Preparation method of qingkailing injection and product thereof

Abstract

The application belongs to the technical field of traditional Chinese medicine, and particularly relates to a preparation method of Qingkailing injection and a product thereof. The preparation method comprises the following steps: firstly, extracting, filtering, and obtaining an extract liquid by adding water to isatis root, gardenia and honeysuckle; secondly, adding water buffalo horn powder and nacre into 8 times of water, adding keratinase and aminopeptidase for enzymolysis, filtering, and obtaining an enzymolysis liquid 1; thirdly, mixing the extract liquid and the enzymolysis liquid 1, adding tannase for enzymolysis, filtering, and obtaining an enzymolysis liquid 2; and finally, adding baicalin into water, mixing the baicalin and the enzymolysis liquid 2, adding into an ethanol solution of cholic acid and hyodeoxycholic acid, mixing, filtering, recovering ethanol from the filtrate, adding water for injection to 1000 ml, treating by activated carbon, filtering, and performing ultrafiltration to obtain the Qingkailing injection. The Qingkailing injection prepared by the preparation method has good stability after being accelerated for 6 months; and pharmacodynamic results show that the Qingkailing injection has certain advantages in treating acute hepatitis.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a preparation method of Qingkailing injection and its product. Background Technology

[0002] Traditional Chinese medicine (TCM) injections are injections made from the extraction of effective substances from Chinese herbs or compound Chinese herbs. They play an important and positive role in the treatment of many serious diseases in clinical practice, and have significant advantages in the treatment of cardiovascular diseases, tumors, and antibacterial and antiviral infections. Currently, the efficacy of TCM injections has been largely resolved from a technical perspective, but their safety is receiving increasing attention from technical personnel, registration personnel, and even the general public in the field of TCM.

[0003] The safety issues of traditional Chinese medicine (TCM) injections mainly include the following aspects: First, the composition of TCM injections is complex, and the active ingredients of many varieties are still unclear. This poses difficulties for product preparation and quality control, seriously affecting the clinical safety of TCM injections. Second, the production process of TCM injections is difficult, and their chemical composition is very complex. They typically contain inorganic salts, alkaloids, amino acids and organic acids, phenols, ketones, saponins, steroidal compounds, terpenoids, proteins, polysaccharides, starch, cellulose, and other components, all of which increase safety risks during production and administration. Third, the administration method, solvent, and storage stability of TCM injections also affect their safety and are important factors contributing to adverse reactions.

[0004] Qingkailing Injection is a modified version of the ancient formula Angong Niuhuang Wan from the *Wenbing Tiaobian* (Treatise on Febrile Diseases). It consists of eight herbs: baicalin, gardenia, cholic acid, deoxycholic acid, honeysuckle, isatis root, buffalo horn, and pearl powder. In this formula, cholic acid and deoxycholic acid replace bezoar as the principal herbs; buffalo horn replaces rhinoceros horn, and baicalin replaces scutellaria, along with honeysuckle, forming the assistant herbs; gardenia, isatis root, and pearl powder are the adjuvant herbs. Together, they clear heat and detoxify, resolve phlegm and unblock the meridians, and awaken the mind and open the orifices. It is used for febrile diseases, delirium, hemiplegia due to stroke, and loss of consciousness; acute hepatitis, upper respiratory tract infection, pneumonia, cerebral thrombosis, and cerebral hemorrhage with the above symptoms.

