Preparation method of Qingkailing injection and product of Qingkailing injection

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a preparation method of a Qingkailing injection and a product thereof. The preparation method comprises the following steps: adding water into the radix isatidis, the fructus gardeniae and the honeysuckle flower, extracting, and filtering to obtain an extracting solution; adding buffalo horn powder and nacre mother powder into 8 times amount of water, mixing, adding keratinase and aminopeptidase for enzymolysis, and filtering to obtain enzymatic hydrolysate 1; mixing the extracting solution with the enzymatic hydrolysate 1, adding tannase for enzymolysis, and filtering to obtain enzymatic hydrolysate 2; and finally, adding water into baicalin, mixing with the enzymatic hydrolysate 2, adding into an ethanol solution of cholic acid and hyodeoxycholic acid, uniformly mixing, filtering, recovering ethanol from filtrate, adding water for injection to 1000ml, treating with activated carbon, filtering, and carrying out ultrafiltration. The Qingkailing injection prepared by the preparation method provided by the invention is accelerated for 6 months and has good stability; pharmacodynamic results show that the traditional Chinese medicine composition has certain advantages in the aspect of treating acute hepatitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a preparation method of Qingkailing injection and a product thereof. Background Art

[0002] Traditional Chinese medicine injections, made from active ingredients extracted from traditional Chinese medicines or compound herbal formulas, have significant positive effects on a wide range of serious clinical conditions, including significant advantages in the treatment of cardiovascular disease, tumors, and bacterial and viral infections. While the effectiveness of traditional Chinese medicine injections has largely been addressed technically, their safety is drawing increasing attention from both technical and registered practitioners in the field, as well as the general public.

[0003] Safety issues with traditional Chinese medicine injections primarily include the following: First, the composition of traditional Chinese medicine injections is complex, and the active ingredients of many varieties remain unknown. This creates difficulties in product preparation and quality control, seriously affecting the clinical safety of traditional Chinese medicine injections. Second, the production process of traditional Chinese medicine injections is difficult, and the chemical composition is very complex, typically containing inorganic salts, alkaloids, amino acids and organic acids, phenols, ketones, saponins, steroids, terpenoids, proteins, polysaccharides, starch, cellulose, and other ingredients, all of which increase safety risks during production and use. Third, the method of administration, solvent, and storage stability of traditional Chinese medicine injections can also affect their safety and are a key factor in the occurrence of adverse reactions.

[0004] Qingkailing Injection is a modified version of the ancient prescription Angong Niuhuang Wan (Angong Niuhuang Wan) from the Treatise on Febrile Diseases. It consists of eight herbs: baicalin, gardenia, cholic acid, hyodeoxycholic acid, honeysuckle, isatis root, buffalo horn, and pearl powder. Cholic acid and hyodeoxycholic acid replace bezoar as the monarch herbs; buffalo horn replaces rhinoceros horn, and baicalin replaces scutellaria, along with honeysuckle, as the assistant herbs. Gardenia, isatis root, and mother-of-pearl serve as adjuvants. Together, they clear heat and detoxify, resolve phlegm, dredge the meridians, and awaken the mind. It is indicated for fever, coma, hemiplegia caused by stroke, and unconsciousness; as well as for acute hepatitis, upper respiratory tract infection, pneumonia, cerebral thrombosis, and cerebral hemorrhage.

