CN107245054B - Amorphous bulleyaconitine A compound and preparation method thereof - Google Patents
Amorphous bulleyaconitine A compound and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of medicinal chemistry, and discloses novel amorphous bulleyaconitine A, a preparation method thereof and a pharmaceutical composition containing the same. The amorphous bulleyaconitine A has an X-ray powder diffraction pattern shown in figure 2 and a differential scanning calorimetry pattern shown in figure 4. The amorphous bulleyaconitine A has good performance in the aspect of solubility. The method for preparing the amorphous bulleyaconitine A has simple operation, is suitable for laboratory development and industrial production, and has wide application. The amorphous bulleyaconitine A product prepared by the method has stable quality and good solubility, and can be directly used for preparing the medicine for treating pain diseases.
Description
Technical Field
The invention relates to the field of medicinal chemistry, in particular to amorphous bulleyaconitine A, a preparation method thereof and a medicinal composition containing the same.
Background
Bulleyaconitine A (Bulleyaconitine A) is called Dianciscolide A and aconitine crassipes (Crassicauline A), is a diterpene diester alkaloid extracted and separated from the root tuber of Aconitum carmichaeli (Aconitum geoorgei Comber) and Aconitum longtonuense T.L.Ming) of Aconitum of Ranunculaceae by Chinese pharmacologists, and has strong analgesic activity and certain anti-inflammatory effect. In earlier researches, the applicant finds a preparation process of bulleyaconitine A and applies for a patent of 'a preparation method of high-purity bulleyaconitine A', and the patent number is ZL 200910094477.X.
The preparation method of bulleyaconitine A disclosed in the prior literature mainly focuses on the extraction, separation and purification of bulleyaconitine A, for example, CN102775349A discloses a preparation method of bulleyaconitine A, CN104326981A discloses a high-efficiency extraction and separation method of bulleyaconitine A, CN101830849A discloses a simplified preparation method of high-purity bulleyaconitine A, CN102924376A discloses a preparation method of high-purity bulleyaconitine A, and the like. By examining these documents, it was found that at the end of these processes, which all involve purification by crystallization, the analysis by the applicant by means of an X-ray powder diffractogram gave an X-ray powder diffractogram substantially as shown in FIG. 1, indicating a certain crystalline structure.
But the preparation of amorphous bulleyaconitine A is only reported. Neither of these references relate to the preparation of amorphous bulleyaconitine A.
However, the effect of the bulleyaconitine A crystal form in the production process of the subsequent medicinal preparation is not ideal, because the dissolution speed of the bulleyaconitine A crystal form in pharmaceutical auxiliaries such as water or 0.9% sodium chloride aqueous solution is slow, the production time of the preparation is prolonged, and the preparation operation is not facilitated.
Solid drug polymorphism is an important matter for studying the existence of a drug, and polymorphism generally exists in most chemical drugs. Generally, the same drug with different crystal forms has differences in appearance, solubility, melting point, dissolution rate, bioavailability, etc., and may even have significant differences, thereby affecting the stability, bioavailability, therapeutic effect, etc. of the drug. Since the physical and chemical properties and biological activity of the drug are affected by the substances in different crystal forms, the existence state of the drug crystal forms should be considered in the aspects of researching raw materials, formulation and the like of the drug.
The amorphous state (amophorus) is a form of polymorphism in a substance, and is also a special crystalline state. It is reported in the literature (Konno T., chem.pharm.Bull., 1990; 38; 2003-2007) that amorphous form-amorphous state in many drugs shows dissolution characteristics and in some cases shows different biological utilization patterns compared to crystalline form. For some treatment modalities, one biological use pattern may be more advantageous than another.
The studies show that (pharmaceutical science, 2009,44(5): 443-. The amorphous state of a solid drug can also exist in different forms, as can a crystalline substance, and this phenomenon is called polymorphism of the amorphous state of a solid substance, also called amorphous polymorphism (polyamorphus). In a broad sense, one solid substance can be obtained as two or more amorphous substances having completely different physical, thermodynamic and kinetic properties due to different preparation methods or storage modes. For example: amorphous felodipine prepared by different cooling rates has different endothermic positions under constant temperature conditions by Differential Scanning Calorimetry (DSC). Luyang and duchenhua (crystal form drug, people's health press, 2009, 10 months) indicate that the reason for forming amorphous polymorphic substances may be related to three factors, namely compound configuration or structural phase, chemical composition and intermolecular force of chemical substances. Amorphous polymorphism increases the opportunity of more choices for drug research, but also increases the difficulty of researching advantageous drug crystal forms, and provides new challenges for preparation processes and control technologies of various amorphous substances.
