CN108938654B - Pulsatillae saponin B4 injection preparation - Google Patents
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Abstract
The invention provides an injection preparation of pulsatilla chinensis saponin B4, which comprises pulsatilla chinensis saponin B4 and pharmaceutically acceptable auxiliary materials, wherein the preparation is a water injection or a freeze-dried powder injection, and more preferably the freeze-dried powder injection. The invention also provides a preparation method of the pulsatilla chinensis saponin B4 water injection and the pulsatilla chinensis saponin B4 freeze-dried powder injection. The injection preparation of the pulsatilla chinensis saponin B4 has low effect dosage for reversing kidney injury caused by cisplatin, obviously improves the medication safety of the pulsatilla chinensis saponin B4, and is expected to provide a new choice for clinically treating the kidney injury and renal failure.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to an injection preparation with pulsatilla saponin B4 as an active ingredient.
Background
The Chinese herbal medicine, Pulsatilla chinensis (Bge.) Regel, is a commonly used Chinese herbal medicine, and is a dried root of Pulsatilla chinensis (Bge.) Regel) of Pulsatilla of Ranunculaceae. It is bitter and cold in nature, has effects of clearing heat and detoxicating, cooling blood and stopping dysentery, eliminating dampness and killing parasite, and can be used for treating toxic heat and bloody dysentery, warm malaria and chills, epistaxis, and bloody hemorrhoid. Modern pharmacological research shows that the Chinese pulsatilla root has wide antibacterial activity and has inhibitory effects on staphylococcus aureus, dysentery bacillus, dermatophyte, saccharomycetes, candida albicans and the like. The anti-tumor effect of pulsatillae radix is also a focus of research. In addition, the pulsatilla chinensis has the effects of resisting inflammation, enhancing the immune function of the organism and the like.
Pulsatillae radix has abundant triterpene saponin components. Pulsatillae saponin B4 belongs to lupane type pentacyclic triterpene saponin, and has structure of formula 1.
The pulsatilla saponin B4 has strong activity, and Chinese patent application with publication number CN105213410A (2016 (1/6) (as published)) discloses application of pulsatilla saponin B4 as an immunomodulator in drugs for treating acute inflammation, wherein the acute inflammation comprises acute kidney injury, acute liver injury and acute lung injury caused by over-expression of inflammatory factors. Also, as disclosed in the chinese patent application publication No. CN105535004A (2016, 5, 4, a), the application of the compound as an EV71 virus inhibitor in the preparation of anti-hand-foot-and-mouth disease drugs is disclosed.
Pulsatillae saponin B4 has five glycosyl groups and good water solubility. It has been shown that if the compound is administered orally (e.g. gavage in animal trials), the effective dose is large, resulting in a narrow safety window. Therefore, it is necessary to research the non-oral administration preparation of the pulsatilla saponin B4 so as to be beneficial to the active effect within the safe dosage range and meet the clinical medical requirements. However, reports related to the injection formulation of the pulsatilla saponin B4 are not found so far.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a preparation of pulsatilla saponin B4 for injection. The preparation has good stability and simple preparation process. Compared with oral administration, the preparation of the pulsatilla saponin B4 for injection has the advantages that the effective dose of the renal injury caused by cisplatin is reduced by 20 times, the medication safety is improved, and a new choice is expected to be provided for clinical treatment of renal injury and renal failure.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a preparation for injection of pulsatilla chinensis saponin B4 comprises pulsatilla chinensis saponin B4 and pharmaceutically acceptable auxiliary materials, and the preparation is water injection or freeze-dried powder injection.
Preferably, the injection preparation of the pulsatilla chinensis saponin B4 is composed of pulsatilla chinensis saponin B4, pharmaceutically acceptable auxiliary materials and inevitable impurities.
Preferably, the injection preparation of the pulsatilla saponin B4 is an intramuscular injection and/or an intravenous injection; more preferably an intravenous injection.
Preferably, the injection preparation of the pulsatilla chinensis saponin B4 is a water injection, and the pharmaceutically acceptable auxiliary material is water for injection.
Preferably, in the water injection, the mass percent of the pulsatilla chinensis saponin B4 is 0.5-5%, and more preferably 2-3%.
The invention also provides a preparation method of the injection, which comprises the steps of dissolving, decoloring by using activated carbon, filtering, fixing the volume, subpackaging and sterilizing.
Preferably, the preparation method of the water injection comprises the following specific operations:
accurately weighing anemonin B4 according to the prescription amount, adding part of water for injection, stirring to completely dissolve anemonin B4, adding activated carbon accounting for 0.1-0.5% of the total mass of the water injection into the solution, heating to boil, stirring for 15min, adding water for injection to constant volume to a final volume, mixing uniformly, and filtering with a 0.22 mu m microporous membrane to remove the activated carbon while the solution is hot to obtain an intermediate liquid medicine; subpackaging and sterilizing to obtain the product.
In the preparation method of the injection, the split charging and the sterilization are carried out by adopting the conventional method in the field. Wherein the sterilization can be performed by autoclaving, and sterilizing at 115 deg.C for 30min, or sterilizing at 121 deg.C for 15 min.
More preferably, the preparation for injection of the pulsatilla saponin B4 is a freeze-dried powder injection, and the pharmaceutically acceptable auxiliary materials are freeze-dried excipients selected from one or more of lactose, sucrose and mannitol in any proportion.
Preferably, in the freeze-dried powder injection, the mass ratio of the pulsatilla saponin B4 to the freeze-dried excipient is as follows: the pulsatilla chinensis saponin B4 is a freeze-dried excipient which is 3: 10-3: 16.
More preferably, in the freeze-dried powder injection, the mass ratio of the pulsatilla saponin B4 to the freeze-dried excipient is as follows: pulsatillae saponin B4 freeze-dried excipient 3: 14.
Also preferably, the lyophilization excipient is mannitol.
