Icaritin methanol solvate and preparation method thereof

Abstract

The application belongs to the field of medicine crystal form, and particularly relates to a icariin methanol solvate and a preparation method thereof. The icariin methanol solvate and the preparation method thereof provided by the application have X-ray diffraction spectra using Cu-K alpha radiation, and diffraction peaks at 6.03+0.2°, 17.59+0.2°, 19.73+0.2°, 24.86+0.2°, 26.34+0.2° and 30.14+0.2° expressed by 2 theta; the icariin methanol solvate provided by the application has excellent properties, high solubility and stability, provides a high-quality raw material selection for the preparation of icariin, and the preparation method is simple to operate, easy to control and suitable for industrial amplification.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical crystal forms, specifically relating to an icariin methanol solvate and its preparation method. Background Technology

[0002] Icaritin (IT), also known as icariin, is a polyhydroxy flavonoid monomer from Epimedium, a plant belonging to the genus Epimedium in the family Berberidaceae. Its structural formula is shown in formula (I) below:

[0003]

[0004] Pharmacological studies have shown that icariin has stronger anti-osteoporosis activity than other flavonoid glycosides in epimedium, and in vitro, it promotes osteoblast activity and inhibits osteoclast activity. Epimedium, a traditional Chinese medicine, is used to tonify the kidneys and strengthen yang, strengthen tendons and bones, dispel wind and dampness, wash sores and kill parasites, and relieve fatigue and pain. As one of its main active components, icariin has attracted the attention of many scholars at home and abroad in recent years, and its pharmacological effects have been extensively and in-depth studied. Current results indicate that the main physiological activities of icariin lie in improving cardiovascular function, enhancing immunity, and regulating endocrine function. It also possesses anti-tumor, anti-hepatotoxic, anti-hypoxia-reoxygenation, and bone-strengthening effects.

[0005] Although icariin has promising clinical application prospects, its poor water solubility greatly limits its clinical application. Current research on improving the solubility of icariin mainly focuses on two aspects: one is to study the preparation of new crystal forms of icariin or its derivatives, and the other, more conventional approach, is to improve its solubility through formulation methods. For example, Jia Dongsheng et al. significantly improved the solubility of the raw material and physical mixture by preparing icariin phospholipid complex (Jia Dongsheng, Zhao Jiangli, Shi Feng et al. Preparation and solid dispersion study of icariin phospholipid complex [J]. Chinese Traditional and Herbal Drugs, 2010, 9(41)). However, this method has the problem that the dissolution medium may destroy the drug structure or evaporate. Wang Jinyan et al. conducted a solubilization study on icariin using microemulsion technology (Jinyan, Chen Yan, Zhang Zhenhai et al. Preliminary study on the intestinal absorption characteristics of icariin self-microemulsion in Caco-2 cell model [J]. Chinese Traditional and Herbal Drugs, 2012, 3(43)). Although it can improve the solubility and absorption of icariin, a large amount of emulsifiers and co-emulsifiers are used in the process, which poses certain quality and safety risks.

[0006] There are few reports on the research on the raw material forms of icariin. Although patent CN101200743A reported the preparation of crystalline icariin, it did not improve its solubility. Patent CN104860958A reported crystal forms A and B of dehydrated icariin, and the research results showed that its solubility was improved to some extent compared with amorphous powdered dehydrated icariin, but not significantly.

[0007] Current reports on icariin mainly focus on its preparation, formulation, physicochemical properties, and pharmacological properties, but none have significantly improved its solubility. Therefore, further research is needed to develop new crystal forms that significantly improve the solubility of icariin, effectively enhance its physicochemical properties, fundamentally solve the solubility problem, minimize the addition of excipients during formulation production, and reduce potential risks associated with medication use. Summary of the Invention

[0008] In view of the poor solubility of icariin in existing technologies and the drawbacks of existing improvement methods, this invention successfully prepared a methanol solvate crystal of icariin through co-crystallization technology. This crystal can significantly improve the solubility characteristics of icariin and has good physicochemical stability.

