Oligopeptide-1 hydrochloride crystal form compound and preparation method thereof

Abstract

The invention relates to the field of crystal forms, in particular to an oligopeptide-1 hydrochloride crystal form compound and a preparation method thereof. The oligopeptide-1 hydrochloride crystal is obtained in a low-temperature crystallization mode, and compared with a conventional liquid or amorphous solid product, the oligopeptide-1 hydrochloride crystal has obvious technical advantages. According to an XRD crystal diffraction result, the oligopeptide-1 hydrochloride prepared by the invention is confirmed to be a crystal; the stability of the crystal form of the oligopeptide-1 hydrochloride is superior to that of amorphous powder, long-term storage of the oligopeptide-1 hydrochloride is facilitated, and application of the oligopeptide-1 hydrochloride in various products such as cosmetics is facilitated; the preparation method of the oligopeptide-1 hydrochloride crystal form is low-temperature solvent devitrification, only an ethanol solution is used, no other organic solvent is used, the cost is low, and amplification is easy.

Description

Technical Field

[0001] The present invention relates to the field of crystal forms, and in particular to an oligopeptide-1 hydrochloride crystal form compound and a preparation method thereof. Background Art

[0002] Oligopeptide-1 is a tripeptide composed of glycine, histidine and lysine, and its sequence is GHK. In 1962, Dr. Stanley Cohen and Rita Levi-Montalcini first isolated it from the mandible of mice, and later it was found to be widely present in human blood, saliva and urine, and its content in the human body gradually decreases with age. Oligopeptide-1 plays an important role in wound healing, immune function and collagen synthesis of the skin. Studies have shown that oligopeptide-1, as a matrikine messenger peptide, acts on the dermis and can promote the synthesis of type I and III collagen, elastin and structural glycoproteins, making the skin firmer and reducing wrinkles. At the same time, oligopeptide-1 can promote the proliferation of keratinocytes, inhibit the secretion of pro-inflammatory factors, and has a soothing and repairing effect on the skin. At present, oligopeptide-1 is widely used in the cosmetics and skin care industries, playing a role in delaying skin aging, whitening and lightening spots.

[0003] Oligopeptide-1 is generally synthesized by chemical or biological methods. At present, oligopeptide-1 related products mostly exist in the form of liquid or amorphous solid, and there is no report on its specific crystal form. Different forms of oligopeptide-1 have significant differences in physical and chemical properties such as appearance, solubility, and stability, which will have different effects on the storage, application, and efficacy of oligopeptide-1.

[0004] In summary, the existing methods for obtaining oligopeptide-1 solids mainly include freeze-drying, rotary evaporation drying or precipitation using poor solvents. However, the solid powder (amorphous) obtained by rotary evaporation drying or freeze-drying process has poor storage stability and is easy to absorb moisture, which puts higher requirements on its transportation and preservation. Compared with amorphous solids, crystals have compact and orderly molecular arrangement, which makes the crystals have better resistance to the external environment and better stability. In order to provide the market with stable, easy-to-transport and preserve oligopeptide-1 solids, it is urgent to develop an efficient and large-scale industrial oligopeptide-1 purification method, and provide oligopeptide-1 crystal solids that are more stable than amorphous solids for use in various products such as cosmetics to meet the needs of the public. Summary of the invention

[0005] In view of this, the present invention provides a stable oligopeptide-1 hydrochloride crystal form and a preparation method thereof. The oligopeptide-1 hydrochloride crystal is obtained by low-temperature crystallization. Compared with amorphous solids, the oligopeptide-1 hydrochloride crystal has better stability, uses less organic solvent, has good cost-effectiveness, and can be produced on a large scale.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] In the first aspect, the present invention provides an oligopeptide-1 hydrochloride crystalline compound, which presents at least 3 of the following characteristic peaks in any combination in the X-ray powder diffraction pattern of Cu-Kα radiation, and the 2θ values ​​of the characteristic peaks include but are not limited to: 6.0728° (±0.2°), 6.2151° (±0.2°), 8.7892° (±0.2°), 11.6106° (±0.2°), 13.1076° (±0.2°), 14.6526° (±0.2°), 17.6190° (±0.2°), 18.8006° (±0.2°), 19.5417° (±0.2°), 20. 4580°(±0.2°), 20.9862°(±0.2°), 22.2112°(±0.2°), 23.7454°(±0.2°), 25.2775°(±0.2°), 26.3296°(±0.2°), 27.1420°(±0.2°), 28.1118°(±0.2°), 28.8980°(±0.2°), 30.2670°(±0.2°), 33.0000°(±0.2°), 35.0295°(±0.2°), 36.4723°(±0.2°) and / or 37.9459°(±0.2°).

