New crystal form of acetyl tetrapeptide-5 as well as preparation method and application thereof
By preparing a new crystalline form of acetyl tetrapeptide-5, the problems of easy moisture absorption and poor thermal stability were solved, enabling stable application in cosmetics and reducing production costs, making it suitable for industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENZHEN READLINE BIOTECH CO LTD
- Filing Date
- 2026-02-06
- Publication Date
- 2026-05-05
AI Technical Summary
Existing acetyl tetrapeptide-5 products suffer from hygroscopicity and poor thermal stability, resulting in stringent storage conditions and high production costs, which limits their widespread use in the cosmetics industry.
A novel crystal form of acetyl tetrapeptide-5 was prepared. The X-ray powder diffraction pattern showed a diffraction peak at 2θ±0.2°. A stable crystal structure was obtained by contact crystallization of acetyl tetrapeptide-5 aqueous solution with acetone, combined with stirring and temperature control.
The newly prepared crystal form exhibits superior moisture resistance and temperature stability compared to commercially available freeze-dried powders, making it suitable for industrial production, applicable to conventional equipment, and reducing production costs.
Smart Images
Figure CN121974973A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of technology, and in particular to a novel crystal form of acetyl tetrapeptide-5, its preparation method, and its applications. Background Technology
[0002] Acetyl tetrapeptide-5 (Ac-βAla-His-Ser-His-OH), also known as eye serine peptide or eye-beautifying peptide, works by inhibiting angiotensin-converting enzyme ACE-1, preventing angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) from being cleaved into angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), which constricts blood vessels. This results in vasodilation and the elimination of edema, eye bags, and dark circles.
[0003] Currently, acetyl tetrapeptide-5 is mainly sold in two formulations: water, butylene glycol, and acetyl tetrapeptide-5, or glycerol, water, and acetyl tetrapeptide-5, which are expensive. It is also sold as a lyophilized powder, but lyophilized powder has inherent problems such as hygroscopicity and poor thermal stability, requiring stringent storage conditions, generally under frozen or refrigerated conditions. Furthermore, the production volume of lyophilized powder is limited by the lyophilization equipment, resulting in small batches and high costs. All of these factors significantly restrict the widespread use of acetyl tetrapeptide-5 in the cosmetics industry. Summary of the Invention
[0004] In view of this, the technical problem to be solved by the present invention is to provide a new crystal form of acetyl tetrapeptide-5, its preparation method and application, wherein the prepared new crystal form of acetyl tetrapeptide-5 has high stability.
[0005] To solve the above technical problems, the present invention provides a new crystal form of acetyl tetrapeptide-5, which has diffraction peaks at 2θ±0.2° in its X-ray powder diffraction pattern, wherein 2θ includes: 8.292°, 16.541°, 18.522°, 21.825° and 24.701°.
[0006] Furthermore, the 2θ includes: 8.292°, 16.541°, 17.96°, 18.522°, 19.981°, 21.53°, 21.825°, 24.701°, and 25.89°.
[0007] Specifically, the X-ray powder diffraction pattern of the new crystal form of acetyl tetrapeptide-5 is as follows: Figure 2 As shown in Table 1, the specific positions of the diffraction peaks are as follows.
[0008] In this invention, the 2θ values of X-ray powder diffraction patterns may vary slightly between machines or samples, with differences of approximately 0.2 units or 0.1 units. Therefore, the values cited should not be interpreted as absolute values. Similarly, it should be understood that peak heights may also vary by approximately 5 units, 4 units, 3 units, 2 units, or 1 unit. Therefore, the XRPD trace intensities included in this invention are illustrative and not intended for absolute comparison.
[0009] This invention provides a method for preparing the novel crystalline form of the above-mentioned acetyl tetrapeptide-5, comprising the following steps:
[0010] An aqueous solution of acetyl tetrapeptide-5 was contacted with acetone to induce crystallization, yielding a new crystalline form of acetyl tetrapeptide-5.
