CN118164999A - A Ginkgolide B crystal form I and its preparation method - Google Patents

Abstract

The invention discloses a bilobalide B sesquihydrate crystal form I, a preparation method and application thereof. The invention provides a bilobalide B sesquihydrate crystal form I, which comprises X-ray diffraction peaks shown in the following 2 theta angles in an X-ray powder diffraction spectrum: 8.15 ° ± 0.2 °, 9.63 ° ± 0.2 °, 12.25 ° ± 0.2 °, 13.41 ° ± 0.2 °, 14.16 ° ± 0.2 °, 21.66 ° ± 0.2 °, 25.08 ° ± 0.2 ° and 25.52 ° ± 0.2 °. The preparation method of the invention is safe and simple, and the prepared bilobalide B sesquihydrate crystal form I has good dissolubility, high stability and excellent physicochemical property, is suitable for drug development, and widens the clinical application of bilobalide B.

Description

Bilobalide B crystal form I and preparation method thereof

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a bilobalide B sesquihydrate and a crystal form thereof, and a preparation method thereof.

Background

Ginkgolide B is diterpenoid compound separated from folium Ginkgo. The Germany first uses solvent extraction method to produce ginkgo leaf extract (EGb) with definite quality standard on a large scale, the EGb contains two main effective components (ginkgetin compounds and ginkgolides compounds), wherein the ginkgolides compounds mainly comprise 4 kinds, namely Ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC) and ginkgolide (BB), and the activity of ginkgolide B is strongest.

The chemical name of the ginkgolide B is 1-hydroxy- (1 beta) -ginkgolide A, the name is ginkgolide B or ginkgolide B, CAS number is 15291-77-7, and the relative molecular mass is 424.40. Ginkgolide B is relatively stable and has a relatively high melting point (about 300 ℃); can be dissolved in organic solvents such as acetone, ethanol, methanol, ethyl acetate, tetrahydrofuran, dioxane and the like; hardly soluble in diethyl ether and water (solubility in water is 2.5X10 ^-4 mol/L); insoluble in cyclohexane, benzene, chloroform and carbon tetrachloride.

Ginkgolide B can be obtained by chemical synthesis, plant tissue culture, extraction, etc. 3 ways. The chemical synthesis and plant tissue culture are limited by the technical difficulty, the high production cost and the like, and the ginkgolide B is mainly extracted and mainly separated from ginkgo leaves. Modern researches have found that bilobalide B has the effects of resisting platelet activating factor, resisting inflammation, resisting oxidation, resisting shock, scavenging free radicals, resisting allergy and the like, protecting cerebral ischemia injury, dilating coronary artery, enhancing myocardial contractility, treating senile dementia and the like, and recently researches have found that the bilobalide B also has the anti-tumor activity and is gradually paid attention to domestic and foreign scholars. As the component with the strongest activity in the ginkgo leaf terpene lactones, the ginkgo lactone B has wide application prospect in clinic, but is greatly limited in practical application due to the characteristics of low solubility, short half-life and the like.

The material is affected by various factors during crystallization, so that the bonding mode of molecules or molecules is changed, and molecules or atoms are arranged differently in lattice space, so that different crystal structures are formed. Although only one crystal form is thermodynamically stable at a given temperature and pressure, many crystalline drugs exhibit polymorphism because the transition from metastable to steady state is typically very slow. Different crystal forms of the same medicine may be significantly different in appearance, solubility, melting point, dissolution rate, bioavailability and the like, thereby affecting the stability, bioavailability and curative effect of the medicine. Drug polymorphism is one of the important factors affecting drug quality and clinical efficacy. The crystal form of the medicine is related to the crystal system, the lattice structure and the molecular structure of the medicine, and is also related to the solvent type, the solution concentration, the cooling, the evaporation rate, the drying method and the like used in the crystallization process during the preparation.

Disclosure of Invention

In the prior art, the bilobalide B has poor dissolution and stability, and in order to overcome the defects, the invention provides a bilobalide B sesquihydrate, a bilobalide B sesquihydrate crystal form I and a preparation method thereof.

The first aim of the invention is to provide a bilobalide B crystal form, in particular to a bilobalide B sesquihydrate which is named as a crystal form I and has high dissolution rate and good chemical stability.

The second aim of the invention is to provide a preparation method of bilobalide B sesquihydrate, which has simple preparation process and is suitable for drug development.

The third object of the invention is to provide a pharmaceutical composition containing bilobalide B sesquihydrate and the application of the crystal form in preparing medicines for treating diseases.