[0008] In some specific embodiments of the present invention, the X-ray powder diffraction pattern of Cu-Kα radiation also includes at least 3 of the following characteristic peaks in any combination, and the 2θ values ​​of the characteristic peaks are not limited to: 6.0728° (± 0.2°), 6.2151° (± 0.2°), 8.7892° (± 0.2°), 11.6106° (± 0.2°), 13.1076° (± 0.2°), 14.6526° (± 0.2°), 17.6190° (± 0.2°), 18.8006° (± 0.2°), 19.5417° (± 0.2°), 20.458 0°(±0.2°), 20.9862°(±0.2°), 22.2112°(±0.2°), 23.7454°(±0.2°), 25.2775°(±0.2°), 26.3296°(±0.2°), 27.1420°(±0.2°), 28.1118°(±0.2°), 28.8980°(±0.2°), 30.2670°(±0.2°), 33.0000°(±0.2°), 35.0295°(±0.2°), 36.4723°(±0.2°) and / or 37.9459°(±0.2°).

[0009] In some specific embodiments of the present invention, in the X-ray powder diffraction pattern of Cu-Kα radiation, the parameters of the characteristic peaks are as follows:

[0010]

[0011]

[0012] In a second aspect, the present invention also provides a method for preparing the oligopeptide-1 hydrochloride crystalline compound, comprising the following steps:

[0013] Step 1: preparing an aqueous solution of oligopeptide-1 hydrochloride;

[0014] Step 2: spray-drying the oligopeptide-1 hydrochloride aqueous solution obtained in step 1 to obtain oligopeptide-1 hydrochloride solid;

[0015] Step 3: dissolving the oligopeptide-1 hydrochloride solid obtained in step 2 in water, adjusting the pH value, filtering, and collecting the filtrate to obtain the oligopeptide-1 hydrochloride filtrate;

[0016] Step 4: Under stirring conditions, the oligopeptide-1 hydrochloride filtrate obtained in step 1 is mixed with ethanol to obtain an oligopeptide-1 hydrochloride mixed solution;

[0017] Step 5: Take the oligopeptide-1 hydrochloride mixed solution obtained in step 4, cool it while stirring, precipitate oligopeptide-1 hydrochloride crystals, and grow the crystals;

[0018] Step 6: Filter and wash the oligopeptide-1 hydrochloride crystals, and vacuum dry to obtain.

[0019] In some specific embodiments of the present invention, the inlet air temperature and outlet air temperature of the spray drying in step 2 are 130° C. and 80° C. respectively, and the flow rate is 20 ml / min.

[0020] In some specific embodiments of the present invention, the pH value in step 3 is 3 to 7, preferably 3 to 5;

[0021] The concentration of the oligopeptide-1 hydrochloride filtrate is 200-300 g / L, preferably 200-250 g / L.

[0022] In some specific embodiments of the present invention, the stirring speed in step 4 is 120RPM, and the volume ratio of ethanol to the oligopeptide-1 hydrochloride filtrate is (2-4):1, preferably (2-3):1.

[0023] In some specific embodiments of the present invention, the stirring speed in step 5 is 120RPM, the initial temperature is 25°C, the temperature is cooled to 5°C, the cooling rate is 5°C / h, and the crystal growing time is 10 to 20 hours, preferably 10 to 15 hours.

[0024] In some specific embodiments of the present invention, the filtration in step 6 is carried out using 0.22 μm filter paper, the washing is carried out using an ethanol aqueous solution, the concentration of ethanol in the ethanol aqueous solution is 70%, the temperature of the vacuum drying is 45° C., the vacuum degree is -0.1 MPa, and the time is 8 to 10 hours.

[0025] In a third aspect, the present invention also provides an oligopeptide-1 hydrochloride crystalline compound obtained by the preparation method.

