A polypeptide sodium hyaluronate composite solution for injection and preparation method thereof

Abstract

The present application relates to the technical field of biomedical materials and dressing or cosmetic preparations, and specifically discloses a polypeptide sodium hyaluronate composite solution for injection and a preparation method thereof. The polypeptide sodium hyaluronate composite solution is composed of a solvent and a solute, the solvent is a buffer, and the solute mainly includes the following raw materials: sodium hyaluronate 5-50 mg / mL, L-carnosine 1-10 mg / mL, acetyl hexapeptide-8 0.05-0.15 mg / mL, acetyl tetrapeptide-5 0.05-0.1 mg / mL, tripeptide-1 derivative 0.03-0.1 mg / mL; the tripeptide-1 derivative is obtained by treating tripeptide-1 with ethyl hexadecanoate and astaxanthin. The polypeptide sodium hyaluronate composite solution, through the synergistic enhancement between the raw materials, shows a better anti-wrinkle and skin tightening effect, and also has moisturizing, hydrating, and antioxidant effects, which can improve the fullness of the skin and make the skin healthier, higher quality, and more natural.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical materials and dressing or cosmetic preparations, and more specifically, to an injectable polypeptide sodium hyaluronate composite solution and a preparation method thereof. Background Art

[0002] With the development of society and the improvement of living standards, people are paying more and more attention to the problem of skin aging. After a person is born, the skin tissue is relatively developed and active. With the increase of age, the skin metabolism gradually slows down, the synthesis of collagen and elastin decreases and is lost, and the skin gradually loses elasticity, becomes loose, and wrinkles appear. Especially after the age of 35, the skin will gradually enter the aging period. Although skin aging is a natural law that cannot be stopped, we can improve the state of skin aging through scientific methods. At this time, in the field of beauty, anti-wrinkle and skin firming have become one of the key needs. At present, there are many anti-wrinkle and skin firming products on the market, such as adding anti-free radicals, plant extracts, Chinese herbal extracts, etc. However, there are many types of anti-free radicals, and the extraction methods of plant extracts or Chinese herbal extracts are different, which makes these products have different effects, and there is still room for improvement in anti-wrinkle and skin firming. In view of this, how to improve the anti-wrinkle and skin firming effect has become one of the technical problems that need to be solved in this field. Summary of the invention

[0003] In order to improve the anti-wrinkle and skin-firming effects, the present application provides an injectable polypeptide sodium hyaluronate composite solution and a preparation method thereof.

[0004] In the first aspect, the present application provides a polypeptide sodium hyaluronate composite solution for injection, which adopts the following technical solution:

[0005] A polypeptide sodium hyaluronate composite solution for injection, the polypeptide sodium hyaluronate composite solution consists of a solvent and a solute, the solvent is a buffer solution, the solute mainly comprises the following raw materials: 5-50 mg / mL of sodium hyaluronate, 1-10 mg / mL of L-carnosine, 0.05-0.15 mg / mL of acetyl hexapeptide-8, 0.05-0.1 mg / mL of acetyl tetrapeptide-5, and 0.03-0.1 mg / mL of a tripeptide-1 derivative; the tripeptide-1 derivative is obtained by treating tripeptide-1 with ethyl hexadecanoate and astaxanthin.

[0006] The polypeptide sodium hyaluronate composite solution of the present application, through the interaction of raw materials, has an elastase inhibition rate of >62% and a type I collagen upregulation rate of >34%, making the polypeptide sodium hyaluronate composite solution show good effects in reducing skin wrinkles and promoting collagen synthesis. Moreover, when the polypeptide sodium hyaluronate composite solution is applied to improve the skin, the wrinkle improvement rate is >33% and the elasticity index improvement rate is >97%, making the polypeptide sodium hyaluronate composite solution show better anti-wrinkle and skin tightening effects.

[0007] The polypeptide sodium hyaluronate composite solution of the present application has excellent moisturizing and antioxidant properties. The sodium hyaluronate and L-carnosine, acetyl hexapeptide-8, acetyl tetrapeptide-5, and tripeptide-1 derivatives therein play a synergistic role to improve the anti-wrinkle and skin-firming effects, and can also promote skin hydration, enhance nutrition to dermal cells, improve skin fullness, increase elasticity and tighten skin. It is suitable for moisturizing and hydrating, making the skin healthier, higher quality, and more natural.

