A freeze-dried preparation of salvianolic acid C, a preparation method and application thereof

Abstract

The present application relates to a kind of salvianolic acid C freeze-dried preparation and preparation method and application, the salvianolic acid C freeze-dried preparation includes salvianolic acid C, pH adjusting agent, stabilizer and excipient.The salvianolic acid C freeze-dried preparation provided in the present application is traditional Chinese medicine extract, and each additive is suitable for administration route, component is safe, and good stability can be achieved, suitable for long-term storage, process is simple, reproducibility is good, easy to large-scale industrial production.The salvianolic acid C freeze-dried preparation of the present application can be injected to administration, also can be atomized to give human respiratory tract, provide a kind of new treatment means for anti respiratory tract influenza virus such as influenza virus A H1N1, influenza virus A H3N2, influenza virus B, parainfluenza virus, respiratory syncytial virus, adenovirus, etc., and have good curative effect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a lyophilized formulation of salvianolic acid C, its preparation method, and its application. Background Technology

[0002] Danshen (Salvia miltiorrhiza Bge.), a plant in the Lamiaceae family, is a dried root and rhizome that possesses various medicinal properties, including promoting blood circulation, removing blood stasis, relieving pain, clearing the mind and relieving irritability, cooling the blood and reducing swelling. Its active ingredients are mainly lipid-soluble diterpenoid quinones and water-soluble phenolic acids, as well as flavonoids, triterpenes, and sterols. The water-soluble components of Danshen mainly include tanshinone, protocatechuic aldehyde, rosmarinic acid, shikonin, and salvianolic acid A, B, C, D, E, F, G, H, and I. Studies have shown that salvianolic acid C can inhibit NF-κB activation through the AMPK / Nrf2 signaling pathway, thereby achieving anti-inflammatory effects and inhibiting the production of lipopolysaccharide-induced inflammatory cytokines. Furthermore, activation of the Nrf2 signaling pathway can increase HO-1 and NQO1 levels, promoting the clearance of intracellular ROS and mitochondrial superoxide; other literature reports that salvianolic acid C effectively inhibits SARS-CoV-2 infection by blocking the formation of the spike protein hexahelical bundle core.

[0003] The chemical substances in Danshen have the following characteristics: (1) The polyphenolic acid components in Danshen are generally unstable to heat, acidity, alkalinity and solution state. High temperature, acid and alkali treatment and aqueous solution preparation in the preparation process have a great influence on the changes and degradation of the water-soluble components of Danshen, resulting in great differences in the chemical components or effective components contained in the final drug; (2) The phenolic acid compounds contained in Danshen are rich in phenolic hydroxyl groups, and are prone to chemical changes such as hydrolysis and oxidation during the extraction and separation process; (3) The phenolic acid components in Danshen are unstable, especially in aqueous solution, they are prone to degradation and interconversion.

[0004] Due to the aforementioned physicochemical properties, the stability of salvianolic acid C preparations is a key technology in their preparation. Currently, there is limited research on salvianolic acid C preparations, and their formulations and processes are not publicly available; furthermore, no salvianolic acid C preparations are currently on the market. Therefore, developing a stable and effective salvianolic acid C preparation to realize its clinical application value and fill the current market gap is an urgent problem to be solved. Summary of the Invention

[0005] One of the objectives of this invention is to overcome the shortcomings of the prior art and provide a lyophilized preparation of salvianolic acid C, which can be used safely in clinical practice and has stable quality and can be stored for a long time.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a lyophilized formulation of salvianolic acid C, which comprises salvianolic acid C, a pH adjuster, a stabilizer, and an excipient.

[0008] Preferably, the content of salvianolic acid C in the unit dose of the salvianolic acid C lyophilized preparation is 0.2-50 mg, such as 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, etc., and more preferably 5-50 mg.

[0009] Preferably, the water content of the lyophilized Danshensu C preparation is below 7%, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, etc., more preferably below 5%, and more preferably below 3%.

[0010] By optimizing the water content of the lyophilized salvianolic acid C formulation, this invention can further improve the product's properties and enhance its storage stability.

[0011] Preferably, the pH adjuster includes an inorganic acid or an organic acid-base buffer, with organic acid-base buffers being more preferred.

[0012] Preferably, the inorganic acid includes any one or at least a combination of two of hydrochloric acid, phosphoric acid, or sulfuric acid.

[0013] Preferably, the organic acid-base buffer comprises a combination of tartaric acid and sodium tartrate, a combination of citric acid and sodium citrate, or a combination of phosphate and sodium dihydrogen phosphate, with the combination of tartaric acid and sodium tartrate or the combination of citric acid and sodium citrate being more preferred.

[0014] This invention can further improve sample properties and storage stability by selecting specific pH adjusters.

[0015] Preferably, the pH of the lyophilized Danshensu C preparation after reconstitution is 3.0-5.5, such as 3.5, 4.0, 4.5, 5.0, etc., preferably 4.0-5.0.

[0016] This invention further improves the storage stability of the product by optimizing the pH value of the reconstituted lyophilized formulation. If the pH value is below 3.0, the pH change during storage increases; if the pH value exceeds 6.0, some degradation will occur. The formulation exhibits optimal stability when the pH is in the range of 4.0-5.0.

[0017] Preferably, the stabilizer comprises disodium edetate and / or sodium bisulfite, with disodium edetate being more preferred.

[0018] The present invention preferably uses disodium edetate as a stabilizer. Compared with other stabilizers, it can achieve higher stability with a smaller amount added, and the impurity content is lower after being placed at 25°C for 5 days.

[0019] Preferably, the amount of stabilizer used is 0.5-3 mg / mL, based on the volume of the solvent used to prepare the salvianolic acid C lyophilized agent.

[0020] Preferably, the excipient comprises sugar alcohols or amino acid compounds.

[0021] Preferably, the sugar alcohol compound includes mannitol.

[0022] Preferably, the amino acid compound includes glycine or arginine.

[0023] Preferably, the excipient comprises any one or at least two combinations of mannitol, arginine, or glycine, with mannitol and / or glycine being more preferred.

[0024] This invention further optimizes the types of excipients to improve the formability of lyophilized salvianolic acid C formulations, with mannitol or glycine showing the best results.

[0025] Preferably, the lyophilized formulation of salvianolic acid C further contains a solubilizer.

[0026] Preferably, the solubilizer comprises polyol compounds or amino acid compounds.

[0027] Preferably, the polyol compound includes propylene glycol or glycerol.

