A tripeptide-1 manganese and preparation method thereof
By reacting manganese salt with dimethyl sulfoxide to generate active manganese complex, and ligand exchange with tripeptide-1, combined with pH adjustment, tripeptide-1 manganese was successfully prepared, solving the problem of lack of synthesis methods in the prior art and achieving an efficient and simple production process.
Patent Information
- Application Number
- CN202411266827.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-10
- Publication Date
- 2025-04-01
AI Technical Summary
There is a lack of effective synthetic methods in the prior art to prepare tripeptide-1 manganese (GHK-Mn), which has a significant facial skin repair function, but its production method has not been reported.
GHK-Mn hydrochloride or acetate is prepared by mixing the manganese salt with dimethyl sulfoxide and reacting with heat to form an unstable, more reactive manganese complex, and then ligand exchanged with tripeptide-1 by adjusting the pH value using hydrochloric acid or acetic acid.
The method has short synthesis steps, simple operation, high yield, high purity, simple post-processing, suitable for large-scale production, and can effectively prepare high-purity tripeptide-1 manganese.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a tripeptide-1 manganese and a preparation method thereof. Background Art
[0002] Tripeptide-1 (peptide sequence: H-Gly-His-Lys-OH, abbreviated as GHK) is a signal peptide. It was first isolated from human serum by Dr. Loren Pickart of UCSF in the United States in 1973. Studies have found that it can act on the dermis, promote the synthesis of extracellular matrix, promote wound recovery and tissue renewal and reconstruction, and thus has the effect of making the skin firm and smooth and reducing wrinkles.
[0003] Subsequent studies have found that GHK can form stable complexes with metal ions. Among them, the stable complex formed with copper ions, tripeptide-1 copper (GHK-Cu for short), also known as blue copper peptide, has better overall efficacy than GHK. Therefore, it has attracted much attention in the functional skin care market and has become one of the most popular functional ingredients in the current cosmetics market.
[0004] In addition to forming a stable complex with copper ions, according to literature reports, GHK forms a stable complex with manganese ions, and tripeptide-1 manganese (GHK-Mn for short) can be used as a cosmetic raw material, which has excellent facial skin repair function. The study recruited 14 women aged between 40 and 70 years old, whose facial skin had moderate photodamage and pigmentation. After using a cream containing GHK-Mn for 12 weeks, all subjects had significant improvements in pigmentation, sallowness, freckles and dry skin.
[0005] Although GHK-Mn has excellent facial skin repairing function and can be used as a cosmetic functional raw material, there is currently no report on the synthesis method of GHK-Mn. Summary of the invention
[0006] The object of the present invention is to provide a tripeptide-1 manganese and a preparation method thereof. The preparation method of the present invention has short synthesis steps, simple operation, high yield, high purity, simple post-treatment, and is suitable for large-scale production.
[0007] The present invention provides a method for preparing tripeptide-1 manganese, comprising the following steps:
[0008] A) mixing a manganese salt and dimethyl sulfoxide and heating them to react, cooling and crystallizing after the reaction to obtain a manganese complex;
[0009] B) dissolving the tripeptide-1 and the manganese complex in water, heating to react, cooling to 20-25° C. after the reaction, adding acid to adjust the pH to 3-6, and concentrating to obtain a concentrated solution;
[0010] C) adding an alcohol solvent to the concentrated solution for crystallization to obtain tripeptide-1 manganese.
[0011] Preferably, the manganese salt is one or more of manganese halide, manganese acetate, manganese nitrate and manganese sulfate.
[0012] Preferably, the mass ratio of the manganese salt to dimethyl sulfoxide is 1:(4-8).
[0013] Preferably, the reaction temperature in step A) is 50-90° C., and the reaction time in step A) is 2-5 hours.
[0014] Preferably, the molar ratio of the tripeptide-1 to the manganese salt is 1:(0.5-0.55).
