An anti-aging and repairing composition containing collagen and its preparation method

Abstract

This invention provides an anti-aging and repairing composition containing collagen and its preparation method, belonging to the technical field of medical, dental, or cosmetic products. Its components consist of hydrolyzed collagen, recombinant type III human collagen, artichoke leaf extract, and fermented blueberry seed oil. This invention uses recombinant type III human collagen and hydrolyzed collagen as its core, overcoming the limitations of traditional collagen through biotechnology, and supplementing it with double-fermented plant ingredients to solve the bottlenecks of transdermal absorption and anti-oxidation. The overall design conforms to the anti-aging triangle logic of "repair, defense, and moisturizing," and has good market prospects.

Description

Technical Field

[0001] This invention belongs to the technical field of medical, dental or cosmetic products, and particularly relates to an anti-aging and repairing composition containing collagen and its preparation method. Background Technology

[0002] Skin aging is the result of the combined effects of endogenous natural aging and exogenous environmental damage. Endogenous aging is mainly regulated by genes, manifested as a decline in collagen synthesis capacity (1%-3% loss per year after age 25), degeneration of dermal elastic fibers, and atrophy of subcutaneous fat, leading to thinning, sagging skin and fine lines.

[0003] Exogenous aging is the dominant factor, with ultraviolet radiation (photoaging) contributing over 80% of the causes: ultraviolet rays (especially UVA) penetrate the dermis, damaging collagen structure, activating matrix metalloproteinases (MMPs) to accelerate collagen degradation, and inducing oxidative stress. Other external factors such as PM2.5 pollutants, staying up late, and smoking can trigger inflammatory responses through skin penetration, exacerbating free radical accumulation and barrier damage.

[0004] Skincare products slow down aging by targeting aging pathways, working from multiple dimensions including protection, repair, and regeneration.

[0005] Sunscreen: The primary defense against photoaging. Sunscreen agents (physical / chemical) protect against UVA / UVB and reduce DNA damage and collagen breakdown.

[0006] Moisturizing and barrier repair: Contains ingredients that enhance the stratum corneum's ability to retain moisture and prevent wrinkles caused by dryness.

[0007] Antioxidant: Vitamins C / E, coenzyme Q10, astaxanthin and other ingredients scavenge reactive oxygen species (ROS), block the chain reaction of oxidative stress, and protect collagen and cellular DNA.

[0008] Retinol promotes collagen regeneration and metabolism: It stimulates fibroblast proliferation, accelerates collagen synthesis, and repairs photoaging wrinkles.

[0009] Anti-inflammatory and cell protection: For example, green tea extract inhibits inflammatory factors (IL-1, TNF-α) and blocks the vicious cycle of inflammation and oxidation. Summary of the Invention

[0010] The purpose of this invention is to provide an anti-aging and repairing composition containing collagen, which has excellent anti-aging effects; it contains the following ingredients in parts by weight:

[0011] Hydrolyzed collagen 2-5

[0012] Recombinant Type III Human Collagen 1-3

[0013] artichoke leaf extract 5-10

[0014] Fermented blueberry seed oil 25-50

[0015] The artichoke leaf extract is obtained by enzymatic hydrolysis of artichoke leaves, followed by fermentation with plant lactic acid bacteria, and then purification.

[0016] The fermented blueberry seed oil is obtained by purifying blueberry seed oil that has been fermented using bumblebee Candida albicans.

[0017] Preferably, the preparation method of the artichoke leaf extract is as follows: take fresh artichoke leaves, wash and crush them; enzymatically hydrolyze them using pectinase and cellulase; inactivate the enzymes after enzymatic hydrolysis, filter and collect the filtrate;

[0018] The filtrate was inoculated with activated Lactobacillus plantarum and anaerobic fermented at 36-38℃ and pH 5.5-6.0 for at least 48 hours.

[0019] After fermentation, the mixture is sterilized, centrifuged, and the supernatant is collected. Ethanol is added to the supernatant, and then it is passed through an 800 Da filter membrane. The filtered solution is then dried into a solid.

[0020] Preferably, when using pectinase and cellulase for enzymatic hydrolysis, the temperature is controlled at 45-50℃ and the pH is 4.5-5.0.

[0021] Preferably, during the anaerobic fermentation of the filtrate, an auxiliary carbon source, an auxiliary nitrogen source, dipotassium hydrogen phosphate, manganese sulfate, and an emulsifier are added.