[0008] Optionally, the total content of the acetyl hexapeptide-8, acetyl tetrapeptide-5, and tripeptide-1 derivatives is greater than 0.15 mg / mL.

[0009] By adopting the above technical solution, the total content of acetyl hexapeptide-8, acetyl tetrapeptide-5, and tripeptide-1 derivatives is limited to ensure the efficacy of the polypeptide sodium hyaluronate composite solution.

[0010] Optionally, the weight average molecular weight of the sodium hyaluronate is 200,000-2,000,000 Daltons. Preferably, the weight average molecular weight of the sodium hyaluronate is 500,000-1,500,000 Daltons. More preferably, the weight average molecular weight of the sodium hyaluronate is 1,200,000 Daltons.

[0011] In multiple embodiments, the weight average molecular weight of sodium hyaluronate is 1.2 million Daltons. The weight average molecular weight can also be set to 200,000 Daltons, 500,000 Daltons, 800,000 Daltons, 1 million Daltons, 1.3 million Daltons, 1.5 million Daltons, 1.8 million Daltons, or 2 million Daltons as needed, but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0012] Optionally, the buffer is a phosphate buffer, and the pH value of the phosphate buffer is 6.6-7.6 and the osmotic pressure is 270-330 mOsm / L.

[0013] By adopting the above technical solution, the solute is diluted with a buffer solution, which not only facilitates the preparation of the polypeptide sodium hyaluronate composite solution, but also the buffer solution is a phosphate buffer solution with a pH value of 6.6-7.6 and an osmotic pressure of 270-330 mOsm / L, so that the buffer solution adapts to the skin and increases biocompatibility. It can also promote the diffusion of the solute in the skin, improve the utilization rate of the solute, and ensure the safety, stability and efficacy of the polypeptide sodium hyaluronate composite solution.

[0014] Optionally, the phosphate buffer is prepared from disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and water.

[0015] In multiple embodiments, the pH value of the phosphate buffer is 6.68 and the osmotic pressure is 300 mOsm / L. The pH value can also be set to 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6 as needed. The osmotic pressure can also be set to 270 mOsm / L, 280 mOsm / L, 290 mOsm / L, 300 mOsm / L, 310 mOsm / L, 320 mOsm / L, or 330 mOsm / L as needed, but is not limited to the listed values, and other values ​​not listed within the numerical range are equally applicable.

[0016] Optionally, the phosphate buffer is prepared by the following method: water, disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride are mixed, filtered, and the filtrate is collected to obtain a phosphate buffer.

[0017] Optionally, the filtration is performed using a water-based filter membrane, the pore size of which is 0.1-0.5 μm. Preferably, the pore size of which is 0.2-0.3 μm. More preferably, the pore size of which is 0.22 μm.

[0018] Optionally, the tripeptide-1 derivative is prepared by the following method: an organic solvent and ethyl hexadecanoate are mixed, and then a tripeptide-1 ethanol dilution is added dropwise, and after the addition is complete, the reaction is continued with stirring for 17-23 hours, and then an astaxanthin ethanol dilution is added dropwise, and after the addition is complete, the reaction is continued with stirring for 27-33 hours, and the mixture is concentrated under reduced pressure and dried to obtain a tripeptide-1 derivative.

[0019] Optionally, the weight ratio of hexadecanoic acid ethyl ester, tripeptide-1 in the ethanol dilution of tripeptide-1, and astaxanthin in the ethanol dilution of astaxanthin is (4-6): (5-9): (4-6).

[0020] By adopting the above technical scheme, the ester group in ethyl hexadecanoate and the amine group in tripeptide-1 are first reacted to form an intermediate to achieve the grafting of ethyl hexadecanoate, and the intermediate contains hexadecyl and amine groups. Then, the amine group in the intermediate reacts with the carbon-carbon double bond in astaxanthin to obtain a tripeptide-1 derivative to achieve the grafting of astaxanthin. Tripeptide-1 is treated with ethyl hexadecanoate and astaxanthin, and the synergistic effect between them can be used to improve the elastase inhibition rate and the type I collagen upregulation rate, so that the tripeptide-1 derivative acts on the dermis, reduces the depth and area of ​​wrinkles, promotes the synthesis of collagen and glycosaminoglycans, helps the skin retain moisture, restores skin elasticity, and enhances the anti-wrinkle and firming effects.