[0028] Preferably, the amino acid compound includes lysine hydrochloride, glycine, or arginine.

[0029] Preferably, the solubilizer comprises any one or at least two combinations of glycerol, propylene glycol, lysine hydrochloride, arginine, or glycine, more preferably any one or at least two combinations of propylene glycol, lysine hydrochloride, arginine, or glycine, and preferably propylene glycol and / or glycine.

[0030] By further optimizing the type of solubilizer, this invention can further improve the solubility performance, which not only enables the material to dissolve quickly and completely during the preparation of lyophilized formulations, but also facilitates the rapid completion of lyophilized agent reconstitution.

[0031] Preferably, the amount of excipient used is 15-50 mg / mL, based on the volume of the solvent used to prepare the salvianolic acid C lyophilized agent.

[0032] Preferably, the amount of solubilizer used is 15-40 mg / mL, based on the volume of the solvent used to prepare the salvianolic acid C lyophilizer.

[0033] A second objective of this invention is to provide a method for preparing the lyophilized formulation of salvianolic acid C as described in one objective, the method comprising the following steps:

[0034] (1) Add solvent to the dispensing container, slowly add the prescribed amount of stabilizer and excipient, and stir until they are completely dissolved;

[0035] (2) Add the prescribed amount of buffer to adjust the pH of the system;

[0036] (3) Add the prescribed amount of salvianolic acid C and stir until it is completely dissolved; add solvent to make up to the total prescribed amount and stir to mix it evenly.

[0037] (4) Aseptic filtration, filling, and transfer to a freeze dryer;

[0038] (5) Pre-freeze;

[0039] (6) Sublimation: Vacuuming begins while heating is being carried out;

[0040] (7) Desorption and drying: Continuous vacuuming, heating, and desorption;

[0041] (8) Plug in the cap, roll in the lid, and end the freeze-drying process.

[0042] Preferably, step (1) further includes adding a prescribed amount of solubilizer to the solvent.

[0043] Preferably, in step (2), the pH value of the system is adjusted to 3.0-5.5, more preferably 4.0-5.0.

[0044] Preferably, the solvent used in steps (1) and (3) is water for injection.

[0045] Preferably, the temperature of the solution preparation process in steps (1) to (3) is controlled at 15-35℃, such as 20℃, 25℃, 30℃, etc.

[0046] Preferably, in step (4), the aseptic filtration includes: primary filtration using a 0.45μm to 0.8μm filter membrane and fine filtration using a 0.22μm to 0.45μm filter membrane.

[0047] Preferably, in step (4), the aseptic filtration includes: primary filtration using a 0.45 μm filter membrane and fine filtration using a 0.22 μm filter membrane.

[0048] Preferably, in step (5), the pre-freezing temperature is -50℃ to -40℃, such as -42℃, -44℃, -46℃, -48℃, etc., and more preferably -50℃ to -45℃.

[0049] Preferably, in step (5), the pre-freezing time is 4 to 8 hours, such as 5 hours, 6 hours, 7 hours, etc., and preferably 6 to 8 hours.

[0050] Preferably, in step (6), the temperature is raised to -25℃ to -15℃, for example -16℃, -17℃, -18℃, -19℃, -20℃, -21℃, -22℃, -23℃, -24℃, etc., preferably -20℃ to -15℃.

[0051] Preferably, in step (6), the sublimation time is 15-30 hours, such as 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, etc., and more preferably 20-30 hours.

[0052] Preferably, in step (7), the temperature is raised to 20℃~30℃, for example 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, etc., and more preferably 25℃~30℃.

[0053] Preferably, in step (7), the drying time is 6-10 hours, such as 7 hours, 8 hours, 9 hours, etc., and more preferably 8-10 hours.

[0054] Preferably, the preparation method includes the following steps:

[0055] (1) Add solvent to the dispensing container, slowly add the prescribed amount of stabilizer, excipient and solubilizer, and stir until they are completely dissolved;

[0056] (2) Add the prescribed amount of buffer to adjust the pH of the system to 3.0-5.5;

[0057] (3) Add the prescribed amount of salvianolic acid C and stir until it is completely dissolved; add solvent to make up to the total prescribed amount and stir to mix it evenly.

[0058] (4) Perform primary filtration with a 0.45μm filter membrane, fine filtration with a 0.22μm filter membrane, and after aseptic filtration, fill and transfer to a freeze dryer;

[0059] (5) Pre-freezing: Pre-freeze at -50℃~-40℃ for 4~8 hours;

[0060] (6) Sublimation: While heating, vacuuming is started, and the temperature is raised to -25℃~-15℃, with a total time of 15-30h.

[0061] (7) Desorption and drying: Continue vacuuming and heat to 20℃~30℃ for 6-10 hours;

[0062] (8) Stopper, cap, and end freeze-drying;

[0063] The solvent used in steps (1) and (3) is sterile water for preparation, and the temperature is controlled at 15-35℃ during the solution preparation process in steps (1) to (3).

[0064] Preferably, the preparation method includes the following steps:

[0065] (1) Add solvent to the dispensing container, slowly add the prescribed amount of stabilizer, excipient and solubilizer, and stir until they are completely dissolved;

[0066] (2) Add the prescribed amount of buffer to adjust the pH of the system to 4.0-5.0;

[0067] (3) Add the prescribed amount of salvianolic acid C and stir until it is completely dissolved; add solvent to make up to the total prescribed amount and stir to mix it evenly.

[0068] (4) Perform primary filtration with a 0.45μm filter membrane, fine filtration with a 0.22μm filter membrane, and after aseptic filtration, fill and transfer to a freeze dryer;

[0069] (5) Pre-freezing: Pre-freeze at -50℃~-45℃ for 6~8 hours;

[0070] (6) Sublimation: While heating, vacuuming is started, and the temperature is raised to -20℃~-15℃ for a total of 20-30 hours;

[0071] (7) Desorption and drying: Continue vacuuming and heat to 25℃~30℃ for 8-10 hours;

[0072] (8) Stopper, cap, and end freeze-drying;

[0073] The solvent used in steps (1) and (3) is water for injection, and the temperature of the solution preparation process in steps (1) to (3) is controlled at 15-35℃.

[0074] A third objective of this invention is to provide an inhalation system or injection drug delivery system for nebulizing a salvianolic acid C solution into the human respiratory tract, comprising 1 ml to 5 ml of salvianolic acid C solution after reconstitution of the lyophilized salvianolic acid C agent described in one objective.