[0005] Traditional Qingkailing injection is obtained by extracting the extract using water extraction, alcohol precipitation, and heating concentration. Specifically, Isatis root is decocted twice with water, 1 hour each time. The decoctions are combined, filtered, and the filtrate is concentrated to 200 ml. Ethanol is added to bring the alcohol content to 60%, and the solution is refrigerated, filtered, and the ethanol is recovered from the filtrate. Water is added, and the solution is refrigerated for later use. Gardenia is decocted twice with water, the first time for 1 hour and the second time for 0.5 hours. The decoctions are combined, filtered, and the filtrate is concentrated to 25 ml. Ethanol is added to bring the alcohol content to 60%, and the solution is refrigerated, filtered, and the ethanol is recovered from the filtrate. Water is added, and the solution is refrigerated for later use. Honeysuckle is decocted twice with water, 0.5 hours each time. The decoctions are combined, filtered, and the filtrate is concentrated to 60 ml. Ethanol is added to bring the alcohol content to 75%, filtered, and the pH of the filtrate is adjusted to 8.0. The solution is refrigerated, the ethanol is recovered, and then ethanol is added again to bring the alcohol content to 85%. The solution is refrigerated, filtered, and the ethanol is recovered from the filtrate. Water is added, and the solution is refrigerated for later use. Buffalo horn powder was hydrolyzed with barium hydroxide solution, and mother-of-pearl powder was hydrolyzed with sulfuric acid for 7–9 hours. The solutions were filtered, the filtrates were combined, the pH was adjusted to 3.5–4.0, and the solutions were filtered again. Ethanol was added to the filtrate to achieve an alcohol content of 60%, and the solution was refrigerated. The solutions were filtered again, the ethanol was recovered from the filtrate, water was added, and the solution was refrigerated for later use. Gardenia extract, Isatis root extract, and a mixture of hydrolyzed buffalo horn and mother-of-pearl were combined and added to a 75% ethanol solution of cholic acid and deoxycholic acid. The mixture was mixed, ethanol was added to achieve an alcohol content of 75%, the pH was adjusted to 7.0, and the solution was refrigerated. The solutions were filtered, the ethanol was recovered from the filtrate, water was added, and the solution was refrigerated for later use. Baicalin was dissolved in water for injection, the pH was adjusted to 7.5, honeysuckle extract was added, and the mixture was mixed. This mixture was then combined with the prepared solutions, mixed again, and water for injection was added to a final volume of 1000 ml. The solution was then treated with activated carbon, refrigerated, filled, and sterilized.

[0006] This method is inefficient due to its high alcohol and energy consumption, and the quality of the resulting product is also unstable. Therefore, it is necessary to conduct secondary development and in-depth research on Qingkailing injection to improve the safety of Qingkailing product and at the same time improve its therapeutic effect. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for preparing Qingkailing injection and the product thereof.

[0008] To achieve the objectives of this invention, the following technical solution is adopted:

[0009] A method for preparing Qingkailing injection includes the following steps:

[0010] (1) Add water to extract Isatis root, gardenia, and honeysuckle, filter, and obtain the extract;

[0011] (2) Add buffalo horn powder and pearl powder to 8 times the amount of water, then add keratinase and aminopeptidase for enzymatic hydrolysis, filter, and obtain hydrolysate 1;

[0012] (3) Mix the extract and hydrolysate 1, add tannin enzyme for enzymatic hydrolysis, filter, and obtain hydrolysate 2;

[0013] (4) Add water to baicalin and mix with enzyme hydrolysate 2. Add the mixture to an ethanol solution of cholic acid and deoxycholic acid, mix well, filter, recover the ethanol from the filtrate, add water for injection to 1000 ml, treat with activated carbon, filter, and then ultrafilter to obtain the product.

[0014] Preferably, the amount of water added in step (1) is 5-10 times the total mass of Isatis root, gardenia, and honeysuckle, and the extraction is performed by heating and reflux extraction for 1-2 hours.

[0015] Preferably, in step (1), after extraction and filtration, ethanol is added to the filtrate to make the alcohol content reach 60%, then filtered, and ethanol is added again to make the alcohol content reach 85%, then filtered, the filtrate is concentrated, the ethanol is recovered, and 1 times the amount of water is added to obtain the extract.

[0016] Preferably, the enzymatic hydrolysis temperature in step (2) is 40-50℃, the pH of the enzymatic hydrolysis is 6.5-7.5, and the hydrolysis time is 2-4h.

[0017] Preferably, the total mass of the keratinase and aminopeptidase is 0.1-0.5% of the mass of the water added in step (2).

[0018] Preferably, the mass ratio of keratinase to aminopeptidase is 1:5-10.