[0015] Preferably, the reaction temperature in step B) is 40-80° C., and the reaction time in step B) is 6-18 hours.
[0016] Preferably, hydrochloric acid or acetic acid is added in step B) to adjust the pH.
[0017] Preferably, the mass of the alcohol solvent in step C) is 8 to 10 times the mass of tripeptide-1.
[0018] Preferably, in the step C), the crystallization is specifically:
[0019] First stir at 35~50℃ for 8~12 hours, then cool to 0~5℃ and continue stirring for 12~16 hours.
[0020] The present invention provides tripeptide-1 manganese prepared by the preparation method described above.
[0021] The present invention provides a preparation method of tripeptide-1 manganese, comprising the following steps: A) mixing a manganese salt and dimethyl sulfoxide, heating and reacting, cooling and crystallizing after the reaction, to obtain a manganese complex; B) dissolving tripeptide-1 and the manganese complex in water, heating and reacting, cooling to 20-25°C after the reaction, adding acid to adjust the pH to 3-6, and concentrating to obtain a concentrated solution; C) adding an alcohol solvent to the concentrated solution, crystallizing, and obtaining tripeptide-1 manganese. The present invention first converts the manganese salt into an unstable, more reactive Mn complex containing a DMSO (dimethyl sulfoxide) ligand, and then performs ligand exchange with GHK, and adjusts the pH by adding hydrochloric acid or acetic acid to prepare the corresponding GHK-Mn hydrochloride or acetate. The method has short synthesis steps, simple operation, high yield, high purity, simple post-treatment, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] 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 will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0023] Figure 1 This is the mass spectrum of the compound prepared in Example 1 of the present invention;
[0024] Figure 2 This is the mass spectrum of the compound prepared in Comparative Example 1 of the present invention. DETAILED DESCRIPTION
[0025] The present invention provides a method for preparing tripeptide-1 manganese, comprising the following steps:
[0026] A) mixing a manganese salt and dimethyl sulfoxide and heating them to react, cooling and crystallizing after the reaction to obtain a manganese complex;
[0027] B) dissolving the tripeptide-1 and the manganese complex in water, heating to react, cooling to 20-25° C. after the reaction, adding acid to adjust the pH to 3-6, and concentrating to obtain a concentrated solution;
[0028] C) adding an alcohol solvent to the concentrated solution for crystallization to obtain tripeptide-1 manganese.
[0029] The method of the present invention is also applicable to common commercially available manganese salts, such as one or more of manganese halides, manganese acetates, manganese nitrates and manganese sulfates. Specifically, in the embodiments of the present invention, it can be one or more of manganese dichloride, manganese acetate, manganese nitrate hydrate and manganese sulfate; the mass ratio of the manganese salt to dimethyl sulfoxide is preferably 1: (4-8), more preferably 1: (5-7), such as 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, preferably a range value with any of the above values as the upper or lower limit.
[0030] In the present invention, the reaction temperature is preferably 50-90°C, more preferably 60-80°C, such as 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, preferably a range value with any of the above values as the upper or lower limit; the reaction time is preferably 2-5 hours, more preferably 3-4 hours.
[0031] Until the manganese salt is completely dissolved, cool to 20~25℃ to precipitate crystals, stir for 1~2 hours to allow the product to be fully precipitated. After crystallization is completed, filter a small amount of ice water to rinse the filter cake twice, collect the solid, and immediately use it for the next step of synthesis.
[0032] After obtaining the manganese complex, the present invention stirs and mixes tripeptide-1 and water, stirs at 20-25° C. until completely dissolved, then adds the manganese complex in batches, and after the addition is completed, heats and stirs to react. After the reaction is completed, the temperature is lowered to 20-25° C., and the pH is adjusted to 3-6 with acid, and then part of the water is removed under reduced pressure until the reaction system is concentrated to about 2-3 times the mass of tripeptide-1 remaining, to obtain a concentrated solution.
