A recombinant humanized collagen gel preparation and its preparation method and application

Abstract

The invention discloses a recombinant humanized collagen gel preparation and a preparation method and application thereof, and relates to the technical field of daily chemicals. The invention uses grape seed extract and silanized hyaluronic acid ester (SHAC) in combination on the basis of recombinant humanized collagen, which can supplement collagen fibers in the skin, promote the proliferation of human skin keratinocytes and fibroblasts and collagen synthesis, inhibit collagen degradation, repair the skin, enhance the skin barrier and moisturize; inhibit the cross-linking effect of collagenase and elastase on connective tissue, improve skin immunity, and maintain skin elasticity; and has good antioxidant properties, can neutralize free radicals, effectively prevent skin aging, thereby obtaining excellent moisturizing and anti-wrinkle properties. The recombinant humanized collagen gel preparation of the invention can promote fibroblast proliferation, has excellent moisturizing properties, and can significantly improve skin elasticity, and can be used in various moisturizing and anti-wrinkle skin care products.

Description

Technical Field

[0001] The invention belongs to the technical field of daily chemicals, and in particular relates to a recombinant humanized collagen gel preparation and a preparation method and application thereof. Background Art

[0002] The appearance of wrinkles is a manifestation of skin aging and oxidation. Skin aging is divided into endogenous aging and exogenous aging, both of which can cause premature wrinkles. From the appearance, aging skin will show decreased elasticity, sagging, fine wrinkles, etc.; from the physiological function point of view, skin aging is mainly caused by the loss of collagen, the reduction of elastic fibers, and the thinning and atrophy of subcutaneous tissue.

[0003] Collagen is a functional protein, accounting for 30% of all proteins in the body. It is mainly found in tissues such as skin, cartilage, and tendons, and plays an important role in maintaining the normal physiological functions of cells, tissues, and organs. In human skin, about 75% of the protein is collagen, which maintains the firmness of the skin. However, with age, the proliferation activity of epidermal cells in the skin gradually declines, and the junction of the true epidermis becomes flat, causing the skin to lose elasticity and wrinkles; the poor function of the stratum corneum, low water content, and the resulting thickening of the stratum corneum cause the skin to lose its softness and wrinkles. Ultraviolet radiation can penetrate the skin, denature elastin, break and lose elastic fibers, and cause low skin elasticity. Long-wave ultraviolet rays can also penetrate the dermis and act on fibroblasts to cause sunburn, erythema, aging, and wrinkles.

[0004] Recombinant humanized collagen has good transdermal properties. It can penetrate the hair follicles into the dermis and diffuse into the collagen fiber layer, replenishing collagen fibers in the skin. It has a significant proliferation effect on human skin keratinocytes and fibroblasts. It acts on epidermal keratinocytes and dermal fibroblasts, promotes collagen synthesis, inhibits collagen degradation, enhances skin barrier and moisturizing, and has significant anti-wrinkle effects. Skin care products based on recombinant humanized collagen have been widely studied.

[0005] The journal article ("Effect of recombinant humanized type III collagen on the expression of genes related to skin function", Fan Ting et al., "Daily Chemical Industry", Issue 12, 2022) explored the effect of recombinant humanized type III collagen on reducing skin wrinkles. By testing its effect on human skin fibroblasts and keratinocytes, its anti-wrinkle effect was verified at the level of skin functional genes. The results showed that 5mg / mL RHCⅢ acting on human skin fibroblasts can significantly promote the expression of collagen synthesis-related genes CollagenⅠ, CollagenⅢ and Smad3; at the same time, under UVA stimulation, it can significantly inhibit the expression of collagen degradation-related genes MMP-1, MMP-3, c-jun and c-fos. Acting on keratinocytes can significantly promote the expression of skin barrier repair and moisturizing-related genes LOR, TGM1, IVL, OCLN1, DSG1, FLG, Caspase14 and K1. The sample recombinant humanized type III collagen has anti-wrinkle effect at a dosage mass concentration of 5mg / mL.