[0019] Preferably, the pH of the enzymatic hydrolysis in step (3) is 5.5-6.0, the temperature of the enzymatic hydrolysis is 30-40℃, and the time of the enzymatic hydrolysis is 1-3h.

[0020] Preferably, the amount of tanninase used in step (3) is 0.2-0.6% of the total mass of the extract and the enzyme hydrolysate 1.

[0021] Preferably, the ethanol in step (4) is an aqueous solution of ethanol with a mass concentration of 70-80%, and the ultrafiltration is obtained by filtration with a 0.22μm filter membrane.

[0022] Another object of the present invention is to provide a Qingkailing injection product prepared by the preparation method described above.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) The Qingkailing injection prepared by this invention has significant efficacy in treating acute hepatitis. Comparative studies have shown that the combined decoction of Isatis root, Gardenia fruit, and Honeysuckle has better efficacy than the decoction of each alone.

[0025] (2) In this invention, buffalo horn powder and mother-of-pearl powder are first enzymatically hydrolyzed with keratinase and aminopeptidase in a certain mass ratio, which can maximize the enzymatic hydrolysis of proteins in buffalo horn powder and mother-of-pearl powder. Finally, the extract and enzymatic hydrolysate are enzymatically hydrolyzed with tannins to reduce protein and tannin impurities, reduce side reactions, increase active components, improve the stability of the injection and the efficacy of treating acute hepatitis.

[0026] (3) The Qingkailing injection prepared by the preparation method of the present invention has good stability after 6 months of accelerated reaction. The product is safe, effective and of controllable quality, and has good application prospects. Detailed Implementation

[0027] The present invention will be described below through specific embodiments to make the technical solution of the present invention easier to understand and master, but the present invention is not limited thereto. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are all commercially available.

[0028] Among them, keratinase, 100,000 U / g, was produced by Shandong Fengtai Technology Biotechnology Co., Ltd.; serine protease, 200,000 U / g, was produced by Xi'an Darwen Biotechnology Co., Ltd.; aminopeptide protease, 100,000 U / g, was produced by Nanning Dongheng Huadao Biotechnology Co., Ltd.; and tanninase, 250 U / g, was produced by Nanning Dongheng Huadao Biotechnology Co., Ltd.

[0029] Example 1

[0030] Prescription: Bile acid 3.25g, pearl powder 50.0g, deoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0031] The preparation method steps are as follows:

[0032] (1) Add 8 times the amount of water to Isatis root, gardenia, and honeysuckle and heat under reflux for 1 hour. Filter the solution and add ethanol to the filtrate to make the alcohol content reach 60%. Filter the solution and continue to add ethanol to make the alcohol content reach 85%. Filter the solution, concentrate the filtrate, recover the ethanol, and add 1 times the amount of water to obtain the extract.

[0033] (2) Add buffalo horn powder and pearl powder to 8 times the amount of water and mix them. Then add 0.3% of the water mass of the compound enzyme (the compound enzyme is composed of keratinase and aminopeptidase in a mass ratio of 1:5). Enzymatically hydrolyze at pH 7 and temperature 45℃ for 3 hours, boil for 2 minutes, filter, and obtain enzyme hydrolysate 1.

[0034] (3) Mix the extract and hydrolysate 1, add 0.4% of tanninase (total mass of extract and hydrolysate 1) and hydrolyze at pH 5.5 and temperature 35℃ for 2 hours, boil for 2 minutes, filter to obtain hydrolysate 2.

[0035] (4) Add water to baicalin and mix with enzyme hydrolysate 2. Add the mixture to a 75% ethanol solution of cholic acid and deoxycholic acid, mix well, filter, recover the ethanol from the filtrate, add water for injection to 1000ml, treat with activated carbon, filter, then use a 0.22μm filter membrane for ultrafiltration, and sterilize to obtain the product.

[0036] Example 2

[0037] Prescription: Bile acid 3.25g, pearl powder 50.0g, deoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0038] The preparation method steps are as follows:

[0039] (1) Add 5 times the amount of water to Isatis root, gardenia, and honeysuckle and heat under reflux for 1 hour. Filter the solution and add ethanol to the filtrate to make the alcohol content reach 60%. Filter the solution and continue to add ethanol to make the alcohol content reach 85%. Filter the solution, concentrate the filtrate, recover the ethanol, and add 1 times the amount of water to obtain the extract.

