Anti-aging composition containing recombinant collagen as well as preparation method and application of anti-aging composition

Abstract

The invention discloses an anti-aging composition containing recombinant collagen. The composition comprises the following components: (1) 2-5g of recombinant collagen; (2) 1-2 g of hyaluronic acid; (3) 0.2 to 0.5 g of a collagen degradation product; (4) 0.2 to 0.3 g of an anti-TGF (Transforming Growth Factor)-beta 1 monoclonal antibody; (5) 0.2 to 0.5 g of vitamin C; (6) 0.2 to 0.3 g of vitamin E; (7) 0.5-1 g of a green tea extract; and (8) adding the aqueous solution to 100 g. The anti-aging composition can effectively delay the skin aging process, improve the skin elasticity and reduce generation of wrinkles and fine wrinkles. The composition not only can be used in daily skin care products, but also can be used for treating skin problems caused by aging, such as skin laxity, fine wrinkles and darkness, and has a remarkable market prospect.

Description

Technical Field

[0001] The present invention relates to an anti-aging composition containing recombinant collagen, in particular to an anti-aging cosmetic or pharmaceutical composition and a preparation method thereof that can effectively improve the skin aging process and delay skin aging. The composition can be used in skin care, anti-aging treatment and related skin care products. Background Art

[0002] As we age, collagen in the skin is gradually lost, causing the skin to lose elasticity and develop aging phenomena such as sagging, wrinkles and fine lines. Collagen is an important component of the skin matrix, accounting for about 70% of the dry weight of the skin. It plays a key role in maintaining the structure and function of the skin. As collagen decreases, the support and tension of the skin decrease, causing the skin to show signs of aging. In order to combat skin aging, collagen supplementation has become an important anti-aging strategy. Traditionally, collagen used on the market mainly comes from animal-derived extracts, such as skin, bones and other tissues of cows, pigs or fish. Collagen from these sources may cause allergic reactions or infection risks, and its composition and quality are difficult to strictly control.

[0003] In recent years, genetic recombination technology has provided a new solution to this problem. The production of recombinant collagen through genetic engineering technology can not only avoid the potential risks of animal-derived ingredients, but also optimize the structure and function of collagen at the molecular level and improve its biological activity. Recombinant collagen has a high degree of purity and controllable molecular weight distribution, and can better combine with the natural collagen of the skin to promote skin repair and regeneration. In addition, collagen degradation products (such as collagen peptides) have also been shown to have the effect of promoting skin cell vitality and enhancing skin barrier function. Therefore, the development of an anti-aging composition containing recombinant collagen and its degradation products can more effectively improve skin aging problems and has become a research hotspot in the field of cosmetics and pharmaceuticals today. Summary of the invention

[0004] The purpose of the present invention is to provide an anti-aging composition containing recombinant collagen, which has excellent skin repair and anti-aging effects.

[0005] Therefore, the present invention discloses an anti-aging composition containing recombinant collagen, which comprises the following components:

[0006] (1) Recombinant collagen: 2-5g;

[0007] (2) Hyaluronic acid: 1-2 g;

[0008] (3) Collagen degradation products: 0.2-0.5 g;

[0009] (4) Anti-TGF-β1 monoclonal antibody: 0.2-0.3 g;

[0010] (5) Vitamin C: 0.2-0.5g;

[0011] (6) Vitamin E: 0.2-0.3 g;

[0012] (7) Green tea extract: 0.5-1g;

[0013] (8) Aqueous solution to 100 g.

[0014] Preferably, the collagen degradation product of the present invention is prepared by degrading recombinant type I collagen using collagenase, wherein the mixing ratio during the degradation reaction is 1 mg recombinant type I collagen: 1 U collagenase.

[0015] Preferably, 1U of collagenase of the present invention further comprises 5mM glucosamine and 50μM vitamin C.

[0016] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TGF-β1 monoclonal antibody of the present invention are as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0017] Preferably, the contents of tea polyphenols and catechins in the green tea extract of the present invention are 0.92 mg / mL and 9.2 mg / g, respectively.

[0018] In one aspect, the present invention further discloses a skin care product containing recombinant collagen, wherein the skin care product comprises the following components:

[0019] (1) Recombinant collagen: 3 g;

[0020] (2) Hyaluronic acid: 1.5 g;

[0021] (3) Collagen degradation products: 0.3 g;

[0022] (4) Anti-TGF-β1 monoclonal antibody: 0.25 g;

[0023] (5) Vitamin C: 0.3 g;

[0024] (6) Vitamin E: 0.25 g;

[0025] (7) Green tea extract: 0.5 g;

[0026] (8) Aqueous solution to 100 g.