The invention also provides a preparation method of the freeze-dried powder injection, which comprises a preparation step of intermediate liquid medicine and a freeze-drying step.
Preferably, the preparation of the intermediate liquid medicine comprises the following specific operations:
dissolving pulsatilla chinensis saponin B4 in water for injection, stirring to dissolve, adding the freeze-drying excipient according to the mass ratio, stirring, adjusting the pH to 6.0-8.5, adding activated carbon accounting for 0.05-0.1% of the mass of the intermediate liquid medicine, heating and stirring at 100 ℃ for 10-20 min, cooling to room temperature, adding water for injection until the concentration of pulsatilla chinensis saponin B4 is 0.5-5%, and filtering with a 0.22-micron microporous membrane to obtain the intermediate liquid medicine.
More preferably, the preparation step of the intermediate liquid medicine comprises the following specific operations:
dissolving pulsatilla chinensis saponin B4 in water for injection, stirring to dissolve, adding the freeze-drying excipient according to the mass ratio, stirring, adjusting the pH to 6.5-8.0, adding active carbon accounting for 0.05% of the mass of the intermediate liquid medicine, heating and stirring at 100 ℃ for 10-20 min, cooling to room temperature, and adding water for injection until the concentration of pulsatilla chinensis saponin B4 is 2-3%; filtering with 0.22 μm microporous membrane to obtain the intermediate medicinal liquid.
Preferably, the freeze-drying step comprises prefreezing, sublimation, and resolution drying; the pre-freezing temperature is-30 ℃ to-15 ℃; the vacuum pressure of the sublimation stage and the desorption drying stage is 0.5-10 Pa; the sublimation temperature is-10 ℃ to 0 ℃; the temperature for desorption and drying is 25-30 ℃.
More preferably, the vacuum pressure in the sublimation stage and the desorption drying stage is 5-10 Pa.
More preferably, the sublimation temperature is-10 ℃.
As a preferred embodiment, the freeze-drying step is specifically performed by:
subpackaging the intermediate liquid medicine, transferring the intermediate liquid medicine into refrigeration equipment, cooling to-30 ℃ to-15 ℃ at the speed of 3.0-5.0 ℃/min or 1.0-1.5 ℃/min, and keeping the temperature for freezing for 8-10 hours; and then placing the mixture in a closed container, raising the temperature to-10-0 ℃ at a constant speed at the speed of 25-30 ℃/3h under the vacuum pressure of 0.5-10 Pa, preserving the heat for 8-10 h, keeping the vacuum degree unchanged, raising the temperature to 25-30 ℃ at the speed of 1.0-1.5 ℃/min, and preserving the heat for 4-6 h.
As a more preferred embodiment, the freeze-drying step is specifically performed by:
subpackaging the intermediate liquid medicine, transferring the intermediate liquid medicine into refrigeration equipment, cooling to-30 ℃ to-20 ℃ at the speed of 3.0-5.0 ℃/min or 1.0-1.5 ℃/min, and keeping the temperature for freezing for 8 hours; then placing the mixture into a closed container, raising the temperature to-10 ℃ at a constant speed at the speed of 25-30 ℃/3h under the vacuum pressure of 5-10 Pa, preserving the heat for 8h, keeping the vacuum degree unchanged, raising the temperature to 25-30 ℃ at the speed of 1.0-1.5 ℃/min, and preserving the heat for 4 h.
The invention also provides the pulsatilla chinensis saponin B4 freeze-dried powder injection prepared by the method.
In addition, the invention also provides an injection preparation of the pulsatilla saponin B4, or an application of the pulsatilla saponin B4 water injection or the pulsatilla saponin B4 freeze-dried powder injection prepared by the method in preparation of medicines for treating acute kidney injury, chronic renal failure and/or renal insufficiency.
The 'inevitable impurities' described in the specification of the invention comprise substances generated by a small amount of degradation of pulsatilla saponin B4 and a small amount of moisture in the pulsatilla saponin B4 freeze-dried powder injection. The above inevitable impurities, although present, should be present in amounts that comply with the limits set in the pharmacopoeia.
The injection preparation of the pulsatilla chinensis saponin B4 is applied to mammals, particularly to human beings. When the subject to which the preparation is administered is a human, the daily dose is 2 to 120mg per an adult having a body weight of 60 kg.
The pulsatilla chinensis saponin B4 water injection or pulsatilla chinensis saponin B4 freeze-dried powder injection realizes intramuscular injection or intravenous injection administration of pulsatilla chinensis saponin B4, ensures that active ingredients enter a circulatory system in an original form and are distributed to organs in vivo, particularly kidney, lung and the like, and realizes that pulsatilla chinensis saponin B4 exerts ideal drug effect at a lower dose, thereby enlarging the safety window of treatment and ensuring the safety of medication.
The pulsatilla chinensis saponin B4 has good water solubility and is convenient to prepare into water injection; but its stability in different media varies, so the solvent for injection solution needs to be carefully studied and selected.
The pulsatilla saponin B4 is sensitive to temperature and illumination as well as aqueous solution thereof; for example, when the pulsatilla saponin B4 aqueous solution is placed at 60 ℃ for 14 days, the purity of pulsatilla saponin B4 is reduced from 98.90% to about 93% on day 0, and the total impurities are increased from 0.64% to 0.94% on day 0. The freeze-dried powder injection is a preferable injection formulation of the pulsatilla saponin B4 because the water content is low and the stability of the preparation is more favorable.
