An anti-aging composition containing recombinant collagen, and its preparation method and application

Abstract

The present invention discloses an anti-aging composition containing recombinant collagen, the composition comprising the following components: (1) recombinant collagen: 2-5g; (2) hyaluronic acid: 1-2g; (3) collagen degradation product: 0.2-0.5g; (4) anti-TGF-β1 monoclonal antibody: 0.2-0.3g; (5) vitamin C: 0.2-0.5g; (6) vitamin E: 0.2-0.3g; (7) green tea extract: 0.5-1g; (8) aqueous solution to 100g. The anti-aging composition of the present invention can effectively delay the skin aging process, improve skin elasticity, and reduce 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 sagging skin, fine lines, dullness, etc., and has significant market prospects.

Description

Technical Field

[0001] The present invention relates to an anti-aging composition containing recombinant collagen, particularly an anti-aging cosmetic or pharmaceutical composition that can effectively improve and delay skin aging, and a preparation method thereof. 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 gradually disappears, causing the skin to lose elasticity and develop signs of aging, such as sagging, wrinkles, and fine lines. Collagen is a key component of the skin matrix, accounting for approximately 70% of the skin's dry weight. It plays a key role in maintaining the skin's structure and function. As collagen decreases, the skin's support and tension decrease, leading to signs of aging. To combat skin aging, collagen supplementation has become an important anti-aging strategy. Traditionally, collagen used on the market has mainly come from animal-derived extracts, such as skin, bones, and other tissues from cows, pigs, or fish. Collagen from these sources may trigger 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 a controllable molecular weight distribution, which can better combine with the skin's natural collagen and 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, in one aspect, the present invention discloses an anti-aging composition containing recombinant collagen, wherein the composition comprises the following components:

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

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

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

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

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

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

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

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

[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 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, the skin care product comprising the following components:

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

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

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

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

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

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

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

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

[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 slow the aging process, improve skin elasticity, and reduce the formation of wrinkles and fine lines. The composition can be used not only in daily skin care products but also for treating skin problems caused by aging, such as sagging skin, fine lines, and dullness, 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 anti-TGF-β1 monoclonal antibody. DETAILED DESCRIPTION

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

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

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

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

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

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

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

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

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

[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: Dissolve the above-mentioned recombinant type I collagen (98% purity, 10 mg) in PBS buffer at a concentration of 2 mg / mL and stir gently until 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 at a ratio of 1 mg recombinant type I collagen: 1 U collagenase. After mixing, divide the mixture into multiple experimental groups. Set different degradation conditions, and set up 3 replicates for 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 + 5 mM glucosamine, 50 μM 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 with appropriate molecular weight ranges.

[0054] 2.6 Quantification of degradation products: Determine the concentration of degradation products using a spectrophotometer and calculate the concentration 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 a 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 degradation products was measured using a spectrophotometer at 280 nm. 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 dermal fibroblasts (HDFs) were used for cell proliferation testing to evaluate the biological activity of the degradation products. Different groups of degradation products (at a concentration of 50 μg / mL) were co-cultured with HDFs, and cell proliferation was measured using the CCK-8 assay. 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 Grinding and weighing green tea leaves: Weigh 10g of dry green tea leaves (accurate to 0.01g) and grind them. Use a grinder to grind the tea leaves into a coarse powder (approximately 1-2mm in diameter). Sieve using an appropriate mesh to ensure uniformity of the powder.

[0068] 3.2 Tea Extraction: Place a crushed tea sample (10 g) in a 250 mL beaker and add 95% ethanol at a ratio of 1:10 tea leaves to solvent. Stir the tea leaves and ethanol solution using a magnetic stirrer at 40°C, maintaining a constant stirring rate. Continue extraction for 2 hours to ensure full dissolution of the active ingredients in the tea leaves.

[0069] 3.3 Ultrasonic Extraction: Transfer the tea leaves and solvent mixture to an ultrasonic cleaner for ultrasonic treatment. Set the ultrasonic power to 300W and the frequency to 40kHz for 30 minutes. Remove the solution and stir it every 10 minutes to ensure uniform extraction.

[0070] 3.4 Filtration of the Extract: Filter the extract using filter paper to remove tea residue 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. After solvent removal, a concentrated green tea extract was obtained. Finally, vacuum drying (60°C, 24 hours) was used to further concentrate the extract to ensure 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 isolate 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 Testing of Green Tea Extract

[0075] 3.8.1 Determination of Tea Polyphenol Content: Tea polyphenol content was determined using ultraviolet spectrophotometry. The wavelength was set to 765 nm, and quantification was performed using a standard tea polyphenol curve. Standard tea polyphenol solutions were prepared, and Folin-Ciocalteu reagent was added to each standard solution. After a 30-minute reaction, the absorbance was measured. The absorbance of the extract sample was measured, and the tea polyphenol content was calculated based on the standard curve. The calculated tea polyphenol concentration in the extract was 0.92 mg / mL.