The specific technical content of the invention is as follows:

The invention provides a crystal form I of bilobalide B sesquihydrate shown in a formula I, wherein a radiation source of the crystal form I is an X-ray powder diffraction pattern of Cu-K alpha, and the X-ray powder diffraction pattern comprises the following X-ray diffraction peaks shown by 2 theta angles: 8.15 ° ± 0.2 °, 9.63 ° ± 0.2 °, 12.25 ° ± 0.2 °, 13.41 ° ± 0.2 °, 14.16 ° ± 0.2 °, 21.66 ° ± 0.2 °, 25.08 ° ± 0.2 ° and 25.52 ° ± 0.2 °.

Further, the X-ray powder diffraction pattern of the crystal form I of the bilobalide B sesquihydrate also comprises X-ray diffraction peaks shown by the following 2 theta angles: 10.65 ° ± 0.2 °, 11.39 ° ± 0.2 °, 14.94 ° ± 0.2 °, 21.99 ° ± 0.2 °, 29.21 ° ± 0.2 °, 32.13 ° ± 0.2 ° and 34.88 ° ± 0.2 °.

In one embodiment of the present invention, the bilobalide B sesquihydrate has an X-ray powder diffraction pattern as shown in fig. 1 using Cu-ka radiation.

The bilobalide B crystal form I is a hydrate and is characterized in that two molecules of bilobalide B are combined with three molecules of water and have asymmetric units.

Further, the crystal form I of the bilobalide B sesquihydrate has the following single crystal X-ray diffraction data that the radiation source is Cu-K alpha: monoclinic system, space group P2 ₁, unit cell parameters are α＝90°，β＝91.413°，γ＝90°，/>Z=2, dx=1.448 g/cm ³,F(000)＝956.0,μ(Cu Kα)＝1.026mm^-1, and the final deviation factor R ₁＝0.0454,wR₂ =0.1269 [ I > =2σ (I) ], the fly constant is 0.12 (14). The X-ray single crystal diffraction pattern of the crystal form I of the bilobalide B sesquihydrate is shown in figure 2.

The crystal form I of the bilobalide B sesquihydrate is determined by Differential Scanning Calorimetry (DSC), and the differential thermal analysis chart (DSC) has a wide endothermic peak in the range of 60-120 ℃ and is generated by evaporating crystal water; a sharp endothermic peak appears in the range of 309-354 ℃, which indicates that the purity of the ginkgolide B is very high; the bilobalide B sesquihydrate of the crystal form i has a DSC-TGA profile as shown in figure 3.

The invention provides a preparation method of bilobalide B sesquihydrate, which is characterized by comprising the following specific preparation steps:

step (1): adding ethanol into terpene lactone, heating for dissolving, maintaining the temperature, slowly adding water, naturally cooling, stirring for crystallization, filtering, and oven drying the filter cake to obtain refined bilobalide product.

Step (2): adding refined bilobalide into the crystallization solution, heating for dissolving, cooling, and naturally crystallizing at room temperature.

Preferably, the preparation method of the terpene lactone comprises the following steps: extracting folium Ginkgo with ethanol solution, reflux extracting, mixing extractive solutions, and concentrating; loading the concentrated solution on macroporous resin, eluting with water, eluting with ethanol, collecting ethanol eluate, concentrating the eluate until no ethanol exists, and extracting with ethyl acetate for three times to obtain water phase and ester phase respectively; concentrating and drying the ester phase, dissolving in ethanol, adding active carbon, stirring for deacidification twice, concentrating the filtrate, and drying to obtain terpene lactone.

Preferably, the volume ratio of the crude product to the ethanol in the step (1) is 1:2-1:4; more preferably, the volume ratio is 1:3;

The ethanol in the step (1) is 75% -95% ethanol; more preferably, 95% ethanol;

preferably, the heat preservation temperature in the step (1) is 55 ℃;

Preferably, the water in the step (1) is used in an amount which is 1 to 2 times the volume of the dissolution liquid; more preferably 1.5 times;

Preferably, the crystallization time in the step (1) is 2-6h; more preferably, the crystallization time is 3h.

Preferably, the mass-to-volume ratio (g: mL) of the sample to the crystallization solution in the step (2) is 1:5-1:10, more preferably, the mass-to-volume ratio (g: mL) is 1:7, preparing a base material;

Preferably, the crystallization solution in the step (2) is a methanol water/ethanol water solution, and the alcohol water volume ratio is 30% -60%, more preferably, the alcohol water volume ratio is 50%;

Preferably, the crystallization time in the step (2) is 24h-72h, more preferably, the crystallization time is 36h.