[0075] Preferably, the inhalation system for administering the salvianolic acid C solution to the human respiratory tract further includes a liquid nebulizer. After the reconstituted salvianolic acid C solution is nebulized using the liquid nebulizer, its aerodynamic mass has a diameter of 2-4 μm, such as 2 μm, 3 μm, 4 μm, etc., and the percentage of fine particles is 40%-70%, such as 40%, 50%, 60%, 70%, etc.

[0076] Compared with the prior art, the present invention has the following beneficial effects:

[0077] The lyophilized preparation of salvianolic acid C provided by this invention contains salvianolic acid C, which is a traditional Chinese medicine extract. All additives are suitable for the route of administration, the components are safe, and they can achieve good stability, making them suitable for long-term storage. The process is simple, reproducible, and easy for large-scale industrial production.

[0078] The lyophilized formulation of salvianolic acid C of this invention fills the gap in current salvianolic acid C formulations. Furthermore, the lyophilized agent provided by this invention can be administered by injection or nebulized to the human respiratory tract, offering a novel treatment method for respiratory influenza viruses such as H1N1 influenza A virus, H3N2 influenza A virus, influenza B virus, parainfluenza virus, respiratory syncytial virus, and adenovirus, with good efficacy. The nebulized inhalation system for salvianolic acid C solution also provides a safe and effective drug formulation and delivery system for infants and young children without spontaneous inhalation ability and patients with difficulty in active breathing. Detailed Implementation

[0079] The present invention will be further described in detail below through embodiments. These embodiments are only used to illustrate the present invention and do not limit the scope of the present invention.

[0080] Example 1 pH Screening

[0081] This embodiment provides lyophilized formulations of salvianolic acid C prepared under different pH conditions, with the following formulations:

[0082] name Dosage Tanshinone C 1g Citric acid 0.12g Sodium citrate Adjust pH Sodium edetate 0.05g Mannitol 2g Add water for injection to 100ml

[0083] The specific preparation method is as follows:

[0084] Control the water temperature at 30℃, add the prescribed amount of stabilizer and excipient to 80% of the prescribed amount of water for injection, stir, and after dissolving, adjust the pH with a buffer, stir evenly, add the prescribed amount of salvianolic acid C, stir to dissolve, and then add water to the required volume. The resulting solution is initially filtered through a 0.45μm filter membrane and then finely filtered through a 0.22μm filter membrane. The filtered solution is filled into 10ml vials, pre-frozen at -50℃ for 6 hours, then vacuum is applied, and the temperature is raised to -15℃ for sublimation for 20 hours, then raised to 25℃ for desorption for 6 hours, stoppered, capped, and the freeze-drying process is complete.

[0085] Based on the above formulation and preparation method, lyophilized salvianolic acid C with pH values ​​of 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 were prepared using a citrate-sodium citrate buffer system. The solubility of salvianolic acid C in aqueous solutions at different pH values ​​was determined, and the pH value and related substances of the lyophilized salvianolic acid C preparation after reconstitution and placement at 25°C for 8 hours were measured to evaluate the stability of the lyophilized preparation. The results are shown in Table 1.

[0086] Table 1. Solubility and stability of salvianolic acid C in aqueous solutions at different pH values

[0087]

[0088] Experimental results show that the solubility of salvianolic acid C gradually increases with increasing pH value. When pH < 3.0, the pH changes significantly during storage; when pH exceeds 5.5, degradation occurs. Therefore, while ensuring the solubility of salvianolic acid C, the influence of pH value on product stability must be considered. Thus, this invention preferably uses a pH value between 3.0 and 5.5 after reconstitution of the salvianolic acid C lyophilized agent, and more preferably between 4.0 and 5.0.

[0089] Example 2 Stabilizer Screening

[0090] Sodium edetate and sodium bisulfite were added to the formulation as stabilizers to investigate their effects on the stability of the solution after reconstitution of the lyophilized Danshensu C preparation. The preparation method of the lyophilized preparation was the same as that in Example 1. The results are shown in Table 2.

[0091] Table 2 Stabilizer Screening

[0092]

[0093] The results showed that the addition of disodium edetate and sodium bisulfite to the formulation could make salvianolic acid C more stable. Among them, the formulation with disodium edetate had lower impurity content and better stability after being placed at 25°C for 5 days. Therefore, the present invention prefers disodium edetate as a stabilizer.

[0094] Example 3: Screening of Excipients and Solubilizers

[0095] The formulation of the salvianolic acid C lyophilizing agent provided in this embodiment is shown in Table 3, and the preparation method is as follows:

[0096] Controlling the water temperature at 30℃, add the prescribed amount of stabilizer, excipient, and solubilizer to 80% of the prescribed amount of water for injection, stir, and after dissolving, adjust the pH with a buffer, stir evenly, add the prescribed amount of salvianolic acid C, stir to dissolve, and then add water to the required volume. The resulting solution is initially filtered through a 0.45μm filter membrane and then finely filtered through a 0.22μm filter membrane. The filtered solution is filled into 10ml vials, pre-frozen at -50℃ for 6 hours, then vacuum is applied while simultaneously raising the temperature to -15℃ for sublimation for 20 hours, then raising the temperature to 25℃ for desorption for 6 hours, stoppering, capping, and ending the freeze-drying process.

[0097] The properties and reconstitution of each sample after freeze-drying were observed, and the results are shown in Table 3.

[0098] Table 3 Screening of Excipients and Solubilizing Substances

[0099]

[0100]

[0101] Comparative experiments 1, 2, 3, 4, 8, 9, and 10 showed that using glycerol, propylene glycol, lysine hydrochloride, arginine, and glycine as solubilizers, salvianolic acid C could be completely dissolved within 60 minutes. This indicates that glycerol, propylene glycol, lysine hydrochloride, arginine, and glycine all have a solubilizing effect on salvianolic acid C. Among these, using propylene glycol, lysine hydrochloride, arginine, and glycine as solubilizers resulted in a shorter dissolution time, indicating a stronger solubilizing effect. Furthermore, the experimental results show that propylene glycol and glycine are more conducive to obtaining lyophilized agents with superior properties.

[0102] The comparison of experiments 5, 6, 7, 8, 9, and 10 shows that samples can be molded using mannitol, arginine, and glycine as excipients, with mannitol and glycine exhibiting better moldability.