[0011] The concentration of the aqueous solution of acetyl tetrapeptide-5 is 100~1000g / L, more preferably 300~600g / L, and for example, it can be 300, 400, 500, or 600g / L.
[0012] The volume ratio of the aqueous solution of acetyl tetrapeptide-5 to acetone is 1:(1~5), more preferably 1:(2~4), and for example, it can be 1:2, 1:3, or 1:4.
[0013] Preferably, acetone is added to an aqueous solution of acetyl tetrapeptide-5. The method of addition is not particularly limited in this invention, and includes, but is not limited to, dropwise addition, flow addition, etc. Flow addition is preferred. The flow addition rate is preferably 40-60 mL / h, for example, 40, 50, or 60 mL / h.
[0014] Preferably, the addition is carried out under conditions of system stirring. The stirring speed is preferably 100~500 rpm, more preferably 200~400 rpm, and for example, it can be 200, 300, or 400 rpm.
[0015] The preferred temperature for contacting the aqueous solution of acetyl tetrapeptide-5 with acetone is 15~45℃, more preferably 20~30℃, and for example, it can be 20, 25, or 30℃.
[0016] The crystallization temperature is preferably 5~15℃, more preferably 6~10℃, and for example, it can be 6, 7, 8, 9, or 10℃.
[0017] The crystallization time is preferably 8~24h, more preferably 10~20h, and for example, it can be 10, 12, 15, 20h, or 24h.
[0018] Preferably, the crystallization is specifically performed by cooling to 5-15°C at a rate of 5-10°C / h. More preferably, the cooling rate is 6-8°C / h; for example, it can be 6, 7, or 8°C / h.
[0019] After crystallization, the obtained crystals are filtered, washed, and dried.
[0020] In some preferred embodiments, the preparation method includes:
[0021] Prepare 25 mL of 400 g / L acetyl tetrapeptide-5 aqueous solution, stir continuously at 25 °C and 300 rpm, add 3 times the volume of acetone at a rate of 50 mL / h, and after the addition is completed, cool down to 8 °C at a rate of 7 °C per hour for 12 h to crystallize. After filtering and washing the powder with an appropriate amount of acetone, dry under vacuum at 50 °C until the weight is constant to obtain a new crystal form of acetyl tetrapeptide-5.
[0022] The present invention also provides the application of the novel crystalline form of acetyl tetrapeptide-5 or the novel crystalline form of acetyl tetrapeptide-5 prepared by the above preparation method in the preparation of cosmetics.
[0023] The cosmetics mentioned can be any type of cosmetics known to those skilled in the art, including but not limited to serums, face creams, lotions, eye creams, cleansers, face masks, sunscreens, foundations, cushion compacts, etc.
[0024] Based on this, the present invention also provides a cosmetic composition comprising the above-described novel crystalline form of acetyl tetrapeptide-5 or the novel crystalline form of acetyl tetrapeptide-5 prepared by the above-described preparation method.
[0025] Depending on the cosmetic dosage form, the cosmetic composition may also include suitable excipients, which may be added as needed by those skilled in the art, without any special limitations.
[0026] The cosmetic may also include other functional ingredients, including but not limited to whitening, moisturizing, and antioxidant ingredients. Those skilled in the art can add them as needed, and the present invention does not impose any special limitations on this.
[0027] Compared with existing technologies, this invention provides a novel crystal form of acetyl tetrapeptide-5, with diffraction peaks at 2θ ± 0.2° in its X-ray powder diffraction pattern, where 2θ includes 8.292°, 16.541°, 18.522°, 21.825°, and 24.701°. This invention solves the problems of hygroscopicity and poor thermal stability of existing lyophilized acetyl tetrapeptide-5 powders. The prepared crystals exhibit significantly better moisture resistance and temperature stability than commercially available lyophilized powders, and have broad application prospects.