However, in view of the complexity of the freeze-drying dispersion system, it is not easy to obtain the powder injection meeting the injection requirement efficiently and quickly, a mature theory of freeze-drying formula selection has not been formed so far, and each factor which may influence the freeze-drying behavior and the freeze-drying effect needs to be considered separately or jointly. Wherein, excipients and other additives in the freeze-drying formula can interact with main drugs, and the type and concentration of the formula can obviously influence the freezing solidification and sublimation dehydration behaviors of the system, such as the maximum freezing concentration glass transition temperature (an important index for measuring whether the system collapses during drying), the minimum freezing temperature, the cooling rate, the freezing annealing time and the like. In addition, the type and concentration of the additives also significantly affect the time of the drying stage and the properties of the final product. The invention inspects the types and the dosage of the freeze-drying excipient, the pH value of the intermediate liquid medicine, the pre-freezing temperature, the pre-freezing mode, the sublimation temperature, the vacuum pressure, the analysis drying temperature and other parameters respectively or jointly, optimizes the optimal process parameters and obtains the pulsatilla saponin B4 freeze-dried powder injection with good appearance and re-solubility and stable quality.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified. Wherein, the purchase conditions of partial reagents and raw auxiliary materials are as follows:
pulsatillae saponin B4: self-made, the HPLC detection purity is not lower than 98%;
mannitol (injection grade): F949A, cantonese chemical pharmaceutical llc;
glucose (injection grade): p20160118, Beijing Fengjing medicine-seeking medicine GmbH;
sucrose (injection grade): lot number 20150701, j.tbaker;
water for injection: self-making;
a magnetic stirrer: IKA Model: r015PS 25;
a freeze drying oven: GZLY-1.0, Beijing Songyuan Huaxing science and technology development Co.
The following test examples and examples relate to the determination of the content of pulsatilla saponin B4 by the following methods:
chromatographic conditions
A chromatographic column: c18，250mm×4.6mm，5μm
Mobile phase: acetonitrile-water (26:74) (volume ratio)
Flow rate: 1.0ml/min
Wavelength: 201nm
Preparation of control solutions
Precisely weighing appropriate amount of pulsatilla chinensis saponin B4 reference substance, placing in a 10ml measuring flask, adding methanol to dissolve and dilute to scale, shaking, and filtering to obtain solution containing pulsatilla chinensis saponin B4 reference substance 1mg per 1ml solution.
Preparation of test solution
Taking about 25mg of a sample to be measured, precisely weighing, placing in a 25ml measuring flask, adding the mobile phase for dissolving, diluting to scale, shaking up, filtering with a 0.22 mu m microporous membrane, and taking the subsequent filtrate to obtain the final product.
Measurement method
And (4) injecting 10 mu l of test solution into a liquid chromatograph, and recording the chromatogram.
Formula for calculation
Wherein:
Cto pair-control concentration (mg/ml);
Sto pair-the purity of the control;
Ato pair-major peak area of control solution;
Asample (A)-the area of the main peak of the test solution.
Test example 1Comparison of drug effects of pulsatilla saponin B4 by different administration routes
The Chinese pulsatilla root saponin B4 is administrated orally and intravenously in parallel, and the influence of different administration routes on the index of acute nephritis in the serum of mice after Cisplatin (CDDP) stimulation is examined.
1. Test materials
1.1 animals: ICR mice, 16-18g, male, hunan slaik experimental animals ltd, license number: SCXK (Xiang) 2016-.
1.2 reagent: cisplatin, the raw liquid is 5mg/kg, and cisplatin is taken before use and is added with a proper amount of normal saline to prepare 1.5mg/ml solution for standby.
1.3 medicine: pulsatillae saponin B4 (self-made, hereinafter referred to as "B4"), batch No.: 20161107, respectively; dexamethasone, 0.75 g/tablet, anhui jin taiyang biochemical pharmaceutical industry ltd, batch number: 15032521.
the above-mentioned test drugs are all prepared into a solution with a prescribed concentration under the term of "test method" by using double distilled water, and administered according to a prescribed manner.
1.4 instrument desk type low speed centrifuge; full-automatic biochemical analyzer
2. Test method
2.1 preparation before molding all animals were numbered and their weights were weighed.
2.2 the molding method is to mold according to the dose of cisplatin of 15ml/kg, and the normal group is injected with normal saline in the abdominal cavity, and the other groups are injected with cisplatin injection in the abdominal cavity.
2.3 grouping, administration animals were randomized into seven groups of 10-12 animals each:
firstly, normally grouping, and performing intragastric administration and double distilled water with the concentration of 20 ml/kg.weight;
② the model group, tail vein injection equal volume normal saline;
③ filling dexamethasone solution into a dexamethasone positive group (0.5mg/kg body weight) according to the volume of 20ml/kg body weight;
fourthly, B4 intravenous injection of high dose group (5mg/kg body weight) is carried out, and B4 solution is injected into tail vein of 5ml/kg body weight;
IV injecting low dose group B4 (2.5mg/kg body weight) into tail vein of 5ml/kg body weight with B4 solution;
sixthly, B4 intragastric administration high dose group (100mg/kg body weight) is performed according to the intragastric administration B4 solution of 20ml/kg body weight;
seventhly, the B4 intragastric administration low dose group (50mg/kg body weight) is intragastric administered with the B4 solution according to the proportion of 20ml/kg body weight.
The administration was continued for 4 days from the day of molding.
2.4 index detection
Taking urine 0.5 hours after the last administration, and measuring urine protein; the blood is taken from eyeballs after 1 hour of administration, the whole blood of the taken mice is kept still for 2 hours at room temperature, 3500rpm/min and 15min of centrifugation are carried out, and 200 mul of supernatant is taken to detect Total Protein (TP), urea nitrogen (BUN) and creatinine (Cre) by a full-automatic biochemical analyzer.
3. Test results
The detection results of each index of each group are shown in table 1.
TABLE 1B4 Effect on the indices of cisplatin-induced acute Kidney injury in mice (SD. + -. Mean)
#: p is less than 0.05 compared with the normal group; *: p is less than 0.05 compared with the model group.
The data in table 1 show:
compared with the normal group, the levels of urea nitrogen (BUN) and creatinine (Cre) in urine protein and serum of the model group are obviously increased (P is less than 0.05), which indicates that the model building is successful.
② compared with the model group, the urea nitrogen (BUN) and urine protein level of the positive control group is reduced remarkably (P is less than 0.05).
