Tripeptide-29 having a hydrate crystal form and a preparation method thereof

Abstract

This invention discloses a tripeptide-29 with a hydrate crystal form and its preparation method, belonging to the field of peptide crystal form preparation technology. The tripeptide-29 with the hydrate crystal form is shown in the X-ray powder diffraction pattern determined by Cu Kα radiation, as shown in Figure 2. θ Characteristic peaks are observed at 13.273°, 14.105°, 16.6591°, 17.943°, 20.308°, 21.771°, 23.863°, and 26.704°. The tripeptide-29 with a hydrated crystal form described in this invention exhibits excellent high-temperature and moisture stability, and its preparation method is simple and efficient, without using the toxic solvent methanol during the crystallization process.

Description

Technical Field

[0001] This invention relates to the field of peptide crystal form preparation technology, and in particular to a tripeptide-29 having a hydrate crystal form and its preparation method. Background Technology

[0002] Prolonged exposure to ultraviolet radiation can lead to photoaging of the skin, manifesting as roughness, dryness, deepened wrinkles, pigmentation, loss of elasticity, and a sallow complexion, particularly due to the excessive breakdown of collagen and hyaluronic acid, the main components of the extracellular matrix (ECM). Tripeptide-29 (Gly-Pro-Hyp) is the most frequently occurring tripeptide in the collagen sequence; exogenous supplementation can promote the synthesis of collagen and hyaluronic acid, thus playing a role in combating photoaging (Yang et al. 2024).

[0003] The existing process for producing tripeptide-29 lyophilized powder or crystal form has problems such as easy moisture absorption and poor stability, which limits its application in cosmetics and oral beauty products.

[0004] Therefore, researching and developing a tripeptide-29 with a novel crystal form is of great significance for its development and application. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a tripeptide-29 having a hydrate crystal form and a method for preparing the same. The tripeptide-29 having a hydrate crystal form exhibits excellent high-temperature stability and moisture resistance.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a tripeptide-29 with a hydrate crystal form. Its X-ray powder diffraction pattern was determined using Cu Kα radiation, and 2 in the figure... θ Characteristic peaks are present at 13.273°, 14.105°, 16.6591°, 17.943°, 20.308°, 21.771°, 23.863° and 26.704°.

[0008] The tripeptide-29 with hydrate crystal form described in this invention has excellent stability, and its high temperature stability and moisture resistance are significantly higher than those of tripeptide-29 with non-crystalline form and tripeptide-29 lyophilized powder.

[0009] Preferably, the tripeptide-29 with the hydrate crystal form of the present invention has endothermic peaks at 155.0℃±2℃, 214.6℃±2℃ and 273.7℃±2℃ in the differential scanning calorimetry spectrum, and an exothermic peak at 168.3℃±2℃.

[0010] Preferably, the tripeptide-29 with the hydrate crystal form has a moisture content of 11.8%, and the moisture content remains unchanged even with extended drying time.

[0011] Since the moisture content remains unchanged, the tripeptide-29 crystal form contains water of crystallization and is a hydrate crystal form. This invention also provides a method for preparing the above-mentioned tripeptide-29 with a hydrate crystal form, comprising the following steps:

[0012] A mixture of tripeptide-29 aqueous solution and dimethylformamide was prepared, crystallized, cooled, and dried to obtain tripeptide-29 with the hydrate crystal form.

[0013] In the preparation of tripeptide-29 with hydrate crystal form, the concentration, amount, order of addition, and crystallization temperature of the raw materials interact to promote the formation of the hydrate crystal form.

[0014] Preferably, the concentration of the tripeptide-29 aqueous solution is 300-350 g / L; more preferably, it is 333 g / L.

[0015] Preferably, the volume ratio of the tripeptide-29 aqueous solution to dimethylformamide is 1:(1.5-3); more preferably, it is 1:2.

[0016] Preferably, the mixing involves adding dimethylformamide to an aqueous solution of tripeptide-29.

[0017] Preferably, the addition is done by dripping.

[0018] Preferably, the dripping rate is 80-100 mL / h; more preferably, it is 100 mL / h.

[0019] Preferably, the crystal growth time is 0.5-1.5 h; more preferably, it is 1 h.

[0020] Preferably, the conditions for the cooling crystallization in this invention are:

[0021] Cool to 5℃ at a rate of 8-12℃ / h and continue crystallizing for 1-3 h.

[0022] More preferably, the cooling rate is 9-11℃ / h; even more preferably, it is 10℃ / h.

[0023] More preferably, the continued crystallization time is 2 hours.