[0005] Traditional Qingkailing injection is obtained by using a method of water extraction, alcohol precipitation and heating concentration to prepare an extract. Specifically, Radix Isatidis is decocted with water twice, each time for 1 hour, the decoction is combined, filtered, the filtrate is concentrated to 200ml, ethanol is added to increase the alcohol content to 60%, refrigerated, filtered, the filtrate is recovered ethanol, water is added, and refrigerated for later use. Gardenia is decocted with water twice, the first time for 1 hour and the second time for 0.5 hour, the decoction is combined, filtered, the filtrate is concentrated to 25ml, ethanol is added to increase the alcohol content to 60%, refrigerated, filtered, the filtrate is recovered ethanol, water is added, and refrigerated for later use. Honeysuckle is decocted with water twice, each time for 0.5 hour, the decoction is combined, filtered, the filtrate is concentrated to 60ml, ethanol is added to increase the alcohol content to 75%, filtered, the filtrate is adjusted to pH 8.0, refrigerated, ethanol is recovered, ethanol is further added to increase the alcohol content to 85%, refrigerated, filtered, the filtrate is recovered ethanol, water is added, and refrigerated for later use. Buffalo horn powder is hydrolyzed with barium hydroxide solution and mother-of-pearl powder is hydrolyzed with sulfuric acid for 7-9 hours, filtered, and the filtrates are combined. The pH is adjusted to 3.5-4.0, filtered, and the filtrate is added with ethanol to a 60% alcohol content. The solution is refrigerated, filtered, and the ethanol is recovered from the filtrate. Water is added and refrigerated for later use. Gardenia jasminoides extract, Radix Isatidis extract, and the hydrolyzed mixture of buffalo horn and mother-of-pearl are combined and added to a 75% ethanol solution of cholic acid and hyodeoxycholic acid. Mix thoroughly, add ethanol to a 75% alcohol content, adjust the pH to 7.0, refrigerate, filter, and recover the ethanol from the filtrate. Water is added and refrigerated for later use. Baicalin is dissolved in water for injection, the pH is adjusted to 7.5, and honeysuckle extract is added. The solution is mixed thoroughly and combined with the above-mentioned reserved solutions. Water for injection is added to 1000 ml. The solution is then treated with activated carbon, refrigerated, sealed, and sterilized.

[0006] This method has low alcohol and energy consumption efficiency, and the quality of the obtained 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 products and at the same time improve the therapeutic effect of the product. Summary of the Invention In view of the deficiencies in the prior art, the present invention provides a preparation method of Qingkailing injection and a product thereof.

[0007] In order to achieve the purpose of the present invention, the technical solutions adopted are as follows: A method for preparing Qingkailing injection comprises the following steps: (1) Extract the Radix Isatidis, Gardenia jasminoides, and Flos Lonicerae with water, and filter to obtain the extract; (2) Add buffalo horn powder and mother-of-pearl powder to 8 times the amount of water and mix, then add keratinase and aminopeptidase for enzymatic hydrolysis, filter, and obtain enzymatic hydrolyzate 1; (3) Mix the extract and enzymatic hydrolysate 1, add tannin enzyme for enzymatic hydrolysis, and filter to obtain enzymatic hydrolysate 2; (4) Add water to baicalin, mix it with enzymatic hydrolysate 2, add it to the ethanol solution of bile acid and hyodeoxycholic acid, mix it evenly, filter it, recover the ethanol from the filtrate, add water for injection to 1000 ml, treat it with activated carbon, filter it, and then ultrafilter it to obtain the product.

[0008] Preferably, the amount of water added in step (1) is 5-10 times the total mass of Radix Isatidis, Gardenia and Flos Lonicerae, and the extraction is performed by heating and reflux extraction, and the extraction time is 1-2 h.

[0009] Preferably, after the extraction in step (1), filtration is performed, ethanol is added to the filtrate to make the alcohol content reach 60%, filtering is performed, ethanol is further added to make the alcohol content reach 85%, filtering is performed, the filtrate is concentrated, the ethanol is recovered, and 1 times the amount of water is added to obtain an extract.

[0010] Preferably, the enzymatic hydrolysis temperature in step (2) is 40-50° C., the enzymatic hydrolysis pH is 6.5-7.5, and the enzymatic hydrolysis time is 2-4 h.

[0011] Preferably, the total mass of the keratinase and aminopeptidase is 0.1-0.5% of the mass of the water added in step (2).

[0012] Preferably, the mass ratio of keratinase to aminopeptidase is 1:5-10.