The applicant prepares the amorphous bulleyaconitine A after carrying out a great deal of research on the bulleyaconitine A, and the research finds that the amorphous bulleyaconitine A can be dissolved quickly in various pharmaceutical solvents such as water or 0.9 percent sodium chloride aqueous solution, has high solubility, is beneficial to preparation operation, can greatly shorten the production time of a preparation, and reduces the loss of the bulleyaconitine A in the production process of a medicinal preparation, thereby completing the invention.
Disclosure of Invention
The invention aims to research, discover and provide novel amorphous bulleyaconitine A and a preparation method and application thereof by a crystallography method. Compared with the bulleyaconitine A crystal in the prior art, the amorphous bulleyaconitine A has better solubility.
The invention researches, discovers and provides novel amorphous bulleyaconitine A by a crystallography method, wherein the amorphous bulleyaconitine A has an X-ray powder diffraction pattern shown in figure 2.
The invention also adopts Differential Scanning Calorimetry (DSC) to research and characterize the novel amorphous bulleyaconitine A. The invention provides a new and basically pure amorphous bulleyaconitine A.
The amorphous bulleyaconitine A has a differential scanning calorimetry diagram as shown in figure 4.
In a differential scanning calorimetry diagram, the endothermic peak of the amorphous bulleyaconitine A is 160.1 ℃.
Experimental results show that the amorphous bulleyaconitine A has good solubility, stronger bioavailability and better curative effect.
The invention also provides a preparation method of the amorphous bulleyaconitine A.
The preparation method comprises the following steps:
1) dissolving bulleyaconitine A in C1-4 organic solvent to obtain bulleyaconitine A solution;
2) dropwise adding the bulleyaconitine A solution obtained in the step 1) into water while stirring, performing suction filtration after adding, and drying a filter cake to obtain the amorphous bulleyaconitine A.
Wherein, in the step 1), the C1-4The organic solvent is one or more of methanol, ethanol, acetone, dimethyl sulfoxide, N-dimethylformamide or N, N-dimethylacetamideAnd (3) mixing.
In the step 1), the concentration of the bulleyaconitine A solution is 0.05 g/mL-0.5 g/mL.
In the step 2), the volume ratio of the bulleyaconitine A solution to water is 1/50-1/5.
In some embodiments, the method of the invention for preparing amorphous bulleyaconitine A comprises the step of adding C1-4The solvent is one or a mixture of methanol, ethanol, acetone, dimethyl sulfoxide, N-dimethylformamide and N, N-dimethylacetamide.
In some embodiments, said C1-4The organic solvent of (2) is methanol.
In some embodiments, said C1-4The organic solvent of (a) is ethanol.
In some embodiments, the bulleyaconitine A-dissolved C in the method for preparing amorphous bulleyaconitine A according to the present invention1-4The concentration of the bulleyaconitine A in the alcohol solvent is 0.05 g/mL-0.2 g/mL.
In the method for preparing the amorphous bulleyaconitine A, the bulleyaconitine A is obtained by any method in the prior art.
The method for preparing the amorphous bulleyaconitine A has simple operation and high yield of the amorphous bulleyaconitine A, is suitable for laboratory development and industrial production, and has wide application. The amorphous bulleyaconitine A product prepared by the method has stable quality and good solubility, and can be directly used for preparation research and production.
Meanwhile, the amorphous bulleyaconitine A has good solubility, stronger bioavailability and better curative effect.
The invention also provides application of the amorphous bulleyaconitine A in preparing medicaments for treating pain diseases, in particular pain diseases caused by rheumatism or rheumatoid arthritis.
The invention also provides a pharmaceutical preparation, which comprises the amorphous bulleyaconitine A and one or more pharmaceutically acceptable auxiliary materials.