[0027] In one aspect, the present invention also discloses a use of the anti-TGF-β1 monoclonal antibody in the preparation of an anti-aging composition.

[0028] The anti-aging composition of the present invention can effectively delay the skin aging process, improve skin elasticity, and reduce the generation of wrinkles and fine lines. The composition can not only be used in daily skin care products, but also can be used to treat skin problems caused by aging, such as skin sagging, fine lines, dullness, etc., and has significant market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Anti-TGF-β1 monoclonal antibody SDS-PAGE detection results, where 1 is the anti-TGF-β1 monoclonal antibody. DETAILED DESCRIPTION

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0031] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0032] Example 1: Anti-aging composition containing recombinant collagen

[0033] 1. Formula:

[0034] (1) Recombinant collagen: 2-5g;

[0035] (2) Hyaluronic acid: 1-2 g;

[0036] (3) Collagen degradation products: 0.2-0.5 g;

[0037] (4) Anti-TGF-β1 monoclonal antibody: 0.2-0.3 g;

[0038] (5) Vitamin C: 0.2-0.5g;

[0039] (6) Vitamin E: 0.2-0.3 g;

[0040] (7) Green tea extract: 0.5-1g;

[0041] (8) Aqueous solution to 100 g.

[0042] 2. Experimental steps:

[0043] 1. Recombinant collagen: Recombinant type I collagen comes from Shaanxi Benhe Bioengineering Co., Ltd.

[0044] 2. Preparation of collagen degradation products

[0045] 2.1 Preparation of collagen solution: Take the above recombinant type I collagen (purity 98%, 10 mg) and dissolve it in PBS buffer at a concentration of 2 mg / mL, and stir gently until it is completely dissolved.

[0046] 2.2 Preparation of collagenase and excipients: Prepare collagenase solution according to the product instructions at a concentration of 1 U / mL. Add glucosamine (5 mM) and vitamin C (50 μM).

[0047] 2.3 Collagen degradation reaction: Mix the collagen solution with collagenase in the ratio of 1 mg recombinant type I collagen: 1 U collagenase, and divide the mixture into multiple experimental groups. Set different degradation conditions, and set 3 replicate samples in each group. The specific groupings are as follows:

[0048] Group 1 (normal conditions): 37°C, PBS, reaction for 2 hours,

[0049] Group 2: 37°C, PBS + 5mM glucosamine, reaction for 2 hours,

[0050] Group 3: 37°C, PBS + 50μM vitamin C, reaction for 2 hours,

[0051] Group 4: 42°C, PBS+5mM glucosamine, 50μM vitamin C, reaction for 2 hours.

[0052] 2.4 Termination of degradation reaction: The reaction solution was placed in an ice bath to stop the reaction. The reaction product was separated using a refrigerated centrifuge (10,000 rpm, 10 minutes) and the supernatant was collected.

[0053] 2.5 Purification of degradation products: Separate the degradation products using a gel filtration column (Sephadex G-50) and collect products in the appropriate molecular weight range.

[0054] 2.6 Quantification of degradation products: The concentration of degradation products was determined using a spectrophotometer and the concentration was calculated by comparing the absorbance at 280 nm with the standard curve.

[0055] 2.7 Test results

[0056] 2.7.1 SDS-PAGE analysis: SDS-PAGE was used to analyze the degradation products of collagen under different degradation conditions. Electrophoresis was performed on 12% SDS-PAGE gel and stained with Coomassie Brilliant Blue. The experimental results are shown in Table 1:

[0057] Table 1 Summary of SDS-PAGE analysis results

[0058]

[0059] 2.7.2 Collagen degradation product concentration and yield: The concentration of the degradation product was measured at 280 nm using a spectrophotometer. The experimental results are shown in Table 2:

[0060] Table 2 Summary of analysis results of collagen degradation product concentration and yield

[0061]

[0062] 2.7.3 Biological activity test: Human skin fibroblasts (HDFs) were used for cell proliferation test to evaluate the biological activity of the degradation products. Different groups of degradation products (concentration of 50 μg / mL) were co-cultured with HDFs, and the cell proliferation was determined using the CCK-8 method. The control group used undegraded collagen. The experimental results are shown in Table 3:

[0063] Table 3 Summary of biological activity test analysis results

[0064]

[0065] The above results show that by optimizing the collagen degradation conditions, using glucosamine, vitamin C and increasing the reaction temperature, collagen degradation products with good activity were successfully prepared.