[0021] Optionally, the weight ratio of tripeptide-1 to ethanol in the tripeptide-1 ethanol dilution is 1: (3-7), and the weight ratio of astaxanthin to ethanol in the astaxanthin ethanol dilution is 1: (3-7).

[0022] By adopting the above technical scheme, the content of tripeptide-1 in the tripeptide-1 ethanol dilution and the content of astaxanthin in the astaxanthin ethanol dilution are limited, which facilitates the preparation of the tripeptide-1 ethanol dilution and the astaxanthin ethanol dilution.

[0023] The tripeptide-1 ethanol dilution is prepared from tripeptide-1 and ethanol. The astaxanthin ethanol dilution is prepared from astaxanthin and ethanol. In multiple embodiments, the weight ratio of tripeptide-1 and ethanol in the tripeptide-1 ethanol dilution is 1:5, and the weight ratio can also be set to 1:3, 1:4, 1:6, 1:7 as needed, but it is not limited to the listed values, and other values ​​not listed in the numerical range are also applicable. In multiple embodiments, the weight ratio of astaxanthin and ethanol in the astaxanthin ethanol dilution is 1:5, and the weight ratio can also be set to 1:3, 1:4, 1:6, 1:7 as needed, but it is not limited to the listed values, and other values ​​not listed in the numerical range are also applicable.

[0024] Optionally, the weight ratio of the organic solvent to ethyl hexadecanoate is (30-70): (4-6).

[0025] By adopting the above technical scheme, the weight ratio of the organic solvent and ethyl hexadecanoate is optimized, the amount of organic solvent added is limited, and the full contact and reaction of ethyl hexadecanoate, tripeptide-1, and astaxanthin are facilitated. In one embodiment, the weight ratio of the organic solvent and ethyl hexadecanoate is 50:4, in another embodiment, the weight ratio of the organic solvent and ethyl hexadecanoate is 50:5, and in another embodiment, the weight ratio of the organic solvent and ethyl hexadecanoate is 50:6. The weight ratio can also be set to 30:4, 30:5, 30:7, 70:4, 70:5 as needed, but it is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0026] Optionally, the organic solvent is one or more of ethanol, methanol, propanol and isopropanol.

[0027] By adopting the above technical solution, the organic solvent is optimized, which facilitates the selection of the organic solvent. In multiple embodiments, the organic solvent is ethanol, and the organic solvent can also be set to ethanol, methanol, propanol, isopropanol as needed, but is not limited to the listed options, and other options not listed within the selection range are also applicable.

[0028] Optionally, the dripping time of the tripeptide-1 ethanol dilution is 20-40 min, and the dripping time of the astaxanthin ethanol dilution is 20-40 min.

[0029] By adopting the above technical solution, the dripping time of the tripeptide-1 ethanol dilution and the astaxanthin ethanol dilution is limited, which facilitates the contact and reaction of hexadecanoic acid ethyl ester, tripeptide-1 and astaxanthin, and reduces side reactions.

[0030] In a second aspect, the present application provides a method for preparing the polypeptide sodium hyaluronate composite solution for injection, using the following technical solution:

[0031] A method for preparing the polypeptide sodium hyaluronate composite solution for injection comprises the following steps:

[0032] S1. Prepare buffer solution, filter and sterilize to obtain sterile buffer solution;

[0033] S2. Under a sterile environment, sterile buffer, sodium hyaluronate, L-carnosine, acetyl hexapeptide-8, acetyl tetrapeptide-5, and tripeptide-1 derivative are mixed to obtain a polypeptide sodium hyaluronate complex solution.

[0034] By adopting the above technical solution, the preparation of the polypeptide sodium hyaluronate composite solution is facilitated.

[0035] Optionally, after obtaining the polypeptide sodium hyaluronate composite solution in step S2, the polypeptide sodium hyaluronate composite solution is canned under a sterile environment to obtain a canned product.