③ compared with the model group, the 5mg/kg body weight B4 administered by intravenous injection can obviously reduce the urea nitrogen (BUN) and creatinine (Cre) levels (P < 0.05), and the urine protein level has the tendency of decreasing although there is no significant difference.
Compared with the model group, the mice only have obvious reduction of the urea nitrogen (BUN) level (P is less than 0.05) by the intragastric administration of the B4 with the weight of 100 mg/kg.
4. Conclusion of the experiment
The pulsatilla chinensis saponin B4 has the effect of resisting kidney injury caused by cisplatin, and the application of the pulsatilla chinensis saponin B4 in clinical application is suggested to have the prospect of developing drugs for treating acute and chronic nephritis and renal failure. The administration route has important influence on the active effect of the pulsatilla saponin B4, the effect of the intravenous injection of 5 mg/kg-body weight B4 on reversing the abnormal renal function index caused by cisplatin is stronger than that of the intravenous injection of 100 mg/kg-body weight for intragastric administration, and the dosage is reduced by at least 20 times. Therefore, the injection administration, especially the intravenous injection administration, has important significance for improving the curative effect and the safety of the pulsatilla saponin B4.
Example 1Chinese pulsatilla root saponin B4 water injection
The prescription of 100ml of the water injection of the embodiment is as follows:
the preparation method comprises the following steps:
weighing the prescription amount of the pulsatilla chinensis saponin B4 raw material, precisely weighing, adding a part (proper amount) of water for injection, completely dissolving the pulsatilla chinensis saponin B4 raw material under the action of magnetic stirring, adding activated carbon with the mass of 0.10% of the solution into the solution, heating and stirring for 15min under the condition of water bath at 100 ℃, diluting to 100ml with water for injection, shaking uniformly, filtering by a 0.22 mu m microporous membrane to remove the activated carbon, precisely weighing 2ml of intermediate liquid medicine, filling into a 5ml ampoule bottle, and sterilizing for 30min at 115 ℃ to obtain the pulsatilla chinensis saponin B4.
Test example 2Research on Pulsatilla saponin B4 freeze-dried powder process
1.1 compatibility test of raw and auxiliary materials
The purification and related substance conditions of the pulsatilla saponin B4 were examined by sampling on day 0, day 7 and day 14 after uniformly mixing the pulsatilla saponin B4 raw material with each auxiliary material (water for injection, normal saline, mannitol, lactose, sucrose, glucose) according to a mass ratio of 1:5, respectively, and placing under conditions of high temperature (60 ℃, 40 ℃) and illumination (4500Lux +/-500), and the results are shown in tables 2, 3 and 4. .
TABLE 2 raw and auxiliary materials compatibility test 0 day results
*: API means pulsatillae radix saponin B4, the same as below.
The data in table 2 show that, on day 0 of the investigation, the purity of the pasqueoside B4 in each raw and auxiliary material combination was equivalent, and the contents of the related substances were not significantly different.
TABLE 3 raw and auxiliary materials compatibility test 7 days results
The data in Table 3 show that the pasqueflower saponin B4 itself and its mixture with various adjuvants under consideration are sensitive to temperature and light, and its high temperature (60 ℃) stability is particularly affected by water for injection, physiological saline and glucose.
TABLE 4 raw and auxiliary materials compatibility test 14 days results
Compared with the investigation result of the 7 th day, the purity of the saponin in the pulsatilla chinensis saponin B4 and the mixture of the pulsatilla chinensis saponin B4 with mannitol, sucrose and lactose is not further obviously reduced at 60 ℃, which indicates that the saponin tends to be stable; however, in the case of the saponin B4 in water for injection, physiological saline, and a mixture of the saponin B4 and glucose, the purity of the saponin is further decreased and the saponin is still degraded.
The compatibility test result of the raw materials and the auxiliary materials shows that the compatibility of the pulsatilla saponin B4 and each auxiliary material is better under the condition of 40 ℃ and illumination, the purity and related substances have no obvious change, and the stability is relatively high. However, when the anemonin B4 raw material is mixed with water for injection, normal saline or glucose at the high temperature of 60 ℃, the main drugs are degraded to different degrees; therefore, the injection water injection or freeze-dried powder injection of the pulsatilla saponin B4 should avoid long-term high temperature in production, storage and transportation.
1.2 excipient type selection
The common freeze-drying excipient is lactose, mannitol, sucrose and glucose. Weighing pulsatilla chinensis saponin B4, mannitol, lactose, glucose and sucrose according to the table 5, preparing a liquid medicine intermediate according to the following process, and freeze-drying:
dissolving pulsatilla chinensis saponin B4 in a proper amount of water for injection, stirring to dissolve, adding the freeze-dried excipient, stirring, adjusting the pH to 7.0, adding 0.05g of activated carbon, heating and stirring at 100 ℃ for 15min, cooling to room temperature, and adding water for injection to 100 g; filtering with 0.22 μm microporous membrane to obtain the intermediate medicinal liquid. Precisely measuring 2ml of intermediate liquid medicine, filling the intermediate liquid medicine into a 10ml penicillin bottle, cooling to-20 ℃ at the speed of 5.0 ℃/min in a refrigeration device, and keeping the temperature for refrigeration for 8 hours; then placing the mixture into a freeze dryer, raising the temperature to-10 ℃ at a constant speed at a speed of 25 ℃/3h under a vacuum degree of 5Pa, preserving the heat for 8h, keeping the vacuum degree unchanged, raising the temperature to 25 ℃ at a speed of 1.0 ℃/min, and preserving the heat for 6 h.
The properties of the freeze-dried products prepared by the above methods are observed, and the purity and the content of related substances of the pulsatilla saponin B4 are examined, and the results are shown in Table 6.