[0024] Preferably, the drying temperature is 45℃-55℃; more preferably, it is 50℃.

[0025] Compared with the prior art, the tripeptide-29 with a hydrate crystal form provided by the present invention has an X-ray powder diffraction pattern determined by Cu Kα radiation, as shown in Figure 2. θ Characteristic peaks are observed at 13.273°, 14.105°, 16.6591°, 17.943°, 20.308°, 21.771°, 23.863°, and 26.704°. The tripeptide-29 with a hydrated crystal form described in this invention exhibits excellent high-temperature and moisture stability, and its preparation method is simple and efficient, without using the toxic solvent methanol during the crystallization process. Attached Figure Description

[0026] Figure 1 Liquid phase spectrum of the crystalline powder (crystalline form III) prepared in Example 1;

[0027] Figure 2 X-ray powder diffraction pattern of the crystalline powder (crystalline type III) prepared in Example 1;

[0028] Figure 3 The DSC spectrum of the crystalline powder (crystalline form III) prepared in Example 1;

[0029] Figure 4 The liquid phase spectrum of the crystalline powder (crystalline form I) prepared in Comparative Example 1;

[0030] Figure 5 X-ray powder diffraction pattern of the crystalline powder (crystalline form I) prepared in Comparative Example 1;

[0031] Figure 6 The DSC spectrum of the crystalline powder (crystalline form I) prepared in Comparative Example 1;

[0032] Figure 7 The liquid phase spectrum of the crystalline powder (crystalline form II) prepared in Comparative Example 2;

[0033] Figure 8 X-ray powder diffraction pattern of the crystalline powder (crystalline form II) prepared in Comparative Example 2;

[0034] Figure 9 The DSC spectrum of the crystalline powder (crystalline form II) prepared in Comparative Example 2;

[0035] Figure 10 The liquid phase spectrum of tripeptide-29 lyophilized powder;

[0036] Figure 11 X-ray powder diffraction pattern of lyophilized tripeptide-29 powder;

[0037] Figure 12 Photographs of samples of tripeptide-29 lyophilized powder and crystalline powders prepared in Example 1, Comparative Example 1 and Comparative Example 2 after hygroscopicity tests.

[0038] Figure 13 Photographs of samples taken from the high-temperature stability test of the freeze-dried tripeptide-29 powder and the crystalline powders prepared in Example 1, Comparative Example 1 and Comparative Example 2, respectively.

[0039] Figure 14 The liquid phase spectrum of the crystalline powder (crystalline form III) prepared in Example 1 after a high-temperature stability test;

[0040] Figure 15 The liquid phase spectrum of the crystalline powder (crystalline form I) prepared in Comparative Example 1 after high-temperature stability test;

[0041] Figure 16 The liquid phase spectrum of the crystalline powder (crystalline form II) prepared in Comparative Example 2 after high-temperature stability test;

[0042] Figure 17 The liquid phase spectrum of tripeptide-29 lyophilized powder after high-temperature stability test. Detailed Implementation

[0043] To further illustrate the present invention, the tripeptide-29 with hydrate crystal form and its preparation method provided by the present invention are described in detail below with reference to embodiments.

[0044] The effective ingredient content calculated based on anhydrous matter is calculated as follows: (Weight of effective ingredient in sample calculated based on external standard method) / (Sample weight - Measured moisture weight) × 100%.

[0045] The reason why the combined tripeptide-29 and moisture content in the lyophilized peptide-29 powder and the crystalline powders prepared in Examples 1, 1, and 2 is greater than 100% is that the present invention uses the external standard method to determine the content, and the content of the content standard will have errors, as will the peak area ratio based on the test sample / content standard. The reason why the combined tripeptide-29 and moisture content is less than 100% is that degradation impurities were generated during the preparation of the crystalline form.