[0033] In the present invention, the water mixed with tripeptide-1 is preferably purified water, and the amount used is 5 to 6 times the mass of tripeptide-1.
[0034] In the present invention, the manganese complex is preferably added in batches within 25 to 35 minutes, and more preferably within 30 minutes.
[0035] In the present invention, the reaction temperature is preferably 40-80°C, more preferably 50-70°C, such as 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, preferably a range value with any of the above values as the upper or lower limit; the reaction time is preferably 6-18 hours, more preferably 10-15 hours.
[0036] In the present invention, hydrochloric acid or acetic acid is preferably used to adjust the pH value of the system, so that GHK-Mn hydrochloride or GHK-Mn acetate can be prepared for application in the field of cosmetics. The present invention has no particular limitation on the concentration and amount of the acid, as long as the pH value of the system can be adjusted to between 3 and 6.
[0037] In the present invention, the technical effect of achieving concentration by removing the system solvent under reduced pressure is a common technical means in the art, and the present invention will not be repeated here.
[0038] After obtaining the concentrated solution, the present invention heats the concentrated solution system to 35-50° C., starts to drop an alcohol solvent, slowly stirs to perform crystallization, and after the dropwise addition is completed, maintains the temperature at 35-50° C. and slowly stirs for 8-12 hours, then cools the temperature to 0-5° C., continues to slowly stir to perform crystallization for 12-16 hours, quickly filters, collects the solid, and dries in a vacuum drying oven to obtain pure GHK-Mn.
[0039] In the present invention, the concentrated solution is preferably heated to 35-50°C, more preferably 40-45°C, and after crystallization for a period of time, preferably cooled to 0-5°C, more preferably 1-4°C, such as 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, preferably with any of the above values as the upper or lower limit of the range value.
[0040] In the present invention, the alcohol solvent is preferably one or more of methanol, ethanol, isopropanol and tert-butanol; the mass of the alcohol solvent is preferably 8 to 10 times the mass of tripeptide-1.
[0041] In the present invention, the crystallization time at 35-50°C is preferably 8-12 hours, more preferably 9-11 hours, such as 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, preferably a range value with the above arbitrary numerical value as the upper or lower limit; the crystallization time at 0-5°C is preferably 12-16 hours, more preferably 13-15 hours, such as 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, preferably a range value with the above arbitrary numerical value as the upper or lower limit.
[0042] In the present invention, the vacuum drying temperature is preferably 40-60° C., more preferably 45-55° C.; the vacuum drying time is preferably 12-20 hours, more preferably 15-18 hours.
[0043] Based on the above preparation method, the present invention also provides a tripeptide-1 manganese. The tripeptide-1 manganese prepared according to the preparation method of the present invention is tripeptide-1 manganese hydrochloride or tripeptide-1 manganese acetate.
[0044] The tripeptide-1 manganese in the present invention can be used to prepare products with the function of repairing facial skin, and can be used to improve problems such as pigmentation, sallowness, freckles and dry skin.
[0045] The present invention provides a preparation method of tripeptide-1 manganese, comprising the following steps: A) mixing a manganese salt and dimethyl sulfoxide, heating and reacting, cooling and crystallizing after the reaction, to obtain a manganese complex; B) dissolving tripeptide-1 and the manganese complex in water, heating and reacting, cooling to 20-25°C after the reaction, adding acid to adjust the pH to 3-6, and concentrating to obtain a concentrated solution; C) adding an alcohol solvent to the concentrated solution, crystallizing, and obtaining tripeptide-1 manganese. The present invention first converts the manganese salt into an unstable, more reactive Mn complex containing a DMSO (dimethyl sulfoxide) ligand, and then performs ligand exchange with GHK, and adjusts the pH by adding hydrochloric acid or acetic acid to prepare the corresponding GHK-Mn hydrochloride or acetate. The method has short synthesis steps, simple operation, high yield, high purity, simple post-treatment, and is suitable for large-scale production.