[0036] Optionally, the canning container used for canning is one of a prefilled syringe, an ampoule, and a vial.

[0037] By adopting the above technical solution, the polypeptide sodium hyaluronate composite solution is canned to obtain a filled product, which is convenient for the storage and use of the polypeptide sodium hyaluronate composite solution.

[0038] In summary, this application has at least the following beneficial effects:

[0039] 1. The polypeptide sodium hyaluronate composite solution of the present application, in which sodium hyaluronate and L-carnosine, acetyl hexapeptide-8, acetyl tetrapeptide-5, and tripeptide-1 derivatives play a synergistic role, can effectively reduce skin wrinkles and promote collagen synthesis, and the elastase inhibition rate is greater than 62%, the type I collagen upregulation rate is greater than 34%, and the wrinkle improvement rate is greater than 33%, and the elasticity index improvement rate is greater than 97%, showing a better anti-wrinkle and skin firming effect. The polypeptide sodium hyaluronate composite solution of the present application has the effects of moisturizing, hydrating, anti-oxidation, anti-wrinkle and skin firming, can promote skin hydration, enhance nutrition to dermal cells, improve skin plumpness, increase skin elasticity and tighten skin, and make the skin healthier, higher quality and more natural.

[0040] 2. In the preparation method of tripeptide-1 derivatives, ethyl hexadecanoate and tripeptide-1 are first reacted to achieve grafting of ethyl hexadecanoate to obtain an intermediate, and then the intermediate and astaxanthin are reacted to achieve grafting of astaxanthin to obtain tripeptide-1 derivatives. The reaction and grafting are carried out in steps, which not only facilitates the preparation of tripeptide-1 derivatives, but also ensures the quality of tripeptide-1, improves the use effect of tripeptide-1, increases the inhibition rate of elastase, the upregulation rate of type I collagen, and improves the wrinkle improvement rate and elasticity index improvement rate. DETAILED DESCRIPTION

[0041] In order to make the present application easier to understand, the present application will be further described in detail below in conjunction with the examples, which are merely illustrative and are not intended to limit the scope of application of the present application. The raw materials or components used in the present application can be obtained through commercial routes or conventional methods unless otherwise specified.

[0042] Preparation Example

[0043] Preparation Example 1

[0044] A tripeptide-1 derivative is prepared by the following method:

[0045] At a speed of 500 r / min, add 5 g of ethyl hexadecanoate to 50 g of organic solvent and stir for 5 minutes. Then add 42 g of tripeptide-1 ethanol dilution dropwise, and the addition of tripeptide-1 ethanol dilution was completed in 30 minutes. After the addition was completed, continue stirring for 20 hours. Then add 31.2 g of astaxanthin ethanol dilution dropwise, and the addition of astaxanthin ethanol dilution was completed in 30 minutes. After the addition was completed, continue stirring for 30 hours. Then, reduce pressure concentration and dry to obtain a tripeptide-1 derivative.

[0046] Among them, the organic solvent is ethanol; the tripeptide-1 ethanol dilution is prepared from tripeptide-1 and ethanol, and the weight ratio of tripeptide-1 and ethanol in the tripeptide-1 ethanol dilution is 1:5; the astaxanthin ethanol dilution is prepared from astaxanthin and ethanol, and the weight ratio of astaxanthin and ethanol in the astaxanthin ethanol dilution is 1:5.

[0047] Preparation Example 2

[0048] A tripeptide-1 derivative is prepared by the following method:

[0049] At a speed of 500 r / min, add 4 g of ethyl hexadecanoate to 50 g of organic solvent and stir for 5 minutes. Then add 54 g of tripeptide-1 ethanol dilution dropwise, and the addition of tripeptide-1 ethanol dilution was completed in 40 minutes. After the addition was completed, continue stirring for 23 hours. Then add 24 g of astaxanthin ethanol dilution dropwise, and the addition of astaxanthin ethanol dilution was completed in 20 minutes. After the addition was completed, continue stirring for 27 hours. Then, reduce pressure concentration and dry to obtain a tripeptide-1 derivative.

