Collagen-containing anti-aging repairing composition and preparation method thereof

Abstract

The invention provides an anti-aging repairing composition containing collagen and a preparation method of the anti-aging repairing composition, and belongs to the technical field of medical, dental or dressing preparations. The collagen composition is composed of hydrolyzed collagen, recombinant III-type human collagen, cynara scolymus leaf extract and fermented bilberry seed oil. Recombinant III-type human collagen and hydrolyzed collagen are taken as cores, the limitation of traditional collagen is broken through through a biotechnology, the bottleneck of transdermal penetration and oxidation resistance is solved with the assistance of double-fermentation plant components, and the overall design conforms to the anti-aging triangular logic of'repairing, defending and moisturizing '; good market prospects are realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical, dental or cosmetic preparations, and particularly relates to an anti-aging and repair composition containing collagen and a preparation method thereof. BACKGROUND

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

[0003] Exogenous aging is dominant, of which ultraviolet light (photoaging) accounts for more than 80% of the inducement: ultraviolet light (especially UVA) penetrates the dermis, destroys the collagen structure, activates matrix metalloproteinase (MMP) to accelerate collagen degradation, and induces oxidative stress. Other external factors such as PM2.5 pollutants, staying up late, smoking, etc. penetrate the skin to trigger an inflammatory response, exacerbating free radical accumulation and barrier damage.

[0004] Skin care products delay aging in multiple dimensions from protection, repair to regeneration by targeting the aging pathway: Sunscreen: the first line of defense against photoaging, sunscreen agents (physical / chemical) resist UVA / UVB, reduce DNA damage and collagen breakdown.

[0005] Moisturizing and barrier repair: contains ingredients that enhance the water retention capacity of the stratum corneum to prevent wrinkles caused by dryness.

[0006] Antioxidants: ingredients such as vitamin C / E, coenzyme Q10, astaxanthin, etc. scavenge reactive oxygen species (ROS), block the oxidative stress chain reaction, and protect collagen and cellular DNA.

[0007] Promote collagen regeneration and metabolic renewal of retinol: stimulate fibroblast proliferation, accelerate collagen synthesis, and repair photoaging wrinkles.

[0008] Anti-inflammatory and cell protection: such as green tea extract inhibits inflammatory factors (IL-1, TNF-α), and blocks the inflammation-oxidation vicious cycle. SUMMARY

[0009] The purpose of the present application is to provide an anti-aging and repair composition containing collagen, which has excellent anti-aging effect; contains the following ingredients by mass fraction: Hydrolyzed collagen 2-5 Recombinant human collagen type III 1-3 Chrysanthemum leaf extract 5-10 Fermented lingonberry seed oil 25-50 The artichoke leaf extract is obtained by enzymatically hydrolyzing the artichoke leaves, fermenting them with plant lactic acid bacteria, and then purifying them. The fermented bilberry seed oil is obtained by purifying bilberry seed oil fermented with bumblebee Candida.

[0010] Preferably, the preparation method of the artichoke leaf extract is as follows: taking fresh artichoke leaf parts, washing and crushing them; enzymatically hydrolyzing them with pectinase and cellulase; inactivating the enzymes after enzymatic hydrolysis, and filtering and collecting the filtrate; The filtrate is inoculated with activated Lactobacillus plantarum and anaerobically fermented at 36-38°C and pH 5.5-6.0 for at least 48 hours; After fermentation, the mixture is sterilized and centrifuged to obtain the supernatant, ethanol is added to the supernatant, and then the supernatant is passed through an 800Da filter membrane. The filtered solution is then dried to form a solid.

[0011] Preferably, when enzymatic hydrolysis is performed using pectinase and cellulase, the temperature is controlled at 45-50° C. and the pH is 4.5-5.0.

[0012] 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.

[0013] Preferably, the fermented cranberry seed oil is prepared by: taking cranberry seed oil, mixing it with an aqueous medium to emulsify it into a fermentation substrate and inoculating it with activated Candida bumblebee; then fermenting it at 28-32°C, pH 5.0-6.0, and an OD value of at least 40% for at least 72h; After fermentation, the mixture is sterilized and centrifuged to obtain the upper oil phase; the oil phase is dehydrated and then molecularly distilled to collect the main fraction at 160-180°C.