The invention also provides a pharmaceutical composition containing bilobalide B sesquihydrate and application of the crystal form in preparing medicines for treating diseases.

The preparation method of the pharmaceutical composition comprises the following steps: the compounds of the present invention are formulated into useful dosage forms by combining them with pharmaceutically acceptable solid or liquid carriers, and optionally with pharmaceutically acceptable adjuvants and excipients, using standard and conventional techniques.

The pharmaceutical composition comprises spray, tablet, capsule, powder injection, liquid injection, freeze-dried powder injection and other pharmaceutically usable dosage forms.

The following is a detailed description of the invention:

the characteristics of the crystal form I of the bilobalide B sesquihydrate of the invention comprise the following aspects:

1. Powder X-ray diffraction:

Instrument model: PANALYTICAL X ray powder diffractometer

The testing method comprises the following steps: the ground sample (100 mg) is filled in a groove of a glass plate, the plane of the sample is flush with the glass surface by a glass slide, the sample is placed in a PANALYTICAL X-ray powder diffractometer, a 40kV and 40mA copper X-ray source is used, the scanning range is 0-50 degrees (2 theta), and the scanning speed is 8 degrees/min. The scan error is typically + -0.2 degrees (2 theta).

Its main characteristic peaks include:

The X-ray powder diffraction pattern is shown in figure 1.

2. Single crystal unit cell parameters: the unit cell parameters areα＝90°，β＝91.413°，γ＝90°，/>Z=2, dx=1.448 g/cm ³,F(000)＝956.0,μ(Cu Kα)＝1.026mm^-1, and the final deviation factor R ₁＝0.0454,wR₂ =0.1269 [ I > =2σ (I) ], the fly constant is 0.12 (14). The X-ray single crystal diffraction pattern is shown in figure 2.

3. The crystal form I of the bilobalide B sesquihydrate is measured by Differential Scanning Calorimetry (DSC), the differential thermal analysis spectrum (DSC) has a wide endothermic peak in the range of 60-120 ℃, and a sharp endothermic peak appears in the range of 309-354 ℃; the bilobalide B sesquihydrate of the crystal form i has a DSC-TGA profile as shown in figure 3.

The invention further examines the stability and the dissolution rate of the crystal form I of the bilobalide B sesquihydrate, and the crystal form I of the bilobalide B sesquihydrate has good stability and high dissolution rate. Compared with the bilobalide B crystal form F reported in the prior literature, the bilobalide B sesquihydrate has more advantages in the aspect of stability and dissolution.

The invention has the following advantages in the field: the bilobalide B sesquihydrate shown in the formula I and the crystal form I thereof have good dissolution property, good stability and excellent physicochemical property, are suitable for drug development, widen the clinical application of bilobalide B, and have good market prospect.

Drawings

FIG. 1 is a chart of X-ray powder diffraction patterns of the B-sesquihydrate of bilobalide of the invention.

FIG. 2 is a chart of X-ray single crystal diffraction patterns of the B-sesquihydrate of bilobalide of the invention.

FIG. 3 is a DSC-TGA spectrum of the B-sesquihydrate of bilobalide of the present invention.

Detailed Description

The present invention will be further described in the following examples in order to fully understand the present invention by those skilled in the art.

Terpene lactones are prepared according to the method described in examples 1-8 in patent application CN108245538a, a ginkgo leaf extract and its pharmaceutical use.

In the following examples of the present invention, terpene lactones, the starting material for the preparation of bilobalide B sesquihydrate, were prepared according to the procedure of example 1 of CN108245538 a.

Weighing 8kg of ginkgo leaves, extracting with 20% ethanol solution according to the ratio of 1:15, reflux-extracting twice, mixing the extracting solutions, and concentrating to the density of 1.1-1.2. The concentrated solution is put on macroporous resin (D101), the column volume is 1L/kg crude drug, the crude drug is eluted by water for 6 times of volume, then eluted by 60 percent ethanol for 8 times of volume, and the ethanol eluent is collected. Concentrating the eluent until no alcohol exists, extracting with ethyl acetate at a ratio of 1:1, extracting for three times by the same method, and combining ethyl ester phases to obtain an aqueous phase and an ethyl ester phase respectively. Concentrating and drying ethyl ester phase, dissolving with 50% ethanol with volume of 0.05L/kg crude drug, adding active carbon with addition amount of 0.1% of crude drug, stirring at 80deg.C for 1 hr, and deacidifying. And (3) carrying out secondary deacidification under the same condition, concentrating the filtrate, and drying to obtain terpene lactone.