[0050] Among them, the organic solvent is ethanol; the tripeptide-1 ethanol dilution is prepared from tripeptide-1 and ethanol, and the weight ratio of tripeptide-1 and ethanol in the tripeptide-1 ethanol dilution is 1:5; the astaxanthin ethanol dilution is prepared from astaxanthin and ethanol, and the weight ratio of astaxanthin and ethanol in the astaxanthin ethanol dilution is 1:5.

[0051] Preparation Example 3

[0052] A tripeptide-1 derivative is prepared by the following method:

[0053] At a speed of 500 r / min, add 6 g of ethyl hexadecanoate to 50 g of organic solvent and stir for 5 minutes. Then add 30 g of tripeptide-1 ethanol dilution dropwise, and the addition of tripeptide-1 ethanol dilution was completed in 20 minutes. After the addition was completed, continue stirring for 17 hours. Then add 36 g of astaxanthin ethanol dilution dropwise, and the addition of astaxanthin ethanol dilution was completed in 40 minutes. After the addition was completed, continue stirring for 33 hours. Then, reduce pressure concentration and dry to obtain a tripeptide-1 derivative.

[0054] Among them, the organic solvent is ethanol; the tripeptide-1 ethanol dilution is prepared from tripeptide-1 and ethanol, and the weight ratio of tripeptide-1 and ethanol in the tripeptide-1 ethanol dilution is 1:5; the astaxanthin ethanol dilution is prepared from astaxanthin and ethanol, and the weight ratio of astaxanthin and ethanol in the astaxanthin ethanol dilution is 1:5.

[0055] Example

[0056] Table 1 Raw materials of polypeptide sodium hyaluronate complex solution

[0057]

[0058] Example 1

[0059] A polypeptide sodium hyaluronate composite solution for injection, the raw materials and raw material ratios of which are shown in Table 1.

[0060] The weight average molecular weight of sodium hyaluronate is 1.2 million Daltons; the tripeptide-1 derivative is prepared by the method of Preparation Example 1.

[0061] The buffer is a phosphate buffer, the pH value of the phosphate buffer is 6.68, the osmotic pressure is 300 mOsm / L, and the phosphate buffer is prepared from disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and water.

[0062] The phosphate buffer was prepared by the following method: disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride were added to water at a rotation speed of 300 r / min, and stirred for 10 min to obtain a phosphate buffer.

[0063] A method for preparing a polypeptide sodium hyaluronate composite solution for injection comprises the following steps:

[0064] S1. Prepare buffer solution, filter it with a 0.22 μm aqueous filter membrane, take the filtrate, put it into an autoclave, and sterilize it at 121°C for 30 min to obtain a sterile buffer solution.

[0065] S2. Under a sterile environment, at a rotation speed of 300 r / min, add sodium hyaluronate to the sterile buffer and mix for 10 min. Then add L-carnosine, acetyl hexapeptide-8, acetyl tetrapeptide-5, and tripeptide-1 derivatives and mix for 20 min to obtain a polypeptide sodium hyaluronate complex solution.

[0066] S3. Under a sterile environment, the polypeptide sodium hyaluronate complex solution is canned to obtain a canned product. The canning container used for canning is a prefilled syringe.

[0067] Example 2

[0068] A polypeptide sodium hyaluronate composite solution for injection, which is different from Example 1 in that the raw material ratio of the polypeptide sodium hyaluronate composite solution is different, and the raw material ratio of the polypeptide sodium hyaluronate composite solution is shown in Table 1.

[0069] Example 3

[0070] A polypeptide sodium hyaluronate composite solution for injection, which is different from Example 1 in that the raw material ratio of the polypeptide sodium hyaluronate composite solution is different, and the raw material ratio of the polypeptide sodium hyaluronate composite solution is shown in Table 1.

[0071] Example 4

[0072] A polypeptide sodium hyaluronate composite solution for injection, which differs from Example 1 in that the sources of the tripeptide-1 derivative in the raw materials of the polypeptide sodium hyaluronate composite solution are different, and the tripeptide-1 derivative is prepared by the method of Preparation Example 2.