[0006] Patent (CN115487348A) discloses a composition containing recombinant humanized collagen, which comprises the following components by weight percentage: 0.01%-10% recombinant humanized collagen, 0.01%-20% plant extract, 1%-20% surfactant, 0.1%-15% thickener, 0.5%-30% water-soluble moisturizer, 0.1%-30% oil-soluble moisturizer, 0.01%-10% functional component, 0.1%-10% polysiloxane, 0.1%-10% film former, 0.01%-1% preservative, and the balance of water; wherein the plant extract comprises agave leaf extract, Polygonum cuspidatum root extract, Coix lacryma-jobi extract, Scutellaria baicalensis root extract, Algae extract and Ganoderma lucidum extract. The composition containing recombinant humanized collagen has strong moisturizing properties and can also significantly enhance the anti-wrinkle effect.

[0007] Patent (CN114224772A) discloses an anti-wrinkle composition, which comprises, by weight percentage: 0.05%-0.2% recombinant human collagen, 0.003%-0.01% palmitic acid tripeptide-5, 0.003%-0.01% acetyl hexapeptide-8

[0008] 0.003-0.01% and the balance is hexylene glycol and water, and the raw materials are mixed and stirred evenly to obtain the anti-wrinkle composition. The obtained anti-wrinkle composition has multiple functions such as moisturizing, anti-wrinkle, and improving skin elasticity, and can effectively resist skin problems such as dryness, aging, and wrinkles caused by external environmental stimulation, and comprehensively and effectively protect skin health.

[0009] Patent (CN114224768A) discloses a repair and anti-aging composition and its preparation method. The repair and anti-aging composition comprises, by weight percentage: 0.3%-1.5% glycerol glucoside, 0.03%-0.15% recombinant human collagen, 0.0003%-0.0015% arginine / lysine polypeptide, and the balance is preservatives and water, and is prepared by mixing and stirring the components evenly. The repair and anti-aging composition of the present invention has multiple functions such as moisturizing, whitening, anti-wrinkle, anti-aging, anti-allergic and soothing, and can effectively fight against skin problems such as dryness, aging, wrinkles, spots, darkening of skin color, allergies, etc. caused by external environmental stimulation, and comprehensively and effectively protect skin health.

[0010] It can be seen that recombinant human collagen has great advantages in repairing anti-aging, moisturizing and anti-wrinkle skin care products. On this basis, a recombinant humanized collagen gel preparation is further provided, which is combined with recombinant humanized collagen, grape seed extract and silanized hyaluronic acid ester to replenish collagen fibers in the skin, promote fibroblast proliferation and collagen synthesis, inhibit collagen degradation, repair the skin, and strengthen the skin barrier, thereby obtaining excellent moisturizing and anti-wrinkle properties. Summary of the invention

[0011] The present invention aims to solve the problems existing in the prior art and provides a recombinant humanized collagen gel preparation and its preparation method and application. By using recombinant humanized collagen, grape seed extract and silanized hyaluronic acid ester in combination, collagen fibers in the skin are replenished, fibroblast proliferation and collagen synthesis are promoted, collagen degradation is inhibited, skin is repaired, and skin barrier is enhanced, thereby obtaining excellent moisturizing and anti-wrinkle properties.

[0012] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0013] In a first aspect, the present invention provides a recombinant humanized collagen gel preparation, which comprises the following raw materials, by weight: 2-10 parts of collagen, 2-10 parts of silanized hyaluronic acid ester, 1-5 parts of plant extract, 2-10 parts of small molecule polyol and 50-150 parts of water.

[0014] In a preferred embodiment, the collagen is recombinant humanized collagen; the recombinant humanized collagen is selected from at least one of type I recombinant humanized collagen, type II recombinant humanized collagen and type III recombinant humanized collagen.

[0015] In a preferred embodiment, the plant extract is grape seed extract.

[0016] In a preferred embodiment, the grape seed extract preparation process is as follows:

[0017] The grape seeds are crushed by a crusher, passed through a ≤1mm sieve, and an extractant is added to stir and extract for 1-3 hours at 20-40°C, with a solid-liquid ratio of 1:(10-30)g / ml, and the extraction is performed 1-3 times, filtered, the extracts are combined, concentrated under reduced pressure, 4 times the volume of petroleum ether is added, mixed evenly, allowed to stand for 2-3 hours, filtered, and the precipitate is dried to obtain a grape seed extract; wherein the extractant is an ethyl acetate-water mixed solvent.

