AHK-Cu acetate crystal form compound and preparation method thereof

Abstract

The invention provides an AHK-Cu acetate crystal form compound and a preparation method thereof. The preparation method comprises the following steps: a) dissolving an AHK solid in water, adding copper acetate with the same amount of substance, stirring and dissolving to form a clear liquid, and freeze-drying to obtain an AHK-Cu acetate solid; b) dissolving the AHK-Cu acetate solid obtained in the step a) in water, adjusting the pH value to 4-6 by using acetic acid, and filtering by using a water-based filter membrane to obtain an AHK-Cu acetate filtrate; and then separating out AHK-Cu acetate crystals by adopting ethanol, and finally, sequentially filtering, leaching with ethanol and drying to obtain the AHK-Cu acetate crystal form compound. Compared with the prior art, the preparation method provided by the invention adopts specific process steps to realize better overall interaction: the AHK-Cu acetate crystal is obtained by adopting a low-temperature crystallization mode, and compared with an amorphous solid, the AHK-Cu acetate crystal has better stability, less organic solvent is used, the cost benefit is good, and large-scale production can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of crystal form compounds, and more particularly to an AHK-Cu acetate crystal form compound and a preparation method and application thereof. BACKGROUND

[0002] AHK-Cu is a complex copper peptide composed of three amino acids, alanine, histidine and lysine, and complexed with copper (Cu) ions, which is usually present in the blood of most mammals and exhibits the potential to treat hair loss and delay skin aging. Studies have shown that AHK-Cu can inhibit the formation of dihydrotestosterone by regulating growth factor secretion and reducing oxidative stress, thereby playing a role in preventing hair loss and promoting hair growth. In addition, AHK-Cu can stimulate the growth and development of new microcells and stimulate the production of type I collagen, thereby forming an anti-aging function. Currently, AHK-Cu is widely used in the cosmetics and skin care industry, and has the effects of treating hair loss and delaying skin aging.

[0003] AHK-Cu is generally synthesized by solid phase or liquid phase, and then complexed with copper ions to obtain. At present, the number of patents and literatures related to AHK-Cu is small, and most of them are related to the formula and efficacy. In addition, some patents related to the synthesis of AHK disclose a synthesis method of GHK or AHK tripeptide, which obtains AHK in five steps without using condensing agent. The AHK-Cu related products on the market are mostly in the form of liquid or amorphous solid, and there is no report on the specific crystal form thereof. GHK-Cu, as a kind of copper peptide, is similar in structure to AHK-Cu, and the two have certain similarity. At present, there are many patents related to purification and solidification, including: a method for preparing GHK tripeptide and blue copper peptide without condensing agent, the method for obtaining solid GHK-Cu is to slowly add ethanol to GHK-Cu aqueous solution under the condition of 5-10℃ to obtain solid, and it is not mentioned whether the obtained solid is crystal; a synthesis method of GHK copper peptide, the method for obtaining solid GHK-Cu is to directly freeze-dry GHK-Cu aqueous solution, and the obtained solid is amorphous powder; a synthesis method of high-purity GHK-Cu, the method for obtaining solid GHK-Cu is to heat GHK-Cu ethanol aqueous solution to 40-70℃, and then cool to precipitate crystals; a synthesis method of tripeptide-1 and blue copper peptide and application thereof, the method for obtaining solid GHK-Cu is to concentrate GHK-Cu aqueous solution under reduced pressure and cool to 0-5℃ to precipitate, and it is not mentioned whether the obtained solid is crystal.