[0073] Example 5

[0074] A polypeptide sodium hyaluronate composite solution for injection, which differs from Example 1 in that the sources of the tripeptide-1 derivative in the raw materials of the polypeptide sodium hyaluronate composite solution are different, and the tripeptide-1 derivative is prepared by the method of Preparation Example 3.

[0075] Comparative Example

[0076] Comparative Example 1

[0077] A polypeptide sodium hyaluronate composite solution for injection, which differs from Example 1 in that an equal amount of tripeptide-1 is used to replace a tripeptide-1 derivative in the raw materials of the polypeptide sodium hyaluronate composite solution.

[0078] Comparative Example 2

[0079] A polypeptide sodium hyaluronate composite solution for injection, which differs from Example 1 in that, in the raw materials of the polypeptide sodium hyaluronate composite solution, in the preparation method of the tripeptide-1 derivative, an equal amount of ethyl hexadecanoate is used to replace astaxanthin.

[0080] Comparative Example 3

[0081] A polypeptide sodium hyaluronate composite solution for injection, which differs from Example 1 in that, in the raw materials of the polypeptide sodium hyaluronate composite solution, in the preparation method of the tripeptide-1 derivative, an equal amount of astaxanthin is used to replace hexadecanoic acid ethyl ester.

[0082] Comparative Example 4

[0083] A polypeptide sodium hyaluronate composite solution for injection, which is different from Example 1 in that acetyl hexapeptide-8 and tripeptide-1 derivatives are not added to the raw materials of the polypeptide sodium hyaluronate composite solution.

[0084] Comparative Example 5

[0085] A polypeptide sodium hyaluronate composite solution for injection, which differs from Example 1 in that an equal amount of acetyl hexapeptide-8 is used to replace the tripeptide-1 derivative in the raw materials of the polypeptide sodium hyaluronate composite solution.

[0086] Comparative Example 6

[0087] A polypeptide sodium hyaluronate composite solution for injection, which differs from Example 1 in that an equal amount of a tripeptide-1 derivative is used to replace acetyl hexapeptide-8 in the raw materials of the polypeptide sodium hyaluronate composite solution.

[0088] Performance Testing

[0089] (1) According to GB / T16886 "Biological Evaluation of Medical Devices", the polypeptide sodium hyaluronate composite solutions obtained in Examples 1-5 were subjected to skin sensitization test, intradermal irritation test, and cytotoxicity test. After the test, the polypeptide sodium hyaluronate composite solutions obtained in Examples 1-5 were all ≤ Grade 1 in skin sensitization test, ≤ Grade 1 in intradermal irritation test, and ≤ Grade 1 in cytotoxicity test. The polypeptide sodium hyaluronate composite solutions obtained in Examples 1-5 met the safety requirements and had good safety.

[0090] (2) Elastase is mainly synthesized and secreted by fibroblasts, has high selectivity and specificity, and can degrade elastin in the skin, thereby causing skin aging. Therefore, the efficacy of the polypeptide sodium hyaluronate composite solution in reducing skin wrinkles is characterized by the inhibition rate of elastase. The polypeptide sodium hyaluronate composite solutions obtained in Examples 1-5 and Comparative Examples 1-6 were respectively taken to test the elastase inhibition rate, and the test results are shown in Table 2.

[0091] Among them, the elastase inhibitor was used in the following method: 200 μL of polypeptide sodium hyaluronate complex solution and 50 μL of porcine pancreatic elastase solution were added to a 96-well plate at a temperature of 25°C and incubated for 15 min. Then 50 μL of N-succinyl-L-alanyl-L-alanyl-L-alanine solution was added and incubated for 15 min. The absorbance was measured at a wavelength of 410 nm using an enzyme reader, and the elastase inhibition rate was calculated.

[0092] The enzyme activity concentration of the porcine pancreatic elastase solution is 0.171 U / mL; the mass concentration of N-succinyl-L-alanyl-L-alanyl-L-alanine in the N-succinyl-L-alanyl-L-alanine solution is 90%.