[0046] In order to further illustrate the present invention, a tripeptide-1 manganese and a preparation method thereof provided by the present invention are described in detail below in conjunction with examples, but it should not be construed as limiting the scope of protection of the present invention.
[0047] Example 1
[0048] In a 500mL reaction bottle, add 62.9g (0.5mol) of manganese dichloride and 315mL of DMSO, heat to 90℃ and stir for 5h until the manganese salt is completely dissolved, then cool naturally to 25℃ to precipitate crystals, continue stirring at 25℃ for 1h to allow the product to fully precipitate. After crystallization, filter, wash the filter cake twice with 50mL of ice water, collect the solid, and use it immediately for the next step of synthesis.
[0049] In another reactor, add GHK 340.2g (1.0mol) and purified water 1701mL, stir evenly at 20-25℃ until completely dissolved, and then add the above-prepared manganese salt in batches within 30min. After the addition is completed, heat to 40℃ and stir for 18 hours. After the reaction is completed, cool to 20-25℃, adjust the pH to 3.0 with 2N hydrochloric acid, and then remove part of the water under reduced pressure until the reaction system remains about 2-3 times (weight ratio of GHK). After concentration is completed, methanol is added dropwise at 40℃, slowly stirred and crystallized, and the amount of methanol added is 10 times the weight of GHK. After the addition is completed, maintain slow stirring at 40℃ for 12 hours, cool to 0-5℃, continue to slowly stir and crystallize for 16 hours, filter quickly, collect the solid, and dry it in a vacuum drying oven at 60℃ for 12 hours to obtain 380.6g of pure GHK-Mn hydrochloride with a yield of 82.0%.
[0050] Example 2
[0051] In a 500mL reaction bottle, add 62.9g (0.5mol) of manganese dichloride and 315mL of DMSO, heat to 90℃ and stir for 5h until the manganese salt is completely dissolved, then cool naturally to 25℃ to precipitate crystals, continue stirring at 25℃ for 1h to allow the product to fully precipitate. After crystallization, filter, wash the filter cake twice with 50mL of ice water, collect the solid, and use it immediately for the next step of synthesis.
[0052] In another reactor, add GHK 340.2g (1.0mol) and purified water 1701mL, stir evenly at 20-25℃ until completely dissolved, and then add the above-prepared manganese salt in batches within 30min. After the addition is completed, heat to 40℃ and stir for 18 hours. After the reaction is completed, cool to 20-25℃, adjust the pH to 5.0 with glacial acetic acid, and then remove part of the water under reduced pressure until the reaction system remains about 2-3 times (weight ratio of GHK). After concentration is completed, start adding ethanol dropwise at 40℃, slowly stir and crystallize, and the amount of ethanol added is 8 times the weight of GHK. After the addition is completed, maintain slow stirring at 40℃ for 12 hours, cool to 0-5℃, continue to slowly stir and crystallize for 16 hours, filter quickly, collect the solid, and dry it in a vacuum drying oven at 40℃ for 20 hours to obtain 426.9g of pure GHK-Mn acetate with a yield of 82.3%.
[0053] Example 3
[0054] In a 1000mL reaction bottle, add 86.5g (0.5mol) of manganese acetate and 435mL of DMSO, heat to 70℃ and stir for 3h until the manganese salt is completely dissolved, then cool naturally to 25℃ to precipitate crystals, continue stirring at 25℃ for 1h to allow the product to fully precipitate. After crystallization, filter, wash the filter cake twice with 50mL of ice water, collect the solid, and use it immediately for the next step of synthesis.