[0022] Preferably, the preparation method of the fermented blueberry seed oil is as follows: take blueberry seed oil, mix and emulsify it with an aqueous culture medium to prepare a fermentation substrate, and inoculate it with activated *Candida bacillus*; then ferment at 28-32℃, pH 5.0-6.0, and OD value of at least 40% for at least 72 hours;

[0023] After fermentation, the mixture is sterilized and centrifuged to obtain the upper oil phase. After dehydration of the oil phase, the main fraction at 160–180℃ is collected by molecular distillation.

[0024] Preferably, the aqueous culture medium contains at least the following components by mass percentage:

[0025] At least 1% yeast extract;

[0026] At least 2% glucose

[0027] At least 1% peptone

[0028] At least 0.1% magnesium sulfate

[0029] At least 2.5% nonionic lipophilic emulsifier.

[0030] Preferably, the aforementioned anti-aging and repairing composition containing collagen further includes a moisturizer; the moisturizer includes at least one of glycerin, β-glucan, sodium hyaluronate, and polyols.

[0031] This invention also provides the application of the aforementioned collagen-containing anti-aging and repairing composition in the preparation of cosmetics; the minimum addition amount is 1 wt%. Specific dosage forms of the cosmetics include, but are not limited to, serums, facial oils, creams, lotions, etc.

[0032] This invention selects two different types of collagen for complementarity. Hydrolyzed collagen, with its small molecular weight and strong penetrability, directly provides synthetic raw materials for skin fibroblasts, enhancing elastin expression. Recombinant type III human collagen has a 100% identical amino acid sequence to human collagen, eliminating the risk of immune rejection. Its unique flexible triple helix structure allows it to directly adhere to cells, filling collapsed areas of the dermis, improving fine lines and sagging, and promoting epidermal cell migration and proliferation.

[0033] Artichoke leaves contain cynarin and flavonoids (quercetin, kaempferol, etc.); they possess highly effective anti-inflammatory, soothing, and barrier-repairing effects. Specifically, they reduce histamine release and alleviate allergic reactions such as skin redness and swelling by inhibiting the activation of the NF-κB inflammatory pathway. The abundant flavonoids and polyphenols can scavenge free radicals, reduce UV-induced oxidative damage, and delay collagen degradation. This invention has found that these effects can be further enhanced after fermentation with *Lactobacillus plantarum*.

[0034] Bilberry seed oil is primarily known for its powerful antioxidant and lipid repair properties, and is rich in anthocyanins, which can neutralize free radicals induced by ultraviolet radiation and pollution. The oil portion is also rich in Omega-3 and Omega-6, mimicking the skin's natural lipid structure to quickly repair barrier damage and improve dryness and redness.

[0035] Oil-loving microorganisms are a class of microorganisms that can utilize lipids. They use oils as nutrients and employ their own enzyme systems (such as lipases) to break down or reassemble oils into other metabolic products. *Starmerella bombicola* can tolerate low-water, high-sugar environments and actively decomposes, processes, and reuses lipids. This strain is a special type of microorganism that grows in an environment composed of esters, waxes (honeycomb), and sugars (honey). This environment allows them to effectively utilize lipids and sugars and metabolize functional substances such as glycolipids, amino acids, small-molecule sugars, fatty acids, and ceramides. In this invention, fermentation with *Starmerella bombicola* converts high-molecular-weight components in blueberry seed oil that are difficult to absorb through the skin into low-molecular-weight components, improving its transdermal absorption rate. Ideally, molecular distillation purification is performed to remove large molecules and fermentation impurities that cannot be absorbed through the skin, retaining the light oils from the 160-180℃ fraction, resulting in a refreshing feel and enhanced permeability.

[0036] In summary, this invention uses recombinant type III human collagen and hydrolyzed collagen as its core, breaks through the limitations of traditional collagen through biotechnology, and solves the bottlenecks of transdermal absorption and anti-oxidation by supplementing it with double-fermented plant ingredients. The overall design conforms to the anti-aging triangle logic of "repair, defense, and moisturizing" and has good market prospects. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] In the description of this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the stated features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0039] In the description of this invention, the term "for example" is used to mean "used as an example, illustration, or description." Any embodiment described as "for example" in this invention is not necessarily to be construed as being more preferred or advantageous than other embodiments. The following description is provided to enable any person skilled in the art to make and use the invention. Details are set forth in the following description for purposes of explanation. It should be understood that those skilled in the art will recognize that the invention can be made without using these specific details. In other instances, well-known structures and processes will not be described in detail to avoid obscuring the description of the invention with unnecessary detail. Therefore, the invention is not intended to be limited to the embodiments shown, but is consistent with the broadest scope of the principles and features disclosed herein.

