mass ratio of SOD, lactose, mannitol and collagen is 1:2:10:0.5~4.

[0010] On the other hand, the present invention relates to a method for preparing a SOD freeze-dried formulation, which includes: dissolving the raw material in an appropriate amount of solvent and then freeze-drying it; The freeze-drying temperature is set at -40 to -60°C, and the pre-freezing time is 20 to 40 minutes.

[0011] On the other hand, the present invention relates to the application of collagen in the preparation of SOD lyophilized formulations, wherein collagen is added to the raw materials of SOD lyophilized formulations.

[0012] Furthermore, in the application provided by the present invention, the ratio of SOD to collagen protein in the raw materials is 1:0.5~4.

[0013] Furthermore, in the application provided by this invention, collagen enhances the retention of SOD activity during the freeze-drying process.

[0014] Furthermore, in the application provided by the present invention, collagen is added to the raw materials of the SOD lyophilized formulation to improve the thermal stability of the SOD lyophilized formulation.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention achieves a breakthrough improvement in the performance of SOD lyophilized formulations by introducing collagen as a key excipient, in addition to synergistic effects with traditional lyophilization protectants. The addition of collagen not only significantly reduces SOD activity loss during lyophilization, increasing enzyme activity by more than 10% compared to the unadded group, but also greatly enhances the thermal stability of the formulation. After a 24-hour heating test at 70°C, the activity retention rate of the collagen-containing formulation was significantly higher than that of the unadded group and the group replaced by conventional protectants. Furthermore, collagen exhibits a unique regulatory ability, forming a synergistic protective mechanism with lactose / mannitol, achieving a balance between activity retention and thermal stability at the optimal ratio, overcoming the shortcomings of insufficient single protectant effects in existing technologies. Crucially, this protective effect is molecularly specific; it cannot be replaced by sugar-based protectants such as lactose, nor can it be replicated by small peptides such as glutathione. This indicates that collagen solves the technical problem of SOD inactivation during lyophilization and high-temperature storage through a unique pathway (such as forming a physical barrier or a spatially stable structure). Detailed Implementation

[0016] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.

[0017] The SOD used in the following examples was purchased from Gansu Yangtaihe Biotechnology Co., Ltd., 8000U / mg; the collagen used was purchased from Zhejiang Zhuji Juyuan Biotechnology Co., Ltd., catalog number SHH01.

[0018] Example 1 This embodiment provides a preparation process for a SOD lyophilized formulation.

[0019] Step 1: Based on the traditional ratio of SOD, lactose, and mannitol (1:2:10), add a certain proportion of collagen (1:2:10:0.5), add water at a solute concentration of 13%, dissolve completely, dispense into freeze-drying trays, and freeze at -20℃ for 1 hour.

[0020] Step 2: First, turn on the compressor to cool the cold trap. After the temperature is reduced to a certain level, place the sample in the freeze dryer, turn on the vacuum pump to evacuate the air, set the temperature to -55℃, and the temperature inside the cold trap to -55℃. Place the dispensed stock solution into the freeze dryer cold trap (freeze dryer model: Songyuan Huaxing LGJ-18), pre-freeze for 30 minutes, set the "automatic mode" program parameters, and freeze dry. After the freeze dryer is completed, the SOD freeze-dried formulation is obtained.

[0021] Example 2 This embodiment provides a preparation process for a SOD lyophilized formulation.

[0022] Step 1: Based on the traditional ratio of SOD, lactose and mannitol (1:2:10), add a certain proportion of collagen (1:2:10:1), add water at a solute concentration of 13%, dissolve completely, dispense into freeze-drying trays, and freeze at -20℃ for 1 hour.

[0023] Step 2: First, turn on the compressor to cool the cold trap. After the temperature is reduced to a certain level, place the sample in the freeze dryer, turn on the vacuum pump to evacuate the air, set the temperature to -55℃, and the temperature inside the cold trap to -55℃. Place the dispensed stock solution into the freeze dryer cold trap (freeze dryer model: Songyuan Huaxing LGJ-18), pre-freeze for 30 minutes, set the "automatic mode" program parameters, and freeze dry. After the freeze dryer is completed, the SOD freeze-dried formulation is obtained.