[0004] In summary, AHK-Cu related patents and literature are rare, and there is no specific crystal form reported. Referring to the solidification method of GHK-Cu, AHK-Cu can be obtained in solid form by rotary evaporation, freeze-drying or using a poor solvent. However, the solid powder (amorphous) obtained by rotary evaporation or freeze-drying process has poor storage stability and is easy to absorb moisture, which puts higher requirements on its transportation and storage. Compared with amorphous, the molecular structure of crystal is more orderly arranged, which can maintain good stability during storage. In order to popularize and provide AHK-Cu to the market, not only solid AHK-Cu needs to be prepared, but also more stable AHK-Cu crystal product compared with amorphous solid needs to be provided. SUMMARY

[0005] Therefore, the purpose of the present application is to provide an AHK-Cu acetate crystal compound and a preparation method thereof. The preparation method provided by the present application obtains AHK-Cu acetate crystal by low-temperature crystallization, which has better stability than amorphous solid, uses less organic solvent, has good cost-effectiveness, and can be produced on a large scale.

[0006] The present application provides an AHK-Cu acetate crystal compound, wherein the X-ray powder diffraction pattern of the AHK-Cu acetate crystal compound has diffraction peaks at 2θ±0.2° positions of 9.5106°, 11.0680°, 12.4906°, 13.7784°, 14.9704°, 15.3815°, 18.3890°, 20.8134°, 22.2639°, 24.4901° and 25.8507°.

[0007] Preferably, the 2θ of the AHK-Cu acetate crystal compound is 9.5106°, 11.0680°, 12.4906°, 13.7784°, 14.9704°, 15.3815°, 18.3890°, 20.8134°, 22.2639°, 24.4901° and 25.8507°.

[0008] The present application also provides a preparation method of the AHK-Cu acetate crystal compound of the above technical solution, comprising the following steps:

[0009] a) dissolving AHK solid in water, adding an equal amount of copper acetate, stirring to dissolve to form a clear solution, and then freeze-drying to obtain AHK-Cu acetate solid;

[0010] b) dissolving the AHK-Cu acetate solid obtained in step a) in water and adjusting the pH to 4-6 with acetic acid, filtering the AHK-Cu acetate filtrate through a water filter membrane, and then precipitating the AHK-Cu acetate crystals from the filtrate using ethanol, and finally filtering, washing with ethanol, and drying to obtain the AHK-Cu acetate crystal form compound.

[0011] Preferably, the freeze-drying process in step a) is as follows:

[0012] Pre-freezing at -15℃ to -30℃ for 1-3 hours, and then freeze-drying at a vacuum degree of 1 Pa to 15 Pa and a temperature of -30℃ to -50℃ for 10-15 hours.

[0013] Preferably, the water filter membrane in step b) is a water filter membrane with a pore size of 0.20 μm to 0.25 μm.

[0014] Preferably, the concentration of the AHK-Cu acetate filtrate in step b) is 200 g / L to 400 g / L.

[0015] Preferably, the process of precipitating the AHK-Cu acetate crystals from the filtrate using ethanol in step b) is as follows:

[0016] Slowly adding ethanol to the AHK-Cu acetate filtrate at a constant speed at 5℃ to 20℃ and 50 RPM to 150 RPM, and adding the ethanol for 20-30 hours to precipitate the AHK-Cu acetate crystals.

[0017] Preferably, the amount of ethanol added is 4-8 times the volume of the AHK-Cu acetate filtrate.

[0018] Preferably, the washing with ethanol in step b) is performed using pre-cooled ethanol at 4℃ to 6℃.

[0019] Preferably, the drying in step b) is performed using a vacuum drying oven at a temperature of 40℃ to 50℃.

[0020] The application provides an AHK-Cu acetate crystal compound and a preparation method thereof. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Appearance of AHK-Cu acetate amorphous solid in Example 1

[0022] Figure 2 Appearance of AHK-Cu acetate crystal in Example 2

[0023] Figure 3 X-ray diffraction pattern of AHK-Cu acetate crystal in Example 2 DETAILED DESCRIPTION

[0024] The technical solutions of the application will be described clearly and completely below with reference to the embodiments of the application. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all the other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the application.