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

[0077] These results demonstrate that the present invention successfully prepared a high-concentration green tea extract, preserving active ingredients such as tea polyphenols and catechins. Experimental results show that by optimizing the extraction process and using ultrasound-assisted extraction, the yield and active ingredient concentration of green tea extract can be effectively increased. The tea polyphenol and catechin contents in the extract were 0.92 mg / mL and 9.2 mg / g, respectively, meeting experimental expectations. This method is suitable for subsequent testing of biological functions such as antioxidant and anti-aging properties.

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

[0079] TGF-β1 is a cytokine that plays a crucial role in the aging process. It promotes skin repair by boosting 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 preventing excessive collagen accumulation that can lead to excessive hardening or fibrosis of the skin.

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

[0081] 4.2 Spleen Cell Fusion: Two weeks after immunization, spleen cells were isolated from the spleen of mice. Melted myeloma cells (SP2 / 0) were used for cell fusion at a ratio of 1:5. The fused cells were cultured in HAT medium to select for surviving fused cells. After two weeks of screening, the cells were transferred to HT medium for further culture and screening.

[0082] 4.3 Monoclonal Antibody Screening: ELISA tests were performed on the screened monoclonal cells to screen for monoclonal antibodies that specifically recognize the TGF-β1 antigen. Highly efficient antibody-producing clones were selected from the positive clones and amplified on a small scale to extract the antibodies.

[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 that ( Figure 1 ), the SDS-PAGE purity of the monoclonal antibody can reach more than 90%.

[0084] 4.5 Monoclonal Antibody Activity Assay: ELISA experiments were conducted using the anti-TGF-β1 monoclonal antibody prepared by the present invention and two commercial antibodies (ab215715 and ab179695) 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 antibodies at varying concentrations were added. HRP-labeled secondary antibodies were used for incubation, and TMB substrate was added for reaction. The absorbance was measured (450 nm). The results (Table 4) showed that the anti-TGF-β1 monoclonal antibody prepared by the present invention had a low IC50 value (2.1 ng / mL), exhibited strong 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: Sequencing and analysis of the hybridoma cells prepared above revealed that the amino acid sequences of the heavy chain variable region and 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: Combine all components in the correct proportions and stir at 4°C until dissolved. Adjust the pH to 5.5-6.5 to ensure skin compatibility and dissolve in deionized water to the desired concentration. After preparation, perform quality testing, including stability and microbial limit tests.

[0090] 6. Filling and Storage: Fill the prepared anti-aging composition into sterile packaging bottles and sterilize them. Store the packaged composition in a cool, dry environment, away from high temperatures and strong light.

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

[0092] 1. Recipe:

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

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

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

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

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

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

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

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

[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. Stir thoroughly with a magnetic stirrer until uniformly dissolved. During stirring, gradually add buffer to adjust the pH 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 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 allowable standard.

[0107] 3. Experimental Data

[0108] 1. Cell culture experiments

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

[0110] The results showed (Table 5): in experimental group 1, the cell proliferation rate increased by 25% (p<0.05), and collagen synthesis increased by 30% (p<0.01), which were better than those 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 experiment results

[0112]

[0113] 2. Animal experiments (mouse model)

[0114] C57BL / 6J mice were divided into experimental group 1 (composition containing all ingredients), experimental group 2 (composition without anti-TGF-β1 monoclonal antibody), and a control group (basal medium), with 10 mice in each group. The experimental groups were treated daily with the composition containing recombinant collagen, while the control group was treated with the basal medium. The mice were observed for 6 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) were randomly divided into an experimental group (using a combination containing all ingredients) and a control group. The experimental group used the combination containing recombinant collagen daily, while the control group used a basic skincare product without the combination. The patients were observed for eight consecutive weeks.

[0120] The results (Table 7) showed 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] These results demonstrate that the anti-aging composition of the present invention, by combining recombinant collagen, hyaluronic acid, collagen degradation products, and monoclonal antibodies, not only effectively promotes skin repair and reduces signs of aging, but also inhibits TGF-β1 activity, preventing excessive collagen accumulation in the skin, providing a more sustained and stable anti-aging effect. These experimental data fully demonstrate the effectiveness and safety of this composition, demonstrating its broad potential for application.

[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 considered as equivalent replacement methods and are included in the scope of protection 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: 1-2g; (3) Collagen degradation products: 0.2-0.5g; (4) Anti-TGF-β1 monoclonal antibody: 0.2-0.3g; (5) Vitamin C: 0.2-0.5g; (6) Vitamin E: 0.2-0.3g; (7) Green tea extract: 0.5-1g; (8) Aqueous solution to 100 g; 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 of recombinant type I collagen: 1 U of collagenase; the 1 U of collagenase also includes 5 mM glucosamine and 50 μM vitamin C; 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; The contents of tea polyphenols and catechins in the green tea extract are 0.92 mg / mL and 9.2 mg / g, respectively; The recombinant collagen is recombinant type I collagen.

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

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