[0026] In a fourth aspect, the present invention further provides the use of the oligopeptide-1 hydrochloride crystalline compound or the oligopeptide-1 hydrochloride crystalline compound prepared by the preparation method in any of the following items;

[0027] (I), preparing products that promote wound healing;

[0028] (II) Preparation of products for enhancing or improving immunity;

[0029] (III) preparing products that promote the synthesis of type I collagen, type III collagen, elastin and / or structural glycoprotein;

[0030] (IV) preparing products that promote the proliferation of keratinocytes, inhibit the secretion of pro-inflammatory factors and / or soothe and repair the skin;

[0031] (V) preparing cosmetics and / or care products for delaying skin aging and / or whitening and removing spots.

[0032] The present invention provides a stable oligopeptide-1 hydrochloride crystal form and a preparation method thereof, wherein oligopeptide-1 hydrochloride crystals are obtained by low-temperature crystallization, which has obvious technical advantages over previous liquid or amorphous solid products. (1) According to Figure 2 The XRD crystal diffraction results in Table 1 confirm that the oligopeptide-1 hydrochloride prepared by the present invention is crystalline; (2) According to Tables 2 and 3, the stability of the oligopeptide-1 hydrochloride crystal form of the present invention is better than that of the amorphous powder, which is beneficial to the long-term storage of oligopeptide-1 hydrochloride and its application in various products such as cosmetics; (3) The preparation method of the oligopeptide-1 hydrochloride crystal form is low-temperature solvent crystallization, which only uses ethanol solution without other organic solvents, is low-cost and easy to scale up. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0034] Figure 1-2 HPLC chart of oligopeptide-1 prepared in Example 1-2 is shown;

[0035] Figure 3 Shown is the X-ray diffraction pattern of oligopeptide-1 amorphous solid;

[0036] Figure 4 Shown is the X-ray diffraction pattern of oligopeptide-1 hydrochloride crystals;

[0037] Figure 5-13 HPLC chart of oligopeptide-1 prepared in Example 3-11 is shown;

[0038] Fig.14 The HPLC chart of oligopeptide-1 prepared in Comparative Example 1 is shown. DETAILED DESCRIPTION

[0039] The present invention discloses a crystalline form of oligopeptide-1 hydrochloride and a method for preparing the same. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0040] The present invention is based on the oligopeptide-1 hydrochloride solution obtained by the preparation method of oligopeptide-1 hydrochloride in patent CN113801190B, and the oligopeptide-1 hydrochloride solid is obtained by spray drying the solution, which is identified as amorphous. The spray drying process of the oligopeptide-1 hydrochloride solid is: the inlet air temperature and the outlet air temperature are 130°C and 80°C respectively, and the flow rate is 20ml / min.

[0041] Oligopeptide-1 hydrochloride crystals are obtained by low-temperature solvent crystallization, which have better stability than amorphous solids, use less organic solvents, are cost-effective, and can be produced on a large scale.

[0042] The oligopeptide-1 hydrochloride amorphous solid used in the present invention is obtained by spray drying an oligopeptide-1 hydrochloride solution.

[0043] The present invention provides a stable oligopeptide-1 hydrochloride crystal form and a preparation method thereof, which can convert oligopeptide-1 hydrochloride amorphous form into crystals by low-temperature solvent crystallization. The present invention requires the following steps:

[0044] Step 1: prepare an oligopeptide-1 hydrochloride solution, dissolve the oligopeptide-1 hydrochloride amorphous solid in purified water, and after adjusting the pH value of the solution with dilute hydrochloric acid, filter through a 0.22 μm water filter membrane, and collect the oligopeptide-1 hydrochloride filtrate;

[0045] The concentration of the oligopeptide-1 hydrochloride filtrate is 200-300 g / L, preferably 200-250 g / L, and the pH is 3-7, preferably 3-5.

[0046] Step 2: adding ethanol to the oligopeptide-1 hydrochloride filtrate obtained in step 1 under stirring to obtain an oligopeptide-1 hydrochloride mixed solution;

[0047] The stirring speed is 120RPM, and the volume of ethanol is 2-4 times the volume of the oligopeptide-1 hydrochloride filtrate, preferably 2-3 times.

[0048] Step 3: slowly cooling the oligopeptide-1 hydrochloride mixed solution obtained in step 2 while stirring, thereby precipitating oligopeptide-1 hydrochloride crystals, and growing the crystals after cooling;

[0049] The stirring speed is 120RPM, the initial temperature is 25°C, the temperature is lowered to 5°C, the cooling rate is 5°C / h, and the crystal growing time is 10-20h, preferably 10-15h.