Table 5 prescription design
| Prescription composition | F1 | F2 | F3 | F4 |
| API | 1.5g | 1.5g | 1.5g | 1.5g |
| Mannitol | 5.0g | --- | --- | --- |
| Sucrose | --- | 5.0g | --- | --- |
| Lactose | --- | --- | 5.0g | --- |
| Glucose | --- | --- | --- | 5.0g |
| Water for injection | To 100g | To 100g | To 100g | To 100g |
TABLE 6 screening results of excipient types
From the results shown in table 6, it can be seen that, using lactose, sucrose and glucose as excipients, the pulsatis saponin B4 was degraded to different degrees after freeze-drying, and the order of the degradation degree is: glucose > sucrose > lactose; after mannitol is used as an excipient, the content and related substances of the mannitol are not obviously changed. Thus, sucrose, lactose or mannitol are preferred as freeze-drying excipients, with mannitol being most preferred.
1.3 excipient dosage selection
Anemonin B4 and mannitol were weighed according to the recipe shown in Table 7, respectively, and the medicinal liquid intermediate was prepared by the following procedure and lyophilized:
dissolving pulsatilla chinensis saponin B4 in a proper amount of water for injection, stirring to dissolve, adding mannitol, stirring, adjusting pH to 7.0, adding 0.05g of activated carbon, heating and stirring at 100 ℃ for 15min, cooling to room temperature, and adding water for injection to 100 g; filtering with 0.22 μm microporous membrane to obtain the intermediate medicinal liquid. Precisely measuring 2ml of intermediate liquid medicine, filling the intermediate liquid medicine into a 10ml penicillin bottle, cooling to-20 ℃ at the speed of 5 ℃/min in a refrigeration device, and keeping the temperature for refrigeration for 8 hours; then placing the mixture into a freeze dryer, raising the temperature to-10 ℃ at a constant speed at a speed of 25 ℃/3h under a vacuum degree of 5Pa, preserving the heat for 8h, keeping the vacuum degree unchanged, raising the temperature to 25 ℃ at a speed of 1 ℃/min, and preserving the heat for 6 h.
The properties of the intermediate liquid medicine and the lyophilized product prepared in each formula were observed, and the purity of anemonin B4 and the content of the related substances were examined, and the test results are shown in Table 7.
Table 7 excipient dosage screening recipe design and screening results
As is clear from the results shown in Table 7, the amount of mannitol used is not as large as possible, but is within a suitable range. When the mass ratio of the B4 to the mannitol is 3: 10-3: 14, the stability of the B4 tends to be improved along with the increase of the dosage of the mannitol; then, as the amount of mannitol was increased, the degradation of B4 in the lyophilized product was increased. Therefore, the preferred mass ratio range of anemonin B4 and mannitol in the present invention is: 3:10 to 3:16, most preferably 3: 14.
1.4 screening of pH value of intermediate liquid medicine
The pulsatilla chinensis saponin B4 is sensitive to pH value, and the degradation degree of the pulsatilla chinensis saponin B4 changes along with the change of the pH value. Therefore, the degradation conditions of the intermediate solution under different pH conditions are considered, and the optimal pH range is screened out. The specific test steps are as follows:
according to a formula F8 in Table 7, 8 parts of anemonin B4 and mannitol are respectively weighed; dissolving pulsatilla chinensis saponin B4 in appropriate amount of water for injection, stirring to dissolve, adding mannitol, stirring, adjusting pH as shown in Table 8, adding 0.05g of activated carbon, heating at 100 deg.C and stirring for 15min, cooling to room temperature, and adding water for injection to 100 g; filtering with 0.22 μm microporous membrane to obtain the intermediate medicinal liquid. Precisely measuring 2ml of intermediate liquid medicine, filling the intermediate liquid medicine into a 10ml penicillin bottle, cooling to-20 ℃ at the speed of 5 ℃/min in a refrigeration device, and keeping the temperature for refrigeration for 8 hours; then placing the mixture into a freeze dryer, raising the temperature to-10 ℃ at a constant speed at a speed of 25 ℃/3h under a vacuum degree of 5Pa, preserving the heat for 8h, keeping the vacuum degree unchanged, raising the temperature to 25 ℃ at a speed of 1.0 ℃/min, and preserving the heat for 6 h.
The results are shown in Table 8.
TABLE 8 investigation of test results under different pH conditions
From the results shown in table 8, it can be seen that: the pH value of the intermediate liquid medicine is preferably 6.0-8.5, and most preferably 6.5-8.0 by taking the content of related substances as an index.
1.5 screening of the amount of activated carbon
The method inspects the influence of the dosage of the active carbon and the adsorption time on the main drug, and comprises the following specific test steps:
according to a formula F8 in a table 7, 3 parts of pasqueflower saponin B4 and mannitol are respectively weighed; dissolving pulsatilla chinensis saponin B4 in a proper amount of water for injection, stirring to dissolve, adding mannitol, stirring, adding water for injection to 100ml, adjusting pH to 7, and determining the purity of pulsatilla chinensis saponin B4; adding activated carbon as shown in Table 8, heating and stirring at 100 deg.C for 15min, filtering part of the medicinal liquid with 0.22 μm microporous membrane, and measuring pH value and purity of Pulsatillae saponin B4; stirring the rest medicinal liquid at 100 deg.C for 30min, filtering part of the medicinal liquid with 0.22 μm microporous membrane, and measuring pH value and purity of Pulsatillae saponin B4.
The results are shown in Table 9.
TABLE 9 test results of the effects of different active carbon dosages and adsorption times on the principal drugs
The results in Table 9 show that the purity of pulsatillae radix saponin B4 is reduced by increasing the amount of activated carbon and the adsorption time. Therefore, the amount (mass) of the activated carbon is preferably 0.05% to 0.1%, more preferably 0.05%, based on the volume of the intermediate chemical solution.