The amorphous bulleyaconitine A can be directly or indirectly added into various pharmaceutically acceptable common auxiliary materials required by preparing different dosage forms, such as a filling agent, a disintegrating agent, a lubricant, an adhesive and the like, and prepared into common preparations suitable for oral administration, parenteral (intravenous or subcutaneous) administration or nasal administration, such as tablets, capsules, injection, oral liquid, granules, pills, powder, dropping pills, troches, suppositories, creams, ointments, skin gels, suspensions, freeze-dried powder injections and the like by a conventional pharmaceutical preparation method. Wherein the filler is selected from starch, lactose, sucrose, glucose, mannitol or silicic acid; disintegrating agents such as agar-agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates and sodium carbonate, low-substituted hydroxypropylcellulose; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate; binding agents such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose or acacia.
The preparation method of the preparation can refer to the preparation method of the bulleyaconitine A crystal form-containing preparation by the technical personnel in the field.
The dosage and method of administration of the pharmaceutical preparation of the present invention depend on a variety of factors including the age, body weight, sex, physical condition, nutritional status, the activity intensity of the compound, the time of use, the metabolic rate, the severity of the disease course, and the subjective judgment of the treating physician. Those skilled in the art will vary depending on the nature and severity of the disease, the route of administration and the age and weight of the patient. In some embodiments, the dose may vary from 0.4 to 1.2 mg/day in one or more administrations.
The invention also provides a pharmaceutical composition which comprises the amorphous bulleyaconitine A and other drugs for treating pain diseases.
Other drugs for pain disorders such as lidocaine, acetaminophen, etc.
The invention provides a new amorphous bulleyaconitine A. The amorphous bulleyaconitine A has good performance in the aspects of stability and solubility. The method for preparing the amorphous bulleyaconitine A has simple operation and high yield, is suitable for laboratory development and industrial production, and has wide application. The amorphous bulleyaconitine A product prepared by the method has stable quality and good solubility, and can be directly used for preparing the medicine for treating pain diseases.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is an X-ray powder diffraction pattern of a bulleyaconitine A reference, wherein the ordinate represents the diffraction intensity in counts per second (cps) and the abscissa represents the diffraction angle 2 θ in degrees;
FIG. 2 is an X-ray powder diffraction pattern of amorphous bulleyaconitine A obtained in example 1 of the present invention, wherein the ordinate represents diffraction intensity in counts per second (cps) and the abscissa represents diffraction angle 2 θ in degrees;
FIG. 3 is a Differential Scanning Calorimetry (DSC) plot of a bulleyaconitine A control in which the ordinate is the heat flow rate in calories/second; the abscissa is temperature in units of;
FIG. 4 is a Differential Scanning Calorimetry (DSC) plot of amorphous bulleyaconitine A prepared in example 1 of the present invention, wherein the ordinate is the heat flow rate in calories/second; the abscissa is temperature in units of;
FIG. 5 is a PLM of bulleyaconitine A reference sample;
FIG. 6 is a PLM of amorphous bulleyaconitine A prepared in example 1 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Wherein, if the bulleyaconitine A related in the embodiment is provided for China food and drug testing research institute, the calibration purity is 100 wt%, and the batch number is 100530-.
The X-ray powder diffraction spectrum was determined under the following experimental conditions: D/MAX-2200 type diffractometer, temperature control room; the voltage is 40kV, and the current is 40 mA; fixed angle of the diverging slit: 0.9570 degrees; the measurement method comprises the following steps: continuously from 4.5 ° to 50 °, in increments of 0.033 °; measurement time/procedure: 19.7 s; measuring temperature: at 25 ℃.
The Differential Scanning Calorimetry (DSC) profile is determined under the following experimental conditions: the instrument comprises the following steps: DSC 204 (germany) differential scanning calorimeter; sample weight 8.17 mg; the heating rate is as follows: 2 ℃/min; the maximum temperature is 250 ℃; nitrogen flow rate: 20 mL/min.
Example 1 preparation of amorphous bulleyaconitine A
Dissolving 0.1g bulleyaconitine A in 2mL methanol to obtain bulleyaconitine A solution, dripping the bulleyaconitine A solution into 100mL water while stirring, vacuum filtering, and drying the filter cake to obtain the final product. Sample 1 was obtained.