[0040] Unless otherwise specified, the experimental methods used in the specific implementation methods are all conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0041] In this invention, unless otherwise specified, "%" represents a percentage by mass; the raw materials and reagents used are all commercially available products.

[0042] The sources of some of the raw materials used in this invention are shown in Table 1 below:

[0043] Table 1

[0044]

[0045] Example 1: Fermentation Experiment of Artichoke Leaves

[0046] Different strains of microorganisms were used to ferment artichoke leaves, and the differences in the antioxidant properties of the fermentation products from different strains were compared. The preparation method includes the following steps:

[0047] S1: Select fresh artichoke leaves, wash and remove mud and sand; drain and then crush at low temperature to a particle size of 10 mesh;

[0048] S2: Take cellulase (20 U / g artichoke leaf) and pectinase (15 U / g artichoke leaf) and mix them together; adjust the solid-liquid ratio to 1:8 (adjust with water), and enzymatically hydrolyze for 2 hours at 45-50℃ and pH 4.5-5.0 (adjust with citric acid);

[0049] After enzymatic hydrolysis, heat to 85℃ and hold for 10 minutes to inactivate the enzyme; centrifuge (3000g, 15 minutes) and collect the filtrate.

[0050] S3: Prepare the substrate according to the proportions in Table 2, and add an appropriate amount of sterile water to adjust the moisture content; prepare the activated bacterial strain into... The suspension of CFU / mL was inoculated at a ratio of 5%, and then cultured according to the conditions in Table 2.

[0051] S4: After fermentation, sterilize and centrifuge to obtain the supernatant. Add ethanol (anhydrous ethanol, 5%) to the supernatant and then pass it through an 800Da filter membrane. Take the filtered solution and dry it into a solid.

[0052] Table 2

[0053]

[0054] Alternatively, unfermented artichoke leaves are prepared, which includes the following steps:

[0055] S1: Select fresh artichoke leaves, wash and remove mud and sand; drain and then crush at low temperature to a particle size of 10 mesh;

[0056] S2: Take cellulase (20 U / g artichoke leaf) and pectinase (15 U / g artichoke leaf) and mix them together; adjust the solid-liquid ratio to 1:8 (adjust with water), and enzymatically hydrolyze for 2 hours at 45-50℃ and pH 4.5-5.0 (adjust with citric acid);

[0057] After enzymatic hydrolysis, heat to 85℃ and hold for 10 minutes to inactivate the enzyme; centrifuge (3000g, 15 minutes) and collect the filtrate.

[0058] S3: Centrifuge the filtrate to obtain the supernatant. Add ethanol (anhydrous ethanol, 5%) to the supernatant and then pass it through an 800 Da filter membrane. Take the filtered solution and dry it into a solid.

[0059] Example 2: Performance Study of Bilberry Seed Oil

[0060] In skincare products, there are three types of bilberry seed oil with recorded usage: VACCINIUM MACROCARPON seed oil (i.e., cranberry), VACCINIUM MYRTILLUS seed oil (also known as black bilberry), and VACCINIUM VITIS-IDAEA seed oil (also known as red bilberry).

[0061] The preparation of fermented oil includes the following steps:

[0062] S1: Take different oils, mix them with aqueous culture medium at a volume ratio of 1:1, homogenize and emulsify them using a microfluidic homogenizer, and then autoclave them at 121℃ for 20 minutes to prepare fermentation substrate;

[0063] The aqueous culture medium contains: 1% yeast extract, 2% glucose, 1% peptone, 0.1% magnesium sulfate, 2.5% Span 80; the remainder is deionized water.

[0064] S2: Fermentation substrate and inoculation with activated bumblebee Candida albicans;

[0065] Fermentation was carried out at 28-32℃, pH 5.0-6.0, OD value ≥40%, and aeration rate of 0.6 vvm for 72 h.

[0066] S3: After fermentation, inactivate at 100℃ for 15 min; then centrifuge at 9000g for 20 min and take the upper oil phase; dehydrate the oil phase under vacuum; after dehydration, collect the main fraction at 160–180℃ by molecular distillation.