[0024] Example 3 This embodiment provides a preparation process for a SOD lyophilized formulation.

[0025] Step 1: Based on the traditional ratio of SOD, lactose, and mannitol (1:2:10), add a certain proportion of collagen (1:2:10:2), add water at a solute concentration of 13%, dissolve completely, dispense into freeze-drying trays, and freeze at -20℃ for 1 hour.

[0026] Step 2: First, turn on the compressor to cool the cold trap. After the temperature is reduced to a certain level, place the sample in the freeze dryer, turn on the vacuum pump to evacuate the air, set the temperature to -55℃, and the temperature inside the cold trap to -55℃. Place the dispensed stock solution into the freeze dryer cold trap (freeze dryer model: Songyuan Huaxing LGJ-18), pre-freeze for 30 minutes, set the "automatic mode" program parameters, and freeze dry. After the freeze dryer is completed, the SOD freeze-dried formulation is obtained.

[0027] Example 4 This embodiment provides a preparation process for a SOD lyophilized formulation.

[0028] Step 1: Based on the traditional ratio of SOD, lactose, and mannitol (1:2:10), add a certain proportion of collagen (1:2:10:4), add water at a solute concentration of 13%, dissolve completely, dispense into freeze-drying trays, and freeze at -20℃ for 1 hour.

[0029] Step 2: First, turn on the compressor to cool the cold trap. After the temperature is reduced to a certain level, place the sample in the freeze dryer, turn on the vacuum pump to evacuate the air, set the temperature to -55℃, and the temperature inside the cold trap to -55℃. Place the dispensed stock solution into the freeze dryer cold trap (freeze dryer model: Songyuan Huaxing LGJ-18), pre-freeze for 30 minutes, set the "automatic mode" program parameters, and freeze dry. After the freeze dryer is completed, the SOD freeze-dried formulation is obtained.

[0030] Comparative Example 1 This comparative example provides the preparation process of a SOD lyophilized formulation without added collagen.

[0031] Step 1: Based on the traditional ratio of SOD, lactose and mannitol (1:2:10), add water at a solute concentration of 13%, dissolve completely, dispense into freeze-drying trays, and freeze at -20℃ for 1 hour.

[0032] Step 2: First, turn on the compressor to cool the cold trap. After the temperature is reduced to a certain level, place the sample in the freeze dryer, turn on the vacuum pump to evacuate the air, set the temperature to -55℃, and the temperature inside the cold trap to -55℃. Place the dispensed stock solution into the freeze dryer cold trap (freeze dryer model: Songyuan Huaxing LGJ-18), pre-freeze for 30 minutes, set the "automatic mode" program parameters, and freeze dry. After the freeze dryer is completed, the SOD freeze-dried formulation is obtained.

[0033] Comparative Example 2 This comparative experiment aims to illustrate the effect of increasing the amount of conventional lyophilization protectants such as lactose and mannitol on the SOD activity and thermal stability of SOD lyophilized formulations.

[0034] This comparative example is the same as Example 4, except that collagen is replaced with lactose.

[0035] Comparative Example 3 This comparative example aims to illustrate the difference between collagen and oligopeptides in conventional freeze-drying protectants.

[0036] This comparative example is the same as Example 4, except that collagen is replaced with glutathione.

[0037] Example 5 This embodiment provides a test of SOD activity in the above-mentioned SOD lyophilized formulation.

[0038] SOD activity was determined for each freeze-dried sample, following the method specified in GB / T5009.171-2003 "Determination of Superoxide Dismutase (SOD) Activity in Health Foods". The moisture content of the freeze-dried powder was determined using an automated Karl Fischer moisture analyzer. Each experiment was repeated three times. The optimal amount of collagen added was determined based on the activity retention rate and moisture content. The experimental results are shown in Tables 1 and 2.