EXAMPLE 1 preparation of bilobalide B sesquihydrate

And (3) adding 1g of terpene lactone into 3ml of 95% ethanol, heating and dissolving, preserving heat at 55 ℃, slowly adding purified water with the volume of 1.5 times of the volume of the solution, naturally cooling after the water addition, continuously stirring and crystallizing for 3 hours, filtering, and drying a filter cake to obtain 0.38g of ginkgolide refined product.

Adding 0.5g of bilobalide refined product into 3.5ml of crystallization solution (methanol water/ethanol water solution, ethanol water volume ratio of 50%), heating to dissolve, cooling, and naturally crystallizing at room temperature for 36 hr. HPLC purity: 98.86%.

EXAMPLE 2 preparation of bilobalide B sesquihydrate

And (3) taking 1g of terpene lactone, adding 4ml of 95% ethanol, heating and dissolving, preserving heat at 55 ℃, slowly adding purified water with the volume of 2 times of the volume of the solution, naturally cooling after the water addition, continuously stirring and crystallizing for 5 hours, filtering, and drying a filter cake to obtain 0.39g of ginkgolide refined product.

Adding 0.5g of bilobalide refined product into 4.0ml of crystallization solution (methanol water/ethanol water solution, ethanol water volume ratio of 45%), heating to dissolve, cooling, and naturally crystallizing at room temperature for 40 hr. HPLC purity: 98.35%.

EXAMPLE 3 preparation of bilobalide B sesquihydrate

Taking 1g of terpene lactone, adding 2ml of 95% ethanol, heating and dissolving, preserving heat at 55 ℃, slowly adding purified water with the volume of 1.2 times of the volume of the solution, naturally cooling after the water addition, continuously stirring and crystallizing for 3 hours, filtering, and drying a filter cake to obtain 0.36g of ginkgolide refined product.

Adding 0.5g of bilobalide refined product into 5.0ml of crystallization solution (methanol water/ethanol water solution, ethanol water volume ratio of 30%), heating to dissolve, cooling, and naturally crystallizing at room temperature for 32 hr. HPLC purity: 98.53%.

EXAMPLE 4 preparation of bilobalide B sesquihydrate

And (3) adding 1g of terpene lactone into 3ml of 95% ethanol, heating and dissolving, preserving heat at 55 ℃, slowly adding purified water with the volume of 1.5 times of the volume of the solution, naturally cooling after the water addition, continuously stirring and crystallizing for 5 hours, filtering, and drying a filter cake to obtain 0.39g of ginkgolide refined product.

Adding 0.5g of bilobalide refined product into 2.5ml of crystallization solution (methanol water/ethanol water solution, ethanol water volume ratio of 40%), heating to dissolve, cooling, and naturally crystallizing at room temperature for 24 hr. HPLC purity: 98.08%.

Comparative example 1 preparation of bilobalide B solvent-free form F

Reference method (Li Qiuya, sun Ting, wang Dongkai. Preparation, characterization and Property study of bilobalide B polymorph [ J ]. J.Chinese pharmaceutical journal: network edition, 2019 (4): 7 ]) 100mg of sample was weighed, placed in an electrothermal constant temperature air drying oven at 200deg.C, and left for 30min, and bilobalide B was solvent-free to obtain form F.

EXAMPLE 5 stability test of the bilobalide B sesquihydrate of the present invention

1 Part of each of the samples prepared in example 1 and example 2, 1 part of each of the samples prepared in comparative example 1 were stored under a sealed condition at 40 ℃ under a light shielding condition, and stability of each of the samples was examined for 1 month, 2 months, 3 months and 6 months, and the results are shown in table 1.

Purity was measured by HPLC-ELSD.

Liquid phase conditions: instrument: thermo Ultimate3000,3000

Mobile phase: n-propanol: tetrahydrofuran: water = 1:15:84

Column temperature: 30 DEG C

Flow rate: 1ml/min

TABLE 1 stability test results of inventive samples

As can be seen from the above experiments, compared with the crystal of comparative example 1, the bilobalide B sesquihydrate crystal form I prepared by the invention has good chemical stability; further detection shows that the X-ray single crystal diffraction patterns of the samples subjected to the 6-month stability test are unchanged, and the X-ray single crystal diffraction patterns indicate that the bilobalide B sesquihydrate crystal form I provided by the invention is high in stability.

EXAMPLE 6 dissolution test of Bilobalide B sesquihydrate according to the invention

Sample to be tested: the bilobalide B sesquihydrate obtained in example 1 and example 2 is compared with the bilobalide B solvent-free crystal form F prepared in example 1.