[0013] Preferably, the pH of the enzymatic hydrolysis in step (3) is 5.5-6.0, the temperature of the enzymatic hydrolysis is 30-40° C., and the time of the enzymatic hydrolysis is 1-3 h.

[0014] Preferably, the amount of tannase used in step (3) is 0.2-0.6% of the total mass of the extract and the enzymatic hydrolysate 1.

[0015] Preferably, the ethanol in step (4) is an ethanol aqueous solution with a mass concentration of 70-80%, and the ultrafiltration is performed by filtering with a 0.22 μm filter membrane.

[0016] Another object of the present invention is to provide a Qingkailing injection product prepared by the preparation method.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The Qingkailing injection prepared by the present invention has significant efficacy in treating acute hepatitis. Comparative studies have shown that the efficacy of decoction of Radix Isatidis, Gardenia, and Flos Lonicerae is better than that of decoction of each of the three herbs.

[0018] (2) The present invention first enzymatically hydrolyzes buffalo horn powder and mother-of-pearl powder using keratinase and aminopeptidase in a certain mass ratio, which can hydrolyze the protein in buffalo horn powder and mother-of-pearl powder to the greatest extent. Finally, the extract and the hydrolyzate are enzymatically hydrolyzed with tannin, which reduces protein and tannin impurities, reduces side reactions, increases active components, and improves the stability of the injection and the efficacy of treating acute hepatitis.

[0019] (3) The Qingkailing injection prepared by the preparation method of the present invention has good accelerated stability for 6 months. The product is safe, effective, and has controllable quality, and has good application prospects. DETAILED DESCRIPTION

[0020] The present invention is described below by way of specific examples to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0021] Among them, keratinase, 100,000 U / g, Shandong Fengtai Technology and Biology Co., Ltd.; serine protease, 200,000 U / g, Xi'an Darwen Biotechnology Co., Ltd.; aminopeptidase, i.e. aminopeptidase, 100,000 U / g, Nanning Dongheng Huadao Biotechnology Co., Ltd.; tannase, 250 U / g, Nanning Dongheng Huadao Biotechnology Co., Ltd.

[0022] Example 1 Prescription: bile acid 3.25g, mother of pearl powder 50.0g, hyodeoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0023] The preparation steps are as follows: (1) Add 8 times the amount of water to Radix Isatidis, Gardenia, and Flos Lonicerae, heat and reflux to extract for 1 hour, filter, add ethanol to the filtrate to make the alcohol content reach 60%, filter, continue to add ethanol to make the alcohol content reach 85%, filter, concentrate the filtrate, recover the ethanol, and add 1 times the amount of water to obtain the extract.

[0024] (2) Add buffalo horn powder and mother-of-pearl powder to 8 times the amount of water and mix them. Then add 0.3% of the water mass of complex enzyme (the complex enzyme is composed of keratinase and aminopeptidase in a mass ratio of 1:5). Hydrolyze at pH 7 and temperature 45℃ for 3h, boil for 2min, filter and obtain enzymatic solution 1.

[0025] (3) The extract and enzymatic hydrolysate 1 were mixed, 0.4% of the total weight of tannase was added, and enzymatic hydrolysis was carried out at pH 5.5 and 35°C for 2 h, boiled for 2 min, and filtered to obtain enzymatic hydrolysate 2; (4) Add water to baicalin, mix it with enzymatic hydrolysate 2, add it to a 75% ethanol solution of bile acid and hyodeoxycholic acid, mix well, filter, recover ethanol from the filtrate, add water for injection to 1000 ml, treat it with activated carbon, filter, and then ultrafilter it with a 0.22 μm filter membrane to sterilize it.

[0026] Example 2 Prescription: bile acid 3.25g, mother of pearl powder 50.0g, hyodeoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0027] The preparation steps are as follows: (1) Add 5 times the amount of water to Radix Isatidis, Gardenia, and Flos Lonicerae, heat and reflux to extract for 1 hour, filter, add ethanol to the filtrate to make the alcohol content reach 60%, filter, continue to add ethanol to make the alcohol content reach 85%, filter, concentrate the filtrate, recover the ethanol, and add 1 times the amount of water to obtain the extract.