Example 2 preparation of amorphous bulleyaconitine A
Dissolving 0.1g bulleyaconitine A in 3mL ethanol to obtain bulleyaconitine A solution, dripping the bulleyaconitine A solution into 100mL water while stirring, filtering, and drying the filter cake to obtain the final product. Sample 2 was obtained.
Example 3 preparation of amorphous bulleyaconitine A
Dissolving 0.1g of bulleyaconitine A in 3mL of dimethyl sulfoxide to obtain a bulleyaconitine A solution, dripping the bulleyaconitine A solution into 100mL of water while stirring, performing suction filtration, and drying a filter cake to obtain the bulleyaconitine A. Sample 3 was obtained.
Example 4 preparation of amorphous bulleyaconitine A
Dissolving 0.1g bulleyaconitine A in 1ml LN, N-dimethylformamide to obtain bulleyaconitine A solution, adding bulleyaconitine A solution dropwise into 100ml water while stirring, vacuum filtering, and drying filter cake to obtain the final product. Sample 4 was obtained.
Example 5 preparation of amorphous bulleyaconitine A
Dissolving 0.1g bulleyaconitine A in 1ml LN, N-dimethylacetamide to obtain bulleyaconitine A solution, dripping bulleyaconitine A solution into 100ml water, stirring while adding, vacuum filtering, and drying filter cake to obtain the final product. Sample 5 was obtained.
Example 6 preparation of amorphous bulleyaconitine A
Dissolving 0.1g bulleyaconitine A in 1ml LN, N-dimethylacetamide to obtain bulleyaconitine A solution, adding dropwise bulleyaconitine A solution into water (volume ratio of bulleyaconitine A solution to water is 1: 50), stirring while adding, vacuum filtering, and drying the filter cake. Sample 6 was obtained.
Example 7 preparation of amorphous bulleyaconitine A
Dissolving 0.1g bulleyaconitine A in 1ml LN, N-dimethylacetamide to obtain bulleyaconitine A solution, adding dropwise bulleyaconitine A solution into water (wherein the volume ratio of bulleyaconitine A solution to water is 1: 5), stirring while adding, vacuum filtering, and drying the filter cake to obtain the final product. Sample 7 was obtained.
Example 8 preparation of amorphous bulleyaconitine A
Dissolving 0.1g bulleyaconitine A in 1ml LN, N-dimethylacetamide to obtain bulleyaconitine A solution, adding dropwise bulleyaconitine A solution into water (volume ratio of bulleyaconitine A solution to water is 1: 20), stirring while adding, vacuum filtering, and drying the filter cake. Sample 8 was obtained.
Example 9 preparation of amorphous bulleyaconitine A
Dissolving 0.5g of bulleyaconitine A in 1mL of acetone to obtain a bulleyaconitine A solution, dripping the bulleyaconitine A solution into water (wherein the volume ratio of the bulleyaconitine A solution to the water is 1: 30), stirring while adding, filtering, and drying the filter cake to obtain the final product. Sample 9 was obtained.
Example 10 preparation of amorphous bulleyaconitine A
Dissolving 0.2g of bulleyaconitine A in 1mL of acetone to obtain a bulleyaconitine A solution, dripping the bulleyaconitine A solution into water (wherein the volume ratio of the bulleyaconitine A solution to the water is 1: 10), stirring while adding, filtering, and drying the filter cake to obtain the bulleyaconitine A. Sample 10 was obtained.
Example 11 preparation of amorphous bulleyaconitine A tablets
Firstly, dissolving the amorphous bulleyaconitine A prepared in the example 1 by 1ml of 95 percent edible grade or medicinal grade ethanol;
secondly, 20mg of lactose, 30mg of powdered sugar, 55mg of microcrystalline cellulose and 10mg of hydroxypropyl cellulose which are auxiliary materials are mixed for 30 minutes, then bulleyaconitine A ethanol solution is added and mixed for 60 seconds, then a proper amount of purified water is added and mixed for 60 seconds to prepare soft materials, and the soft materials are further prepared into particles and dried for 40 minutes at the temperature of 55-65 ℃;
thirdly, the dried granules are placed to room temperature, then other auxiliary materials of 1mg of magnesium stearate and 25mg of sodium carboxymethyl starch are added into the dried granules, the mixture is mixed for 30 minutes, a tablet machine is used for tabletting, and then 6mg of film-coated polyvinylpyrrolidone (PVP) and 1mg of plasticizer glycerol are coated to obtain the finished product.