[0103] Based on the above results, the preferred solubilizers of the present invention are glycerol, propylene glycol, lysine hydrochloride, arginine, and glycine, more preferably propylene glycol, lysine hydrochloride, arginine, and glycine, and even more preferably propylene glycol or glycine; the preferred excipients are mannitol, arginine, and glycine, more preferably mannitol or glycine.

[0104] Example 4: Screening of pH Adjuster Types

[0105] Citric acid-sodium citrate, tartaric acid-sodium tartrate, phosphate-sodium dihydrogen phosphate buffer systems, and hydrochloric acid were used as pH adjusters, respectively. The solutions were prepared and lyophilized using the same method as in Example 3. The effects of different pH adjusters on the properties of the samples were investigated. The lyophilized samples were placed at 40°C for 30 days to observe the pH changes.

[0106] Table 4 Screening of pH Adjuster Types

[0107]

[0108] The results showed that, compared with hydrochloric acid, pH adjustment using citrate-sodium citrate, tartaric acid-sodium tartrate, and phosphate-sodium dihydrogen phosphate buffer systems resulted in good sample properties and stability, with the citrate-sodium citrate and tartaric acid-sodium tartrate buffer systems exhibiting superior properties.

[0109] Example 5: Freeze-drying process and moisture content study

[0110] Samples were prepared using the same formulation, and the freeze-drying process was adjusted based on the preparation method in Example 3 to obtain samples with different moisture contents. The effects of freeze-drying process on moisture content and the effects of moisture content on sample stability were investigated.

[0111] Prescription composition:

[0112]

[0113]

[0114] Table 5. Moisture content determination results of freeze-dried samples

[0115] Serial Number freeze-drying process Moisture (%) 1 Pre-freezing: -45℃, 4h → Sublimation: -25℃, 12h → Desorption: 15℃, 6h 9.89 2 Pre-freezing: -40℃, 4h → Sublimation: -25℃, 15h → Desorption: 20℃, 8h 6.77 3 Pre-freezing: -50℃, 6h → Sublimation: -15℃, 20h → Desorption: 25℃, 6h 4.96 4 Pre-freezing: -45℃, 8h → Sublimation: -20℃, 25h → Desorption: 30℃, 8h 2.71 5 Pre-freezing: -45℃, 8h → Sublimation: -15℃, 30h → Desorption: 30℃, 10h 1.38

[0116] The above freeze-dried formulations with different moisture content were placed at 40℃-75%RH for 6 months to investigate their stability. The results are shown in Table 6.

[0117] Table 6. Results of stability studies on samples with different moisture content

[0118]

[0119]

[0120] Table 6 shows that during the preparation of the lyophilized formulation of salvianolic acid C, the following steps were performed: pre-freezing at -40℃ to -50℃ for 4-6 hours; sublimation at -15℃ to -25℃ for 15-30 hours; and desorption at 20℃ to 30℃ for 6-10 hours. The resulting lyophilized formulation had a moisture content of less than 7% and exhibited good properties and stability. Based on these results, a moisture content of less than 7% is preferred for the lyophilized formulation of salvianolic acid C, further preferred to be less than 5%, and even more preferred to be less than 3%.

[0121] Example 6: 250 vials of lyophilized Danshenol C formulation

[0122] Prescription composition:

[0123] name Dosage Tanshinone C 0.05g Citric acid 0.6g Sodium citrate Adjust the pH to 4.5. Sodium edetate 0.25g Mannitol 25g Add water for injection to 500ml

[0124] Preparation process:

[0125] Control the water temperature at 15℃, add the prescribed amounts of disodium edetate, citric acid, and mannitol to 80% of the prescribed amount of water for injection, stir, and after dissolving, adjust the pH with sodium citrate aqueous solution, stir evenly, add the prescribed amount of salvianolic acid C, stir to dissolve, and then add water to the required volume. Filter the resulting solution through a sterile filter, fill the filtered solution into 10ml vials, pre-freeze at -40℃ for 6 hours, then begin vacuuming, and simultaneously raise the temperature to -15℃ for sublimation for 20 hours, then raise the temperature to 20℃ for desorption for 6 hours, stopper, crimp, and end the freeze-drying process.

[0126] Example 7: 250 vials of lyophilized Danshenol C formulation

[0127] Prescription composition:

[0128]

[0129]

[0130] Preparation process:

[0131] Control the water temperature at 25℃. Add the prescribed amounts of disodium edetate, tartaric acid, glycerin, and mannitol to 80% of the prescribed amount of water for injection. Stir and dissolve. Adjust the pH with sodium tartrate aqueous solution. Stir until homogeneous. Add the prescribed amount of salvianolic acid C. Stir until dissolved. Add water to the required volume. Filter the resulting solution through a sterile filter. Fill the filtered solution into 10ml vials. Pre-freeze at -45℃ for 6 hours. Then, begin vacuuming and simultaneously raise the temperature to -20℃ for sublimation for 20 hours. Then, raise the temperature to 30℃ for desorption for 6 hours. Stopper and cap the vials to complete the freeze-drying process.

[0132] Example 8: 250 vials of lyophilized Danshenol C formulation

[0133] Prescription composition:

[0134] name Dosage Tanshinone C 5g Citric acid 0.6g Sodium citrate Adjust the pH to 5.0. Propylene glycol 20g Sodium edetate 0.25g Mannitol 10g Add water for injection to 500ml

[0135] Preparation process:

[0136] Control the water temperature at 35℃. Add the prescribed amounts of disodium edetate, citric acid, propylene glycol, and mannitol to 80% of the prescribed amount of water for injection. Stir and dissolve. Adjust the pH with sodium citrate aqueous solution. Stir until homogeneous. Add the prescribed amount of salvianolic acid C. Stir until dissolved. Add water to the required volume. Filter the resulting solution through a sterile filter. Fill the filtered solution into 10ml vials. Pre-freeze at -50℃ for 8 hours. Then, begin vacuuming and simultaneously raise the temperature to -25℃ for sublimation for 30 hours. Then, raise the temperature to 25℃ for desorption for 10 hours. Stopper and cap the vials to complete the freeze-drying process.