[0025] The application provides an AHK-Cu acetate crystal compound, and the X-ray powder diffraction pattern of the AHK-Cu acetate crystal compound has diffraction peaks at 2θ±0.2° positions of 9.5106°, 11.0680°, 12.4906°, 13.7784°, 14.9704°, 15.3815°, 18.3890°, 20.8134°, 22.2639°, 24.4901° and 25.8507°.

[0026] In the present application, the 2theta of the AHK-Cu acetate salt crystal compound is preferably 9.5106°, 11.0680°, 12.4906°, 13.7784°, 14.9704°, 15.3815°, 18.3890°, 20.8134°, 22.2639°, 24.4901°, and 25.8507°.

[0027] The AHK-Cu acetate salt crystal compound provided by the present application is converted from AHK-Cu acetate salt amorphous solid into crystal by low-temperature solvent crystallization, which significantly improves the stability of the product, is conducive to the long-term storage of AHK-Cu acetate salt, and is conducive to the application in various products such as cosmetics. The experimental results show that, compared with the previous liquid or amorphous solid product, the AHK-Cu acetate salt crystal compound provided by the present application has obvious technical advantages: (1) According to the XRD crystal diffraction results, it is confirmed that the AHK-Cu acetate salt prepared by the present application is a crystal. (2) According to Tables 2 and 3, the stability of the AHK-Cu acetate salt crystal provided by the present application is better than that of the amorphous powder, which is conducive to the long-term storage of AHK-Cu acetate salt and the application in various products such as cosmetics. (3) The preparation method of the AHK-Cu acetate salt crystal provided by the present application is low-temperature solvent crystallization, only ethanol solution is used, no other organic solvents are used, the safety is high, the cost is low, and the scale-up is easy.

[0028] The present application also provides a preparation method of the AHK-Cu acetate salt crystal compound of the above technical solution, which comprises the following steps:

[0029] a) dissolving AHK solid in water, adding copper acetate in equal amount, stirring to dissolve to form a clear solution, and then freeze-drying to obtain AHK-Cu acetate salt solid;

[0030] b) dissolving the AHK-Cu acetate salt solid obtained in step a) in water and adjusting the pH to 4-6 with acetic acid, filtering through a water system filter membrane to obtain an AHK-Cu acetate salt filtrate; then precipitating AHK-Cu acetate salt crystals with ethanol, and finally filtering, ethanol leaching and drying in sequence to obtain the AHK-Cu acetate salt crystal compound.

[0031] Firstly, the AHK solid is dissolved in water, and an equal amount of copper acetate is added, stirred to dissolve to form a clear solution, and then freeze-dried to obtain AHK-Cu acetate salt solid.

[0032] The source of the AHK solid is not particularly limited in the present application, and commercially available or self-made products known to those skilled in the art can be used, such as the AHK solid prepared by the synthetic method of GHK or AHK tripeptide in patent CN107098950B. In the present application, the water is pure water, which will not be described again.

[0033] In the present application, the process of freeze-drying is preferably specific as follows:

[0034] first pre-freezing at -15℃ to -30℃ for 1h to 3h; and then freeze-drying at a vacuum degree of 1Pa to 15Pa and a temperature of -30℃ to -50℃ for 10h to 15h;

[0035] More preferably, it is specific as follows:

[0036] first pre-freezing at -20℃ to -25℃ for 2h; and then freeze-drying at a vacuum degree of 5Pa to 10Pa and a temperature of -40℃ to -45℃ for 12h.

[0037] On this basis, the obtained AHK-Cu acetate solid is in amorphous solid form.

[0038] After obtaining the AHK-Cu acetate solid, the present application dissolves the obtained AHK-Cu acetate solid in water and adjusts the pH to 4 to 6 with acetic acid, and then filters the solution through a water filter membrane to obtain an AHK-Cu acetate filtrate; then ethanol is used to precipitate AHK-Cu acetate crystals, and finally, filtration, ethanol elution and drying are sequentially performed to obtain an AHK-Cu acetate crystalline compound.