[0028] (2) Add buffalo horn powder and mother-of-pearl powder to 8 times the amount of water and mix them. Then add 0.1% of the water mass of the complex enzyme (the complex enzyme is composed of keratinase and aminopeptidase in a mass ratio of 1:10). Hydrolyze at pH 7.5 and temperature 50℃ for 4h, boil for 2min, and filter to obtain enzymatic solution 1.

[0029] (3) The extract and enzymatic hydrolysate 1 were mixed, 0.6% of the total weight of tannase was added, and the mixture was incubated at pH 6.0 and 40°C for 3 h, boiled for 2 min, and filtered to obtain enzymatic hydrolysate 2; (4) Add water to baicalin, mix it with enzymatic hydrolysate 2, add it to a 75% ethanol solution of bile acid and hyodeoxycholic acid, mix well, filter, recover ethanol from the filtrate, add water for injection to 1000 ml, treat it with activated carbon, filter, and then ultrafilter it with a 0.22 μm filter membrane to sterilize it.

[0030] Example 3 Prescription: bile acid 3.25g, mother of pearl powder 50.0g, hyodeoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0031] The preparation steps are as follows: (1) Add 10 times the amount of water to Radix Isatidis, Gardenia, and Flos Lonicerae, heat and reflux to extract for 2 hours, filter, add ethanol to the filtrate to make the alcohol content reach 60%, filter, continue to add ethanol to make the alcohol content reach 85%, filter, concentrate the filtrate, recover the ethanol, and add 1 times the amount of water to obtain the extract.

[0032] (2) Add buffalo horn powder and mother-of-pearl powder to 8 times the amount of water and mix them. Then add 0.5% of the water mass of the complex enzyme (the complex enzyme is composed of keratinase and aminopeptidase in a mass ratio of 1:7). Hydrolyze at pH 6.5 and temperature 40℃ for 2h, boil for 2min, filter, and obtain enzymatic solution 1.

[0033] (3) The extract and enzymatic hydrolysate 1 were mixed, 0.2% of the total weight of tannase was added, and the mixture was incubated at pH 5.5 and 30°C for 1 hour, boiled for 2 minutes, and filtered to obtain enzymatic hydrolysate 2; (4) Add water to baicalin, mix it with enzymatic hydrolysate 2, add it to a 75% ethanol solution of bile acid and hyodeoxycholic acid, mix well, filter, recover ethanol from the filtrate, add water for injection to 1000 ml, treat it with activated carbon, filter, and then ultrafilter it with a 0.22 μm filter membrane to sterilize it.

[0034] Comparative Example 1 Prescription: bile acid 3.25g, mother of pearl powder 50.0g, hyodeoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0035] The preparation method of the comparative example Qingkailing injection comprises the following steps: (1) The above eight ingredients are decocted twice with water for 1 hour each time. The decoctions are combined and filtered. The filtrate is concentrated to 200 ml. Ethanol is added to make the alcohol content reach 60%. The filtrate is refrigerated, filtered, and the ethanol is recovered from the filtrate. Water is added and the filtrate is refrigerated for later use.

[0036] (2) Boil Gardenia jasminoides in water twice, the first time for 1 hour and the second time for 0.5 hour. Combine the decoctions, filter, and concentrate the filtrate to 25 ml. Add ethanol to make the alcohol content reach 60%, refrigerate, filter, recover the ethanol in the filtrate, add water, and refrigerate for later use.

[0037] (3) Decoction honeysuckle with water twice, each time for 0.5 h, combine the decoctions, filter, concentrate the filtrate to 60 ml, add ethanol to make the alcohol content reach 75%, filter, adjust the pH value 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 in the filtrate, add water, and refrigerate for later use.