[0046] Example 1

[0047] At 25℃, 50 mL of a 333 g / L aqueous solution was prepared from the lyophilized tripeptide-29 powder synthesized in the liquid phase. The solution was stirred continuously at 240 rpm, and two volumes of dimethylformamide (DMF) were added dropwise at a rate of 100 mL / h. After the addition was complete, the temperature was maintained and stirred for 1 h to allow crystal growth. The temperature was then lowered to 5℃ at a rate of 10℃ per hour, and crystallization continued for 2 h. After filtration, the powder was washed with an appropriate amount of the corresponding antisolvent and then vacuum dried at 50℃ to constant weight to obtain tripeptide-29. Its crystal form was a hydrate, with a tripeptide-29 content of 89.2% and a water content of 11.8%. The water content remained unchanged even with prolonged drying, indicating the presence of water of crystallization and the presence of a hydrate. Because the content was determined using the external standard method, there may be errors in the content of the standard and in the peak area ratio of the sample to the standard. The tripeptide-29 content was calculated to be 101.1% based on anhydrous form, with a purity of 99.8%. Figure 1 The X-ray powder diffraction pattern of the obtained product was determined using CuKα rays, as shown below. Figure 2 As shown, the results indicate that the obtained product has crystal form III, which is crystalline in 2... θ Characteristic peaks were observed at 13.273°, 14.105°, 16.6591°, 17.943°, 20.308°, 21.771°, 23.863°, and 26.704°. Simultaneously, the thermal effect of the crystal powder during the melting process was measured using a differential calorimeter (DSC), and the results are as follows... Figure 3 As shown, Figure 3 The results show three endothermic peaks at 155.0℃, 214.6℃ and 273.7℃, and an exothermic peak at 168.3℃.

[0048] Since the molecular weight of the tripeptide-29 (Gly-Pro-Hyp) is 285.3 g / mol, dividing it by (100% - 11.8%) gives a total molecular weight of 323.5 g / mol, 323.5 - 285.3 = 38.2 g / mol. If we divide the molecular weight of tripeptide-29 by the tripeptide-29 content of 89.2%, the total molecular weight is 319.8 g / mol, 319.8 - 285.3 = 34.5 g / mol. Since a molecular weight of 34.5 - 38.2 g / mol is approximately equivalent to two water molecules, it is presumed that the tripeptide-29 in Example 1 is in a dihydrate form.

[0049] The X-ray powder diffraction results of the product (crystal form III) obtained in Example 1 are shown in Table 1.

[0050] Table 1. X-ray powder diffraction results of the product (crystal form III) obtained in Example 1.

[0051] Comparative Example 1

[0052] At 25℃, 130 mL of a 115 g / L tripeptide-29 solution was prepared using 130 mL of 80% methanol aqueous solution as the solvent. The solution was stirred continuously at 300 rpm. 1.2 times the volume of 1,4-dioxane was added dropwise at a rate of 50 mL / h. After the addition was complete, the temperature was maintained and stirred for 3 h to allow crystal growth. The temperature was then lowered to 5℃ at a rate of 5℃ per hour, and crystallization continued for 6 h. The solution was filtered, and the powder was washed with an appropriate amount of the corresponding anti-solvent. It was then vacuum dried at 50℃ until the moisture content met the requirements, yielding tripeptide-29 of crystal form I. The tripeptide-29 content was 90.0%, the moisture content was 3.8%, and the anhydrous content was 93.6%, with a purity of 99.5%. Figure 4 The X-ray powder diffraction pattern of the obtained product was determined using Cu-Kα rays, as shown below. Figure 5 As shown, it is in 2 θ Characteristic peaks are observed at 8.924°, 13.136°, 15.472°, 16.704°, 18.569°, 20.384°, 20.907°, 21.895°, and 28.679°. (DSC spectrum) Figure 6 The results show two endothermic peaks at 212.9℃ and 270.3℃, and an exothermic peak at 162.3℃.

[0053] Comparative Example 2

[0054] At 25℃, 130 mL of a 115 g / L tripeptide-29 solution was prepared using 72% methanol aqueous solution as the solvent. The solution was stirred continuously at 300 rpm, and an equal volume of isopropyl acetate was added dropwise at a rate of 40 mL / h. After the addition was complete, the temperature was maintained and stirred for 3 h to allow crystal growth. The temperature was then lowered to 5℃ at a rate of 5℃ per hour, and crystallization continued for 6 h. After filtration, the powder was washed with an appropriate amount of the corresponding anti-solvent and then vacuum dried at 50℃ until the moisture content met the requirements, yielding tripeptide-29 of crystal form II. The tripeptide-29 content was 78.9%, the moisture content was 3.2%, the anhydrous content was 81.5%, and the purity was 97.9%. Figure 7 The X-ray powder diffraction pattern of the obtained product was determined using Cu-Kα rays, as shown below. Figure 8 As shown, it is in 2 θ Characteristic peaks are observed at 13.253°, 14.082°, 16.626°, 17.944°, 18.218°, 20.2599°, 21.2297°, 21.7354°, 22.9827°, and 23.818°. (DSC spectrum) Figure 9 The results showed three endothermic peaks at 146.8℃, 213.3℃, ​​and 268.5℃.