[0018] In a preferred embodiment, the water content in the ethyl acetate-water mixed solvent is 6-20%.

[0019] Grape seed extract is rich in proanthocyanidins, which are one of the most effective antioxidants in nature. They can play an antioxidant role by scavenging free radicals, further preventing the formation of skin wrinkles. Proanthocyanidins react with collagen through hydrogen bonds, ionic bonds, covalent bonds, etc., thereby enhancing collagen activity. Proanthocyanidin cross-linking can prevent collagenase from loosening the triple helix structure of collagen, thereby improving the anti-enzymatic performance of collagen and the stability of mechanical properties.

[0020] Skin belongs to connective tissue, and the collagen and elastin contained in it play an important role in the entire structure of the skin. The antioxidant and free radical scavenging capabilities of proanthocyanidins are much higher than those of VE and VC. They have a unique effect on premature skin aging caused by various factors, and can inhibit the cross-linking of connective tissue by collagenase and elastase, thus helping to improve skin immunity, maintain skin elasticity, and play an anti-aging and anti-wrinkle role.

[0021] Silylated hyaluronic acid ester (SHAC) has a strong moisturizing effect on the skin. It can also eliminate the sticky feeling on the skin surface. It has strong stability at high temperatures and can prevent and repair skin damage.

[0022] The present invention uses grape seed extract and silanized hyaluronic acid ester (SHAC) in combination on the basis of recombinant humanized collagen, which can supplement collagen fibers in the skin, promote the proliferation of human skin keratinocytes and fibroblasts and collagen synthesis, inhibit collagen degradation, repair the skin, enhance the skin barrier and moisturize; inhibit the cross-linking effect of collagenase and elastase on connective tissue, improve skin immunity, and maintain skin elasticity; and has good antioxidant properties, can neutralize free radicals, effectively prevent skin aging, thereby obtaining excellent moisturizing and anti-wrinkle properties. In addition, by optimizing the preparation process of grape seed extract, it is found that the grape seed extract obtained by extraction with ethyl acetate-water mixed solvent has better moisturizing, improving skin elasticity and promoting cell proliferation than the grape seed extract obtained by extraction with 70% ethanol.

[0023] In a preferred embodiment, the small molecule polyol is selected from glycerol and / or butanediol.

[0024] In a preferred embodiment, the silanized hyaluronic acid ester uses tetrabutylammonium iodide as a catalyst, uses dimethyl monochlorosilane to react with sodium hyaluronic acid suspended in an organic phase, and the reaction filtrate is precipitated with ethyl acetate to obtain.

[0025] In a second aspect, the present application also provides a method for preparing the above-mentioned recombinant humanized collagen gel preparation, comprising the following steps:

[0026] Weigh water, add it into a reaction pot, heat it to 40-60°C, add collagen, silanized hyaluronic acid ester, plant extract and small molecule polyol in sequence, and stir until uniform.

[0027] In a third aspect, the present application also provides the use of the above-mentioned recombinant humanized collagen gel preparation in moisturizing and anti-wrinkle products.

[0028] In a preferred embodiment, the moisturizing and anti-wrinkle product is selected from at least one of essence water, essence liquid, cream, face cream, and facial mask.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The present invention uses grape seed extract and silanized hyaluronic acid ester (SHAC) in combination on the basis of recombinant humanized collagen, which can replenish collagen fibers in the skin, promote the proliferation of human skin keratinocytes and fibroblasts and collagen synthesis, inhibit collagen degradation, repair the skin, enhance the skin barrier and moisturize; inhibit the cross-linking effect of collagenase and elastase on connective tissue, improve skin immunity, and maintain skin elasticity; and has good antioxidant properties, can neutralize free radicals, and effectively prevent skin aging, thereby obtaining excellent moisturizing and anti-wrinkle properties.

[0031] 2. By optimizing the preparation process of grape seed extract, it was found that the grape seed extract obtained by extraction with ethyl acetate-water mixed solvent has better moisturizing, improving skin elasticity and promoting cell proliferation than the grape seed extract obtained by extraction with 70% ethanol.