[0038] (4) Hydrolyze buffalo horn powder with barium hydroxide solution and mother-of-pearl powder with sulfuric acid for 7-9 hours, filter, combine the filtrates, adjust the pH to 3.5-4.0, filter, add ethanol to the filtrate to make the alcohol content reach 60%, refrigerate, filter, recover ethanol from the filtrate, add water, and refrigerate for later use.

[0039] (5) Combine the Gardenia jasminoides extract, Radix Isatidis extract, and the hydrolyzed mixture of buffalo horn and mother-of-pearl, add them to a 75% ethanol solution of bile acid and hyodeoxycholic acid, mix well, add ethanol to make the alcohol content reach 75%, adjust the pH value to 7.0, refrigerate, filter, recover ethanol from the filtrate, add water, and refrigerate for later use.

[0040] (6) Dissolve baicalin in water for injection, adjust the pH to 7.5, add honeysuckle extract, mix well, combine with the above-mentioned reserve solutions, mix well, and add water for injection to 1000 ml. After treatment with activated carbon, refrigerate, fill, and sterilize.

[0041] Comparative Example 2 Prescription: bile acid 3.25g, mother of pearl powder 50.0g, hyodeoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0042] This comparative example provides a method for preparing Qingkailing injection, comprising the following steps: (1) Add 8 times the amount of water to Radix Isatidis, Gardenia, and Flos Lonicerae, heat and reflux to extract for 1 hour, filter, add ethanol to the filtrate to make the alcohol content reach 60%, filter, continue to add ethanol to make the alcohol content reach 85%, filter, concentrate the filtrate, recover the ethanol, and add 1 times the amount of water to obtain the extract.

[0043] (2) Add buffalo horn powder and mother-of-pearl powder to 8 times the amount of water and mix. Then add keratinase (0.3% by weight of water) and perform enzymolysis at pH 7 and 45°C for 3 hours. Boil for 2 minutes and filter to obtain enzymatic solution 1.

[0044] (3) The extract and enzymatic hydrolysate 1 were mixed, 0.4% of the total weight of tannase was added, and enzymatic hydrolysis was carried out at pH 5.5 and 35°C for 2 h, boiled for 2 min, and filtered to obtain enzymatic hydrolysate 2; (4) Add water to baicalin, mix it with enzymatic hydrolysate 2, add it to a 75% ethanol solution of bile acid and hyodeoxycholic acid, mix well, filter, recover ethanol from the filtrate, add water for injection to 1000 ml, treat it with activated carbon, filter, and then sterilize it by ultrafiltration using a 0.22 μm filter membrane.

[0045] Comparative Example 3 Prescription: bile acid 3.25g, mother of pearl powder 50.0g, hyodeoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0046] A preparation method of Qingkailing injection, comprising the following steps: (1) Add 8 times the amount of water to Radix Isatidis, Gardenia, and Flos Lonicerae, heat and reflux to extract for 1 hour, filter, add ethanol to the filtrate to make the alcohol content reach 60%, filter, continue to add ethanol to make the alcohol content reach 85%, filter, 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 mother-of-pearl powder to 8 times the amount of water and mix. Then add 0.3% aminopeptidase by weight of water and perform enzymolysis at pH 7 and 45°C for 3 hours. Boil for 2 minutes and filter to obtain enzymatic solution 1.

[0048] (3) The extract and enzymatic hydrolysate 1 were mixed, 0.4% of the total weight of tannase was added, and enzymatic hydrolysis was carried out at pH 5.5 and 35°C for 2 h, boiled for 2 min, and filtered to obtain enzymatic hydrolysate 2; (4) Add water to baicalin, mix it with enzymatic hydrolysate 2, add it to a 75% ethanol solution of bile acid and hyodeoxycholic acid, mix well, filter, recover ethanol from the filtrate, add water for injection to 1000 ml, treat it with activated carbon, filter, and then sterilize it by ultrafiltration using a 0.22 μm filter membrane.