[0066] 3. Preparation of green tea extract

[0067] 3.1 Crushing and weighing green tea leaves: Take dry green tea leaves, weigh 10g (accurate to 0.01g), and crush. Use a grinder to grind the tea leaves into coarse powder (coarseness is about 1-2mm). Use an appropriate sieve to sieve to ensure the uniformity of the tea powder.

[0068] 3.2 Extraction of tea leaves: Place the crushed tea leaves sample (10 g) in a 250 mL beaker, add 95% ethanol, and extract at a ratio of 1:10 between tea leaves and solvent. Use a magnetic stirrer to stir the tea leaves and ethanol solution, set the temperature to 40 °C, and keep the stirring speed uniform. Continue the extraction for 2 hours to ensure that the active ingredients in the tea leaves are fully dissolved.

[0069] 3.3 Ultrasonic extraction: Transfer the solution after mixing tea leaves and solvent into an ultrasonic cleaner for ultrasonic treatment. Set the ultrasonic power to 300W and the frequency to 40kHz, and perform ultrasonic extraction for 30 minutes. During the ultrasonic process, take out the solution and stir it every 10 minutes to ensure uniform extraction.

[0070] 3.4 Filtration of the extract: Filter the extract with filter paper to remove tea residues and obtain clear green tea extract. Collect the filtrate into a 100 mL volumetric flask and dilute to 100 mL.

[0071] 3.5 Solvent removal and concentration: The filtered extract was transferred to a rotary evaporator and evaporated at 40°C to remove excess ethanol solvent. After removing the solvent, a concentrated green tea extract was obtained, which was further concentrated using a vacuum drying method (60°C, 24 hours) to ensure that the extract reached a high concentration.

[0072] 3.6 Purification and partitioning: The purity of the extract was tested by thin layer chromatography (TLC). The extract was partitioned using a separatory funnel and an appropriate solvent (chloroform) to further separate and purify the active ingredient.

[0073] 3.7 Storage of green tea extract: Transfer the prepared green tea extract into a sterile glass bottle and store it at 4°C away from light.

[0074] 3.8 Detection of Green Tea Extract

[0075] 3.8.1 Determination of tea polyphenol content: Use ultraviolet spectrophotometry to determine the tea polyphenol content. Set the wavelength to 765nm and use the standard tea polyphenol curve for quantification. Prepare standard tea polyphenol solutions, add Folin-Ciocalteu reagent to each standard solution, react for 30 minutes, and then measure the absorbance. Measure the absorbance of the extract sample and calculate the tea polyphenol content based on the standard curve. After calculation, the tea polyphenol concentration in the extract is 0.92mg / mL.

[0076] 3.8.2 Determination of total catechin content: The catechin content in green tea extract was determined by HPLC. A C18 column was used, and the mobile phase was a gradient solvent system of acetonitrile and water. A standard catechin solution (standard substances: EGCG, EC, EGC, ECG, with a concentration of 1 mg / mL) was prepared, and the standard substances and extract samples were separated by HPLC columns, and the retention time of each component was determined. The total catechin content was calculated based on the ratio of the peak area of ​​the standard substance to the peak area of ​​the sample. After calculation, the total catechin content in the extract was 9.2 mg / g.

[0077] The above results show that the present invention successfully prepared a high-concentration green tea extract, retaining active ingredients such as tea polyphenols and catechins. The experimental results show that by optimizing the extraction process and ultrasonic-assisted extraction, the yield and active ingredient concentration of green tea extract can be effectively improved. The contents of tea polyphenols and catechins in the extract were 0.92 mg / mL and 9.2 mg / g, respectively, which met the experimental expectations. This method is suitable for subsequent biological function tests such as antioxidant and anti-aging.

[0078] 4. Preparation of anti-TGF-β1 monoclonal antibody

[0079] TGF-β1 is a cytokine that plays an important role in the aging process. It can promote skin repair by promoting collagen synthesis and enhancing the stability of the skin matrix. Therefore, anti-TGF-β1 monoclonal antibodies can specifically bind to TGF-β1 and inhibit its activity, thereby regulating collagen synthesis and avoiding excessive hardening or fibrosis of the skin due to excessive collagen accumulation.