The experimental method comprises the following steps: taking 7mg of bilobalide B sesquihydrate, tabletting under the pressure of 40Bar, and keeping the pressure for 30s. The tablets were dissolved in 10ml of dissolution medium (37 ℃), 0.2ml of solution was taken at intervals and the same volume of purified water was replenished. And (3) filtering with a 0.22 mu m filter membrane, and monitoring the solution concentration at each time point by using a high performance liquid phase to finally obtain the dissolution rate of the bilobalide B sesquihydrate. The test was performed in triplicate.

The dissolution rate of the ginkgolide B glycol compound was measured by the same method as above.

Dissolution conditions: instrument: trace dissolution instrument

Dissolution medium: 1% Tween aqueous solution

Stirring speed: 75 rpm

Dissolution temperature: 37 DEG C

Sampling time: 5,10,15,25,40,60,100,120 minutes

Liquid phase conditions: instrument: thermo Ultimate3000,3000

Mobile phase: n-propanol: tetrahydrofuran: water = 1:15:84

Column temperature: 30 DEG C

Flow rate: 1ml/min

The experimental results are shown in Table 2.

TABLE 2 dissolution test results for inventive samples

As is clear from the results of table 2, the dissolution rate of bilobalide B sesquihydrate is significantly improved as compared with the crystal form of comparative example 1.

Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention. The present invention is intended to include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof. The foregoing examples or embodiments are merely illustrative of the invention, which may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims are intended to be encompassed within the scope of the invention.

Claims (9)

1. A crystalline form I of ginkgolide B sesquihydrate, characterized in that its X-ray powder diffraction pattern includes the following X-ray diffraction peaks shown at 2θ angles: 8.15°±0.2°, 9.63°±0.2°, 12.25°±0.2°, 13.41°±0.2°, 14.16°±0.2°, 21.66°±0.2°, 25.08°±0.2° and 25.52°±0.2°. 2. The crystalline form I of ginkgolide B sesquihydrate according to claim 1, characterized in that its X-ray powder diffraction pattern also includes the following X-ray diffraction peaks shown at 2θ angles: 10.65°±0.2°, 11.39°±0.2°, 14.94°±0.2°, 21.99°±0.2°, 29.21°±0.2°, 32.13°±0.2° and 34.88°±0.2°. 3. The crystalline form I of Ginkgolide B sesquihydrate according to claim 1, characterized in that it has an X-ray powder diffraction pattern as shown in Figure 1. 4. The crystalline form I of ginkgolide B sesquihydrate according to claim 1, characterized in that the differential scanning calorimetry (DSC) spectrum of the crystalline form shows an endothermic peak in the range of 309°C to 354°C, and the crystalline form has a DSC-TGA spectrum as shown in FIG3. 5. The method for preparing the Ginkgolide B sesquihydrate crystal form I according to any one of claims 1 to 4, characterized in that it comprises the following steps: Step (1): adding ethanol to the terpene lactone, heating and dissolving, keeping the temperature, slowly adding water, and cooling naturally after the water is added, continuing to stir and crystallize, filtering, and drying the filter cake to obtain a refined ginkgo lactone product; Step (2): Add the refined ginkgolide to the crystallization solution, heat to dissolve, cool, and allow to crystallize naturally at room temperature. 6. The method for preparing the Ginkgolide B sesquihydrate crystal form I according to claim 5, characterized in that the volume ratio of the crude product to ethanol in the step (1) is 1:2-1:4, more preferably, the volume ratio is 1:3; In the step (1), the ethanol is 75% to 95% ethanol, more preferably, 95% ethanol; The insulation temperature in step (1) is 55° C. The amount of water used in step (1) is 1-2 times the volume of the dissolving solution, more preferably, 1.5 times; The crystallization time in step (1) is 2-6 hours, more preferably, the crystallization time is 3 hours. 7. The method for preparing the crystalline form I of Ginkgolide B sesquihydrate according to claim 5, characterized in that the mass volume ratio (g:mL) of the sample to the crystallization solution in the step (2) is 1:5-1:10, more preferably, the mass volume ratio (g:mL) is 1:7; the crystallization solution in the step (2) is methanol water/ethanol water solution, and the volume ratio of alcohol to water is 30%-60%, more preferably, the volume ratio of alcohol to water is 50%; The crystallization time in step (2) is 24h-72h, and more preferably, the crystallization time is 36h. 8. A pharmaceutical composition comprising the Ginkgolide B sesquihydrate crystalline form I according to any one of claims 1 to 7. 9. Use of the Ginkgolide B sesquihydrate crystal form I according to any one of claims 1 to 7 in the preparation of drugs for treating diseases.