[0039] In the present application, the pH adjustment is preferably 4 to 5.

[0040] In the present application, the water filter membrane is preferably a water filter membrane with a pore size of 0.20μm to 0.25μm; the AHK-Cu acetate filtrate is collected, and the concentration of the AHK-Cu acetate filtrate is preferably 200g / L to 400g / L, and more preferably 300g / L to 400g / L.

[0041] In the present application, the process of precipitating AHK-Cu acetate crystals by using ethanol is preferably specific as follows:

[0042] ethanol is slowly and uniformly added to the AHK-Cu acetate filtrate at 5℃ to 20℃ and 50RPM to 150RPM, and the addition is completed in 20h to 30h, so as to precipitate AHK-Cu acetate crystals;

[0043] More preferably, it is specific as follows:

[0044] ethanol is slowly and uniformly added to the AHK-Cu acetate filtrate at 5℃ to 10℃ (crystallization temperature) and 100RPM, and the addition is completed in 24h, so as to precipitate AHK-Cu acetate crystals.

[0045] In the present application, the amount of ethanol added is preferably 4 times to 8 times the volume of the AHK-Cu acetate filtrate, and more preferably 4 times to 6 times the volume of the AHK-Cu acetate filtrate.

[0046] In the present application, the ethanol elution is preferably performed using 4-6℃ pre-cooled ethanol, and more preferably 5℃ pre-cooled ethanol.

[0047] In the present application, the drying is preferably performed using a vacuum drying oven, and the temperature is preferably 40-50℃, and more preferably 45℃.

[0048] The preparation method provided by the present application adopts specific process steps to achieve better overall interaction: the AHK-Cu acetate crystal is obtained by low-temperature crystallization, has better stability compared to amorphous solid, uses less organic solvent, has good cost-effectiveness, and can be produced on a large scale.

[0049] The present application provides an AHK-Cu acetate crystal compound and a preparation method thereof; the preparation method comprises the following steps: a) dissolving AHK solid in water, adding an equal amount of copper acetate, stirring to dissolve to form a clear solution, and then freeze-drying to obtain AHK-Cu acetate solid; b) dissolving the AHK-Cu acetate solid obtained in step a) in water and adjusting the pH to 4-6 with acetic acid, passing through a water system filter membrane to obtain an AHK-Cu acetate filtrate; then precipitating the AHK-Cu acetate crystal with ethanol, and finally filtering, eluting with ethanol and drying in sequence to obtain the AHK-Cu acetate crystal compound. Compared with the prior art, the preparation method provided by the present application adopts specific process steps to achieve better overall interaction: the AHK-Cu acetate crystal is obtained by low-temperature crystallization, has better stability compared to amorphous solid, uses less organic solvent, has good cost-effectiveness, and can be produced on a large scale.

[0050] In order to further illustrate the present application, the following examples are used for detailed description. The reagents and raw materials used in the following examples of the present application are commercially available or self-made; among them, the AHK solid is prepared according to the patent CN107098950B (Example 2) "A synthesis method of GHK or AHK tripeptide".

[0051] Example 1

[0052] The AHK solid is dissolved in pure water, and an equal amount of copper acetate is added, stirred to dissolve to form a clear solution, and the clear solution is freeze-dried to obtain AHK-Cu acetate solid, which is identified as amorphous. The freeze-drying process of the AHK-Cu acetate solid is as follows: (1) pre-freezing at-20 to-25℃ for 2h; (2) freeze-drying at a vacuum degree of 5-10Pa and-40 to-45℃ for 12h.

[0053] The prepared AHK-Cu solid has a purity of 97.5% determined by HPLC. The appearance is shown in the attached Figure 1 picture, and the whole presents a powder shape without typical crystal appearance.