[0050] Step 4: Filter and wash the oligopeptide-1 hydrochloride crystals with ethanol aqueous solution, and dry them in a vacuum drying oven to obtain the oligopeptide-1 hydrochloride crystal product.

[0051] The solution was filtered using 0.22 μm filter paper, the concentration of the ethanol aqueous solution used for washing was 70%, and the temperature of the vacuum drying oven was 45°C.

[0052] In the oligopeptide-1 hydrochloride crystalline compound and the preparation method thereof provided by the present invention, the raw materials and reagents used can be purchased from the market.

[0053] The present invention will be further described below in conjunction with embodiments:

[0054] Example 1 Preparation of oligopeptide-1 solid

[0055] The oligopeptide-1 hydrochloride aqueous solution was obtained by referring to the preparation method of oligopeptide-1 hydrochloride in patent CN113801190B. The spray drying process of oligopeptide-1 hydrochloride solid is as follows: the inlet and outlet air temperatures are 130°C and 80°C, respectively, and the flow rate is 20ml / min. The prepared oligopeptide-1 solid was determined to have a purity of 96.8% by HPLC (see Figure 1 ), with a yield of 80%.

[0056] Example 2

[0057] Take 25g of the oligopeptide-1 solid obtained in Example 1, add purified water, adjust the pH to 5 with dilute hydrochloric acid, make the volume to 100ml, and filter through a 0.22μm water filter membrane to obtain the oligopeptide-1 hydrochloride filtrate with a concentration of 250g / L. Slowly add 3 times of ethanol under stirring conditions at 120RPM, and then keep the stirring state at an initial temperature of 25°C, reduce the temperature to 5°C at a rate of 5°C / h, and grow the crystals for 15h. Finally, filter and wash the oligopeptide-1 hydrochloride crystals with 70% ethanol aqueous solution, place in a 45°C vacuum drying oven and dry to obtain the oligopeptide-1 hydrochloride crystal product, the purity of which is 98.5% as determined by HPLC (see Figure 2 ), with a yield of 90.4%.

[0058] The oligopeptide-1 hydrochloride solid obtained by spray drying in Example 1 and the oligopeptide-1 hydrochloride crystal product obtained in this example were tested by powder X-ray diffraction (PXRD). The testing method is as follows:

[0059] 1. Instrument parameters:

[0060] PANalytical Empyrean X-ray powder diffractometer (PW3040 / 60, PANalytical Analytical Instruments Ltd., the Netherlands), Cu-Kα radiation, wavelength Equipped with Bragg-Brentano High Definition. Incident light path: divergence slit 1 / 8°, Soller slit 0.04rad, light shielding frame Mask 10mm, anti-scatter slit 1 / 2°; diffraction light path: anti-scatter slit P7.5; X-ray sample stage: rotation mode; scanning detector: PIXcel1D-Medipix3; X-ray tube voltage 45kV, X-ray tube current 40mA, scanning range 2-40° (2θ), step length 0.026°, step time: 36.465s. Data acquisition software DataCollector, data viewing software HighScore Plus.

[0061] 2. Sample Preparation

[0062] The samples were not pretreated. A clean stainless steel spoon was used to place the powder sample on a round silicon wafer sample plate without background. The sample was gently collected with a spatula while trying to keep the sample in the middle of the sample plate. Then, it was gently flattened with weighing paper.

[0063] Example 1 The X-ray diffraction pattern of the oligopeptide-1 hydrochloride solid obtained by spray drying is shown in Figure 3 , which is an amorphous solid. The X-ray diffraction pattern of the oligopeptide-1 hydrochloride crystals obtained in this example is shown in Figure 4 , there are characteristic peaks of the crystal, and the peak positions and peak intensities are shown in Table 1.