[0028] In addition, this invention further develops a crystallization process for the stable crystal form of acetyl tetrapeptide-5. This process can complete the solid-liquid separation and drying process in a conventional enamel-lined reactor and vacuum dryer, without the need for a freeze dryer with small batch size and high energy consumption, making it more suitable for industrial production. Attached Figure Description
[0029] Figure 1 High-performance liquid chromatography (HPLC) image of the novel crystalline form of acetyl tetrapeptide-5 prepared in Example 1;
[0030] Figure 2 X-ray powder diffraction pattern of the new crystalline form of acetyl tetrapeptide-5 prepared in Example 1;
[0031] Figure 3 X-ray powder diffraction pattern of lyophilized acetyl tetrapeptide-5 powder. Detailed Implementation
[0032] To further illustrate the present invention, a detailed description is provided below with reference to embodiments. However, it should be understood that these descriptions are merely for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims.
[0033] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.
[0034] Example 1
[0035] Prepare 25 mL of a 400 g / L acetyl tetrapeptide-5 aqueous solution. Stir continuously at 25°C and 300 rpm. Add 3 times the volume of acetone at a rate of 50 mL / h. After the addition is complete, cool the solution at a rate of 7°C per hour until crystallization reaches 8°C for 12 h. After washing the powder with a suitable amount of acetone, dry under vacuum at 50°C until the weight is constant. The yield is 86.8%. High-performance liquid chromatography (HPLC) analysis shows a single peak, no obvious impurities, and a purity of 99.6%. Figure 1 ).
[0036] The crystal powder was sent for X-ray powder diffraction analysis. The instrument was an Ultima IV (equipped with a Cu K-β filter), with an X-ray tube voltage of 40 kV, an X-ray tube current of 40 mA, a scanning range of 3–60° (2θ), and a step size of 0.0200°. See Table 1 and... Figure 2 As shown, the obtained crystal form has characteristic peaks at 2θ = 8.292°, 16.541°, 18.522°, 21.825° and 24.701°, which is a new crystal form of acetyl tetrapeptide-5.
[0037] Table 1
[0038] Comparative Example 1
[0039] Measure 5 ml of each of the solvents listed in Table 2 into a 20 ml glass bottle, and gradually add acetyl tetrapeptide-5 lyophilized powder while stirring. As shown in Table 1, acetyl tetrapeptide-5 lyophilized powder is only well soluble in water and DMSO, and is insoluble in other solvents.
[0040] Table 2
[0041] Comparative Example 2
[0042] Using water as a good solvent, 2 mL of a 175 g / L aqueous solution of acetyl tetrapeptide-5 lyophilized powder was dispensed into 20 mL glass bottles and stirred continuously at 240 rpm at 25°C. Solvents listed in Table 3 were selected as unsuitable solvents, and 2.5 times their volume were added to the acetyl tetrapeptide-5 aqueous solution respectively, and crystallization was observed. After 9 days of observation, only acetone, as an unsuitable solvent, precipitated crystal powder; no solid precipitated with the other solvents. The crystal powder was collected by vacuum filtration, dried under vacuum at 50°C until the weight remained constant, and weighed to 0.085 g.
[0043] Table 3 Poor solvents Crystallization status methanol Clarification, Discard ethanol Clarification, Discard acetone 0.085g Isopropanol Clarification, Discard Tetrahydrofuran Turbid oil, separated into layers upon standing, discard. Acetonitrile Turbid oil, separated into layers upon standing, discard. dimethylamide Clarification, Discard 1,4-Dioxane Turbid oil, separated into layers upon standing, discard.
[0044] Comparative Example 3
[0045] Using DMSO as a good solvent, 150 g / L DMSO solution was prepared by weighing acetyl tetrapeptide-5 lyophilized powder and dispensed into 2 mL aliquots in 20 mL glass bottles. The solution was stirred continuously at 240 rpm and 25 °C. Solvents listed in Table 4 were selected as poor solvents, and 1.5 times their volume were added to the DMSO solution of acetyl tetrapeptide-5, and crystallization was observed. After one day, all samples were either gel-like or the solvents were immiscible, with no solid precipitated.