[0055] In another reactor, add GHK 340.2g (1.0mol) and purified water 1701mL, stir evenly at 20-25℃ until completely dissolved, and then add the above-prepared manganese salt in batches within 30min. After the addition is completed, heat to 80℃ and stir for 6 hours. After the reaction is completed, cool to 20-25℃, adjust the pH to 4.0 with 2N hydrochloric acid, and then remove part of the water under reduced pressure until the reaction system remains about 2-3 times (weight ratio of GHK). After concentration is completed, start adding ethanol dropwise at 40℃, slowly stir and crystallize, and the amount of ethanol added is 8 times the weight of GHK. After the addition is completed, maintain slow stirring at 40℃ for 12 hours, cool to 0-5℃, continue to slowly stir and crystallize for 12 hours, filter quickly, collect the solid, and dry it in a vacuum drying oven at 60℃ for 12 hours to obtain 317.9g of pure GHK-Mn hydrochloride with a yield of 68.5%.
[0056] Example 4
[0057] In a 1000mL reaction bottle, add 95.2g (0.55mol) of manganese acetate and 475mL of DMSO, heat to 70℃ and stir for 3h until the manganese salt is completely dissolved, then cool naturally to 25℃ to precipitate crystals, continue stirring at 25℃ for 1h to allow the product to fully precipitate. After crystallization, filter, wash the filter cake twice with 50mL of ice water, collect the solid, and use it immediately for the next step of synthesis.
[0058] In another reactor, add GHK 340.2g (1.0mol) and purified water 1701mL, stir evenly at 20-25℃ until completely dissolved, and then add the above-prepared manganese salt in batches within 30min. After the addition is completed, heat to 80℃ and stir for 8 hours. After the reaction is completed, cool to 20-25℃, adjust the pH to 4.0 with 2N hydrochloric acid, and then remove part of the water under reduced pressure until the reaction system remains about 2-3 times (weight ratio of GHK). After concentration is completed, methanol is added dropwise at 40℃, slowly stirred and crystallized, and the amount of methanol added is 10 times the weight of GHK. After the addition is completed, maintain slow stirring at 40℃ for 16 hours, cool to 0-5℃, continue to slowly stir and crystallize for 12 hours, filter quickly, collect the solid, and dry it in a vacuum drying oven at 50℃ for 16 hours to obtain 365.7g of pure GHK-Mn hydrochloride with a yield of 78.8%.
[0059] Example 5
[0060] In a 1000mL reaction bottle, add 114.5g (0.5mol) of manganese nitrate hydrate and 575mL of DMSO, heat to 90℃ and stir for 2h until the manganese salt is completely dissolved, then cool naturally to 25℃ to precipitate crystals, continue stirring at 25℃ for 1h to allow the product to fully precipitate. After crystallization, filter, wash the filter cake twice with 50mL of ice water, collect the solid, and use it immediately for the next step of synthesis.
[0061] In another reactor, add GHK 340.2g (1.0mol) and purified water 1701mL, stir evenly at 20-25℃ until completely dissolved, and then add the above-prepared manganese salt in batches within 30min. After the addition is completed, heat to 60℃ and stir for 6 hours. After the reaction is completed, cool to 20-25℃, adjust the pH to 3.0 with 2N hydrochloric acid, and then remove part of the water under reduced pressure until the reaction system remains about 2-3 times (weight ratio of GHK). After concentration is completed, start adding isopropanol at 40℃, slowly stir and crystallize, and the amount of isopropanol added is 8 times the weight of GHK. After the addition is completed, maintain slow stirring at 40℃ for 12 hours, cool to 0-5℃, continue to slowly stir and crystallize for 16 hours, filter quickly, collect the solid, and dry it in a vacuum drying oven at 50℃ for 16 hours to obtain 361.6g of pure GHK-Mn hydrochloride with a yield of 77.9%.
[0062] Example 6
[0063] In a 1000mL reaction bottle, add 126.0g (0.55mol) of manganese nitrate hydrate and 630mL of DMSO, heat to 75℃ and stir for 4h until the manganese salt is completely dissolved, then cool naturally to 25℃ to precipitate crystals, continue stirring at 25℃ for 1h to allow the product to fully precipitate. After crystallization, filter, wash the filter cake twice with 50mL of ice water, collect the solid, and use it immediately for the next step of synthesis.