[0055] Performance testing

[0056] 1. Hygroscopicity test

[0057] The hygroscopicity test method was as follows: Four new weighing bottles were selected, dried in an oven at 105℃ until constant weight, and numbered accordingly; crystalline powders prepared in Example 1, Comparative Example 1, and Comparative Example 2, as well as tripeptide-29 lyophilized powder (liquid phase spectrum of tripeptide-29 lyophilized powder is shown in...) were taken respectively. Figure 10 , X See X-ray powder diffraction pattern Figure 11 Approximately 1g of each sample was then placed into weighing bottles and placed in a constant temperature and humidity chamber (25℃±1℃, relative humidity 80%±2%). After 24 hours, the samples were weighed and photographed. Figure 12 From left to right, the crystal powders are tripeptide-29 lyophilized powder, the crystalline powder prepared in Example 1, the crystalline powder prepared in Comparative Example 2, and the crystalline powder prepared in Comparative Example 1. The calculation results are shown in Table 2.

[0058] Table 2. Hygroscopicity test results of tripeptide-29 lyophilized powder and the crystalline powders prepared in Example 1, Comparative Example 1, and Comparative Example 2, respectively.

[0059]

[0060] The results in Table 2 show that the crystal form III (hydrate crystal form) prepared in Example 1 is more stable in a high humidity environment, and its hygroscopicity is significantly lower than that of the crystal form I prepared in Comparative Example 1, the crystal form II prepared in Comparative Example 1 (both crystal forms I and II are amorphous), and the lyophilized powder.

[0061] 2. High-temperature stability test

[0062] The high-temperature stability test method was as follows: 4-5 g of the crystalline powders prepared in Example 1, Comparative Example 1, and Comparative Example 2, as well as the lyophilized tripeptide-29 powder, were respectively placed in clean glass vials and incubated at 60℃ for 33 days. The powder composition was measured at 5, 11, and 33 days. After the test, samples were taken and photographed. Figure 13 The test results are shown in Table 3.

[0063] Table 3. High-temperature stability test results of tripeptide-29 lyophilized powder and the crystalline powders prepared in Example 1, Comparative Example 1, and Comparative Example 2, respectively.

[0064]

[0065]

[0066] Table 3 shows that crystal form III prepared in Example 1 has better high-temperature stability, and its content and purity are better than those of crystal form I prepared in Comparative Example 1 and crystal form II prepared in Comparative Example 2, and significantly better than the lyophilized powder. Liquid chromatography chromatograms of the tripeptide-29 lyophilized powder and the crystal form powders prepared in Examples 1, Comparative Example 1, and Comparative Example 2 after high-temperature stability testing are shown in Table 3. Figure 14-17 As shown.

[0067] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A tripeptide-29 having a hydrated crystal form, characterized in that, Its X-ray powder diffraction pattern was determined using Cu Kα rays, and 2 in the figure... θ Characteristic peaks are present at 13.273°, 14.105°, 16.6591°, 17.943°, 20.308°, 21.771°, 23.863° and 26.704°.

2. The tripeptide-29 having a hydrated crystal form according to claim 1, characterized in that, The tripeptide-29 with the hydrate crystal form exhibits endothermic peaks at 155.0℃±2℃, 214.6℃±2℃ and 273.7℃±2℃ in the differential scanning calorimetry spectrum, and an exothermic peak at 168.3℃±2℃.

3. The tripeptide-29 having a hydrate crystal form according to claim 1 or 2, characterized in that, The tripeptide-29 with the hydrate crystal form has a moisture content of 11.8%, and the moisture content remains unchanged even after prolonged drying time.

4. The method for preparing tripeptide-29 having a hydrate crystal form according to any one of claims 1-3, characterized in that, Includes the following steps: A mixture of tripeptide-29 aqueous solution and dimethylformamide was prepared, crystallized by cooling, and dried to obtain tripeptide-29 with the hydrate crystal form.

5. The preparation method according to claim 4, characterized in that, The concentration of the tripeptide-29 aqueous solution is 300-350 g / L; The volume ratio of the tripeptide-29 aqueous solution to dimethylformamide is 1:(1.5-3).

6. The preparation method according to claim 4, characterized in that, The mixing process involves adding dimethylformamide to an aqueous solution of tripeptide-29.

7. The preparation method according to claim 6, characterized in that, The addition method is dropwise addition; the dropwise addition rate is 80-100 mL / h.

8. The preparation method according to claim 4, characterized in that, The crystal growth time is 0.5-1.5 h.

9. The preparation method according to claim 4, characterized in that, The conditions for cooling crystallization are as follows: Cool to 5℃ at a rate of 8-12℃ / h and continue crystallizing for 1-3 h.

10. The preparation method according to claim 4, characterized in that, The drying temperature is 45℃-55℃.

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