Example 12 amorphous bulleyaconitine A lyophilized powder for injection
The formula is as follows:
amorphous bulleyaconitine A0.2 g
Mannitol 50g
0.002g of water for injection
The preparation process comprises the following steps: under the condition of 1 ten thousand grades, the amorphous bulleyaconitine A prepared in the example 1 is firstly dissolved in 300 times (about 60ml)) of water for injection to be completely dissolved; adding water for injection to dilute to 600ml, adding 50g of mannitol to completely dissolve, and adding water for injection to 1000 ml; filtering, coarse-filtering the filtrate with filter paper and 0.4 μm filter membrane, and filtering with filter membrane of 0.2 μm or less under 100-grade condition; and (3) feeding the fine filtrate into a packaging machine, packaging according to 1mo per unit, covering a cover with a groove, feeding into a freeze vacuum drier, rapidly cooling to-40 ℃ for 2-3 hours, gradually and slowly heating to 35 ℃ (about 10 hours), plugging the cover, taking out a product, rolling the cover, packaging, and inspecting to obtain 1000 amorphous bulleyaconitine A powder injection finished products with the specification of 0.2 mg.
Example 13 amorphous bulleyaconitine A Soft Capsule
The preparation process comprises the following steps:
(1) weighing gelatin, adding into a 100L gelatin reaction tank, adding appropriate amount of water under stirring, sealing, adding glycerol and appropriate amount of ethyl p-hydroxybenzoate after gelatin is completely dissolved, stirring, vacuum degassing for 2 hr, placing into a gelatin heat-insulating barrel, and standing;
(2) weighing the amorphous bulleyaconitine A prepared in the embodiment 1, dissolving in ethyl acetate at 70 ℃, mixing with a proper amount of solvent, adding alpha-tocopherol after completely mixing, stirring uniformly, and standing at room temperature;
(3) suspending gelatin heat-insulating barrel to a certain height, introducing the prepared medicinal liquid into medicinal liquid hopper, changing mold, starting pressing soft capsule, adjusting soft capsule loading to design range, drying at room temperature, removing capsule with poor appearance quality, washing capsule (removing lubricant oil during pressing pill), drying at 24 deg.C for two days to obtain amorphous bulleyaconitine A soft capsule semi-finished product, quality testing, packaging, labeling, and packaging to obtain amorphous bulleyaconitine A soft capsule finished product.
Example 14 amorphous bulleyaconitine A pharmaceutical composition
The formula is as follows: the pharmaceutical composition consists of the amorphous bulleyaconitine A and the acetaminophen prepared in the embodiment 1, wherein the mass ratio of the amorphous bulleyaconitine A to the acetaminophen is 1: 750.
the composition can be further added with pharmaceutically acceptable auxiliary materials to be prepared into tablets, and can be prepared according to the following formula and dosage:
feeding 10 ten thousand tablets, taking 0.04kg of the amorphous bulleyaconitine A prepared in the example 1, 30kg of acetaminophen and 17.96kg of auxiliary materials, putting the materials into a wet granulator, the adjuvants comprise 3kg of sugar powder as filler, 6.96kg of starch and 4kg of microcrystalline cellulose, 4kg of low-substituted hydroxypropyl cellulose as disintegrant, mixing for 30min, adding 3kg of ethanol as wetting agent and binder, mixing for 30min, granulating with granulator, drying in fluidized bed for 40 min at drying temperature below 60 deg.C, cooling to room temperature, vacuum pumping into mixer, adding magnesium stearate 1kg as lubricant, mixing for 30min, tabletting, coating with coating powder 1kg, and internally and externally coating to obtain the final product. The specification of the finished tablet is 500mg, wherein the effective components of the medicine are 0.4mg of bulleyaconitine A and 300mg of acetaminophen.
The first experimental example: x-ray powder diffraction detection
X-ray diffraction test was performed on a bulleyaconitine A reference substance (provided by the institute of food and drug testing, having a nominal purity of 100 wt%, lot number 100530-.