[0064] In another reactor, add GHK 340.2g (1.0mol) and purified water 1701mL, stir evenly at 20-25℃ until completely dissolved, and then add the above-prepared manganese salt in batches within 30min. After the addition is completed, heat to 80℃ and stir for 5 hours. After the reaction is completed, cool to 20-25℃, adjust the pH to 4.5 with acetic acid, and then remove part of the water under reduced pressure until the reaction system remains about 2-3 times (weight ratio of GHK). After concentration is completed, start adding isopropanol at 40℃, slowly stir and crystallize, and the amount of isopropanol added is 8 times the weight of GHK. After the addition is completed, maintain slow stirring at 40℃ for 12 hours, cool to 0-5℃, continue to slowly stir and crystallize for 14 hours, filter quickly, collect the solid, and dry it in a vacuum drying oven at 50℃ for 18 hours to obtain 368.4g of pure GHK-Mn acetate with a yield of 71.8%.
[0065] Example 7
[0066] In a 1000mL reaction bottle, add 75.5g (0.5mol) of manganese sulfate and 378mL of DMSO, heat to 90℃ and stir for 5h until the manganese salt is completely dissolved, then cool naturally to 25℃ to precipitate crystals, continue stirring at 25℃ for 1h to allow the product to fully precipitate. After crystallization, filter, wash the filter cake twice with 50mL of ice water, collect the solid, and use it immediately for the next step of synthesis.
[0067] In another reactor, add GHK 340.2g (1.0mol) and purified water 1701mL, stir evenly at 20-25℃ until completely dissolved, and then add the above-prepared manganese salt in batches within 30min. After the addition is completed, heat to 50℃ and stir for 18 hours. After the reaction is completed, cool to 20-25℃, adjust the pH to 3.0 with 2N hydrochloric acid, and then remove part of the water under reduced pressure until the reaction system remains about 2-3 times (weight ratio of GHK). After concentration is completed, tert-butanol is added dropwise at 40℃, slowly stirred and crystallized, and the amount of tert-butanol added is 8 times the weight of GHK. After the addition is completed, maintain slow stirring at 40℃ for 12 hours, cool to 0-5℃, continue to slowly stir and crystallize for 14 hours, filter quickly, collect the solid, and dry it in a vacuum drying oven at 40℃ for 20 hours to obtain 374.1g of pure GHK-Mn hydrochloride with a yield of 80.6%.
[0068] Example 8
[0069] In a 1000mL reaction bottle, add 75.5g (0.5mol) of manganese sulfate and 378mL of DMSO, heat to 90℃ and stir for 5h until the manganese salt is completely dissolved, then cool naturally to 25℃ to precipitate crystals, continue stirring at 25℃ for 1h to allow the product to fully precipitate. After crystallization, filter, wash the filter cake twice with 50mL of ice water, collect the solid, and use it immediately for the next step of synthesis.
[0070] In another reactor, add GHK 340.2g (1.0mol) and purified water 1701mL, stir evenly at 20-25℃ until completely dissolved, and then add the above-prepared manganese salt in batches within 30min. After the addition is completed, heat to 50℃ and stir for 18 hours. After the reaction is completed, cool to 20-25℃, adjust the pH to 3.5 with acetic acid, and then remove part of the water under reduced pressure until the reaction system remains about 2-3 times (weight ratio of GHK). After concentration is completed, methanol is added dropwise at 40℃, slowly stirred and crystallized, and the amount of methanol added is 8 times the weight of GHK. After the addition is completed, maintain slow stirring at 40℃ for 12 hours, cool to 0-5℃, continue to slowly stir and crystallize for 15 hours, filter quickly, collect the solid, and dry it in a vacuum drying oven at 40℃ for 20 hours to obtain 418.2g of pure GHK-Mn acetate with a yield of 81.5%.