1.6 study of the Freeze-drying Process
The freeze-drying process directly affects the appearance, reconstitution rate, moisture and stability of the product. Therefore, the parameters such as pre-freezing temperature, pre-freezing mode, sublimation temperature, vacuum pressure, analysis drying temperature and the like are mainly considered, and the specific test steps are as follows:
weighing 4 parts of pasqueflower saponin B4 and mannitol according to a formula F8 in Table 7; dissolving pulsatilla chinensis saponin B4 in a proper amount of water for injection, stirring to dissolve, adding mannitol, stirring, adjusting pH to 7, adding 0.05g of activated carbon, heating and stirring at 100 ℃ for 15min, cooling to room temperature, and adding water for injection to 100 g; filtering with 0.22 μm microporous membrane to obtain the intermediate medicinal liquid. Precisely measuring 2ml of intermediate liquid medicine, filling the intermediate liquid medicine into a 10ml penicillin bottle, and pre-freezing in a freezing device according to the parameters and conditions shown in the table 10; then, the mixture was placed in a freeze dryer and subjected to sublimation and desorption drying under the parameters and conditions shown in Table 10. And respectively observing the appearance of each freeze-dried product, measuring the content of related substances in the freeze-dried products, carrying out a redissolution test, and recording the redissolution time. The results are shown in Table 10.
TABLE 10 Effect test results on the product of different lyophilization Process parameters
From the results shown in table 10, it can be seen that the vacuum pressure during sublimation and desorption drying is a key parameter, and the vacuum pressure is too low (0.1 Pa of process 3), so that the obtained freeze-dried product has poor appearance and high moisture content, and the content of related substances is obviously higher than that of the products of other processes, which indicates that the pulsatilla saponin B4 is degraded more. Therefore, the vacuum pressure is preferably 0.5 to 10Pa, and more preferably 5 to 10 Pa.
Preferably, the freeze-drying step of the present invention specifically comprises the following steps:
subpackaging the intermediate liquid medicine, cooling to-30 ℃ to-15 ℃ at the speed of 3-5 ℃/min or 1.0-1.5 ℃/min, keeping the temperature and freezing for 8-10 hours; and then placing the mixture in a closed container, raising the temperature to-10-0 ℃ at a constant speed at the speed of 25-30 ℃/3h under the vacuum pressure of 0.5-10 Pa, preserving the heat for 8-10 h, keeping the vacuum degree unchanged, raising the temperature to 25-30 ℃ at the speed of 1.0-1.5 ℃/min, and preserving the heat for 4-6 h.
More preferably, the freeze-drying step is specifically performed by:
subpackaging the intermediate liquid medicine, cooling to-30 ℃ to-20 ℃ at the speed of 3.0-5.0 ℃/min or 1.0-1.5 ℃/min in a refrigeration device, and keeping the temperature for freezing for 8 hours; then placing the mixture into a closed container, raising the temperature to-10 ℃ at a constant speed at the speed of 25-30 ℃/3h under the vacuum pressure of 5-10 Pa, preserving the heat for 8h, keeping the vacuum degree unchanged, raising the temperature to 25-30 ℃ at the speed of 1.0-1.5 ℃/min, and preserving the heat for 4 h.
According to the results of the test example, the pulsatilla chinensis saponin B4 freeze-dried powder injection comprises pulsatilla chinensis saponin B4 and a freeze-drying excipient, wherein the freeze-drying excipient is selected from one or more of mannitol, sucrose or lactose in any proportion, and preferably mannitol; the mass ratio of the pulsatilla saponin B4 to the freeze-drying excipient is 3: 10-3: 16, and the preferred mass ratio is 3: 14.
Preferably, the pulsatilla chinensis saponin B4 freeze-dried powder injection comprises pulsatilla chinensis saponin B4 and mannitol in a mass ratio of 3: 10-3: 16; and may also contain small amount of inevitable impurities, such as water, and degradation product of Pulsatillae saponin B4.
The preparation method of the pulsatilla chinensis saponin B4 freeze-dried powder injection comprises the following steps:
I. preparation of intermediate liquid medicine
Dissolving pulsatilla chinensis saponin B4 in a proper amount of water for injection, stirring to dissolve, adding the freeze-drying excipient according to a ratio, stirring, adjusting the pH to 6.0-8.5, adding activated carbon accounting for 0.05-0.1% of the mass of the intermediate liquid medicine, heating and stirring at 100 ℃ for 10-20 min, cooling to room temperature, and adding water for injection until the concentration of pulsatilla chinensis saponin B4 is 0.5-5%; filtering with 0.22 μm microporous membrane to obtain the intermediate medicinal liquid;
freeze drying of
Subpackaging the intermediate liquid medicine prepared in the step I; transferring the mixture into refrigeration equipment, cooling the mixture to minus 30 ℃ to minus 15 ℃ at the speed of 3.0 to 5.0 ℃/min or 1.0 to 1.5 ℃/min, and keeping the temperature for freezing for 8 to 10 hours; and then placing the mixture in a closed container, raising the temperature to-10-0 ℃ at a constant speed at the speed of 25-30 ℃/3h under the vacuum pressure of 0.5-10 Pa, preserving the heat for 8-10 h, keeping the vacuum degree unchanged, raising the temperature to 25-30 ℃ at the speed of 1.0-1.5 ℃/min, and preserving the heat for 4-6 h.
The preparation method preferably adopts the technical scheme that:
I. preparation of intermediate liquid medicine
Dissolving pulsatilla chinensis saponin B4 in water for injection, stirring to dissolve, adding the freeze-drying excipient according to the mass ratio, stirring, adjusting the pH to 6.5-8.0, adding active carbon accounting for 0.05% of the mass of the intermediate liquid medicine, heating and stirring at 100 ℃ for 10-20 min, cooling to room temperature, and adding water for injection until the concentration of pulsatilla chinensis saponin B4 is 2-3%; filtering with 0.22 μm microporous membrane to obtain the intermediate medicinal liquid;
freeze drying of
Subpackaging the intermediate liquid medicine prepared in the step I, transferring the intermediate liquid medicine into refrigeration equipment, cooling to-30 ℃ to-20 ℃ at the speed of 3.0-5.0 ℃/min or 1.0-1.5 ℃/min, and keeping the temperature for freezing for 8 hours; then placing the mixture into a closed container, raising the temperature to-10 ℃ at a constant speed at the speed of 25-30 ℃/3h under the vacuum pressure of 5-10 Pa, preserving the heat for 8h, keeping the vacuum degree unchanged, raising the temperature to 25-30 ℃ at the speed of 1.0-1.5 ℃/min, and preserving the heat for 4 h.