[0093] (3) Type I collagen is one of the main components of the extracellular matrix of dermal cells. Type I procollagen is synthesized intracellularly by dermal fibroblasts and secreted to the extracellular space. Under the action of terminal procollagen peptidase, the terminal peptides are separated and polymerized to form collagen fibers. Therefore, human dermal fibroblasts are used as a cell model to study the effect of polypeptide sodium hyaluronate composite solution on improving the content of type I collagen, and the effect of polypeptide sodium hyaluronate composite solution on promoting collagen synthesis is characterized by the upregulation rate of type I collagen. The polypeptide sodium hyaluronate composite solutions obtained in Examples 1-5 and Comparative Examples 1-6 were taken respectively, and the increase rate of type I collagen was tested. The test results are shown in Table 2.

[0094] Table 2 Detection results of elastase inhibition rate and type I collagen upregulation rate

[0095]

[0096] As can be seen from Table 2, the polypeptide sodium hyaluronate composite solution of the present application has a higher elastase inhibition rate and type I collagen upregulation rate, with an elastase inhibition rate of 62.8-65.3% and a type I collagen upregulation rate of 34.9-36.7%. The polypeptide sodium hyaluronate composite solution exhibits good effects in reducing skin wrinkles and promoting collagen synthesis, and can play an excellent skin tightening and anti-wrinkle effect, increase skin elasticity, and improve skin condition.

[0097] Comparing Example 1 with Comparative Examples 1-3, tripeptide-1 is added to the raw materials of the polypeptide sodium hyaluronate composite solution of Comparative Example 1; tripeptide-1 derivatives are added to the raw materials of the polypeptide sodium hyaluronate composite solution of Comparative Example 2, and the tripeptide-1 derivatives are obtained by treating tripeptide-1 with ethyl hexadecanoate; tripeptide-1 derivatives are added to the raw materials of the polypeptide sodium hyaluronate composite solution of Comparative Example 3, and the tripeptide-1 derivatives are obtained by treating tripeptide-1 with astaxanthin; tripeptide-1 derivatives are added to the raw materials of the polypeptide sodium hyaluronate composite solution of Example 1, and the tripeptide-1 derivatives are obtained by treating tripeptide-1 with ethyl hexadecanoate and astaxanthin. It can be seen that grafting ethyl hexadecanoate and astaxanthin into tripeptide-1 can effectively improve the inhibition rate of elastase and the upregulation rate of type I collagen.

[0098] Comparing Example 1 with Comparative Examples 4-6, acetyl hexapeptide-8 and tripeptide-1 derivatives were not added to the raw materials of the polypeptide sodium hyaluronate composite solution of Comparative Example 4; acetyl hexapeptide-8 was added to the raw materials of the polypeptide sodium hyaluronate composite solution of Comparative Example 5; tripeptide-1 derivatives were added to the raw materials of the polypeptide sodium hyaluronate composite solution of Comparative Example 6; acetyl hexapeptide-8 and tripeptide-1 derivatives were added to the raw materials of the polypeptide sodium hyaluronate composite solution of Example 1. It can be seen that by adding acetyl hexapeptide-8 and tripeptide-1 derivatives to the raw materials at the same time, and utilizing the synergistic enhancement between them, the elastase inhibition rate and type I collagen upregulation rate can be significantly increased, and the polypeptide sodium hyaluronate composite solution can show a better use effect.

[0099] (4) A discussion group consisting of 10 professional medical staff was formed to recruit 70 female volunteers aged 35-45. All volunteers signed a written informed consent form and were randomly divided into 8 groups, with 10 people in each group. Group 1 used the polypeptide sodium hyaluronate composite solution obtained in Example 1, and groups 2-7 used the polypeptide sodium hyaluronate composite solutions obtained in Comparative Examples 1-6, respectively. The improvement rate of wrinkle depth, wrinkle area, elasticity index R2, elasticity index R5, and elasticity index R7 of the volunteers before and after use were recorded. The test results are shown in Table 3.

[0100] Table 3 Test results of skin anti-wrinkle and skin firming improvement

[0101]

[0102] As can be seen from Table 3, the polypeptide sodium hyaluronate composite solution of the present application has a wrinkle depth improvement rate of 33.8%, a wrinkle area improvement rate of 41.3%, an elasticity index R2 improvement rate of 99.6%, an elasticity index R5 improvement rate of 98.3%, and an elasticity index R7 improvement rate of 97.1%. The polypeptide sodium hyaluronate composite solution of the present application shows a better anti-wrinkle and skin-tightening effect, and can also promote skin hydration, enhance nutrition to dermal cells, improve skin plumpness, increase elasticity and firm skin, and make the skin healthier, higher quality, and more natural.