[0040] (2) Add buffalo horn powder and pearl powder to 8 times the amount of water and mix them. Then add 0.1% of the water mass of the compound enzyme (the compound enzyme is composed of keratinase and aminopeptidase in a mass ratio of 1:10). Enzymatically hydrolyze at pH 7.5 and temperature 50℃ for 4 hours, boil for 2 minutes, filter, and obtain enzyme hydrolysate 1.

[0041] (3) Mix the extract and hydrolysate 1, add 0.6% of tanninase (total mass of extract and hydrolysate 1), incubate at pH 6.0 and 40°C for 3 hours, boil for 2 minutes, filter, and obtain hydrolysate 2.

[0042] (4) Add water to baicalin and mix with enzyme hydrolysate 2. Add the mixture to a 75% ethanol solution of cholic acid and deoxycholic acid, mix well, filter, recover the ethanol from the filtrate, add water for injection to 1000ml, treat with activated carbon, filter, then use a 0.22μm filter membrane for ultrafiltration, and sterilize to obtain the product.

[0043] Example 3

[0044] Prescription: Bile acid 3.25g, pearl powder 50.0g, deoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0045] The preparation method steps are as follows:

[0046] (1) Add 10 times the amount of water to Isatis root, gardenia, and honeysuckle and heat under reflux for 2 hours to extract. Filter the solution and add ethanol to the filtrate to make the alcohol content reach 60%. Filter the solution and continue to add ethanol to make the alcohol content reach 85%. Filter the solution, concentrate the filtrate, recover the ethanol, and add 1 times the amount of water to obtain the extract.

[0047] (2) Add buffalo horn powder and pearl powder to 8 times the amount of water and mix them. Then add 0.5% of the water mass of the compound enzyme (the compound enzyme is composed of keratinase and aminopeptidase in a mass ratio of 1:7). Enzymatically hydrolyze at pH 6.5 and temperature 40℃ for 2 hours, boil for 2 minutes, filter, and obtain enzyme hydrolysate 1.

[0048] (3) Mix the extract and hydrolysate 1, add 0.2% of tanninase (total mass of extract and hydrolysate 1), incubate at pH 5.5 and temperature 30℃ for 1 h, boil for 2 min, filter, and obtain hydrolysate 2.

[0049] (4) Add water to baicalin and mix with enzyme hydrolysate 2. Add the mixture to a 75% ethanol solution of cholic acid and deoxycholic acid, mix well, filter, recover the ethanol from the filtrate, add water for injection to 1000ml, treat with activated carbon, filter, then use a 0.22μm filter membrane for ultrafiltration, and sterilize to obtain the product.

[0050] Comparative Example 1

[0051] Prescription: Bile acid 3.25g, pearl powder 50.0g, deoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0052] The preparation method of this comparative Qingkailing injection is as follows:

[0053] (1) The above eight ingredients, add water to Isatis root and decoct twice, 1 hour each time, combine the decoctions, filter, concentrate the filtrate to 200ml, add ethanol to make the alcohol content reach 60%, refrigerate, filter, recover ethanol from the filtrate, add water, refrigerate for later use.

[0054] (2) Gardenia is decocted twice with water, the first time for 1 hour and the second time for 0.5 hours. The decoctions are combined, filtered, and the filtrate is concentrated to 25 ml. Ethanol is added to make the alcohol content reach 60%, and the decoction is refrigerated. The filtrate is filtered, the ethanol is recovered, water is added, and the decoction is refrigerated for later use.

[0055] (3) Decoction of honeysuckle twice with water, 0.5h each time, combine the decoctions, filter, concentrate the filtrate to 60ml, add ethanol to make the alcohol content reach 75%, filter, adjust the pH of the filtrate to 8.0, refrigerate, recover the ethanol, add ethanol again to make the alcohol content reach 85%, refrigerate, filter, recover the ethanol from the filtrate, add water, refrigerate for later use.