[0080] 4.1 Immunization of mice: Select healthy 6-8 week old Balb / c mice and feed them appropriately. Prepare the immunogen by mixing TGF-β1 recombinant protein (ab50036) with Freund's complete adjuvant (1:1). Immunize mice by subcutaneous injection, inject 50 μg of TGF-β1 recombinant protein each time (total immunization dose is 200 μg), once every two weeks, for a total of four times. Two weeks after the last round of immunization, evaluate the intensity of the immune response by detecting the antibody titer in the serum (such as ELISA).

[0081] 4.2 Spleen cell fusion: Take the spleen of mice two weeks after immunization and separate the spleen cells. Use melted myeloma cells (SP2 / 0) to fuse with spleen cells at a ratio of 1:5. The fused cells are cultured in HAT medium to select surviving fused cells. After two weeks of screening, transfer to HT medium for further culture and screening.

[0082] 4.3 Monoclonal antibody screening: ELISA test is performed on the screened monoclonal cells to screen out monoclonal antibodies that can specifically recognize TGF-β1 antigen. From the positive clones, clones with high antibody production efficiency are selected, amplified on a small scale, and antibodies are extracted.

[0083] 4.4 Antibody purification: The supernatant was collected from the culture medium and the antibody was purified by Protein A affinity chromatography. The antibody purity was analyzed by SDS-PAGE. The results showed ( Figure 1 ), the SDS-PAGE purity of the monoclonal antibody can reach more than 90%.

[0084] 4.5 Monoclonal antibody activity detection: The anti-TGF-β1 monoclonal antibody prepared by the present invention and two commercial antibodies (ab215715 and ab179695) were used to conduct ELISA experiments to determine their affinity for TGF-β1. The specific results are as follows:

[0085] A 96-well plate was coated with TGF-β1 recombinant protein (1 μg / mL) overnight. Anti-TGF-β1 monoclonal antibody of different concentrations was added. HRP-labeled secondary antibody was used for incubation, and TMB substrate was added for reaction. The absorbance value (450 nm) was measured. The results showed (Table 4): The anti-TGF-β1 monoclonal antibody prepared by the present invention had a lower IC50 value (2.1 ng / mL), exhibited a stronger affinity, and was superior to commercially available antibodies.

[0086] Table 4 Summary of monoclonal antibody activity test results

[0087]

[0088] 4.6 Monoclonal antibody activity detection: By sequencing and analyzing the hybridoma cells prepared as described above, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TGF-β1 monoclonal antibody prepared in the present invention are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0089] 5. Preparation of the composition: Mix the components in proportion and stir at 4°C until dissolved evenly. Adjust the pH value to 5.5-6.5 to ensure skin compatibility and use deionized water to dissolve to the required concentration. After preparation, conduct quality inspections, including stability, microbial limit and other tests.

[0090] 6. Filling and storage: Fill the prepared anti-aging composition into a sterile packaging bottle and sterilize it. Store the packaged composition in a cool and dry environment, avoiding high temperature and strong light.

[0091] Example 2: Skin care products containing recombinant collagen

[0092] 1. Formula:

[0093] (1) Recombinant collagen: 3 g;

[0094] (2) Hyaluronic acid: 1.5 g;

[0095] (3) Collagen degradation products: 0.3 g;

[0096] (4) Anti-TGF-β1 monoclonal antibody: 0.25 g;

[0097] (5) Vitamin C: 0.3 g;

[0098] (6) Vitamin E: 0.25 g;

[0099] (7) Green tea extract: 0.5 g;

[0100] (8) Aqueous solution to 100 g.

[0101] 2. Experimental steps:

[0102] 1. Preparation of the composition: Add recombinant collagen, hyaluronic acid, collagen degradation products, monoclonal antibodies, vitamin C and glycerol to an appropriate amount of deionized water according to the formula ratio, and stir thoroughly with a magnetic stirrer until dissolved evenly. During the stirring process, gradually add buffer to adjust the pH value to 5.5-6.0.

[0103] 2. After preparation, filling is performed: the skin care product composition is filled into sterile bottles to ensure that contamination is avoided during the packaging process.

[0104] 3. Quality control: Conduct quality testing, including but not limited to stability testing, microbial limit testing and skin irritation testing.

[0105] (1) Stability test: The product was stored at 40°C for 6 months, and the changes in its appearance, pH value, viscosity and other indicators were observed.

[0106] (2) Microbial limit test: in compliance with international pharmacopoeia standards, the microbial content in the product is lower than the permitted standard.