[0064] Table 1: Characteristic peaks of X-ray diffraction of oligopeptide-1 hydrochloride crystals

[0065]

[0066]

[0067] Example 3

[0068] Take 20g of the oligopeptide-1 solid obtained in Example 1, add purified water, adjust the pH to 5 with dilute hydrochloric acid, make the volume to 100ml, and filter through a 0.22μm water filter membrane to obtain the oligopeptide-1 hydrochloride filtrate with a concentration of 200g / L. Slowly add 2 times ethanol under stirring conditions at 120RPM, and then keep the stirring state at an initial temperature of 25°C, reduce the temperature to 5°C at a rate of 5°C / h, and grow the crystals for 15h. Finally, filter and wash the oligopeptide-1 hydrochloride crystals with 70% ethanol aqueous solution, place in a 45°C vacuum drying oven and dry to obtain the oligopeptide-1 hydrochloride crystal product, whose X-ray diffraction pattern is the same as the crystal form obtained in Example 2, and the purity determined by HPLC is 98.7% (see Figure 5 ), with a yield of 80.1%.

[0069] Example 4

[0070] Take 20g of the oligopeptide-1 solid obtained in Example 1, add purified water, adjust the pH to 5 with dilute hydrochloric acid, make the volume to 100ml, and filter through a 0.22μm water filter membrane to obtain the oligopeptide-1 hydrochloride filtrate with a concentration of 200g / L. Slowly add 3 times of ethanol under stirring conditions at 120RPM, and then keep the stirring state at an initial temperature of 25°C, reduce the temperature to 5°C at a rate of 5°C / h, and grow the crystals for 15h. Finally, filter and wash the oligopeptide-1 hydrochloride crystals with 70% ethanol aqueous solution, place in a 45°C vacuum drying oven and dry to obtain the oligopeptide-1 hydrochloride crystal product, whose X-ray diffraction pattern is the same as the crystal form obtained in Example 2, and the purity determined by HPLC is 98.8% (see Figure 6 ), with a yield of 85.4%.

[0071] Example 5

[0072] Take 30g of the oligopeptide-1 solid obtained in Example 1, add purified water, adjust the pH to 5 with dilute hydrochloric acid, make the volume to 100ml, and filter through a 0.22μm water filter membrane to obtain the oligopeptide-1 hydrochloride filtrate with a concentration of 300g / L. Slowly add 3 times ethanol under stirring conditions at 120RPM, and then keep the stirring state at an initial temperature of 25°C, reduce the temperature to 5°C at a rate of 5°C / h, and grow the crystals for 15h. Finally, filter and wash the oligopeptide-1 hydrochloride crystals with 70% ethanol aqueous solution, place in a 45°C vacuum drying oven and dry to obtain the oligopeptide-1 hydrochloride crystal product, whose X-ray diffraction pattern is the same as the crystal form obtained in Example 2, and the purity determined by HPLC is 97.3% (see Figure 7), with a yield of 93.2%.

[0073] Example 6

[0074] Take 25g of the oligopeptide-1 solid obtained in Example 1, add purified water, adjust the pH to 3 with dilute hydrochloric acid, make the volume to 100ml, and filter through a 0.22μm water filter membrane to obtain the oligopeptide-1 hydrochloride filtrate with a concentration of 250g / L. Slowly add 3 times of ethanol under stirring conditions at 120RPM, and then keep the stirring state at an initial temperature of 25°C, reduce the temperature to 5°C at a rate of 5°C / h, and grow the crystals for 15h. Finally, filter and wash the oligopeptide-1 hydrochloride crystals with 70% ethanol aqueous solution, place in a 45°C vacuum drying oven and dry to obtain the oligopeptide-1 hydrochloride crystal product, whose X-ray diffraction pattern is the same as the crystal form obtained in Example 2, and the purity determined by HPLC is 98.3% (see Figure 8 ), with a yield of 85.2%.

[0075] Example 7

[0076] Take 25g of the oligopeptide-1 solid obtained in Example 1, add purified water, adjust the pH to 7 with dilute hydrochloric acid, make the volume to 100ml, and filter through a 0.22μm water filter membrane to obtain the oligopeptide-1 hydrochloride filtrate with a concentration of 250g / L. Slowly add 3 times of ethanol under stirring conditions at 120RPM, and then keep the stirring state at an initial temperature of 25°C, reduce the temperature to 5°C at a rate of 5°C / h, and grow the crystals for 15h. Finally, filter and wash the oligopeptide-1 hydrochloride crystals with 70% ethanol aqueous solution, place in a 45°C vacuum drying oven and dry to obtain the oligopeptide-1 hydrochloride crystal product, whose X-ray diffraction pattern is the same as the crystal form obtained in Example 2, and the purity determined by HPLC is 98.4% (see Fig. 9 ), with a yield of 87.3%.