[0056] (4) Hydrolyze buffalo horn powder with barium hydroxide solution and pearl powder with sulfuric acid for 7-9 hours respectively. Filter, combine the filtrates, adjust the pH value to 3.5-4.0, filter, add ethanol to the filtrate to make the alcohol content reach 60%, refrigerate, filter, recover the ethanol from the filtrate, add water, and refrigerate for later use.

[0057] (5) Combine the gardenia extract, isatis root extract and the hydrolyzed mixture of buffalo horn and mother-of-pearl, add it to a 75% ethanol solution of cholic acid and porcine deoxycholic acid, mix well, add ethanol to make the alcohol content reach 75%, adjust the pH value to 7.0, refrigerate, filter, recover the ethanol from the filtrate, add water, and refrigerate for later use.

[0058] (6) Dissolve baicalin in water for injection, adjust the pH to 7.5, add honeysuckle extract, mix well, combine with the above-mentioned prepared solutions, mix well, add water for injection to 1000ml, then treat with activated carbon, refrigerate, fill and sterilize to obtain the product.

[0059] Comparative Example 2

[0060] Prescription: Bile acid 3.25g, pearl powder 50.0g, deoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0061] The comparative example describes a method for preparing Qingkailing injection, with the following steps:

[0062] (1) Add 8 times the amount of water to Isatis root, gardenia, and honeysuckle and heat under reflux for 1 hour. Filter the solution and add ethanol to the filtrate to make the alcohol content reach 60%. Filter the solution and continue to add ethanol to make the alcohol content reach 85%. Filter the solution, concentrate the filtrate, recover the ethanol, and add 1 times the amount of water to obtain the extract.

[0063] (2) Add buffalo horn powder and pearl powder to 8 times the amount of water and mix. Then add 0.3% of water mass of keratinase and hydrolyze at pH 7 and temperature 45℃ for 3 hours. Boil for 2 minutes and filter to obtain hydrolysate 1.

[0064] (3) Mix the extract and hydrolysate 1, add 0.4% of tanninase (total mass of extract and hydrolysate 1) and hydrolyze at pH 5.5 and temperature 35℃ for 2 hours, boil for 2 minutes, filter to obtain hydrolysate 2.

[0065] (4) Add water to baicalin and mix with enzyme hydrolysate 2. Add the mixture to a 75% ethanol solution of cholic acid and deoxycholic acid, mix well, filter, recover the ethanol from the filtrate, add water for injection to 1000ml, treat with activated carbon, filter, and then sterilize by ultrafiltration with a 0.22μm filter membrane.

[0066] Comparative Example 3

[0067] Prescription: Bile acid 3.25g, pearl powder 50.0g, deoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0068] A method for preparing Qingkailing injection, comprising the following steps:

[0069] (1) Add 8 times the amount of water to Isatis root, gardenia, and honeysuckle and heat under reflux for 1 hour. Filter the solution and add ethanol to the filtrate to make the alcohol content reach 60%. Filter the solution and continue to add ethanol to make the alcohol content reach 85%. Filter the solution, concentrate the filtrate, recover the ethanol, and add 1 times the amount of water to obtain the extract.

[0070] (2) Add buffalo horn powder and pearl powder to 8 times the amount of water and mix. Then add 0.3% aminopeptidase by weight of water and hydrolyze at pH 7 and temperature 45℃ for 3 hours. Boil for 2 minutes and filter to obtain hydrolysate 1.

[0071] (3) Mix the extract and hydrolysate 1, add 0.4% of tanninase (total mass of extract and hydrolysate 1) and hydrolyze at pH 5.5 and temperature 35℃ for 2 hours, boil for 2 minutes, filter to obtain hydrolysate 2.

[0072] (4) Add water to baicalin and mix with enzyme hydrolysate 2. Add the mixture to a 75% ethanol solution of cholic acid and deoxycholic acid, mix well, filter, recover the ethanol from the filtrate, add water for injection to 1000ml, treat with activated carbon, filter, and then sterilize by ultrafiltration with a 0.22μm filter membrane.