[0032] 3. The recombinant humanized collagen gel preparation of the present invention can promote the proliferation of fibroblasts, has excellent moisturizing properties, and can significantly improve skin elasticity, and can be used in various moisturizing and anti-wrinkle skin care products. DETAILED DESCRIPTION

[0033] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0034] Among them, type III recombinant humanized collagen: provided by Jiyuan Biotechnology Co., Ltd.

[0035] Silylated hyaluronic acid ester: self-made, using tetrabutylammonium iodide as a catalyst, using dimethyl monochlorosilane to react with sodium hyaluronate suspended in an organic phase, and the reaction filtrate is precipitated with ethyl acetate to obtain silanized hyaluronic acid ester (SHAC). For the specific process, please refer to the prior art ("Preparation of silanized hyaluronic acid ester and research on its skin care effect", Li Yan et al., "Daily Chemical Industry", Issue 9, 2014) Section 1.2.1.

[0036] Preparation Example 1 Grape seed extract 1

[0037] The preparation process is as follows:

[0038] The grape seeds were crushed with a grinder, passed through a ≤1 mm sieve, added with an ethyl acetate-water mixed solvent (water content 8%), stirred and extracted for 3 h at 20°C, with a solid-liquid ratio of 1:25 g / ml, filtered, the filtrate was concentrated under reduced pressure, 4 times the volume of petroleum ether was added and mixed evenly, allowed to stand for 2 h, filtered, and the precipitate was dried to obtain grape seed extract 1.

[0039] Preparation Example 2 Grape Seed Extract 2

[0040] The preparation process is as follows:

[0041] The grape seeds were crushed with a crusher, passed through a ≤1 mm sieve, added with an ethyl acetate-water mixed solvent (water content 10%), stirred and extracted at 25°C for 2 hours, with a solid-liquid ratio of 1:20 g / ml, extracted twice, filtered, the filtrate was combined, concentrated under reduced pressure, 4 times the volume of petroleum ether was added and mixed evenly, allowed to stand for 2 hours, filtered, and the precipitate was dried to obtain grape seed extract 2.

[0042] Example 1-3 Recombinant humanized collagen gel preparation

[0043] The formula of Examples 1-3 is shown in Table 1, and the preparation process is as follows:

[0044] Weigh water, add it into a reaction pot, heat it to 40°C, add type III recombinant humanized collagen, silanized hyaluronic acid ester, grape seed extract and small molecule polyols in sequence, and stir until uniform.

[0045] Table 1

[0046]

[0047] Comparative Example 1

[0048] The same procedures as in Example 2 were followed except that grape seed extract (Preparation Example 1) was not added and 8 parts of silanized hyaluronic acid ester were added.

[0049] Comparative Example 2

[0050] The same procedures as in Example 2 were followed except that 8 parts of grape seed extract (Preparation Example 1) was added and silanized hyaluronic acid ester was not added.

[0051] Comparative Example 3

[0052] Except for using 70% ethanol as the extraction solvent, the rest is the same as Example 2.

[0053] That is, the preparation process of grape seed extract is as follows:

[0054] The grape seeds were crushed with a grinder, passed through a ≤1mm sieve, added with 70% ethanol and stirred for extraction at 20°C for 3 hours, with a solid-liquid ratio of 1:25g / ml, filtered, the filtrate was concentrated under reduced pressure, 4 times the volume of petroleum ether was added and mixed evenly, allowed to stand for 2 hours, filtered, and the precipitate was dried to obtain grape seed extract.

[0055] Test example

[0056] The performance of the recombinant humanized collagen gel preparations of Examples 1-3 and Comparative Examples 1-3 was verified.

[0057] 1. Irritation test

[0058] 50 female volunteers, aged 18-50 years old, were selected to conduct the Human Skin Patch Test on the products with reference to the Technical Specifications for Safety of Cosmetics (2015 Edition), specifically: closed patch test method, the test ingredients were removed after 24 hours, and the skin reactions were observed at 0.5 hours, 24 hours, and 48 hours after the removal of the test ingredients. The skin reaction grading standards for the human skin closed patch test are shown in Table 2, and the experimental results are shown in Table 3.