[0071] Figure 1 This is the mass spectrum of the compound synthesized in Example 1. The molecular weight of GHK-Mn is 393.11. The mass spectrum shows that the molecular weight of the synthesized compound is 394.3100, proving that the synthesized compound is GHK-Mn.
[0072] Comparative Example 1
[0073] In a 3000mL reaction bottle, add 340.2g (1.0mol) of GHK and 1701mL of purified water, stir evenly at 20-25℃ until completely dissolved, adjust the pH to 3.0 with hydrochloric acid, and then slowly add 63mL of 1N manganese dichloride (0.5 mol) aqueous solution at 20-25℃. After the addition is complete, heat to 40-45℃ and stir for 18 hours. As the reaction proceeds, a brown-black solid gradually precipitates from the system, which is identified as non-GHK-Mn.
[0074] Figure 2 This is the mass spectrum of the compound synthesized in Comparative Example 1. From the mass spectrum results, it can be seen that the mass spectrum signal is messy and the main peak is not obvious, that is, the compound obtained in Comparative Example 1 is not GHK-Mn.
[0075] Comparative Example 2
[0076] In a 3000mL reaction bottle, add 340.2g (1.0mol) of GHK and 1701mL of purified water, stir evenly at 20-25℃ until completely dissolved, adjust the pH to 4.0 with hydrochloric acid, and then slowly add 63mL of 1N manganese acetate (0.5 mol) aqueous solution at 40-45℃. After the addition is complete, heat to 40-45℃ and stir for 18 hours. As the reaction proceeds, a brown-black solid gradually precipitates from the system, which is identified as non-GHK-Mn.
[0077] It can be seen that according to the existing GHK-Cu synthesis method (Comparative Example 1 and Comparative Example 2), GHK-Mn cannot be successfully synthesized. It is necessary to first prepare a Mn complex containing a DMSO (dimethyl sulfoxide) ligand with higher reaction activity, and then perform ligand exchange with GHK to synthesize the corresponding GHK-Mn hydrochloride or acetate.
[0078] 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. A method for preparing tripeptide-1 manganese, comprising the following steps: A) mixing a manganese salt and dimethyl sulfoxide and heating them to react, cooling and crystallizing after the reaction to obtain a manganese complex; B) dissolving the tripeptide-1 and the manganese complex in water, heating to react, cooling to 20-25° C. after the reaction, adding acid to adjust the pH to 3-6, and concentrating to obtain a concentrated solution; C) adding an alcohol solvent to the concentrated solution for crystallization to obtain tripeptide-1 manganese.
2. The preparation method according to claim 1, characterized in that: The manganese salt is one or more of manganese halide, manganese acetate, manganese nitrate and manganese sulfate.
3. The preparation method according to claim 1, characterized in that: The mass ratio of the manganese salt to dimethyl sulfoxide is 1:(4-8).
4. The preparation method according to claim 1, characterized in that: The reaction temperature in step A) is 50-90° C., and the reaction time in step A) is 2-5 hours.
5. The preparation method according to claim 1, characterized in that: The molar ratio of the tripeptide-1 to the manganese salt is 1:(0.5-0.55).
6. The preparation method according to claim 1, characterized in that: The reaction temperature in step B) is 40-80° C., and the reaction time in step B) is 6-18 hours.
7. The preparation method according to claim 1, characterized in that: In the step B), hydrochloric acid or acetic acid is added to adjust the pH.
8. The preparation method according to claim 1, characterized in that: The mass of the alcohol solvent in step C) is 8 to 10 times the mass of tripeptide-1.
9. The preparation method according to claim 1, characterized in that: In the step C), the crystallization is specifically: First stir at 35~50℃ for 8~12 hours, then cool to 0~5℃ and continue stirring for 12~16 hours.
10. Tripeptide-1 manganese obtained by the preparation method according to any one of claims 1 to 9.