[0009] The specific technical content of this invention is as follows:

[0010] In a first aspect, the present invention provides an icariin methanol solvate, characterized in that the molar ratio of icariin to methanol in the crystal unit structure is 1:1.

[0011] Preferably, the icariin methanol solvate, when subjected to Cu-Kα radiation, exhibits characteristic peaks in its 2θ X-ray diffraction pattern at 6.03±0.2°, 17.59±0.2°, 19.73±0.2°, 24.86±0.2°, 26.34±0.2°, and 30.14±0.2°.

[0012] Preferably, the icariin methanol solvate, when subjected to Cu-Kα radiation, has the following main crystallographic parameters: triclinic crystal system, space group P-1; cell parameters are: α = 93.3430(10)°, β = 93.3430(10)°, γ = 91.8490(10)°, cell volume

[0013] Preferably, the icariin methanol solvate, when subjected to Cu-Kα radiation, exhibits characteristic peaks with... Figure 1 The X-ray powder diffraction pattern shown is shown.

[0014] In a second aspect, the present invention provides a method for preparing icariin methanol solvate, comprising the following steps: dissolving icariin in a mixed solvent of methanol and organic solvent A, heating and stirring, cooling to crystallize, filtering, and drying to obtain icariin methanol solvate.

[0015] Preferably, the organic solvent A is selected from one or more of acetonitrile, tetrahydrofuran, acetone, and isopropanol, and more preferably from one or two of tetrahydrofuran and isopropanol.

[0016] Preferably, the mass-to-volume ratio of icariin to methanol is 11–20:1, mg / mL; more preferably, it is 14–17:1, mg / mL.

[0017] Preferably, the volume ratio of methanol to organic solvent A is 5 to 7:1.

[0018] Preferably, the heating and stirring temperature is 45–65°C, more preferably 50–55°C.

[0019] Preferably, the cooling and crystallization temperature is 0–15°C; more preferably, it is 5–10°C.

[0020] Preferably, the crystallization time is 25 to 72 hours.

[0021] The drying method described is a conventional drying method in the field, such as vacuum drying at 50-60°C.

[0022] Preferably, the specific steps of the preparation method of the icariin methanol solvate are as follows: dissolve icariin in a mixed solvent of methanol / organic solvent A, heat and stir at 45-65°C until completely dissolved, cool to 0-15°C to crystallize for 25-72 hours, filter, and dry the filter cake to obtain the icariin methanol solvate.

[0023] In a third aspect, the present invention provides a pharmaceutical composition comprising the icariin methanol solvate of the present invention and other pharmaceutically acceptable components.

[0024] Preferably, the pharmaceutical composition is prepared by means of: using standard and conventional techniques, combining the icariin methanol solvate with a pharmaceutically acceptable solid or liquid carrier, and optionally combining it with pharmaceutically acceptable excipients and excipients to prepare a pharmaceutical dosage form.

[0025] Preferably, the other pharmaceutically acceptable components include, but are not limited to, pharmaceutically active ingredients, excipients, fillers, etc., that can be used in combination.

[0026] Preferably, the dosage form of the pharmaceutical composition includes, but is not limited to, sprays, tablets, capsules, powder for injection, and injections.

[0027] In a fourth aspect, the present invention provides the application of icariin methanol solvate as an active ingredient in the preparation of drugs for treating liver injury, liver fibrosis, tumors, etc.