[0049] Comparative Example 4 Prescription: bile acid 3.25g, mother of pearl powder 50.0g, hyodeoxycholic acid 3.75g, gardenia 25.0g, buffalo horn powder 25.0g, isatis root 200.0g, baicalin 5.0g, honeysuckle 60.0g.

[0050] A preparation method of Qingkailing injection, comprising the following steps: (1) Add 8 times the amount of water to Radix Isatidis, Gardenia, and Flos Lonicerae, heat and reflux to extract for 1 hour, filter, add ethanol to the filtrate to make the alcohol content reach 60%, filter, continue to add ethanol to make the alcohol content reach 85%, filter, concentrate the filtrate, recover the ethanol, and add 1 times the amount of water to obtain the extract.

[0051] (2) Add buffalo horn powder and mother-of-pearl powder to 8 times the amount of water and mix, then add 0.3% of the water mass of complex enzyme (the complex enzyme is composed of serine protease and aminopeptidase in a mass ratio of 1:5) and perform enzymolysis at pH 7 and temperature of 45℃ for 3h, boil for 2min, filter and obtain enzymatic solution 1.

[0052] (3) Add water to baicalin, mix it with enzymatic hydrolysate 1, add it to a 75% ethanol solution of bile acid and hyodeoxycholic acid, mix it evenly, filter it, recover the ethanol from the filtrate, add water for injection to 1000 ml, treat it with activated carbon, filter it, and then sterilize it by ultrafiltration using a 0.22 μm filter membrane.

[0053] Pharmacodynamics Experiment---Pharmacodynamic Verification of the Qingkailing Injection of the Present Invention in Treating Acute Hepatitis 1.1 Experimental animals SPF Kunming mice, half male and half female, weighing 20±2g, were used. All animals had free access to water and food and were acclimated to feeding for one week.

[0054] 1.2 Animal grouping and drug administration 80 KM mice, half male and half female, were randomly divided into a blank group, a model group, a positive group, and Example 1-3 and Comparative Example 1-4 groups, with 8 mice in each group. The blank and model groups were given intraperitoneal injections of normal saline for 7 days, the Example 1-3 and Comparative Example 1-4 groups were given 10 ml / kg of Qingkailing injection prepared in the corresponding group, and the positive group was given an intraperitoneal injection of silymarin 200 mg / kg for 7 consecutive days. One hour after the last administration, all groups except the blank group were intraperitoneally injected with 0.1% CCl4 olive oil solution (10 mL / kg). The blank group was intraperitoneally injected with an equal volume of olive oil. Afterwards, all mice were fasted but not watered. After 24 hours, blood was collected and centrifuged at 3000 r / min for 10 min. Serum was carefully aspirated with a pipette, and AST and ALT levels were measured according to the kit procedures. The liver was stored at -80℃ for later use. 50 mg of the same part of the liver tissue was taken and ground with 9 times the amount of PBS solution. The solution was centrifuged and the supernatant was collected. The MDA and GSH contents were determined according to the kit instructions.

[0055] 1.3 Experimental Results The results are shown in Tables 1 and 2.

[0056] SPSS 27.0 software was used to analyze the experimental data. When P < 0.05, the difference was considered statistically significant. The data were expressed as number ± standard deviation (x ± s), and one-way ANOVA was used for comparison among multiple groups, which was consistent with normal distribution and homogeneity of variance.

[0057] Table 1

[0058] Note: Compared with the control group, **P<0.01, compared with the model group, ##P<0.01, #P<0.05, compared with the Example 1 group, &P<0.05.

[0059] Results from Table 1 show that the model group showed significant differences in AST and ALT levels compared to the control group (P < 0.01). Furthermore, the experimental groups showed significant differences compared to the model group (P < 0.05 and P < 0.01, respectively), indicating that AST and ALT levels improved to some extent in each experimental group after treatment with the corresponding injection. Comparative Examples 1-4 showed less improvement than Example 1 (P < 0.05).