[0014] Preferably, the aqueous culture medium contains at least the following components in percentage by mass: At least 1% yeast extract; At least 2% glucose At least 1% peptone At least 0.1% magnesium sulfate At least 2.5% non-ionic lipophilic emulsifier.

[0015] Preferably, the aforementioned anti-aging repair composition containing collagen further comprises a moisturizer; the moisturizer comprises at least one of glycerin, β-glucan, sodium hyaluronate, and polyol.

[0016] The present invention also provides the use of the aforementioned anti-aging and repairing composition containing collagen in the preparation of cosmetics; the minimum addition amount is 1 wt %. Specific dosage forms of the cosmetics include, but are not limited to, essences, essence oils, creams, and lotions.

[0017] This invention utilizes two different collagens for complementary effects. Hydrolyzed collagen has a low molecular weight and strong penetrating power, directly providing raw materials for the synthesis of skin fibroblasts, thereby enhancing elastin production. Recombinant human type III collagen is 100% identical to the human amino acid sequence, eliminating the risk of immune rejection. Its unique, flexible triple-helix structure allows for direct cell adhesion, filling collapsed areas of the dermis, improving fine lines and sagging, and promoting epidermal cell migration and proliferation.

[0018] Cardoon leaves contain cynarin and flavonoids (such as quercetin and kaempferol), which have potent anti-inflammatory, soothing, and barrier-repairing effects. Specifically, they inhibit activation of the NF-κB inflammatory pathway, reduce histamine release, and alleviate allergic reactions such as skin redness and swelling. The flavonoids and polyphenols rich in them can scavenge free radicals, reduce UV-induced oxidative damage, and slow collagen degradation. The present inventors discovered that these effects are further enhanced after fermentation with Lactobacillus plantarum.

[0019] Bilberry seed oil's primary benefits are its powerful antioxidant and lipid-repair properties. Rich in anthocyanins, it neutralizes free radicals induced by UV rays and pollution. Its oily components are rich in omega-3 and omega-6, mimicking the skin's natural lipid structure to rapidly repair barrier defects and improve dryness and redness.

[0020] Oleophilic microorganisms are microorganisms that can utilize lipids. They use lipids as nutrients, using their own enzyme systems (such as lipase) to break down or reassemble lipids into other metabolites. Starmerella bombicola can tolerate low-water, high-sugar environments and actively decompose, process, and reuse lipids. This strain thrives in an environment composed of esters, wax (honeycomb), and sugar (honey). This unique environment allows them to effectively utilize lipids and sugars, metabolizing them into beneficial substances such as glycolipids, amino acids, small molecule sugars, fatty acids, and ceramides. In this invention, fermentation with Starmerella bombicola is used to convert high-molecular-weight components in bilberry seed oil that are difficult to absorb transdermally into low-molecular-weight components, thereby improving transdermal absorption. Molecular distillation is then preferably used to purify the oil, removing large molecules and fermentation impurities that are not transdermally absorbable, while retaining the lightweight oil in the 160-180°C fraction, resulting in a refreshing skin feel and enhanced permeability.

[0021] In summary, the present invention takes recombinant human type III collagen and hydrolyzed collagen as its core, breaks through the limitations of traditional collagen through biotechnology, and uses double-fermented plant ingredients to solve the bottlenecks of transdermal penetration and anti-oxidation. The overall design conforms to the anti-aging triangle logic of "repair, defense, and moisturizing" and has good market prospects. DETAILED DESCRIPTION

[0022] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0023] In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include one or more of the specified features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0024] In the description of the present invention, the term "for example" is used to mean "used as an example, illustration or illustration". Any embodiment of the present invention described as "for example" is not necessarily to be construed as being more preferred or advantageous than other embodiments. The following description is given to enable any person skilled in the art to implement and use the present invention. In the following description, details are listed for the purpose of explanation. It should be understood that a person of ordinary skill in the art can recognize that the present invention can be implemented without using these specific details. In other examples, well-known structures and processes are not elaborated in detail to avoid obscuring the description of the present invention with unnecessary details. Therefore, the present invention is not intended to be limited to the embodiments shown, but is consistent with the widest scope consistent with the principles and features disclosed herein.