[0028] Confirmation of crystal structure:

[0029] X-ray crystal data for this invention were collected on a Rigaku XtaLAB Synergy instrument in Japan at a test temperature of 293(2) K. CuKa radiation was used, and data were collected via ω-scan and Lp correction was performed. The crystal structure was calculated using the ShelXT program in the O1ex2 software. The structure parameters and atom types were corrected using the least squares method with the ShelXL program. The positions of all hydrogen atoms were obtained using geometric calculation and the Fourier difference method. The goodness-of-fit (goof value) was 1.019, close to 1.0, indicating that the weighting scheme was appropriate and the structure was accurate. The crystallographic data obtained from the icariin methanol solvate prepared in this invention are shown in Table 1. The ORTEP diagram of the icariin methanol solvate of this invention is attached. Figure 2 As shown, icariin and methanol are linked by intramolecular hydrogen bonds; the crystal packing diagram of the icariin methanol solvate is attached. Figure 3 As shown.

[0030] Table 1. Main crystallographic data of icariin methanol solvate

[0031]

[0032]

[0033] The X-ray powder diffraction testing instrument and conditions in this invention are as follows: X-ray powder diffractometer: PANalytical EMPYREAN; Cu-Kα; sample stage: flat plate; incident light path: BBHD; diffraction light path: PLXCEL; voltage: 45kV; current: 40mA; divergence slit: 1 / 4; anti-scattering slit: 1; Solar slit: 0.04rad; step size: 0.5s; scanning range: 3~50°. Based on crystallographic data, the characteristic peaks in the corresponding X-ray powder diffraction pattern (Cu-Kα) are detailed in the appendix. Figure 1 And Table 2.

[0034] Table 2. Major PXRD peaks of icariin methanol solvate

[0035]

[0036]

[0037] The samples prepared in Examples 1 to 5 all conform to X-ray powder diffraction patterns.

[0038] The beneficial effects of this invention are:

[0039] The icariin methanol solvate provided by this invention exhibits good stability in both solid and solution states, and significantly improves the solubility of icariin, providing a high-quality raw material option for the formulation production of icariin. Furthermore, its preparation method is simple to operate, the crystallization process is easy to control, it has good reproducibility, and the prepared crystals have high purity, making it suitable for industrial production. Attached Figure Description

[0040] Figure 1 X-ray powder diffraction pattern of icariin methanol solvate.

[0041] Figure 2 ORTEP plot of icariin methanol solvate

[0042] Figure 3 : Packing diagram of icariin methanol solvate. Detailed Implementation

[0043] The present invention will be further illustrated by the following embodiments. It should be understood that the embodiments of the present invention are merely for illustrating the present invention and are not intended to limit the present invention. Therefore, any simple improvements to the present invention under the premise of the method of the present invention are within the scope of protection claimed by the present invention.

[0044] The raw material of this invention, icariin, can be prepared by any method in the prior art or purchased from commercially available products (purity ≥98%), and the other reagents and materials are all commercially available.

[0045] I. Crystal Form Preparation

[0046] Example 1

[0047] 368.4 mg of icariin was dissolved in a mixed solvent of 24 mL methanol and 4 mL tetrahydrofuran. The solution was stirred at 50–55 °C until completely dissolved. The solution was then slowly cooled to 5–10 °C and allowed to stand for about 48 hours to crystallize. The solution was filtered, and the filter cake was dried under vacuum at 60 °C to obtain the icariin methanol solvate. The yield was 97.6% and the purity was 99.97%.

[0048] Example 2

[0049] 368.5 mg of icariin was dissolved in a mixed solvent of 26.3 mL methanol and 5.3 mL isopropanol. The solution was stirred at 55–60 °C until completely dissolved. The solution was then slowly cooled to 10–15 °C and allowed to stand for about 72 hours to crystallize. The solution was filtered, and the filter cake was dried under vacuum at 50 °C to obtain the icariin methanol solvate. The yield was 96.0% and the purity was 99.96%.

[0050] Example 3

[0051] 368.4 mg of icariin was dissolved in a mixed solvent of 21.7 mL methanol and 3 mL acetonitrile. The solution was stirred at 60–65 °C until completely dissolved. The solution was then slowly cooled to 0–5 °C and allowed to stand for about 25 hours to crystallize. The solution was filtered, and the filter cake was dried under vacuum at 60 °C to obtain the icariin methanol solvate. The yield was 96.5% and the purity was 99.94%.