[0054] Example 2

[0055] Take the AHK-Cu acetate solid obtained in Example 1, 30 g, add purified water, adjust the pH to 5 with acetic acid, make up to 100 ml, pass through a 0.22 μm water filter membrane, and obtain an AHK-Cu acetate filtrate with a concentration of 300 g / L. At 10°C, 100 RPM, slowly add 6 times the amount of ethanol at a constant speed, and add for 24 h, thereby precipitating AHK-Cu acetate crystals. Finally, filter and rinse the AHK-Cu acetate crystals with a 5°C pre-cooled ethanol solution, and dry in a 45°C vacuum drying oven to obtain an AHK-Cu acetate crystal product; the purity is 99.5% as determined by HPLC, and the yield is 81.2%. The appearance is shown in FIG. 2, and the overall appearance is uniform granular with a typical crystal appearance. Figure 2

[0056] The AHK-Cu acetate crystal product obtained in Example 2 was detected by powder X-ray diffraction (PXRD); the detection method is as follows:

[0057] (1) Instrument parameters:

[0058] PANalytical Empyrean sharp X-ray powder diffractometer (PW3040 / 60, PANalytical Analytical Instruments Co., Ltd., Netherlands), Cu-Ka radiation, wavelength equipped with Bragg-Brentano High Definition. Incident light path: divergence slit 1 / 8°, Soller slit 0.04 rad, light shield Mask 10 mm, anti-scattering slit 1 / 2°; diffraction light path: anti-scattering slit P7.5; X-ray sample stage: rotation mode; scanning detector: PIXcel1D-Medipix3; X-ray tube voltage 45 kV, X-ray tube current 40 mA, scanning range 2-40° (2θ), step size 0.026°, step time: 36.465 s. Data acquisition software DataCollector, data viewing software HighScore Plus.

[0059] (2) Sample preparation:

[0060] The sample was not pretreated, and the powder sample was placed on a clean stainless steel spoon in a background-free circular silicon sample disc, and a spatula was used to gently collect the sample to keep it in the middle of the sample disc, and then a weighing paper was used to press it flat.

[0061] The X-ray diffraction pattern of the AHK-Cu acetate crystal of Example 2 is shown in FIG. 3, which has characteristic peaks of crystals, and the peak position and peak intensity are shown in Table 1. Figure 3

[0062] ​​Table 1 AHK-Cu acetate crystal X-ray diffraction characteristic peaks

[0063]

[0064]

[0065] Example 3

[0066] Take the AHK-Cu acetate solid obtained in Example 1, 30 g, add purified water, adjust pH to 5 with acetic acid, make up to 100 ml, pass through a 0.22 μm water filter membrane, obtain AHK-Cu acetate filtrate, concentration is 300 g / L. At 20 °C, 100 RPM, slowly add 6 times ethanol at a constant speed, 24 h complete, thereby precipitate AHK-Cu acetate crystals. Finally filter and use 5 °C pre-cooled ethanol solution to rinse the AHK-Cu acetate crystals, place in a 45 °C vacuum drying oven to dry to obtain AHK-Cu acetate crystal product; its X-ray diffraction pattern is the same as the crystal form obtained in Example 2, HPLC determination of purity is 99.1%, yield is 71.5%.

[0067] Example 4

[0068] Take the AHK-Cu acetate solid obtained in Example 1, 30 g, add purified water, adjust pH to 5 with acetic acid, make up to 100 ml, pass through a 0.22 μm water filter membrane, obtain AHK-Cu acetate filtrate, concentration is 300 g / L. At 5 °C, 100 RPM, slowly add 6 times ethanol at a constant speed, 24 h complete, thereby precipitate AHK-Cu acetate crystals. Finally filter and use 5 °C pre-cooled ethanol solution to rinse the AHK-Cu acetate crystals, place in a 45 °C vacuum drying oven to dry to obtain AHK-Cu acetate crystal product; its X-ray diffraction pattern is the same as the crystal form obtained in Example 2, HPLC determination of purity is 98.9%, yield is 82.5%.