[0059] Table 2 Skin reaction grading standards

[0060]

[0061] Table 3 Irritation test results

[0062]

[0063]

[0064] According to Table 3, the recombinant humanized collagen gel preparations of Examples 1-3 and Comparative Examples 1-3 are non-irritating and highly safe.

[0065] 2. Stability determination

[0066] Heat resistance test: Place at 40℃ for 24h, take out and cool to room temperature, and observe whether there is oil-water separation before and after the test.

[0067] Cold resistance test: Place at -6℃ for 24h, take out and cool to room temperature, and observe whether there is oil-water separation before and after the test.

[0068] Hot and cold alternating test: Place at 40℃ for 24 hours, then place at room temperature for 12 hours, and then place at -6℃ for 24 hours. Observe whether there is oil-water separation before and after the test.

[0069] Centrifugal test: 20℃, 3000r / min centrifuge for 15min, observe whether there is oil-water separation before and after the test.

[0070] The experimental results are shown in Table 4.

[0071] Table 4 Stability test results

[0072]

[0073] 3. Moisture test

[0074] 3.1 Experimental procedures

[0075] (1) Place the prepared saturated potassium acetate solution and saturated ammonium sulfate solution into a sealed box respectively, and wait for the sealed box to reach the required humidity.

[0076] (2) Use an analytical balance to weigh the mass of the glass plate with 3 cm of tape on it (M0).

[0077] (3) Use a glass rod to evenly spread the sample on a glass plate covered with 3 cm adhesive tape, and weigh the mass (M1) using an analytical balance. Then place it in a desiccator at the required constant humidity.

[0078] (4) After being placed in a desiccator at a relative humidity of 85% for 4 h and 8 h, the glass plates were taken out and their mass (M2) was weighed to test their moisture retention rate. After weighing, the glass plates were immediately placed in a desiccator at a constant humidity.

[0079] 3.2 Moisture Retention Calculation Formula

[0080] Moisture retention rate = (M2-M0) / (M1-M0)×100%

[0081] Wherein: M0 is the mass of the empty plate (g); M1 is the mass of the glass plate after adding the sample (g); M2 is the mass after being placed in the dryer for several hours (g). Among them, the comparative example 5 was stirred until uniform and then immediately subjected to the smearing test. The results are shown in Table 5.

[0082] Table 5 Moisture retention test results of the composition (%)

[0083]

[0084] It can be seen that the recombinant humanized collagen gel preparations of Examples 1-3 of the present application have a good moisturizing effect, which is significantly better than Comparative Examples 1-3 and type III recombinant humanized collagen. Comparing Example 1 with Comparative Examples 1-2, it can be seen that the combination of grape seed extract and silanized hyaluronic acid ester has a better moisturizing effect; comparing Example 1 with Comparative Example 3, it can be seen that the extraction process of grape seed extract has a great influence on the performance of grape seed extract, and the grape seed extract obtained by extraction with ethyl acetate-water mixed solvent has better moisturizing properties.

[0085] 4. Skin elasticity test

[0086] Test method: 60 volunteers (aged 28-45 years old) were randomly divided into groups, with 10 people in each group. The left and right cheekbones and cheeks of the face were used as test areas. The test volunteers took 2g of samples (Examples 1-3 and Comparative Examples 1-3) each time and applied them on the face. The test products were applied twice a day in the morning and evening. The total experimental period was 28 days. During the experiment, the subjects could not apply any other cosmetics on the experimental site. The skin elasticity meter MPA580 (CK Company, Germany) was used for detection. The changes in skin elasticity of the test area were tested before use (Day 0), in the first week of use, and in the fourth week of use. During the measurement, the probe repeated the measurement 3 times in the same tested area, and the average value was taken. The test of the same volunteer was completed by the same measurement personnel. The improvement rate calculation formula is as follows. The test results are shown in Table 6:

[0087]

[0088] Table 6 Skin elasticity R2 value test results

[0089]

[0090] It can be seen from Table 6 that the recombinant humanized collagen gel preparations of Examples 1-3 of the present invention can significantly improve skin elasticity.