Example 2Pulsatillae saponin B4 freeze-dried powder injection
Prescription of intermediate liquid medicine:
the preparation method comprises the following steps:
I. preparation of intermediate liquid medicine
Taking the prescribed amount of pulsatilla saponin B4Precisely weighing raw materials, adding appropriate amount of water for injection, and magnetically stirring to obtain anemonin B4After the raw materials are completely dissolved, adding mannitol with the amount of the prescription into the solution, stirring to completely dissolve the mannitol, continuously stirring for about 10 minutes, adding 0.05g of activated carbon, heating and stirring for 15 minutes under the condition of 100 ℃ water bath, cooling to room temperature, adding water for injection to 100ml, shaking up, and filtering through a 0.22 mu m microporous filter membrane to remove the activated carbon to obtain the injection solution;
freeze drying of
Subpackaging the intermediate liquid medicine prepared in the step I: precisely measuring 2ml of intermediate liquid medicine and filling the intermediate liquid medicine into a 10ml penicillin bottle; transferring to a refrigeration device, cooling to-20 ℃ at full speed, and keeping the temperature for 8 h; then transferring to a freeze dryer, raising the temperature to-10 ℃ at a constant speed of 25 ℃/3h under the vacuum pressure of 5Pa, keeping the temperature at-10 ℃ for 8h, raising the temperature to 25 ℃ at a constant speed, and keeping the temperature for 4h to obtain the freeze-dried powder.
The prepared pulsatilla chinensis saponin B4 freeze-dried powder injection is a white fluffy block, the water redissolving time is 6s, the water content is 1.3%, the purity of pulsatilla chinensis saponin B4 is 99.74%, the maximum single impurity content is 0.12%, and the total impurity content is 0.26%.
Example 3Pulsatillae saponin B4 freeze-dried powder injection
Prescription of intermediate liquid medicine:
the preparation method comprises the following steps:
I. preparation of intermediate liquid medicine
Taking a prescription amount of the pulsatilla chinensis saponin B4 raw material, precisely weighing, adding a proper amount of water for injection, completely dissolving the pulsatilla chinensis saponin B4 raw material under the action of magnetic stirring, adding a prescription amount of lactose into the solution, stirring to completely dissolve the lactose, continuously stirring for about 10 minutes, adding 0.1g of activated carbon, heating and stirring for 20 minutes under the condition of 100 ℃ water bath, cooling to room temperature, adding water for injection to 100ml, shaking uniformly, and filtering by a 0.22 mu m microporous filter membrane to remove the activated carbon to obtain the pulsatilla chinensis saponin B4;
freeze drying of
Subpackaging the intermediate liquid medicine prepared in the step I: precisely measuring 2ml of intermediate liquid medicine and filling the intermediate liquid medicine into a 10ml penicillin bottle; transferring to a refrigeration device, cooling to-30 ℃ at the speed of 3 ℃/min, and keeping the temperature for 10 h; then transferring to a freeze dryer, raising the temperature to-10 ℃ at a constant speed at a speed of 25 ℃/3h under a vacuum pressure of 10Pa, keeping the temperature at-10 ℃ for 10h, raising the temperature to 30 ℃ at a speed of 1.5 ℃/min, and keeping the temperature for 6h to obtain the freeze-dried powder.
The prepared pulsatilla chinensis saponin B4 freeze-dried powder injection is a white fluffy block, the water redissolving time is 6s, the water content is 1.07%, the purity of pulsatilla chinensis saponin B4 is 99.61%, the maximum single impurity content is 0.28%, and the total impurity content is 0.39%.
Example 4Pulsatillae saponin B4 freeze-dried powder injection
Prescription of intermediate liquid medicine:
the preparation method comprises the following steps:
I. preparation of intermediate liquid medicine
Taking a prescription amount of the pulsatilla chinensis saponin B4 raw material, precisely weighing, adding a proper amount of water for injection, completely dissolving the pulsatilla chinensis saponin B4 raw material under the action of magnetic stirring, adding a prescription amount of sucrose into the solution, stirring to completely dissolve the sucrose, continuously stirring for about 10 minutes, adding 0.15g of activated carbon, heating and stirring for 15 minutes under the condition of 100 ℃ water bath, cooling to room temperature, adding water for injection to 100ml, shaking uniformly, and filtering by a 0.22 mu m microporous filter membrane to remove the activated carbon to obtain the pulsatilla chinensis saponin B4;
freeze drying of
Subpackaging the intermediate liquid medicine prepared in the step I: precisely measuring 2ml of intermediate liquid medicine and filling the intermediate liquid medicine into a 10ml penicillin bottle; transferring to a refrigeration device, cooling to-15 ℃ at the speed of 5 ℃/min, and keeping the temperature for 9 h; then transferring to a freeze dryer, raising the temperature to 0 ℃ at a constant speed of 30 ℃/3h under the vacuum pressure of 0.5Pa, keeping the temperature at 0 ℃ for 8h, raising the temperature to 25 ℃ at the speed of 1 ℃/min, and keeping the temperature for 5h to obtain the product.
The prepared pulsatilla chinensis saponin B4 freeze-dried powder injection is a white fluffy block, the water redissolving time is 6s, the water content is 1.39%, the purity of pulsatilla chinensis saponin B4 is 99.68%, the maximum single impurity content is 0.22%, and the total impurity content is 0.32%.