[0069] Example 5

[0070] Take the AHK-Cu acetate solid obtained in Example 1, 30 g, add purified water, adjust pH to 5 with acetic acid, make up to 100 ml, pass through a 0.22 μm water filter membrane, obtain AHK-Cu acetate filtrate, concentration is 300 g / L. At 10 °C, 100 RPM, slowly add 4 times ethanol at a constant speed, 24 h complete, thereby precipitate AHK-Cu acetate crystals. Finally filter and use 5 °C pre-cooled ethanol solution to rinse the AHK-Cu acetate crystals, place in a 45 °C vacuum drying oven to dry to obtain AHK-Cu acetate crystal product; its X-ray diffraction pattern is the same as the crystal form obtained in Example 2, HPLC determination of purity is 99.3%, yield is 73.2%.

[0071] Example 6

[0072] Take the AHK-Cu acetate solid 30g obtained in Example 1, add purified water, adjust pH to 5 with acetic acid, constant volume to 100ml, pass through 0.22μm water filter membrane, get AHK-Cu acetate filtrate, concentration is 300g / L. At 10°C, 100RPM conditions, uniform speed slowly add 8 times ethanol, 24h add complete, thus precipitate AHK-Cu acetate crystal. Finally filter and use 5°C pre-cooled ethanol solution to rinse AHK-Cu acetate crystal, place in 45°C vacuum drying oven to dry to get AHK-Cu acetate crystal product; its X-ray diffraction pattern is same as the crystal form obtained in Example 2, HPLC determination purity is 99.2%, yield is 82.2%.

[0073] Example 7

[0074] Take the AHK-Cu acetate solid 30g obtained in Example 1, add purified water, adjust pH to 4 with acetic acid, constant volume to 100ml, pass through 0.22μm water filter membrane, get AHK-Cu acetate filtrate, concentration is 300g / L. At 10°C, 100RPM conditions, uniform speed slowly add 6 times ethanol, 24h add complete, thus precipitate AHK-Cu acetate crystal. Finally filter and use 5°C pre-cooled ethanol solution to rinse AHK-Cu acetate crystal, place in 45°C vacuum drying oven to dry to get AHK-Cu acetate crystal product; its X-ray diffraction pattern is same as the crystal form obtained in Example 2, HPLC determination purity is 99.1%, yield is 72.1%.

[0075] Example 8

[0076] Take the AHK-Cu acetate solid 30g obtained in Example 1, add purified water, adjust pH to 6 with acetic acid, constant volume to 100ml, pass through 0.22μm water filter membrane, get AHK-Cu acetate filtrate, concentration is 300g / L. At 10°C, 100RPM conditions, uniform speed slowly add 6 times ethanol, 24h add complete, thus precipitate AHK-Cu acetate crystal. Finally filter and use 5°C pre-cooled ethanol solution to rinse AHK-Cu acetate crystal, place in 45°C vacuum drying oven to dry to get AHK-Cu acetate crystal product; its X-ray diffraction pattern is same as the crystal form obtained in Example 2, HPLC determination purity is 99.1%, yield is 65.2%.

[0077] Example 9

[0078] Take the AHK-Cu acetate solid obtained in Example 1, 20 g, add purified water, adjust pH to 5 with acetic acid, constant volume to 100 ml, pass through 0.22 μm water filter membrane, obtain AHK-Cu acetate filtrate, concentration is 200 g / L. At 10°C, 100 RPM, slowly add 6 times of ethanol at a constant speed, 24 h to complete, thereby precipitating AHK-Cu acetate crystals. Finally filter and use 5°C pre-cooled ethanol solution to rinse the AHK-Cu acetate crystals, place in a 45°C vacuum drying oven to dry to obtain AHK-Cu acetate crystal product; its X-ray diffraction pattern is the same as the crystal form obtained in Example 2, HPLC determination of purity is 99.6%, yield is 64.4%.