[0073] Comparative Example 4

[0074] Prescription: Bile acid 3.25g, pearl powder 50.0g, deoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0075] A method for preparing Qingkailing injection, comprising the following steps:

[0076] (1) Add 8 times the amount of water to Isatis root, gardenia, and honeysuckle and heat under reflux for 1 hour. Filter the solution and add ethanol to the filtrate to make the alcohol content reach 60%. Filter the solution and continue to add ethanol to make the alcohol content reach 85%. Filter the solution, concentrate the filtrate, recover the ethanol, and add 1 times the amount of water to obtain the extract.

[0077] (2) Add buffalo horn powder and pearl powder to 8 times the amount of water and mix them. Then add 0.3% of the water mass of the compound enzyme (the compound enzyme is composed of serine protease and aminopeptidase in a mass ratio of 1:5). Enzymatically hydrolyze at pH 7 and temperature 45℃ for 3 hours, boil for 2 minutes, filter, and obtain enzyme hydrolysate 1.

[0078] (3) Add water to baicalin and mix with enzyme hydrolysate 1. Add the mixture to a 75% ethanol solution of cholic acid and deoxycholic acid, mix well, filter, recover the ethanol from the filtrate, add water for injection to 1000ml, treat with activated carbon, filter, and then sterilize by ultrafiltration with a 0.22μm filter membrane.

[0079] Pharmacodynamic Experiment---Pharmacodynamic Verification of Qingkailing Injection in the Treatment of Acute Hepatitis

[0080] 1.1 Laboratory Animals

[0081] SPF-grade Kunming mice, half male and half female, weighing 20±2g, were used. All animals had free access to water and food and were adapted to the feeding program for one week.

[0082] 1.2 Animal grouping and administration

[0083] Eighty KM mice, half male and half female, were randomly divided into a blank control group, a model group, a positive control group, and groups for Examples 1-3 and Comparative Examples 1-4, with eight mice in each group. The blank control group and the model group were given intraperitoneal injections of physiological saline for 7 days. The groups for Examples 1-3 and Comparative Examples 1-4 were given 10 ml / kg of Qingkailing injection prepared for their respective groups. The positive control group was given intraperitoneal injections of silymarin 200 mg / kg. The administration was continued for 7 days. One hour after the last administration, except for the blank control group, all other groups were given intraperitoneal injections of 0.1% CCl4 olive oil solution (10 mL / kg). The blank control group was given an equal volume of olive oil. All mice were then fasted but allowed free access to water. Blood was collected 24 hours later. The serum was centrifuged at 3000 r / min for 10 min, and the serum was carefully aspirated using a pipette. The AST and ALT levels were measured according to the kit instructions. The liver tissue was stored at -80℃ for later use. 50 mg of the same part of the liver tissue was taken, ground with 9 times the amount of PBS solution, centrifuged, and the supernatant was collected. The MDA and GSH contents were determined according to the kit instructions.

[0084] 1.3 Experimental Results

[0085] The results are shown in Tables 1-2.

[0086] SPSS 27.0 software was used to analyze the experimental data. A p-value < 0.05 was considered statistically significant. Data were presented as mean ± standard deviation.

[0087] The value ± standard deviation (x ± s) indicates that one-way ANOVA is used for comparisons among multiple groups and conforms to normal distribution and homogeneity of variance.

[0088] Table 1

[0089]

[0090] Note: Compared with the control group, **P<0.01; compared with the model group, ##P<0.01, #P<0.05; compared with Example 1 group, &P<0.05.

[0091] Results analysis, as shown in Table 1, revealed highly significant differences in AST and ALT levels between the model group and the control group (**P<0.01). Compared to the model group, each experimental group showed significant differences (#P<0.05, ##P<0.01), indicating that the AST and ALT levels in each experimental group improved to some extent after treatment with the corresponding injection solution. Comparative Examples 1-4 showed inferior results compared to Example 1 (&P<0.05).

[0092] Table 2

[0093]

[0094] Note: Compared with the control group, ** P < 0.01. Compared with the model group, ## P < 0.01, # P < 0.05. Compared with Example 1, & P < 0.05.