[0077] Example 8

[0078] Take 25g of the oligopeptide-1 solid obtained in Example 1, add purified water, adjust the pH to 5 with dilute hydrochloric acid, make the volume to 100ml, and filter through a 0.22μm water filter membrane to obtain the oligopeptide-1 hydrochloride filtrate with a concentration of 250g / L. Slowly add 4 times ethanol under stirring conditions at 120RPM, and then keep the stirring state at an initial temperature of 25°C, reduce the temperature to 5°C at a rate of 5°C / h, and grow the crystals for 15h. Finally, filter and wash the oligopeptide-1 hydrochloride crystals with 70% ethanol aqueous solution, place in a 45°C vacuum drying oven and dry to obtain the oligopeptide-1 hydrochloride crystal product, whose X-ray diffraction pattern is the same as the crystal form obtained in Example 2, and the purity determined by HPLC is 98.1% (see Fig.10 ), with a yield of 91.2%.

[0079] Example 9

[0080] Take 25g of the oligopeptide-1 solid obtained in Example 1, add purified water, adjust the pH to 5 with dilute hydrochloric acid, make the volume to 100ml, and filter through a 0.22μm water filter membrane to obtain the oligopeptide-1 hydrochloride filtrate with a concentration of 250g / L. Slowly add 4 times ethanol under stirring conditions at 120RPM, and then keep the stirring state at an initial temperature of 25°C, reduce the temperature to 5°C at a rate of 5°C / h, and grow the crystals for 15h. Finally, filter and wash the oligopeptide-1 hydrochloride crystals with 70% ethanol aqueous solution, place in a 45°C vacuum drying oven and dry to obtain the oligopeptide-1 hydrochloride crystal product, whose X-ray diffraction pattern is the same as the crystal form obtained in Example 2, and the purity determined by HPLC is 99.2% (see Fig.11 ), with a yield of 86.2%.

[0081] Example 10

[0082] Take 25g of the oligopeptide-1 solid obtained in Example 1, add purified water, adjust the pH to 5 with dilute hydrochloric acid, make the volume to 100ml, and filter through a 0.22μm water filter membrane to obtain the oligopeptide-1 hydrochloride filtrate with a concentration of 250g / L. Slowly add 3 times ethanol under stirring conditions at 120RPM, and then keep the stirring state at an initial temperature of 25°C, reduce the temperature to 5°C at a rate of 5°C / h, and grow the crystals for 10h. Finally, filter and wash the oligopeptide-1 hydrochloride crystals with 70% ethanol aqueous solution, place in a 45°C vacuum drying oven and dry to obtain the oligopeptide-1 hydrochloride crystal product, whose X-ray diffraction pattern is the same as the crystal form obtained in Example 2, and the purity determined by HPLC is 98.8% (see Fig.12 ), with a yield of 89.5%.

[0083] Embodiment 11

[0084] Take 25g of the oligopeptide-1 solid obtained in Example 1, add purified water, adjust the pH to 5 with dilute hydrochloric acid, make the volume to 100ml, and filter through a 0.22μm water filter membrane to obtain the oligopeptide-1 hydrochloride filtrate with a concentration of 250g / L. Slowly add 3 times of ethanol under stirring conditions at 120RPM, and then keep the stirring state at an initial temperature of 25°C, reduce the temperature to 5°C at a rate of 5°C / h, and grow the crystals for 20h. Finally, filter and wash the oligopeptide-1 hydrochloride crystals with 70% ethanol aqueous solution, place in a 45°C vacuum drying oven and dry to obtain the oligopeptide-1 hydrochloride crystal product, whose X-ray diffraction pattern is the same as the crystal form obtained in Example 2, and the purity determined by HPLC is 98.1% (see Fig.13 ), with a yield of 90.8%.

[0085] Example 12 Hygroscopicity Determination

[0086] The detection method is as follows:

[0087] Step 1: Place multiple glass culture dishes in a light-proof constant temperature and humidity incubator at 25±0.5℃ and 70±2% relative humidity for 6 hours, take them out and weigh them accurately, and repeat the test until constant weight is reached;

[0088] Step 2: Weigh the oligopeptide-1 hydrochloride solid obtained by spray drying in Example 1 and the oligopeptide-1 hydrochloride crystals obtained in the other examples respectively and place them in the glass culture dish treated with constant weight in Step 1, and continue to place them in a light-proof constant temperature and humidity incubator at 25±0.5°C and a relative humidity of 70±2% for 10 days, and observe the appearance and quality changes of the samples. The hygroscopic appearance results of all crystals are consistent, and the quality changes are not much different. The hygroscopic results of all examples are listed here, and the results are recorded in Table 2.