[0137] Example 9: 100 vials of lyophilized Danshenol C formulation

[0138] Prescription composition:

[0139]

[0140]

[0141] Preparation process:

[0142] Control the water temperature at 30℃. Add the prescribed amounts of disodium edetate, citric acid, and glycine to 80% of the prescribed amount of water for injection, stir, and after dissolving, adjust the pH with sodium citrate aqueous solution. Stir evenly, add the prescribed amount of salvianolic acid C, stir to dissolve, and then add water to the required volume. Filter the resulting solution through a sterile filter, fill the filtered solution into 20ml vials, pre-freeze at -50℃ for 8 hours, then begin vacuuming, and simultaneously raise the temperature to -15℃ for sublimation for 30 hours. Then raise the temperature to 30℃ for desorption for 10 hours, stopper, crimp, and end the freeze-drying process.

[0143] Example 10 Stability Study

[0144] The freeze-dried formulations prepared according to Examples 6, 7, 8 and 9 of this application were placed at 40℃-75%RH and 25℃-60%RH for 6 months to examine their stability. The results are shown in Table 7.

[0145] Table 7. Results of stability tests

[0146]

[0147]

[0148] As shown in Table 8, the formulations prepared according to Examples 6, 7, 8 and 9 showed no significant changes in appearance, pH and related substances, indicating good stability.

[0149] Example 11 Atomization Characteristics Measurement

[0150] The aerodynamic particle size distribution of the samples in Examples 7, 8, and 9 was determined using a NeoPatek particle size analyzer; the total delivery amount and fine particle dose were determined using an NGI pharmaceutical impactor. The results are as follows:

[0151] Table 8 Results of atomization characteristic measurement

[0152]

[0153] Note: X50 represents the aerodynamic mass median diameter.

[0154] The results showed that after reconstitution and atomization, the median diameter (X50) of the salvianolic acid C lyophilizer of the present invention was between 2 and 4 μm, and the percentage of fine particles was between 40% and 70%.

[0155] Example 12: Inhibitory effect of salvianolic acid C on respiratory viruses in A549 cells cultured in vitro.

[0156] 1. Experimental Materials

[0157] 1.1 Test Sample

[0158]

[0159] 1.2 Virus strains and cell strains

[0160] 1.2.1 Virus strains: Influenza A virus strains FM1 (H1N1 subtype), PR8 (H1N1 subtype), brisbane / 10 / 2009 (H3N2 subtype), Influenza B virus strain Jiangxi Xiushui / 32 / 2009, parainfluenza virus, respiratory syncytial virus, and adenovirus were all purchased from the American Type Culture Collection (ATCC) with TCID50 values ​​of 10⁻⁴, 10⁻⁴, 10⁻⁴, 10⁻⁴, 10⁻⁴, 10⁻⁴, 10⁻⁶, and 10⁻⁴. These strains were passaged in our laboratory and stored at -80°C for later use.

[0161] 1.2.2 Cell line: Human lung cancer cells (A549) were purchased from Beijing Beina Chuanglian Biotechnology Research Institute, passaged in our laboratory, and stored in liquid nitrogen for future use.

[0162] 1.3 Test Reagents

[0163] Reagent Name Manufacturer batch number Reagent Uses fetal bovine serum SIGMA-ALDRICH, USA 2300496P Cell culture DMEM medium Gibco USA 8120350 A549 cell culture 0.25% trypsin-EDTA Gibco USA 2186974 Cell culture Penicillin-Streptomycin NewSaiMei Biotechnology Co., Ltd. 20201016 Cell culture

[0164] 1.4 Test Instruments

[0165] Instrument Name Manufacturer model Instrument Applications Type A2 biosafety cabinet Thermo Technologies, Inc. 1374 Virus infection operation <![CDATA[CO2 Incubator]]> Thermo Technologies, Inc. 371 Cell culture after viral infection Inverted microscope Olympus, Inc. (USA) CKX53 Observation of lesions after viral infection Clean bench Jinan Xinbeixi Biotechnology Co., Ltd. BBS-SDC Cell culture <![CDATA[CO2 Incubator]]> Thermo Technologies, Inc. 371 Cell culture Inverted fluorescence microscope Olympus, Inc. (USA) IX73 Observation of cytopathic effects Small benchtop high-speed centrifuge Eppendorf, Germany 5430 Cell passage Handheld cell counter Millipore, USA Scepter 2.0 Cell Count

[0166] 2. Experimental methods and results

[0167] Take culture plates that have grown into monolayers of cells, discard the culture medium, and inoculate them with different dilutions of influenza virus solution: FM1 strain 10⁻² dilution, PR8 strain 10⁻² dilution, Jiangxi Xiushui / 32 / 2009 strain 10⁻² dilution, parainfluenza virus 10⁻² dilution, respiratory syncytial virus 10⁻⁴ dilution, adenovirus 10⁻² dilution, and brisbane / 10 / 2009 10⁻² dilution, 100 μL of each strain per well. Incubate at 37°C in a 5% CO₂ incubator for 1 hour for adsorption. Discard the virus solution, rinse the cell surface three times with cell maintenance medium, and then add 100 μL of the corresponding dilution of salvianolic acid C per well. Normal cell control and virus control are set up, and ribavirin and oseltamivir phosphate capsules (Tamiflu) are used as positive controls. Incubate at 37℃ in a 5% CO2 incubator. Observe the cytopathic effect of the cells daily under an inverted microscope. Record the experimental results when the cytopathic effect of the virus control cells reaches ++++.

[0168] Cellular lesions are judged according to a 6-grade standard:

[0169] - Cell growth is normal, and no lesions are observed;

[0170] ±: Cellular lesions account for less than 10% of the entire monolayer;

[0171] +: Cytopathic effects account for less than 25% of the entire monolayer of cells;

[0172] ++: Cytopathic effects account for less than 50% of the entire monolayer of cells;

[0173] +++: Cytopathic effects account for less than 75% of the entire monolayer of cells;

[0174] ++++: Cytopathic effects account for more than 75% of the entire monolayer of cells.

[0175] The 50% inhibitory concentration (IC50) was calculated using the Reed-Muench method. 50 ) and the therapeutic index (TI), TI = TC 50 / IC 50

[0176] Table 9. Inhibitory effects of salvianolic acid C on A549 cell cytopathic effects induced by different viruses.

[0177]

[0178] Drug concentration unit: mg·mL -1 Table 10. Inhibitory effect of salvianolic acid C on virus in A549 cells cultured in vitro.

[0179]

[0180] IC 50 mg·mL -1 Note: "FM1" refers to the FM1 strain of influenza A virus (H1N1 subtype).

[0181] "PR8" refers to the PR8 strain of influenza A virus (H1N1 subtype).

[0182] The strain identified as "Jiangxi Xiushui" is the Jiangxi Xiushui / 32 / 2009 strain of influenza B virus.