[0095] Results analysis, as shown in Table 2, revealed that compared to the control group, the levels of MDA and MDA in the model group were significantly different. ** P<0.01, indicating a highly significant difference; compared with the model group, #P<0.05 and ##P<0.01, indicating that the MDA and MDA levels of each experimental group were improved to some extent after treatment with the corresponding injection solution. The improvement effects of Examples 1-3 were generally better than those of Comparative Examples 1-4.

[0096] Experiment 2: Stability Study of Qingkailing Injection

[0097] Stability tests were conducted according to the guidelines for drug stability testing in the Chinese Pharmacopoeia. The Qingkailing injection prepared in Example 1 was placed under accelerated testing conditions for 6 months, and its stability at 3 and 6 months was examined. The results are shown in Table 3, where the content item represents the percentage compared to 0 months. This indicates that the improved process results in better stability of the injection.

[0098] Table 3

[0099]

[0100] The results, as shown in Table 3, indicate that the injection solutions prepared in Examples 1-3 were more stable after 6 months of accelerated stability testing. Comparative Example 1 showed a significant darkening of color and a decrease in the content of key components after 6 months of accelerated stability testing. The injection solutions prepared in Comparative Examples 2-4 were prone to turbidity with prolonged storage, indicating a significant decrease in active ingredients and insufficient stability.

[0101] In summary, macromolecules are the main material basis for safety issues in traditional Chinese medicine (TCM) injections. Improving the safety of TCM injections while maintaining or even enhancing their material basis and main efficacy is a pressing technical problem in this field. This study, through process improvement, not only effectively extracts the active ingredients and enhances drug activity but also removes macromolecules such as proteins and condensed tannins, thereby enhancing the stability of TCM injections and demonstrating promising application prospects.

[0102] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for preparing Qingkailing injection, characterized in that, Includes the following steps: (1) Add water to extract Isatis root, gardenia, and honeysuckle, filter, and obtain the extract; (2) Mix buffalo horn powder and mother-of-pearl powder with water, then add keratinase and aminopeptidase for enzymatic hydrolysis, filter, and obtain hydrolysate 1; (3) Mix the extract and hydrolysate 1, add tannin enzyme for enzymatic hydrolysis, filter, and obtain hydrolysate 2; (4) Add water to baicalin and mix with enzyme hydrolysate 2. Add the mixture to an ethanol solution of cholic acid and deoxycholic acid, mix well, filter, recover the ethanol from the filtrate, add water for injection, treat with activated carbon, filter, and then ultrafilter to obtain the product. The extraction described in step (1) is a heating reflux extraction; After extraction and filtration as described in step (1), ethanol is added to the filtrate to make the alcohol content reach 60%, and then filtered. Ethanol is added again to make the alcohol content reach 85%, and then filtered. The filtrate is concentrated, the ethanol is recovered, and 1 times the amount of water is added to obtain the extract. The total mass of keratinase and aminopeptidase in step (2) is 0.1-0.5% of the mass of water added in step (2); the mass ratio of keratinase to aminopeptidase is 1:5-10; The amount of tanninase used in step (3) is 0.2-0.6% of the total mass of the extract and enzyme hydrolysate 1.

2. The preparation method according to claim 1, characterized in that, The amount of water added in step (1) is 5-10 times the total mass of Isatis root, gardenia, and honeysuckle, and the extraction time is 1-2 hours.

3. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis temperature in step (2) is 40-50℃, the pH of the enzymatic hydrolysis is 6.5-7.5, and the hydrolysis time is 2-4h.

4. The preparation method according to claim 1, characterized in that, The pH of the enzymatic hydrolysis in step (3) is 5.5-6.0, the temperature of the enzymatic hydrolysis is 30-40℃, and the time of the enzymatic hydrolysis is 1-3h.

5. The preparation method according to claim 1, characterized in that, The ethanol mentioned in step (4) is an aqueous solution of ethanol with a mass concentration of 70-80%, and the ultrafiltration is obtained by filtering with a 0.22μm filter membrane.

6. A Qingkailing injection solution prepared by the preparation method according to any one of claims 1-5.

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