[0089] Table 2: Appearance and mass changes of different oligopeptides-1

[0090]

[0091]

[0092] The spray-dried oligopeptide-1 hydrochloride solid is more hygroscopic than the crystal, and agglomerates after 10 days of storage, with a more obvious mass increase, indicating that the oligopeptide-1 hydrochloride crystal is more suitable for long-term storage.

[0093] Example 13 Stability Determination

[0094] The detection method is as follows:

[0095] Step 1: Place multiple glass culture dishes in a light-proof constant temperature and humidity incubator at 35±0.5℃ and 70±2% relative humidity for 6 hours, take them out and weigh them accurately, and repeat the test until constant weight is reached;

[0096] Step 2: Weigh the oligopeptide-1 hydrochloride solid obtained by spray drying in Example 1 and the oligopeptide-1 hydrochloride crystals obtained in the other examples respectively and place them in the glass culture dish treated with constant weight in Step 1, and continue to place them in a light-proof constant temperature and humidity incubator at 35±0.5°C and a relative humidity of 70±2%. Observe the changes in sample purity every 5 days for 10 days, and the results are recorded in Table 3.

[0097] Table 3: Purity variation of different oligopeptides-1

[0098]

[0099] Comparative Example 1:

[0100] Take 25g of the oligopeptide-1 solid obtained in Example 1, add purified water without adjusting the pH, and make the volume to 100ml, and filter through a 0.22μm water filter membrane to obtain an oligopeptide-1 hydrochloride filtrate with a concentration of 250g / L and a pH of 8. Slowly add 3 times of ethanol under stirring conditions at 120RPM, and then keep stirring at an initial temperature of 25°C, and reduce the temperature to 5°C at a rate of 5°C / h for 15h. Finally, filter and wash the oligopeptide-1 hydrochloride solid with 70% ethanol aqueous solution, place it in a 45°C vacuum drying oven and dry it to obtain an oligopeptide-1 hydrochloride solid product, whose X-ray diffraction pattern is the same as the amorphous solid obtained in Example 2, and the purity determined by HPLC is 97.3% (see Fig.14 ), with a yield of 84.6%.

[0101] The stability and solubility in water (5 g dissolved in 20 ml water) of the oligopeptide-1 hydrochloride solid prepared above and the oligopeptide-1 hydrochloride crystals of Examples 6 and 7 were compared and investigated. The results are recorded in Table 4.

[0102] Table 4

[0103]

[0104] From the comparison results in Table 4, it can be seen that the oligopeptide-1 prepared in the comparative example has poor solubility. Since it is an amorphous solid, its purity decreases more significantly as the observation time increases. The purity of the oligopeptide-1 crystals of Examples 6 and 7 changes little, indicating that it is an oligopeptide-1 solid with better stability and is more conducive to long-term storage.

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An oligopeptide-1 hydrochloride crystalline compound, characterized in that: In the X-ray powder diffraction pattern of Cu-Kα radiation, it presents at least 3 of the following characteristic peaks in any combination, and the 2θ values ​​of the characteristic peaks include but are not limited to: 6.0728° (±0.2°), 6.2151° (±0.2°), 8.7892° (±0.2°), 11.6106° (±0.2°), 13.1076° (±0.2°), 14.6526° (±0.2°), 17.6190° (±0.2°), 18.8006° (±0.2°), 19.5417° (±0.2°), 20.4580° (±0.2°) , 20.9862°(±0.2°), 22.2112°(±0.2°), 23.7454°(±0.2°), 25.2775°(±0.2°), 26.3296°(±0.2°), 27.1420°(±0.2°), 28.1118°(±0.2°), 28.8980°(±0.2°), 30.2670°(±0.2°), 33.0000°(±0.2°), 35.0295°(±0.2°), 36.4723°(±0.2°) and / or 37.9459°(±0.2°).