[0183] "Brisbane10" refers to the Brisbane / 10 / 2009 strain of influenza A (H3N2 subtype).

[0184] "Parasyl influenza" refers to the parainfluenza virus.

[0185] "RSV" refers to respiratory syncytial virus.

[0186] "Ad3" refers to Ad3 adenovirus.

[0187] Tables 9 and 10 show that salvianolic acid C had a slight inhibitory effect on cytopathic effects caused by influenza A strain brisbane / 10 / 2009 (H3N2 subtype) in the A549 cell model. Its IC50 was calculated to be [value missing]. 50 The concentration was 0.089 mg·mL⁻¹, with a TI value of 2.0; it showed significant inhibitory effect on respiratory syncytial virus-induced cytopathic effects, and its IC₂O₃ value was calculated to be 0.089 mg·mL⁻¹. 50The concentration was 0.045 mg·mL⁻¹, with a TI value of 4.0. It showed significant inhibitory effects on influenza A virus strain FM1 (H1N1 subtype), PR8 (H1N1 subtype), influenza B virus strain Jiangxi Xiushui / 32 / 2009, and adenovirus cytopathic effects. The calculated IC₂ value was [value missing]. 50 All were 0.022 mg·mL⁻¹, with a TI value of 8.0; they showed significant inhibitory effects on parainfluenza virus-induced cytopathic effects, and their IC₂ values ​​were calculated to be [value missing]. 50 The concentration was 0.011 mg·mL⁻¹, and the TI value was 16.0.

[0188] Example 13: Therapeutic effect of lyophilized Danshensu C formulation on a mouse model of pneumonia induced by respiratory virus infection.

[0189] 1. Experimental Materials

[0190] 1.1 Test Sample

[0191] Samples were prepared based on Example 7, and the specific information is as follows:

[0192] Sample Name batch number Specification Production date Lyophilized preparation of salvianolic acid C 230502 10mg 2023.05.25

[0193] 1.2 Virus strain

[0194] The H1N1 influenza A virus FM1 strain (ATCC: VR-1754) and the H3N2 influenza A virus (ATCC: VR-1811) were both obtained from the ATCC Virus Collection in the United States; the parainfluenza virus and respiratory syncytial virus were both obtained from the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. These were passaged in our laboratory and stored at -80°C for future use.

[0195] 1.3 Experimental Animals

[0196] ICR mice, SPF grade, 13-15g, 40 mice, half male and half female, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0197] NIH mice, SPF grade, 12-14g, 40 mice, half male and half female, were purchased from Speford (Beijing) Biotechnology Co., Ltd.

[0198] BALB / c mice, SPF grade, 13-15g, 40 mice, half male and half female, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0199] 1.4 Test Reagents

[0200]

[0201]

[0202] 1.5 Test Instruments

[0203] Instrument Name Manufacturer model Instrument Applications Type A2 biosafety cabinet Shandong Boke Biotechnology Co., Ltd. 11229BBC86 Mouse infection procedure Type A2 biosafety cabinet Shandong Boke Biotechnology Co., Ltd. 11229BBC86 Mouse nebulized protection Type A2 biosafety cabinet Thermo Technologies, Inc. 1379 Mouse dissection Type A2 biosafety cabinet Thermo Technologies, Inc. 1374 Viral nucleic acid testing IVC rat cage Tennessee SMARTFLOW Mouse feeding Animal oral-nasal inhalation exposure system Shanghai Meili Experimental Technology Co., Ltd. InhalogicNIES Nebulized drug delivery to mice atomizer German PARI Blue Core Nebulized drug delivery to mice Electronic balance Sartorius BSA3202S-CW Weigh the mouse Electronic balance Sartorius BSA323S-CW Weigh the mouse lungs High-throughput tissue homogenizer Ningbo Xinzhi Scientz-48 Lung tissue grinding Tabletop high-speed refrigerated centrifuge Eppendorf 5810R Separate the supernatant Real-Time PCR Instrument Appliedbiosystems QuantStudio5 Viral nucleic acid testing Eight-tube mini centrifuge Kylin-BellLabInstruments LX-300 Viral nucleic acid testing

[0204] 2. Experimental methods and results

[0205] 2.1 Experimental grouping and drug administration

[0206] 2.1.1 Therapeutic effects on mouse models of pneumonia induced by H1N1 influenza A virus FM1 strain and H3N2 influenza A virus infection.

[0207] Forty SPF-grade ICR mice, weighing 13–15 g (half male and half female), were randomly divided into four groups according to weight class: a normal control group, a model control group, a salvianolic acid C intravenous injection group, and a salvianolic acid C nebulized inhalation group, with ten mice in each group. Except for the normal control group, all other groups of mice were lightly anesthetized with isoflurane and infected with 15 LD50 influenza virus solution via nasal instillation (35 μl per mouse). Treatment began in all groups 4 hours after infection, once daily for 4 consecutive days. Dissection was performed on day 5.

[0208] 2.1.2 Therapeutic effect on a mouse model of respiratory syncytial virus-induced pneumonia

[0209] Forty NIH mice (SPF grade, 12-14g, half male and half female) were randomly divided into four groups according to weight class: normal control group, model control group, salvianolic acid C intravenous injection group, and salvianolic acid C nebulized inhalation group, with 10 mice in each group. Except for the normal control group, the mice in the other groups were lightly anesthetized with isoflurane and injected with 100 TCID45. 50 Parainfluenza virus was administered via nasal drops, 50 μl per animal. Treatment began in all groups 4 hours after infection, once daily for 4 consecutive days. Dissection took place on day 5.

[0210] 2.1.3 Therapeutic effect on a mouse model of respiratory syncytial virus-induced pneumonia

[0211] Forty BALB / c mice (SPF grade, 13-15g, half male and half female) were randomly divided into four groups according to weight class: normal control group, model control group, intravenous injection group of salvianolic acid C, and nebulized inhalation group of salvianolic acid C, with 10 mice in each group. Except for the normal control group, mice in the other groups were lightly anesthetized with isoflurane and infected nasally with 10000 TCID50 respiratory syncytial virus solution (50 μL / mouse). Treatment began 4 hours after infection in all groups, once daily for 4 consecutive days. On day 4, except for the normal control group, mice in the other groups were infected nasally with 10000 TCID50 respiratory syncytial virus solution (50 μL / mouse). Dissection was performed on day 5.