[0067] Example 3 Antioxidant Performance Test

[0068] The test method is as follows:

[0069] DPPH methanol solution is violet in color and has a strong absorbance at 517 nm. If it binds to the sample, it will reduce the absorbance at 517 nm, thus determining the sample's ability to scavenge DPPH free radicals.

[0070] The specific method is as follows:

[0071] (1) Take samples from Example 1, Example 2, and commercially available samples, mix them with sterile water, and prepare equal volumes (2 mL) of test solutions with different concentrations and 2 × 10⁻⁶ ppm. -4 Mix the mol / L DPPH solution thoroughly (Al);

[0072] (2) Take equal volumes of anhydrous ethanol (the solvent for the analyte) and 2×10 -4 Mix the mol / L DPPH solution thoroughly (A2);

[0073] (3) Take an equal volume of anhydrous ethanol and mix it with the test solution (A3);

[0074] (4) After reacting for 40 min, the absorbance values ​​of tubes A1, A2 and A3 were measured at 517 nm.

[0075] The formula for calculating the clearance rate is: Clearance rate (%) = [1 - (A1 - A3) / A2] × 100%

[0076] The test results are shown in Table 3.

[0077] Table 3. Antioxidant test results

[0078]

[0079] Table 3 shows that the blueberry seed oil samples contained a certain amount of emulsifier (Tween 40); the unfermented artichoke leaves were from the sample prepared from the "unfermented artichoke leaves" in Example 1.

[0080] Example 4

[0081] The preparation of the anti-aging composition includes the following steps:

[0082] Weigh each raw material according to the mass fractions recorded in Table 4; mix thoroughly and then seal for storage.

[0083] Table 4. Formulation of Anti-aging Compositions

[0084]

[0085] The anti-aging composition prepared in Example 4 was subjected to performance testing, specifically as follows:

[0086] (a) Allergy testing

[0087] The 10 samples obtained were tested using the human skin patch test as specified in the "2022 Cosmetic Safety Technical Specifications";

[0088] The method for closed patch testing is as follows: 50 individuals aged 18-60 years are selected, and patches with an area not exceeding 50 mm are used. 2 A qualified patch test apparatus with a depth of approximately 1 mm was used. 0.020 mL of the prepared product (diluted to a concentration of 10%) from the example was placed in the small chamber of the patch test apparatus. The control well was a blank control (without any substance). The patch test apparatus containing the prepared product was applied to the flexor side of the subject's forearm using hypoallergenic adhesive tape. Gently press with the palm of the hand to ensure even application to the skin, and leave for 24 hours. Skin reactions were observed at 30 minutes (after the indentation disappeared), 24 hours, and 48 hours after removal of the product, and the results were recorded.

[0089] All subjects showed a "-" response level, or a level 0 response; that is, a negative response, with no allergic reaction observed.

[0090] (ii) Antioxidant test

[0091] The experimental method was the same as in Example 3, and the results are shown in Table 5 below.

[0092] Table 5. Antioxidant Test Results

[0093]

[0094] (III) Test for promoting type I collagen

[0095] The testing method is as follows:

[0096] Elastic fibers, composed of elastin and microfibrils, are distributed in the dermis and subcutaneous tissue, giving the skin its elasticity. Environmental factors such as ultraviolet radiation, stress, and pollution can promote the production of elastase in the body. Elastase, a member of the chymotrypsin family, degrades elastin, causing the loss of connective tissue in the epidermis, thus leading to skin aging, wrinkles, and photoaging. This experiment used porcine pancreatic elastase as the research subject and N-succinyl-alanine-alanine-p-nitroaniline (AAAPVN) as the substrate. Porcine pancreatic elastase can hydrolyze AAAPVN, and its hydrolysis products can cause an increase in absorbance at a wavelength of 420 nm. Measuring the absorbance using an enzyme-linked immunosorbent assay (ELISA) reader allows for the evaluation of the skin's firming and anti-wrinkle effects.

[0097] 1. Solution preparation

[0098] (1) Sample concentration setting: The anti-aging composition prepared in Example 4 was selected and the test concentration was 1%.

[0099] (2) Prepare Tris-HCl buffer (0.1M pH=8.0): Weigh 2.42g Tris into a beaker, add 200mL of ultrapure water, dissolve completely, and then adjust the pH to 8.0 with concentrated HCl.

[0100] (3) Preparation of positive control tea polyphenol solution (1 mg / ml, 0.1%): Weigh 5 mg of tea polyphenol and dissolve it in 5 mL of Tris-HCl buffer.