[0107] 3. Experimental Data

[0108] 1. Cell culture experiment

[0109] Fibroblasts (HDF) were divided into experimental group 1 (addition of a composition containing all ingredients), experimental group 2 (addition of a composition without anti-TGF-β1 monoclonal antibody) and control group (addition of basal medium). Each group was cultured for 48 hours, and cell proliferation (measured by CCK-8 method) and collagen synthesis level (measured by amino acid analysis or Hydroxyproline) were detected.

[0110] The results showed (Table 5): the cell proliferation rate in experimental group 1 increased by 25% (p<0.05), and collagen synthesis increased by 30% (p<0.01), which was better than that in experimental group 2, indicating that the addition of anti-TGF-β1 monoclonal antibody had a good effect.

[0111] Table 5 Summary of cell culture experimental results

[0112]

[0113] 2. Animal experiment (mouse model)

[0114] C57BL / 6J mice were used and divided into experimental group 1 (composition containing all ingredients), experimental group 2 (composition without anti-TGF-β1 monoclonal antibody) and control group (basal medium), with 10 mice in each group. The experimental group was smeared with the composition containing recombinant collagen every day, and the control group was smeared with the basic solution. The mice were observed for 6 consecutive weeks.

[0115] The results showed (Table 6): Experiment 1: The collagen content of mouse skin increased by 20% (p<0.05), the skin thickness increased by 15% (p<0.05), and the inflammatory factors IL-6 and TNF-α decreased significantly, which was better than the effect of experimental group 2, indicating that the addition of anti-TGF-β1 monoclonal antibody has a good effect.

[0116] Table 6 Summary of mouse experimental results

[0117]

[0118] 3. Skin repair effect experiment

[0119] Thirty healthy women (aged 25-45 years old) were selected and randomly divided into an experimental group (composition containing all ingredients) and a control group. The experimental group used the composition containing recombinant collagen every day, and the control group used basic skin care products without the composition. The observation lasted for 8 weeks.

[0120] The results showed (Table 7) that the skin elasticity of the experimental group increased by an average of 22% (p<0.01), wrinkle depth decreased by 15% (p<0.05), and skin hydration increased by 30% (p<0.01). There was no significant change in the control group.

[0121] Table 7 Summary of experimental results of skin repair effect

[0122]

[0123] The above results show that by combining recombinant collagen, hyaluronic acid, collagen degradation products and monoclonal antibodies, the anti-aging composition of the present invention can not only effectively promote skin repair and reduce signs of aging, but also inhibit the activity of TGF-β1 to avoid excessive accumulation of skin collagen, providing a more lasting and stable anti-aging effect. The experimental data fully proves the effectiveness and safety of the composition, and has broad application prospects.

[0124] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An anti-aging composition containing recombinant collagen, characterized in that: The composition comprises the following components: (1) Recombinant collagen: 2-5g; (2) Hyaluronic acid: 1-2 g; (3) Collagen degradation products: 0.2-0.5 g; (4) Anti-TGF-β1 monoclonal antibody: 0.2-0.3 g; (5) Vitamin C: 0.2-0.5g; (6) Vitamin E: 0.2-0.3 g; (7) Green tea extract: 0.5-1g; (8) Aqueous solution to 100 g.

2. The composition according to claim 1, characterized in that The collagen degradation product is prepared by degrading recombinant type I collagen using collagenase, wherein the mixing ratio during the degradation reaction is 1 mg recombinant type I collagen: 1 U collagenase.

3. The composition according to claim 2, characterized in that The 1U collagenase also includes 5mM glucosamine and 50μM vitamin C.

4. The composition according to claim 1, characterized in that The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TGF-β1 monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.

2.

5. The composition according to claim 1, characterized in that The contents of tea polyphenols and catechins in the green tea extract are 0.92 mg / mL and 9.2 mg / g respectively.

6. A skin care product containing recombinant collagen, characterized in that: The skin care product comprises the following components: (1) Recombinant collagen: 3 g; (2) Hyaluronic acid: 1.5 g; (3) Collagen degradation products: 0.3 g; (4) Anti-TGF-β1 monoclonal antibody: 0.25 g; (5) Vitamin C: 0.3 g; (6) Vitamin E: 0.25 g; (7) Green tea extract: 0.5 g; (8) Aqueous solution to 100 g.

7. Use of the anti-TGF-β1 monoclonal antibody according to claim 4 in the preparation of an anti-aging composition.

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