[0212] 2.2 Detection Indicators

[0213] ① After weighing the mice, they were dissected, and the whole lungs were removed and weighed. The lung index and inhibition rate were calculated. The results were statistically analyzed by the t-test for intergroup comparison.

[0214] ②Left lung lobe of mice in each group was taken for viral nucleic acid content detection, and the results were statistically analyzed by t-test for intergroup comparison.

[0215] 2.3 Effects on mouse lung index

[0216] Lung index (%) = Lung wet weight (g) × 100% / Body weight (g)

[0217]

[0218] Table 11 Effects on a mouse model of pneumonia induced by respiratory virus infection.

[0219]

[0220] Note: Compared with the normal control group ## P<0.01; compared with the model control group, *P<0.05, **P<0.01.

[0221] The results showed that after mice were infected with influenza A H1N1 virus FM1 strain, influenza A H3N2 virus, parainfluenza virus and respiratory syncytial virus, the lung index of mice in the model control group was significantly increased, which was significantly different from that of the normal control group (P<0.01). After 4 days of treatment with salvianolic acid C starting on the day of infection, both the salvianolic acid C nebulization group and the intravenous injection group could significantly reduce the lung index of mice, and the nebulization group had a better effect.

[0222] 2.4 Effect on viral load in mouse lung tissue

[0223] Nucleic acid detection in lung tissue (RT-PCR method)

[0224] ① Nucleic acid lysis treatment

[0225] Mouse lung tissue was removed from a -80°C freezer and placed in a clean mortar. A small amount of liquid nitrogen was added, and the tissue was ground into powder using a pestle. The powder was collected in a 1.5 ml centrifuge tube, and 1 ml of TRIzol was immediately added. Reagent: Gently tap the bottom of the tube to quickly resuspend the sample; place the centrifuge tube horizontally at room temperature and incubate for 20 min; centrifuge at 12000 rpm for 10 min at 4°C; transfer the clear supernatant to a new 1.5 ml centrifuge tube; add 0.2 ml chloroform, tighten the cap, shake the centrifuge tube vigorously for 15 s, and incubate at room temperature for 2-3 min until the liquid separates into layers; centrifuge at 12000 rpm for 15 min at 4°C; carefully transfer the clear supernatant to a new 1.5 ml centrifuge tube, add 0.5 ml isopropanol, mix well, and incubate at room temperature for 30 min; centrifuge at 12000 rpm for 10 min at 4°C; discard the supernatant, and gently wash the precipitate with 1 ml 75% ethanol (until the white precipitate floats gently); centrifuge at 7500 rpm for 5 min at 4°C; aspirate the supernatant and briefly dry the RNA precipitate for 5-10 min; dissolve the precipitate in 20 μl DEPC water and store at -80°C.

[0226] ② Nucleic acid testing

[0227] Nucleic acid treatment of control standards: DEPC-H2O was used as a negative control. Positive control standards were prepared by serial dilution at concentrations of 10⁷, 10⁶, 10⁵, and 10⁴ copies / ml, for a total of four concentrations.

[0228] Reagent preparation: Take n×18μl of IFVA nucleic acid fluorescence PCR detection mixture, n×1μl of internal control, and n×1μl of RT-PCR enzyme (n is the number of reaction tubes), vortex to mix for a few seconds, and centrifuge at 3000rpm for a few seconds.

[0229] Sample addition: Take 20 μl of the above mixture and place it in a PCR tube. Then add 5 μl each of the sample nucleic acid extract, DEPC-H2O, and positive control to the PCR tube. Tighten the cap, centrifuge for a few seconds to bring all liquids to the bottom, and immediately perform the PCR amplification reaction.

[0230] PCR amplification: The reaction tubes were placed on a quantitative PCR instrument, and the cycling parameters were set as follows: 45℃×10min; 95℃×15min; then 95℃×15sec→60℃×60sec, for a total of 40 cycles; single-point fluorescence detection was performed at 60℃, and the reaction volume was 25μl.

[0231] Fluorescence channel selection: FAM and HEX / VIC / JOE channels are selected.

[0232] Note: When using an ABI series PCR instrument, please be sure to select "none" for both passive reference and quencher.

[0233] Calculation method:

[0234] (1) Therapeutic effects on mouse models of pneumonia induced by H1N1 influenza A virus FM1 strain, H3N2 influenza A virus, and respiratory syncytial virus infection.

[0235] Standard curves were plotted based on the CT values ​​of positive control standards at different concentrations, and the viral nucleic acid concentration of the samples was calculated based on the CT values ​​of the samples.

[0236] (2) Therapeutic effect on parainfluenza virus-induced pneumonia model in mice

[0237] This experiment used a relative quantification method, with the VIC channel as an internal control and the normal group sample (Con) as the calibrator. The calculation method is as follows:

[0238] ConΔCt=Con FAM Ct-Con VIC Ct

[0239] Sample ΔCt = Sample FAM Ct - Sample VIC Ct

[0240] Sample ΔCt = Sample ΔCt - ConΔCt

[0241] The change in relative content = 2 - ΔΔCt, which is the fold change in gene expression relative to Calibrator in each treatment group.

[0242] The results were statistically analyzed using t-tests.

[0243] Table 12 Effects on a mouse model of pneumonia induced by respiratory virus infection.

[0244]

[0245] Note: Compared with the normal control group ## P<0.01; compared with the model control group, *P<0.05, **P<0.01.

[0246] The results showed that no viral nucleic acid was expressed in the lung tissue of mice in the normal control group. After mice were infected with influenza A H1N1 virus FM1 strain, influenza A H3N2 virus, parainfluenza virus and respiratory syncytial virus, viral nucleic acid expression was significantly observed in the lung tissue of mice in the model control group, which was significantly different from that in the normal control group (P<0.01). Four days after the onset of treatment with Tanshinone C on the day of infection, the expression of viral nucleic acid in the lung tissue of mice in the Tanshinone C nebulization group and the intravenous injection group was significantly reduced, and the nebulization group showed better results.

[0247] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A freeze-dried preparation of salvianolic acid C, characterized in that, The lyophilized formulation of salvianolic acid C contains salvianolic acid C, a pH adjuster, a stabilizer, and an excipient.