[0060] Table 2

[0061] Note: **P<0.01 compared with the control group. ##P<0.01, #P<0.05 compared with the model group, and &P<0.05 compared with Example 1.

[0062] Results analysis: From Table 2, we can see that compared with the control group, the levels of MDA and MDA in the model group were ** P < 0.01, showing a highly significant difference; compared with the model group, each experimental group showed # P < 0.05, ## P < 0.01, indicating that after treatment with the corresponding injection, the MDA and MDA levels of each experimental group were improved to a certain extent. The improvement effects of Examples 1-3 were generally better than those of Comparative Examples 1-4.

[0063] Experiment 2: Stability Study of Qingkailing Injection A stability test was conducted in accordance with the Chinese Pharmacopoeia's drug stability test guidelines. The Qingkailing injection prepared in Example 1 was subjected to accelerated test conditions for 6 months. Its stability after 3 and 6 months of accelerated testing was investigated. The test results are shown in Table 3, where the content is expressed as a percentage compared to the 0-month test. This indicates that the improved process results in a more stable injection.

[0064] Table 3

[0065] Results from Table 3 show that the injections prepared in Examples 1-3 were more stable after an accelerated 6-month period. Comparative Example 1 showed a significant darkening of color and a decrease in the content of its indicative components after an accelerated 6-month period. The injections prepared in Comparative Examples 2-4 showed a tendency to become turbid with extended storage, a significant decrease in the content of its active ingredients, and insufficient stability.

[0066] In summary, macromolecules are the primary material basis for safety issues in traditional Chinese medicine injections. Improving the safety of traditional Chinese medicine injections while maintaining or even enhancing their primary efficacy remains a pressing technical challenge in this field. This study, through improved process development, not only effectively extracts active ingredients and enhances drug activity, but also removes macromolecules such as proteins and condensed tannins, enhancing the stability of traditional Chinese medicine injections and promising application prospects.

[0067] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for preparing Qingkailing injection, characterized in that: The steps include: (1) Extract the Radix Isatidis, Gardenia jasminoides, and Flos Lonicerae with water, and filter to obtain the extract; (2) Buffalo horn powder and mother-of-pearl powder are mixed with water, and then keratinase and aminopeptidase are added for enzymatic hydrolysis, and the mixture is filtered to obtain enzymatic hydrolyzate 1; (3) Mix the extract and enzymatic hydrolysate 1, add tannin enzyme for enzymatic hydrolysis, and filter to obtain enzymatic hydrolysate 2; (4) Add water to baicalin, mix it with enzymatic hydrolysate 2, add it to the ethanol solution of bile acid and hyodeoxycholic acid, mix it evenly, filter it, recover the ethanol from the filtrate, add water for injection, treat it with activated carbon, filter it, and then ultrafilter it to obtain the product.

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 Radix Isatidis, Gardenia and Flos Lonicerae. The extraction is performed by heating and reflux extraction, and the extraction time is 1-2 hours.

3. The preparation method according to claim 1, characterized in that After the extraction in step (1), filtration is performed, ethanol is added to the filtrate to make the alcohol content reach 60%, filtering is performed, ethanol is further added to make the alcohol content reach 85%, filtering is performed, the filtrate is concentrated, ethanol is recovered, and 1 times the amount of water is added to obtain an extract.

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

5. The preparation method according to claim 4, characterized in that The total mass of the keratinase and aminopeptidase is 0.1-0.5% of the mass of the water added in step (2).

6. The preparation method according to claim 4, characterized in that The mass ratio of keratinase to aminopeptidase is 1:5-10.

7. 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° C., and the time of the enzymatic hydrolysis is 1-3 h.

8. The preparation method according to claim 1, characterized in that The amount of tannase used in step (3) is 0.2-0.6% of the total mass of the extract and the enzymatic hydrolysate 1.

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

10. A Qingkailing injection product prepared by the preparation method according to any one of claims 1 to 9.

Images