[0052] Example 4

[0053] 368.4 mg of icariin was dissolved in a mixed solvent of 18.5 mL methanol and 3.7 mL acetone. The solution was stirred at 45–50 °C until completely dissolved. The solution was then slowly cooled to 5–10 °C and allowed to stand for about two days to crystallize. The solution was filtered, and the filter cake was dried under vacuum at 60 °C to obtain the icariin methanol solvate. The yield was 95.2% and the purity was 99.92%.

[0054] Example 5

[0055] 368.5 mg of icariin was dissolved in a mixed solvent of 33.5 mL methanol, 3 mL tetrahydrofuran, and 3.5 mL isopropanol. The solution was stirred at 50–55 °C until completely dissolved. The solution was then slowly cooled to 5–10 °C and allowed to stand for about two days to crystallize. The solution was filtered, and the filter cake was dried under vacuum at 50 °C to obtain the icariin methanol solvate. The yield was 95.6%, and the purity was 99.94%.

[0056] Comparative Example 1

[0057] 368.4 mg of icariin was dissolved in 30 mL of acetone, filtered, and approximately 15 mL of distilled water was added to the filtrate. The solution was refluxed at 75 °C and allowed to crystallize at 20 °C. After crystallization for 24 hours, the crystals were filtered to obtain pale yellow crystals. The obtained crystals were continuously dried at 80 °C until the weight of the crystals no longer changed, yielding icariin crystals with a yield of 91.5% and a purity of 99.88%.

[0058] Comparative Example 2

[0059] 15 mg of icariin crystals obtained in Comparative Example 1 were dissolved in 1.2 mL of methanol to obtain a suspension. The suspension was stirred at 20 °C for 4 days, centrifuged, and the collected crystals were 1 / 3 methanol solvate of icariin, with a yield of 88.6% and a purity of 99.84%.

[0060] Comparative Example 3

[0061] Weigh 15 mg of the crystals obtained in Comparative Example 1 into a 3 mL vial. Take a 20 mL vial and add 4 mL of methanol solvent to it. Place the 3 mL vial into the 20 mL vial, seal it, and let it stand at 20°C for 5 days. The collected crystals are icariin methanol solvate, with a yield of 90.3% and a purity of 99.85%.

[0062] II. Verification Implementation Examples

[0063] 1. Stability test

[0064] The stability testing method of this invention was carried out in accordance with the guidelines for stability studies in Part IV of the Chinese Pharmacopoeia (2020 edition). The high-temperature test conditions were 60℃, the strong light irradiation test conditions were 4500 lx ± 500 lx, and the high humidity test conditions were 92.5%. Purity was determined by HPLC, with three parallel experiments performed and the average value taken. Specific test results are shown in Table 3.

[0065] Table 3. Stability test results of icariin methanol solvate under light, high temperature and high humidity conditions.

[0066]

[0067] Meanwhile, the investigation revealed that Examples 1 to 5 of this application have similar stability test results.

[0068] 2. Solubility test

[0069] Method: Measure 10 ml of medium (water, 0.01 mol / L HCl solution) into a vial, add excess sample, seal the vial and place it in a 25°C constant temperature water bath and stir for 1 hour. Filter through a filter membrane and collect the filtrate. Measure the absorbance at a wavelength of 270 nm. Calculate the solubility by measuring the absorbance of the standard reference.

[0070] Table 4. Solubility of icariin methanol solvate in different media (μg / mL)

[0071]

[0072] Meanwhile, the investigation revealed that Examples 1 to 5 of this application have similar solubility test results.

[0073] III. Bioactivity Experiment

[0074] 1. Materials

[0075] 1.1 Medicines

[0076] Example 1 and Comparative Examples 1-3: Self-made crystal forms.