[0046] Table 4
[0047] Application Example 1: Influencing Factors in High Humidity Test
[0048] Six new 20 mL glass bottles were selected, dried in an oven at 105℃ until constant weight, and numbered. The novel acetyl tetrapeptide-5 crystalline form prepared in Example 1 and the commercially available acetyl tetrapeptide-5 lyophilized powder were dried to constant weight, and 1 g of powder was placed in each glass bottle, which were then placed open in a constant temperature and humidity chamber (25℃, 90% ± 5% humidity). The samples were weighed on days 5 and 10, and the moisture gain was calculated. The results are shown in Table 5. The novel acetyl tetrapeptide-5 crystalline form prepared in this invention is more stable in high humidity environments than the lyophilized powder.
[0049] Table 5
[0050] Application Example 2: Influencing Factors in High Temperature Tests
[0051] The novel crystalline form of acetyl tetrapeptide-5 prepared in Example 1 and commercially available lyophilized acetyl tetrapeptide-5 powder were each placed in glass vials (1g each), sealed, and incubated at 60°C. The purity of the samples was determined by high-performance liquid chromatography (HPLC) on days 5, 10, and 30. The results are shown in Table 6. The novel crystalline form of acetyl tetrapeptide-5 prepared in this invention exhibits better temperature stability compared to the lyophilized powder.
[0052] Table 6
[0053] As can be seen from the results in Tables 5 and 6, the new crystalline form of acetyl tetrapeptide-5 prepared in this invention exhibits significantly better moisture resistance and temperature stability than commercially available lyophilized powder.
[0054] 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 novel crystalline form of acetyl tetrapeptide-5, characterized in that, The X-ray powder diffraction pattern shows diffraction peaks at 2θ ± 0.2°, where 2θ includes 8.292°, 16.541°, 18.522°, 21.825°, and 24.701°.
2. The novel crystalline form of acetyl tetrapeptide-5 according to claim 1, characterized in that, The 2θ includes: 8.292°, 16.541°, 17.96°, 18.522°, 19.981°, 21.53°, 21.825°, 24.701° and 25.89°.
3. The novel crystalline form of acetyl tetrapeptide-5 according to claim 1, characterized in that, The X-ray powder diffraction pattern is shown in Figure 2.
4. A method for preparing a novel crystalline form of acetyl tetrapeptide-5, comprising the following steps: An aqueous solution of acetyl tetrapeptide-5 was contacted with acetone to induce crystallization, yielding a new crystalline form of acetyl tetrapeptide-5.
5. The preparation method according to claim 4, characterized in that, The concentration of the aqueous solution of acetyl tetrapeptide-5 is 100~1000g / L.
6. The preparation method according to claim 4, characterized in that, The volume ratio of the aqueous solution of acetyl tetrapeptide-5 to acetone is 1:(1~5).
7. The preparation method according to claim 4, characterized in that, The aqueous solution of acetyl tetrapeptide-5 is contacted with acetone by dropwise addition or continuous addition.
8. The preparation method according to claim 4, characterized in that, The aqueous solution of acetyl tetrapeptide-5 is in contact with acetone at a temperature of 15~45℃; The crystallization temperature is 5~15℃; The crystallization time is 8~24h.
9. The preparation method according to claim 4, characterized in that, The crystallization process specifically involves cooling the temperature to 5-15℃ at a rate of 5-10℃ / h to induce crystallization.
10. The use of the novel crystalline form of acetyl tetrapeptide-5 according to any one of claims 1 to 3 or the novel crystalline form of acetyl tetrapeptide-5 prepared by the preparation method according to any one of claims 4 to 9 in the preparation of cosmetics.