2. The freeze-dried preparation of salvianolic acid C according to claim 1, characterized in that, The content of salvianolic acid C in the unit dose of the lyophilized preparation of salvianolic acid C is 0.2-50 mg, preferably 5-50 mg. 3.The freeze-dried preparation of salvianolic acid C according to claim 1, characterized in that, The water content of the lyophilized preparation of salvianolic acid C is less than 7%, preferably less than 5%, and more preferably less than 3%. 4.The freeze-dried preparation of salvianolic acid C according to claim 1, characterized in that, The pH adjuster includes inorganic acids or organic acid-base buffers, preferably organic acid-base buffers; Preferably, the inorganic acid includes any one or at least a combination of two of hydrochloric acid, phosphoric acid, or sulfuric acid; Preferably, the organic acid-base buffer comprises a combination of tartaric acid and sodium tartrate, a combination of citric acid and sodium citrate, or a combination of phosphate and sodium dihydrogen phosphate, with the combination of tartaric acid and sodium tartrate or the combination of citric acid and sodium citrate being more preferred. Preferably, the pH of the lyophilized Danshensu C preparation after reconstitution is 3.0-5.5, more preferably 4.0-5.

0.

5. The lyophilized formulation of salvianolic acid C according to claim 1, characterized in that, The stabilizer includes disodium edetate and / or sodium bisulfite, preferably disodium edetate; Preferably, the amount of stabilizer used is 0.5-3 mg / mL, based on the volume of the solvent used to prepare the salvianolic acid C lyophilized agent.

6. The lyophilized formulation of salvianolic acid C according to claim 1, characterized in that, The excipients include sugar alcohols or amino acid compounds; Preferably, the sugar alcohol compound includes mannitol; Preferably, the amino acid compound includes glycine or arginine; Preferably, the excipient comprises any one or at least two combinations of mannitol, arginine, or glycine, with mannitol and / or glycine being more preferred. Preferably, the amount of excipient used is 15-50 mg / mL, based on the volume of the solvent used to prepare the salvianolic acid C lyophilized agent.

7. The lyophilized formulation of salvianolic acid C according to claim 1, characterized in that, The lyophilized formulation of salvianolic acid C also contains a solubilizer; Preferably, the solubilizer comprises polyol compounds or amino acid compounds; Preferably, the polyol compound includes propylene glycol or glycerol; Preferably, the amino acid compound includes lysine hydrochloride, glycine, or arginine; Preferably, the solubilizer comprises any one or at least two combinations of glycerol, propylene glycol, lysine hydrochloride, arginine or glycine, more preferably any one or at least two combinations of propylene glycol, lysine hydrochloride, arginine or glycine, preferably propylene glycol and / or glycine. Preferably, the amount of solubilizer used is 15-40 mg / mL, based on the volume of the solvent used to prepare the salvianolic acid C lyophilizer.

8. A method for preparing a lyophilized formulation of salvianolic acid C according to any one of claims 1-7, characterized in that, The preparation method includes the following steps: (1) Add solvent to the dispensing container, slowly add the prescribed amount of stabilizer and excipient, and stir until they are completely dissolved; (2) Add the prescribed amount of buffer to adjust the pH of the system; (3) Add the prescribed amount of salvianolic acid C and stir until it is completely dissolved; add solvent to make up to the total prescribed amount and stir to mix it evenly. (4) Aseptic filtration, filling, and transfer to a freeze dryer; (5) Pre-freeze; (6) Sublimation: Vacuuming begins while heating is being carried out; (7) Desorption and drying: Continuous vacuuming, heating, and desorption; (8) Plug in the cap, roll in the lid, and end the freeze-drying process.

9. The preparation method according to claim 8, characterized in that, Step (1) also includes adding the prescribed amount of solubilizer to the solvent; Preferably, in step (2), the pH of the system is adjusted to 3.0-5.5, more preferably 4.0-5.0; Preferably, the solvent used in steps (1) and (3) is water for injection; Preferably, the temperature during the solution preparation process in steps (1) to (3) is controlled at 15-35℃; Preferably, in step (4), the aseptic filtration includes: primary filtration using a 0.45μm to 0.8μm filter membrane and fine filtration using a 0.22μm to 0.45μm filter membrane; Preferably, in step (5), the pre-freezing temperature is -50℃ to -40℃, more preferably -50℃ to -45℃; Preferably, in step (5), the pre-freezing time is 4 to 8 hours, more preferably 6 to 8 hours; Preferably, in step (6), the temperature is raised to -25℃ to -15℃, more preferably -20℃ to -15℃; Preferably, in step (6), the sublimation time is 15-30 hours, more preferably 20-30 hours; Preferably, in step (7), the temperature is raised to 20°C to 30°C, more preferably 25°C to 30°C; Preferably, in step (7), the drying time is 6-10 hours, more preferably 8-10 hours.

10. The preparation method according to claim 8, characterized in that, The preparation method includes the following steps: (1) Add solvent to the dispensing container, slowly add the prescribed amount of stabilizer, excipient and solubilizer, and stir until they are completely dissolved; (2) Add the prescribed amount of buffer to adjust the pH of the system to 4.0-5.0; (3) Add the prescribed amount of salvianolic acid C and stir until it is completely dissolved; add solvent to make up to the total prescribed amount and stir to mix it evenly. (4) Perform primary filtration with a 0.45μm filter membrane, fine filtration with a 0.22μm filter membrane, and after aseptic filtration, fill and transfer to a freeze dryer; (5) Pre-freezing: Pre-freeze at -50℃~-45℃ for 6~8 hours; (6) Sublimation: While heating, vacuuming is started, and the temperature is raised to -20℃~-15℃ for a total of 20-30 hours; (7) Desorption and drying: Continue vacuuming and heat to 25℃~30℃ for 8-10 hours; (8) Stopper, cap, and end freeze-drying; The solvent used in steps (1) and (3) is water for injection, and the temperature of the solution preparation process in steps (1) to (3) is controlled at 15-35℃.

11. An inhalation system or injection delivery system for nebulizing a salvianolic acid C solution to the human respiratory tract, comprising 1 ml to 5 ml of salvianolic acid C solution reconstituted from the lyophilized salvianolic acid C agent according to any one of claims 1-7.

12. The inhalation system or injection drug delivery system for administering salvianolic acid C solution via nebulization to the human respiratory tract according to claim 11, characterized in that, The inhalation system for administering salvianolic acid C solution to the human respiratory tract via nebulization also includes a liquid nebulizer. After the reconstituted salvianolic acid C solution is nebulized using a liquid nebulizer, its aerodynamic mass has a diameter of 2-4 μm and the percentage of fine particles is 40%-70%.

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