[0101] (4) Prepare substrate solution AAAPVN (2mM): Weigh 4.51mg N-succinyl-alanine-alanine-alanine-p-nitroaniline and dissolve it in 5ml Tris-HCl buffer.

[0102] (5) Prepare porcine pancreatic elastase solution (0.171 U / mL): Dissolve 280 μL in 10 mL Tris-HCl buffer.

[0103] 2. Sample addition

[0104] The experiment was divided into four groups: sample group, positive control group, blank control group, and model control group. Each group had four replicates at the same concentration. The amounts of each solution added are shown in Table 6.

[0105] Table 6

[0106]

[0107] 3. Measurement

[0108] The reaction was allowed to proceed at room temperature for 15 minutes, and the absorbance was measured at 420 nm using an ELISA reader.

[0109] 4. Result Calculation

[0110]

[0111] Where: A0 -- the average absorbance of the blank control wells;

[0112] A1 -- The average absorbance of the sample well;

[0113] A2 -- The average absorbance of the model control well.

[0114] 5. Data Statistics

[0115] Statistical analysis was performed using SPSS 22.0 statistical software to conduct descriptive statistics on the measured values ​​in the test area. Changes in the analyzed values ​​and differences between the control group and the sample group were calculated. If the test data were normally distributed, the independent t-test was used for statistical analysis; if the test data were not normally distributed, the rank-sum test was used. All statistical methods employed two-tailed tests with a significance level of α=0.05.

[0116] 6. Result Determination

[0117] Compared with the model control group, the absorbance of the sample group showed a significant positive difference, indicating that the tested sample had an inhibitory effect on porcine pancreatic elastase. Conversely, if the absorbance was significantly lower, the sample group had no such effect.

[0118] 7. The experimental results are shown in Table 7.

[0119] Table 7 Results of detection of porcine pancreatic elastase inhibition rate

[0120]

[0121] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. An anti-aging and repairing composition containing collagen, characterized in that, It consists of the following components, calculated by parts by weight: Hydrolyzed collagen 2-5 Recombinant Type III Human Collagen 1-3 artichoke leaf extract 5-10 Fermented blueberry seed oil 25-50 The preparation method of the artichoke leaf extract is as follows: take fresh artichoke leaves, wash and crush them; enzymatically hydrolyze them using pectinase and cellulase; inactivate the enzymes after enzymatic hydrolysis, filter and collect the filtrate; inoculate the filtrate with activated Lactobacillus plantarum, and anaerobic ferment it at 36-38℃ and pH 5.5-6.0 for 45 hours; sterilize after fermentation, centrifuge and collect the supernatant, add ethanol to the supernatant, and then pass it through an 800Da filter membrane. Collect the filtered solution and dry it into a solid. The preparation method of the fermented blueberry seed oil is as follows: take blueberry seed oil, mix and emulsify it with aqueous culture medium to prepare a fermentation substrate, and inoculate it with activated bumblebee Candida albicans; then ferment at 28-32℃, pH 5.0-6.0, OD value of at least 40% for at least 72h; after fermentation, sterilize, centrifuge and take the upper oil phase; after dehydration of the oil phase, molecular distill to collect the main fraction at 160-180℃.

2. The anti-aging and repairing composition containing collagen according to claim 1, characterized in that, When using pectinase and cellulase for enzymatic hydrolysis, the temperature should be controlled at 45-50℃, the pH at 4.5-5.0, and the hydrolysis time at least 2 hours.

3. The anti-aging and repairing composition containing collagen according to claim 1, characterized in that, During the anaerobic fermentation of the filtrate, auxiliary carbon source, auxiliary nitrogen source, dipotassium hydrogen phosphate, manganese sulfate and emulsifier are added.

4. The anti-aging and repairing composition containing collagen according to claim 1, characterized in that, The aqueous culture medium contains at least the following components by mass percentage: At least 1% yeast extract; At least 2% glucose At least 1% peptone At least 0.1% magnesium sulfate At least 2.5% nonionic lipophilic emulsifier.

5. The anti-aging and repairing composition containing collagen according to claim 1, characterized in that, It also includes a moisturizer; the moisturizer includes at least one of glycerin, beta-glucan, sodium hyaluronate, and polyols.

6. The use of the collagen-containing anti-aging and repairing composition according to any one of claims 1-5 in the preparation of cosmetics.

7. The application in the preparation of cosmetics according to claim 6, characterized in that, The minimum addition amount is 1 wt%.

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