Sample 1 was subjected to X-ray powder diffraction testing and the results are shown in figure 2. The result shows that the sample 1 is in an amorphous state and has different X-ray diffraction patterns with the bulleyaconitine A reference substance.
X-ray powder diffraction measurements were performed on samples 2-10 and the results were similar to those of figure 2.
Experiment example two: differential scanning calorimetry
DSC detection is carried out on a reference substance (provided by China food and drug testing research institute, calibration purity is 100 wt%, lot number is 100530-.
The DSC measurement of sample 1 is shown in FIG. 4. The results in fig. 4 show that the differential scanning calorimetry curve of sample 1 has an endothermic peak at about 212.4 ℃, indicating that the amorphous bulleyaconitine a provided by the present invention has different DSC results from the bulleyaconitine a control.
Differential scanning calorimetry was performed on samples 2-10 and the results were similar to those of FIG. 4.
Experiment example three: solubility test
A proper amount of the sample prepared in the above examples 1 to 4 and a proper amount of a reference sample (provided by the institute for testing and testing of food and drug, having a nominal purity of 100 wt%, lot number 100530-.
TABLE 1 comparison of solubility tests for bulleyaconitine A (mg/mL)
| Sample numbering | Water (W) | 0.9% aqueous sodium chloride solution |
| Sample 1 | 0.47 | 0.66 |
| |
0.58 | 0.69 |
| |
0.49 | 0.68 |
| Sample No. 4 | 0.41 | 0.61 |
| Sample No. 5 | 0.57 | 0.70 |
| Reference substance | 0.05 | 0.09 |
As can be seen from the results in Table 1, the solubility of the amorphous bulleyaconitine A samples prepared in examples 1 to 4 of the invention in water is above 0.4mg/mL, the solubility in 0.9% sodium chloride aqueous solution is above 0.6mg/mL, the solubility of the bulleyaconitine A reference substance in water is 0.05mg/mL, and the solubility in 0.9% sodium chloride aqueous solution is 0.09mg/mL, which indicates that the solubility of the amorphous bulleyaconitine A of the invention is significantly better than that of the bulleyaconitine A sample.
The above tests were also carried out on samples 6-10, which gave similar results.
Experimental example four: purity detection
Instruments and reagent reagents: agilent1260 high performance liquid chromatograph.
A chromatographic column: cosmosil cholesterol 250X 4.6 mm.
Acetonitrile is chromatographic grade, and HPLC water is redistilled distilled water.
Comparison products: raw material samples (provided by China food and drug testing research institute, calibration purity of 100 wt%, lot number of 100530-;
detection products are as follows: sample 1, sample 2, sample 3, sample 4, sample 5.
Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; using 0.2% triethylamine water solution (pH value is adjusted to 3.1 plus or minus 0.1 by phosphoric acid) -acetonitrile (60: 40) as mobile phase; the detection wavelength was 260 nm. The number of theoretical plates is not less than 3000 calculated according to bulleyaconitine A peak.
And (3) determination: weighing about 20mg of the product, accurately weighing, placing in a 100ml measuring flask, adding mobile phase for dissolving and diluting to scale, shaking, accurately weighing 5ml, placing in a 50ml measuring flask, diluting to scale with mobile phase, shaking, accurately weighing 20 μ l, injecting into a liquid chromatograph, and recording chromatogram. Calculating according to the peak area by an external standard method to obtain the product. The results are shown in Table 2.
TABLE 2 purity test results
| Sample batch number | Sample 1 | |
|
Sample No. 4 | Sample No. 5 |
| Content (wt%) | 99.31 | 98.95 | 99.18 | 98.82 | 99.23 |
As can be seen from the results in Table 2, the content of the amorphous bulleyaconitine A prepared in the examples 1 to 4 of the invention is more than 98.8%.
The above tests were also carried out on samples 6-10, which gave similar results.
Experimental example five: observation with polarizing microscope
A suitable amount of the sample 1 prepared in example 1 and a suitable amount of the control sample (provided by the institute for testing and testing of Chinese food and drug; having a nominal purity of 100 wt% and lot number of 100530-200501) were taken and observed under a polarizing microscope (PLM), and the PLM patterns of the two samples are shown in FIG. 5 and FIG. 6.