[0039] Table 1: Effect of collagen on SOD activity in SOD lyophilized formulations

[0040] As shown in Table 1, the addition of collagen significantly enhanced the enzyme activity of the SOD lyophilized formulation. All examples containing collagen exhibited significantly higher activity than Comparative Example 1 (without collagen), with peak activity achieved at collagen ratios of 1:2:10:1 and 1:2:10:2. Notably, replacing collagen with lactose or glutathione did not result in a significant difference in activity compared to the basic formulation, demonstrating that the protective effect of collagen is specific and cannot be replaced by conventional lyophilization protectants or oligopeptides (collagen analogs).

[0041] Table 2: Effect of collagen on moisture content in SOD lyophilized formulations

[0042] As shown in Table 2, within the range of SOD to collagen protein ratio of 1:0.5 to 4 given in this invention, the moisture content of the prepared SOD freeze-dried preparation is not higher than 3%, which meets the moisture content requirements of pharmaceutical freeze-dried products and avoids the problem of product moisture absorption, decomposition and shrinkage caused by excessive moisture content in freeze-dried products.

[0043] Example 6 This embodiment provides a thermal stability analysis of the above-mentioned SOD lyophilized formulation.

[0044] Weigh an appropriate amount of lyophilized powder and dissolve it thoroughly in water to make its initial activity 10000 U / mL. Keep it at 70℃ and take samples at 1h, 2h, 4h, 8h, 16h and 24h for activity determination. Calculate the activity retention rate and determine the differences in SOD activity retention among different formulations.

[0045] Table 3: Activity retention rate (%) of different formulations

[0046] Table 3 shows that collagen significantly enhances the stability of SOD lyophilized formulations at high temperatures. After incubation at 70°C for 24 hours, the activity retention rates of all collagen-containing examples were significantly higher than those of Comparative Example 1 without collagen, with Example 2, containing a collagen ratio of 1:2:10:1, showing the best performance. Although high-proportion collagen exhibits better short-term (1-4h) stability, its long-term (16-24h) retention rate significantly decreases, indicating that excessive addition may weaken the protective effect. The thermal stability curves of the lactose or glutathione substitution groups almost perfectly overlap with the basic formulation, further confirming the irreplaceable role of collagen in heat protection.

[0047] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A SOD lyophilized formulation, wherein the SOD lyophilized formulation is obtained by lyophilizing a raw material dissolved in an appropriate amount of solvent, characterized in that, The raw materials include: SOD and collagen; The ratio of SOD to collagen protein is 1:0.5~4.

2. The SOD lyophilized formulation according to claim 1, characterized in that, The raw materials also include freeze-drying protectants.

3. The SOD lyophilized formulation according to claim 1, characterized in that, The raw materials also include a freeze-drying protectant, which is lactose and / or mannitol.

4. The SOD lyophilized formulation according to claim 3, characterized in that, The mass ratio of SOD to the freeze-drying protectant is 1:0.1~30.

5. The SOD lyophilized formulation according to claim 4, characterized in that, The raw materials include: SOD, lactose, mannitol and collagen; The mass ratio of SOD, lactose, mannitol and collagen is 1:2:10:0.5~4.

6. The method for preparing the SOD lyophilized formulation according to any one of claims 1 to 5, characterized in that, include: The raw material is dissolved in an appropriate amount of solvent and then freeze-dried. The freeze-drying temperature is set at -40 to -60°C, and the pre-freezing time is 20 to 40 minutes.

7. The application of collagen in the preparation of SOD lyophilized formulations, characterized in that, Collagen is added to the raw materials of SOD freeze-dried formulations.

8. The application according to claim 7, characterized in that, In the raw materials, the ratio of SOD to collagen protein is 1:0.5~4.

9. The application according to claim 7, characterized in that, Collagen enhances the retention of SOD activity during freeze-drying.

10. The application according to claim 7, characterized in that, Adding collagen to the raw materials of SOD lyophilized formulations improves the thermal stability of the SOD lyophilized formulations.

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