In a word, the invention provides a preparation of pulsatilla chinensis saponin B4 for injection, which comprises a water injection and a freeze-dried powder injection. Compared with oral administration, the injection preparation provided by the invention obviously reduces the effective dose of the pulsatilla saponin B4 for reversing kidney injury caused by cisplatin, and improves the medication safety. In addition, through the optimization of preparation process parameters, all indexes of the injection preparation are stable and controllable.
Claims (9)
1. A preparation for injection of Pulsatillae saponin B4 comprises Pulsatillae saponin B4, pharmaceutically acceptable adjuvants and inevitable impurities; the preparation for injection of the pulsatilla chinensis saponin B4 is a freeze-dried powder injection, and the pharmaceutically acceptable auxiliary materials are freeze-dried excipients selected from one or more of lactose, sucrose and mannitol in any proportion;
in the freeze-dried powder injection, the mass ratio of the pulsatilla chinensis saponin B4 to the freeze-dried excipient is as follows: the pulsatilla chinensis saponin B4 is a freeze-dried excipient which is 3: 10-3: 16;
the freeze-dried powder injection is prepared by the following steps:
preparing an intermediate liquid medicine: dissolving pulsatilla chinensis saponin B4 in water for injection, stirring for dissolving, adding the freeze-drying excipient according to the mass ratio, stirring, adjusting the pH to 6.0-8.5, adding activated carbon accounting for 0.05-0.1% of the mass of the intermediate liquid medicine, heating and stirring for 10-20 min at 100 ℃, cooling to room temperature, adding water for injection until the concentration of pulsatilla chinensis saponin B4 is 0.5-5%, and filtering with a 0.22-micron microporous membrane to obtain the intermediate liquid medicine;
and (3) freeze drying: subpackaging the intermediate liquid medicine, transferring the intermediate liquid medicine into refrigeration equipment, cooling to-30 ℃ to-15 ℃ at the speed of 3.0-5.0 ℃/min or 1.0-1.5 ℃/min, and keeping the temperature for freezing for 8-10 hours; and then placing the mixture in a closed container, raising the temperature to-10-0 ℃ at a constant speed at the speed of 25-30 ℃/3h under the vacuum pressure of 0.5-10 Pa, preserving the heat for 8-10 h, keeping the vacuum degree unchanged, raising the temperature to 25-30 ℃ at the speed of 1.0-1.5 ℃/min, and preserving the heat for 4-6 h.
2. The preparation of claim 1, wherein the mass ratio of the pulsatilla saponin B4 to the freeze-dried excipient in the freeze-dried powder injection is as follows: pulsatillae saponin B4 freeze-dried excipient 3: 14.
3. The injectable preparation of pulsatilla saponin B4 according to claim 1 or 2, wherein the lyophilized excipient is mannitol.
4. A method for preparing lyophilized powder for injection of Pulsatillae saponin B4, wherein the lyophilized powder for injection of Pulsatillae saponin B4 is as defined in any one of claims 1-3, and comprises the steps of preparing intermediate liquid medicine and lyophilizing;
the preparation method of the intermediate liquid medicine comprises the following specific operations:
dissolving pulsatilla chinensis saponin B4 in water for injection, stirring for dissolving, adding the freeze-drying excipient according to the mass ratio, stirring, adjusting the pH to 6.0-8.5, adding activated carbon accounting for 0.05-0.1% of the mass of the intermediate liquid medicine, heating and stirring for 10-20 min at 100 ℃, cooling to room temperature, adding water for injection until the concentration of pulsatilla chinensis saponin B4 is 0.5-5%, and filtering with a 0.22-micron microporous membrane to obtain the intermediate liquid medicine;
the freeze drying step comprises the following specific operations:
subpackaging the intermediate liquid medicine, transferring the intermediate liquid medicine into refrigeration equipment, cooling to-30 ℃ to-15 ℃ at the speed of 3.0-5.0 ℃/min or 1.0-1.5 ℃/min, and keeping the temperature for freezing for 8-10 hours; and then placing the mixture in a closed container, raising the temperature to-10-0 ℃ at a constant speed at the speed of 25-30 ℃/3h under the vacuum pressure of 0.5-10 Pa, preserving the heat for 8-10 h, keeping the vacuum degree unchanged, raising the temperature to 25-30 ℃ at the speed of 1.0-1.5 ℃/min, and preserving the heat for 4-6 h.
5. The preparation method according to claim 4, wherein the preparation of the intermediate chemical solution comprises the following steps:
dissolving pulsatilla chinensis saponin B4 in water for injection, stirring to dissolve, adding the freeze-drying excipient according to the mass ratio, stirring, adjusting the pH to 6.5-8.0, adding active carbon accounting for 0.05% of the mass of the intermediate liquid medicine, heating and stirring at 100 ℃ for 10-20 min, cooling to room temperature, and adding water for injection until the concentration of pulsatilla chinensis saponin B4 is 2-3%; filtering with 0.22 μm microporous membrane to obtain the intermediate medicinal liquid.
6. The method according to claim 4, wherein the vacuum pressure in the freeze-drying step, the sublimation step and the desorption-drying step is 5 to 10 Pa.
7. The method according to claim 4, wherein the sublimation temperature is-10 ℃.
8. The preparation method according to claim 4, wherein the freeze-drying step is carried out by:
subpackaging the intermediate liquid medicine, transferring the intermediate liquid medicine into refrigeration equipment, cooling to-30 ℃ to-20 ℃ at the speed of 3.0-5.0 ℃/min or 1.0-1.5 ℃/min, and keeping the temperature for freezing for 8 hours; then placing the mixture into a closed container, raising the temperature to-10 ℃ at a constant speed at the speed of 25-30 ℃/3h under the vacuum pressure of 5-10 Pa, preserving the heat for 8h, keeping the vacuum degree unchanged, raising the temperature to 25-30 ℃ at the speed of 1.0-1.5 ℃/min, and preserving the heat for 4 h.
9. A pulsatilla saponin B4 freeze-dried powder injection prepared by the preparation method of any one of claims 4 to 8.
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