[0079] Example 10

[0080] Take the AHK-Cu acetate solid obtained in Example 1, 40 g, add purified water, adjust pH to 5 with acetic acid, constant volume to 100 ml, pass through 0.22 μm water filter membrane, obtain AHK-Cu acetate filtrate, concentration is 400 g / L. At 10°C, 100 RPM, slowly add 6 times of ethanol at a constant speed, 24 h to complete, thereby precipitating AHK-Cu acetate crystals. Finally filter and use 5°C pre-cooled ethanol solution to rinse the AHK-Cu acetate crystals, place in a 45°C vacuum drying oven to dry to obtain AHK-Cu acetate crystal product; its X-ray diffraction pattern is the same as the crystal form obtained in Example 2, HPLC determination of purity is 98.1%, yield is 88.2%.

[0081] Hygroscopicity determination, detection method as follows:

[0082] Step 1: Place multiple glass culture dishes in a light-proof constant temperature and humidity incubator at 25±0.5°C, relative humidity 70±2% for 6 h, take out and weigh accurately, repeat the test until the weight is constant;

[0083] Step 2: Respectively take the AHK-Cu acetate solid obtained by freeze-drying in Example 1 and the AHK-Cu acetate crystal obtained in Example 2, place them in the glass culture dishes treated in Step 1 to constant weight, continue to place them in a light-proof constant temperature and humidity incubator at 25±0.5°C, relative humidity 70±2%, for 7 days, observe the appearance and mass change of the samples, and record the results in Table 2.

[0084] Table 2 Appearance and mass change of different AHK-Cu acetate

[0085]

[0086] Stability determination, detection method as follows:

[0087] Step 1: Put a plurality of glass culture dishes in a light-proof constant temperature and humidity incubator at 35±0.5℃ and 70±2% relative humidity for 6h, take out and weigh accurately, and repeat the test until the weight is constant;

[0088] Step 2: Put the AHK-Cu acetate salt solid obtained in Example 1 and the AHK-Cu acetate salt crystal obtained in Example 2 into the glass culture dishes treated by constant weight in Step 1, and continue to be placed in a light-proof constant temperature and humidity incubator at 35±0.5℃ and 70±2% relative humidity, observe the purity change of the sample every 7 days for 14 days, and record the results in Table 3.

[0089] Table 3: Purity change of different AHK-Cu acetate salts

[0090]

[0091] Comparative Example 1

[0092] Take 10g of the AHK-Cu acetate salt solid obtained in Example 1, add purified water, adjust the pH to 5 with acetic acid, make up to 100ml, pass through a 0.22μm water filter membrane, and obtain an AHK-Cu acetate salt filtrate with a concentration of 100g / L. Under the condition of 10℃ and 100RPM, 6 times of ethanol is slowly and uniformly added, and the addition is completed in 24h. The solution is clear, and no solid is precipitated.

[0093] Comparative Example 2

[0094] Take 30g of the AHK-Cu acetate salt solid obtained in Example 1, add purified water, adjust the pH of the solution to 8 with sodium hydroxide solution, make up to 100ml, pass through a 0.22μm water filter membrane, and obtain an AHK-Cu acetate salt filtrate with a concentration of 300g / L. Under the condition of 10℃ and 100RPM, 6 times of ethanol is slowly and uniformly added, and the addition is completed in 24h. Oil is precipitated, and no solid is precipitated.

[0095] Comparative Example 3

[0096] Take 50g of the AHK-Cu acetate salt solid obtained in Example 1, add purified water, adjust the pH to 7 with acetic acid, make up to 100ml, pass through a 0.22μm water filter membrane, and obtain an AHK-Cu acetate salt filtrate with a concentration of 500g / L. Under the condition of 10℃ and 100RPM, 8 times of ethanol is slowly and uniformly added, and the addition is completed in 24h. The precipitated solid is amorphous.