2. The oligopeptide-1 hydrochloride crystalline compound according to claim 1, characterized in that: The X-ray powder diffraction pattern of Cu-Kα radiation also includes at least 3 of the following characteristic peaks in any combination, and the 2θ values ​​of the characteristic peaks include but are not limited to: 6.0728° (± 0.2°), 6.2151° (± 0.2°), 8.7892° (± 0.2°), 11.6106° (± 0.2°), 13.1076° (± 0.2°), 14.6526° (± 0.2°), 17.6190° (± 0.2°), 18.8006° (± 0.2°), 19.5417° (± 0.2°), 20.4580° (± 0.2°), °), 20.9862°(±0.2°), 22.2112°(±0.2°), 23.7454°(±0.2°), 25.2775°(±0.2°), 26.3296°(±0.2°), 27.1420°(±0.2°), 28.1118°(±0.2°), 28.8980°(±0.2°), 30.2670°(±0.2°), 33.0000°(±0.2°), 35.0295°(±0.2°), 36.4723°(±0.2°) and / or 37.9459°(±0.2°).

3. The oligopeptide-1 hydrochloride crystalline compound according to claim 1 or 2, characterized in that: In the X-ray powder diffraction spectrum of Cu-Kα radiation, the parameters of the characteristic peaks are as follows:

4. The method for preparing the oligopeptide-1 hydrochloride crystalline compound according to any one of claims 1 to 3, characterized in that: The steps include: Step 1: preparing an aqueous solution of oligopeptide-1 hydrochloride; Step 2: spray-drying the oligopeptide-1 hydrochloride aqueous solution obtained in step 1 to obtain oligopeptide-1 hydrochloride solid; Step 3: dissolving the oligopeptide-1 hydrochloride solid obtained in step 2 in water, adjusting the pH value, filtering, and collecting the filtrate to obtain the oligopeptide-1 hydrochloride filtrate; Step 4: Under stirring conditions, the oligopeptide-1 hydrochloride filtrate obtained in step 1 is mixed with ethanol to obtain an oligopeptide-1 hydrochloride mixed solution; Step 5: Take the oligopeptide-1 hydrochloride mixed solution obtained in step 4, cool it while stirring, precipitate oligopeptide-1 hydrochloride crystals, and grow the crystals; Step 6: Filter and wash the oligopeptide-1 hydrochloride crystals, and vacuum dry to obtain.

5. The preparation method according to claim 4, characterized in that: The inlet and outlet air temperatures of the spray drying in step 2 are 130° C. and 80° C., respectively, and the flow rate is 20 ml / min; the pH value in step 3 is 3 to 7, preferably 3 to 5; The concentration of the oligopeptide-1 hydrochloride filtrate is 200-300 g / L, preferably 200-250 g / L.

6. The preparation method according to any one of claims 4 or 5, characterized in that: The stirring speed in step 4 is 120RPM, and the volume ratio of ethanol to the oligopeptide-1 hydrochloride filtrate is (2-4):1, preferably (2-3):

1.

7. The preparation method according to any one of claims 4 to 6, characterized in that: In step 5, the stirring speed is 120RPM, the initial temperature is 25°C, the temperature is lowered to 5°C, the cooling rate is 5°C / h, and the crystal growing time is 10 to 20 hours, preferably 10 to 15 hours.

8. The preparation method according to any one of claims 4 to 7, characterized in that: The filtration in step 6 is carried out using 0.22 μm filter paper, the washing is carried out using ethanol aqueous solution, the concentration of ethanol in the ethanol aqueous solution is 70%, the temperature of the vacuum drying is 45° C., the vacuum degree is -0.1 MPa, and the time is 8 to 10 hours.

9. The oligopeptide-1 hydrochloride crystalline compound obtained by the preparation method according to any one of claims 4 to 8.

10. Use of the oligopeptide-1 hydrochloride crystalline compound according to any one of claims 1 to 3 or the oligopeptide-1 hydrochloride crystalline compound according to claim 9 in any of the following; (I) preparing products that promote wound healing; (II) Preparation of products for enhancing or improving immunity; (III) preparing products that promote the synthesis of type I collagen, type III collagen, elastin and / or structural glycoprotein; (IV) preparing products that promote the proliferation of keratinocytes, inhibit the secretion of pro-inflammatory factors and / or soothe and repair the skin; (V) preparing cosmetics and / or care products for delaying skin aging and / or whitening and removing spots.

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