From the PLM chart, under the same magnification condition of 400 times, the reference substance is bulk crystal and has agglomeration; example 1 is a fine particle, smaller in particle size than the control, and uniformly distributed.
Experimental example six: comparative analgesic test
1. Animals: SPF mice (Kun medicine group, Ltd., animal quality certificate number: SCXK (Dian) K2014-0001, body weight about 18-22 g.
2. The method comprises the following steps: 80 male mice were taken and randomly divided into 8 groups: a vehicle control group; positive control (aspirin 200mg/kg) group; amorphous bulleyaconitine A (sample 1 prepared in example 1)0.2, 0.4, 0.8mg/kg and bulleyaconitine A reference substance (provided by China food and drug testing institute, calibration purity 100 wt%, batch number 100530-; each group had 10. The animals of each group were administered once by intragastric administration at the dose, and the vehicle control group was administered 1% CMC-Na at 20 mL/kg. 30min after administration, 0.6% glacial acetic acid solution 0.1mL/10g is intraperitoneally injected, the times of body writhing of each mouse in 15min are observed and recorded, and the inhibition rates of two different crystal forms of bulleyaconitine A on the body writhing caused by acetic acid are compared.
The inhibition rate (%) was (mean number of twists in vehicle group-mean number of twists in drug group)/mean number of twists in vehicle group × 100%.
The results are shown in Table 3.
TABLE 3 Effect of bulleyaconitine A on acetic acid-induced pain in mice
P < 0.05 compared to the 1% CMC-Na group; p < 0.01; from the results, the positive control group aspirin and the bulleyaconitine A middle and high dose (0.4mg/kg, 0.8mg/kg) groups can inhibit the painful writhing frequency of the mice caused by acetic acid, and have statistical significance (P is less than 0.05 or P is less than 0.01) compared with the vehicle control group. Compared with the bulleyaconitine A control group, the inhibition rate of the amorphous bulleyaconitine A group on the painful writhing of mice is better than that of the bulleyaconitine A control group.
Similar results were obtained for the amorphous bulleyaconitine A samples prepared according to other examples of the invention.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. The amorphous bulleyaconitine A is characterized by having an X-ray powder diffraction pattern as shown in figure 2.
2. The amorphous bulleyaconitine A as claimed in claim 1, wherein the amorphous bulleyaconitine A has a differential scanning calorimetry trace as shown in figure 4.
3. The amorphous bulleyaconitine A as claimed in claim 2, wherein the endothermic peak of the amorphous bulleyaconitine A in differential scanning calorimetry is 160.1 ℃.
4. A process for the preparation of amorphous bulleyaconitine a as claimed in any of claims 1 to 3, which comprises:
1) dissolving bulleyaconitine A in C1-4 organic solvent to obtain bulleyaconitine A solution;
2) dropwise adding the bulleyaconitine A solution obtained in the step 1) into water, stirring while adding, performing suction filtration after adding, and drying a filter cake to obtain the amorphous bulleyaconitine A;
in the step 1), the C1-4 organic solvent is one or a mixture of methanol, ethanol, acetone, dimethyl sulfoxide, N-dimethylformamide or N, N-dimethylacetamide; the concentration of the bulleyaconitine A solution is 0.05 g/mL-0.5 g/mL;
in the step 2), the volume ratio of the bulleyaconitine A solution to water is 1/50-1/5.
5. Use of amorphous bulleyaconitine A according to any one of claims 1 to 3 in the preparation of a medicament for the treatment of pain.
6. The use of amorphous bulleyaconitine A according to claim 5 in the preparation of a medicament for the treatment of painful conditions caused by rheumatic or rheumatoid arthritis.
7. A pharmaceutical formulation comprising amorphous bulleyaconitine a as claimed in any of claims 1 to 3 and one or more pharmaceutically acceptable excipients.
8. A pharmaceutical composition comprising amorphous bulleyaconitine a as claimed in any of claims 1 to 3 and other drugs for the treatment of pain.
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| CN101239947A (en) * | 2008-03-07 | 2008-08-13 | 中国科学院昆明植物研究所 | Method for preparing cryptotanshinone |
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