[0091] 5. Fibroblast proliferation activity assay

[0092] 5.1 Cell inoculation: L929 cells were revived and cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells in the logarithmic growth phase were taken and digested with trypsin-EDTA solution to adjust the cell concentration to 5×10 4 The cells were inoculated into 96-well plates at 100 μL / well and cultured for 24 h.

[0093] 5.2 Solution preparation and grouping and drug addition The drug was dissolved in DMEM basal culture medium without fetal bovine serum, and the cells in the 96-well plate were divided into 6 drug addition groups (each group had 10 replicate wells) and 1 negative control group (36 replicate wells). The original culture medium in the replicate wells corresponding to the drug addition group in the 96-well plate was discarded, and 100 μL of the recombinant humanized collagen gel preparation of Examples 1-3 and Comparative Examples 1-3 was added to each well according to the experimental grouping scheme, and the replicate wells (36 replicate wells) of the negative control group retained the original culture medium.

[0094] 5.3 Determination of relative cell proliferation rate by MTT method After 48 hours of drug addition and culture, add 20 μL of 5 mg / mL MTT solution to each well of the cell culture plate. Incubate in a dark incubator for 4 hours. Aspirate and discard the supernatant, and add 1000 μL of DMSO to each well. Incubate in a dark incubator for 20 minutes at a low speed. Use an ELISA reader to measure the light absorption value A at a wavelength of 570 nm and calculate the relative cell proliferation rate.

[0095] Relative proliferation rate = (A n -A0) / (Ay-A0)×100%(A n : light absorption value of the experimental group, Ay: absorption value of the negative control group, A0: absorption value of the blank control group)

[0096] Table 7 Relative survival rate of cells after sample treatment

[0097]

[0098]

[0099] Note: * represents P < 0.05 compared with the negative control group.

[0100] Table 7 characterizes the anti-wrinkle performance of the recombinant humanized collagen gel preparation of the present invention by cell biology method. It can be seen from Table 7 that after treating L929 cells with the recombinant humanized collagen gel preparations of Examples 1-3 and Comparative Examples 1-3, their survival rate can be improved and cell proliferation can be promoted, thereby playing an anti-wrinkle role. Among them, the degree of promoting cell proliferation of Examples 1-3 is significantly better than that of Comparative Examples 1-3.

[0101] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A recombinant humanized collagen gel preparation, characterized in that: The composition comprises the following raw materials in parts by weight: 2-10 parts of collagen, 2-10 parts of silanized hyaluronic acid ester, 1-5 parts of plant extract, 2-10 parts of small molecule polyol and 50-150 parts of water; The collagen is type III recombinant humanized collagen; The plant extract is grape seed extract; the preparation process of the grape seed extract is as follows: The grape seeds are crushed by a crusher, passed through a ≤ 1mm sieve, and an extractant is added to stir and extract for 1-3 hours at 20-40°C, with a solid-liquid ratio of 1: (10-30) g / ml, and extracted 1-3 times, filtered, and the extracts are combined, concentrated under reduced pressure, and 4 times the volume of petroleum ether is added to mix evenly, and then allowed to stand for 2-3 hours, filtered, and the precipitate is dried to obtain a grape seed extract; wherein the extractant is an ethyl acetate-water mixed solvent.

2. The recombinant humanized collagen gel preparation according to claim 1, characterized in that: The water content in the ethyl acetate-water mixed solvent is 6-20%.

3. The recombinant humanized collagen gel preparation according to claim 1, characterized in that: The small molecule polyol is selected from glycerol and / or butanediol.

4. The method for preparing the recombinant humanized collagen gel preparation according to any one of claims 1 to 3, characterized in that: The steps include: Weigh water, add it into a reaction pot, heat it to 40-60°C, add collagen, silanized hyaluronic acid ester, plant extract and small molecule polyol in sequence, and stir until uniform.

5. Use of the recombinant humanized collagen gel preparation according to any one of claims 1 to 3 or the recombinant humanized collagen gel preparation prepared by the preparation method according to claim 4 in the preparation of moisturizing and anti-wrinkle products.

6. The use according to claim 5, characterized in that The moisturizing and anti-wrinkle product is selected from at least one of essence water, cream, and facial mask.

7. The use according to claim 5, characterized in that The moisturizing and anti-wrinkle product is selected from at least one of essence and cream.

Images