[0103] Comparing Example 1 with Comparative Examples 1-3, it can be seen that grafting ethyl hexadecanoate and astaxanthin into tripeptide-1 is beneficial to improving the use effect of tripeptide-1 derivatives. Combined with Comparative Examples 4-6, it can be seen that adding acetyl hexapeptide-8 and tripeptide-1 derivatives to the raw materials at the same time and utilizing the synergistic effect between them can significantly improve the wrinkle depth, wrinkle area and elasticity index, and enhance the anti-wrinkle and skin firming effect of the polypeptide sodium hyaluronate composite solution.

[0104] It should be noted that the embodiments described above are only used to explain the present application and do not constitute any limitation to the present application. The present application is described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present application may be modified as specified within the scope of the claims of the present application, and the present invention may be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same functions.

Claims

1. A polypeptide sodium hyaluronate composite solution for injection, characterized in that: The polypeptide sodium hyaluronate composite solution is composed of a solvent and a solute, wherein the solvent is a buffer solution, and the solute mainly comprises the following raw materials: sodium hyaluronate 5-50 mg / mL, L-carnosine 1-10 mg / mL, acetyl hexapeptide-8 0.05-0.15 mg / mL, acetyl tetrapeptide-5 0.05-0.1 mg / mL, and tripeptide-1 derivative 0.03-0.1 mg / mL; the tripeptide-1 derivative is obtained by treating tripeptide-1 with ethyl hexadecanoate and astaxanthin; The tripeptide-1 derivative is prepared by the following method: an organic solvent and ethyl hexadecanoate are mixed, and then a tripeptide-1 ethanol dilution is added dropwise, and after the addition is complete, the reaction is continued with stirring for 17-23 hours, and then an astaxanthin ethanol dilution is added dropwise, and after the addition is complete, the reaction is continued with stirring for 27-33 hours, and the mixture is concentrated under reduced pressure and dried to obtain a tripeptide-1 derivative; The weight ratio of hexadecanoic acid ethyl ester, tripeptide-1 in the tripeptide-1 ethanol dilution, and astaxanthin in the astaxanthin ethanol dilution is (4-6): (5-9): (4-6); the dripping time of the tripeptide-1 ethanol dilution is 20-40 min, and the dripping time of the astaxanthin ethanol dilution is 20-40 min; The total content of the acetyl hexapeptide-8, acetyl tetrapeptide-5 and tripeptide-1 derivatives is greater than 0.15 mg / mL.

2. The polypeptide sodium hyaluronate composite solution for injection according to claim 1, characterized in that: The buffer is a phosphate buffer, and the pH value of the phosphate buffer is 6.6-7.6 and the osmotic pressure is 270-330 mOsm / L.

3. The polypeptide sodium hyaluronate composite solution for injection according to claim 1, characterized in that: The weight ratio of tripeptide-1 to ethanol in the tripeptide-1 ethanol dilution is 1: (3-7), and the weight ratio of astaxanthin to ethanol in the astaxanthin ethanol dilution is 1: (3-7).

4. The polypeptide sodium hyaluronate composite solution for injection according to claim 1, characterized in that: The weight ratio of the organic solvent to ethyl hexadecanoate is (30-70): (4-6).

5. The polypeptide sodium hyaluronate composite solution for injection according to claim 1, characterized in that: The organic solvent is one or more of ethanol, methanol, propanol and isopropanol.

6. A method for preparing the polypeptide sodium hyaluronate composite solution for injection as claimed in any one of claims 1 to 5, characterized in that: The steps include: S1. Prepare buffer solution, filter and sterilize to obtain sterile buffer solution; S2. Under a sterile environment, sterile buffer, sodium hyaluronate, L-carnosine, acetyl hexapeptide-8, acetyl tetrapeptide-5, and tripeptide-1 derivative are mixed to obtain a polypeptide sodium hyaluronate complex solution.

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