[0097] In summary, the present application provides a stable AHK-Cu acetate salt crystal form and a preparation method thereof. The AHK-Cu acetate salt crystal is obtained by low-temperature solvent crystallization, which has obvious technical advantages compared with the previous liquid or amorphous solid product: (1) according to the attached Figure 3Compared with the XRD crystal diffraction results in Table 1, it is confirmed that the AHK-Cu acetate prepared in the application is a crystal (2) According to Table 2 and Table 3, the stability of the AHK-Cu acetate crystal form provided in the application is better than that of the amorphous powder, which is beneficial to the long-term storage of the AHK-Cu acetate and the application in various products such as cosmetics; (3) The preparation method of the AHK-Cu acetate crystal form provided in the application is low-temperature solvent crystallization, only ethanol solution is used, no other organic solvents are used, the safety is high, the cost is low, and the scale-up is easy.

[0098] The above description of disclosed embodiments enables one skilled in the art to make or use the application. Numerous modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An AHK-Cu acetate crystalline compound, characterized in that, The X-ray powder diffraction pattern of the AHK-Cu acetate crystal compound shows diffraction peaks at 2θ ± 0.2°, where 2θ is 9.5106°, 11.0680°, 12.4906°, 13.7784°, 14.9704°, 15.3815°, 18.3890°, 20.8134°, 22.2639°, 24.4901°, and 25.8507°.

2. The AHK-Cu acetate crystal compound according to claim 1, characterized in that, The 2θ values ​​of the AHK-Cu acetate crystal form compounds are 9.5106°, 11.0680°, 12.4906°, 13.7784°, 14.9704°, 15.3815°, 18.3890°, 20.8134°, 22.2639°, 24.4901°, and 25.8507°.

3. A method for preparing the AHK-Cu acetate crystalline compound according to any one of claims 1 to 2, comprising the following steps: a) Dissolve solid AHK in water and add an equal amount of copper acetate. Stir to dissolve and form a clear liquid, then freeze dry to obtain solid AHK-Cu acetate. b) Dissolve the AHK-Cu acetate solid obtained in step a) in water and adjust the pH to 4-6 with acetic acid. Filter the solution through an aqueous filter membrane to obtain AHK-Cu acetate filtrate. Then precipitate AHK-Cu acetate crystals with ethanol. Finally, filter, wash with ethanol, and dry sequentially to obtain the AHK-Cu acetate crystalline compound.

4. The preparation method according to claim 3, characterized in that, The freeze-drying process described in step a) is specifically as follows: First, pre-freeze at -15℃ to -30℃ for 1 to 3 hours; then freeze-dry at a vacuum of 1 Pa to 15 Pa and a temperature of -30℃ to -50℃ for 10 to 15 hours.

5. The preparation method according to claim 3, characterized in that, The aqueous filter membrane mentioned in step b) is an aqueous filter membrane with a diameter of 0.20 μm to 0.25 μm.

6. The preparation method according to claim 3, characterized in that, The concentration of the AHK-Cu acetate filtrate in step b) is 200 g / L to 400 g / L.

7. The preparation method according to claim 3, characterized in that, The process of precipitating AHK-Cu acetate crystals with ethanol in step b) is specifically as follows: Ethanol was slowly and uniformly added to the AHK-Cu acetate filtrate at 5℃~20℃ and 50RPM~150RPM for 20h~30h, thereby precipitating AHK-Cu acetate crystals.

8. The preparation method according to claim 7, characterized in that, The amount of ethanol added is 4 to 8 times the volume of the AHK-Cu acetate filtrate.

9. The preparation method according to claim 1, characterized in that, The ethanol rinsing described in step b) is performed using pre-cooled ethanol at 4°C to 6°C.

10. The preparation method according to claim 1, characterized in that, The drying process described in step b) uses a vacuum drying oven at a temperature of 40℃~50℃.

Images