[0077] 1.2 Animals

[0078] Thirty Beagle experimental dogs, half male and half female, weighing 12-15 kg, were provided by Guangzhou Pharmaceutical Research Institute Co., Ltd.

[0079] 2. Pharmacokinetics in Beagle dogs

[0080] Thirty Beagle dogs were acclimatized for two weeks and then quarantined. Twenty-four healthy dogs were divided into four groups and administered the crystal forms of Examples 1 and 1-3 (comparative Examples 1-3) orally at a dose of 15 mg / kg. Blood samples were collected from peripheral veins at 0, 0.5, 1, 2, 4, 6, 8, and 12 hours, and plasma was obtained by centrifugation at 2-8°C for 10 minutes. The concentration of icariin in Beagle dog plasma was determined by β-glucuronidase hydrolysis and LC-MS / MS (refer to Huang Ying, Liu Yanjun, Hu Lei, et al. Determination of Acoradin Concentration in Beagle Plasma by LC-MS / MS [C] Proceedings of the 7th National Toxicology Conference of the Chinese Society of Toxicology and the 8th Hubei Science and Technology Forum. 2015.). The obtained pharmacokinetic parameters are shown in Table 5. The area under the plasma concentration-time curve (AUC) was calculated using the trapezoidal method, and the elimination half-life (t) was calculated. 1 / 2 The peak concentration (C) was calculated using the Best Fit method. max ) and peak time (T) max Actual measured values ​​are used.

[0081] Table 5 Pharmacokinetic parameters of Beagle dogs after oral administration ( n=6)

[0082]

[0083] Note: Group 1 is the first example of drug administration; Group 2 is the second example of drug administration; Group 3 is the third example of drug administration; Group 4 is the fourth example of drug administration.

Claims

1. An icariin methanol solvate, characterized in that, Using Cu-Kα radiation, the X-ray diffraction pattern, expressed as 2θ, shows characteristic peaks at 6.03±0.2°, 17.59±0.2°, 19.73±0.2°, 24.86±0.2°, 26.34±0.2°, and 30.14±0.2°. The molar ratio of icariin to methanol in the crystal unit structure is 1:

1.

2. The icariin methanol solvate according to claim 1, characterized in that, Using Cu-Kα radiation, its main crystallographic parameters are: triclinic crystal system, space group P-1; cell parameters are: a=7.62490(10)Å, b=8.61620(10)Å, c=15.4242(2)Å, α=96.9620(10)°, β=93.3430(10)°, γ=91.8490(10)°, cell volume V=1003.36(2)Å. 3 .

3. The icariin methanol solvate according to claim 1, characterized in that, Its characteristic peaks have the X-ray powder diffraction pattern shown in Figure 1.

4. A method for preparing the icariin methanol solvate according to any one of claims 1-3, characterized in that, Includes the following steps: Icariin was dissolved in a mixed solvent of methanol and organic solvent A, heated and stirred, cooled to crystallize, filtered and dried to obtain icariin methanol solvate.

5. The preparation method according to claim 4, characterized in that, The organic solvent A is selected from one or more of acetonitrile, tetrahydrofuran, acetone, and isopropanol.

6. The preparation method according to claim 4, characterized in that, The mass-to-volume ratio of icariin to methanol is 11–20:1, mg / mL.

7. The method according to claim 4, characterized in that, The volume ratio of methanol to organic solvent A is 5 to 7:

1.

8. The method according to claim 4, characterized in that, The heating and stirring temperature is 45–65°C.

9. The preparation method according to claim 4, characterized in that, The specific steps are as follows: Icariin is dissolved in a mixed solvent of methanol and organic solvent A, heated and stirred at 45-65°C until completely dissolved, cooled to 0-15°C for crystallization for 25-72 hours, filtered, and the filter cake is dried to obtain icariin methanol solvate.

10. The use of the icariin methanol solvate according to any one of claims 1-3 as an active ingredient in the preparation of drugs for treating liver injury, liver fibrosis, and tumors.

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