[0025] Unless otherwise specified, the experimental methods used in the specific embodiments are all conventional methods, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0026] In the present invention, unless otherwise specified, “%” represents mass percentage; the raw materials, reagents, etc. used are all conventional commercially available products.

[0027] The sources of some raw materials used in the present invention are shown in Table 1 below: Table 1

[0028] Example 1 Fermentation experiment of cardoon leaves Different strains of bacteria were used to ferment artichoke leaves, and the antioxidant properties of the fermentation products of different fermentation strains were compared. The preparation method includes the following steps: S1: Select fresh cardoon leaves, wash and remove sediment; drain and crush at low temperature to a particle size of 10 mesh; S2: Prepare a mixture of cellulase (20 U / g artichoke leaves) and pectinase (15 U / g artichoke leaves). Adjust the solid-liquid ratio to 1:8 (using water) and perform enzymatic hydrolysis at 45-50°C and pH 4.5-5.0 (using citric acid) for 2 h. After enzymatic hydrolysis, the temperature was raised to 85°C and maintained for 10 minutes to inactivate the enzyme; the filtrate was collected by centrifugation (3000 g, 15 minutes).

[0029] S3: Prepare the substrate according to the ratio in Table 2 and add an appropriate amount of sterile water to adjust the water content; prepare the activated bacteria into 1×10 7 -1×10 8 CFU / mL suspension was inoculated at a ratio of 5% and then cultured according to the conditions in Table 2.

[0030] S4: After the fermentation is completed, the fermentation is sterilized and centrifuged to obtain the supernatant. Ethanol (absolute ethanol, the addition amount is 5%) is added to the supernatant, and then it is passed through an 800Da filter membrane. The filtered solution is taken and dried to a solid.

[0031] Table 2

[0032] Another method for preparing unfermented artichoke leaves includes the following steps: S1: Select fresh cardoon leaves, wash and remove sediment; drain and crush at low temperature to a particle size of 10 mesh; S2: Prepare a mixture of cellulase (20 U / g artichoke leaves) and pectinase (15 U / g artichoke leaves). Adjust the solid-liquid ratio to 1:8 (using water) and perform enzymatic hydrolysis at 45-50°C and pH 4.5-5.0 (using citric acid) for 2 h. After enzymatic hydrolysis, the temperature was raised to 85°C and maintained for 10 minutes to inactivate the enzyme; the filtrate was collected by centrifugation (3000 g, 15 minutes).

[0033] S3: The filtrate was centrifuged to obtain the supernatant, ethanol (absolute ethanol, added in an amount of 5%) was added to the supernatant, and then the supernatant was passed through an 800Da filter membrane. The filtered solution was dried to a solid.

[0034] Example 2 Study on the properties of blueberry seed oil In skin care products, there are three types of Vaccinium seed oil that have been used in records: Vaccinium macrocarpon seed oil (also known as cranberry), Vaccinium myrtillus seed oil (also known as black bilberry), and Vaccinium vitis-idaea seed oil (also known as red bilberry). The preparation of fermented oil comprises the following steps: S1: Different oils were mixed with aqueous medium in a 1:1 volume ratio and homogenized by a microfluidizer, and then sterilized by autoclaving at 121°C for 20 min to prepare the fermentation substrate. The aqueous medium contains the following ingredients: 1% yeast extract, 2% glucose, 1% peptone, 0.1% magnesium sulfate, 2.5% Span 80; the balance is deionized water; S2: fermentation of substrate and inoculation with activated Candida bombus; Fermentation was carried out at 28-32°C, pH 5.0-6.0, OD value ≥ 40%, and aeration rate of 0.6 vvm for 72 h; S3: After fermentation, the mixture was inactivated at 100°C for 15 minutes, followed by centrifugation at 9000 g for 20 minutes to collect the upper oil phase. The oil phase was vacuum dehydrated, and after dehydration, the main fraction at 160–180°C was collected by molecular distillation.

[0035] Example 3 Antioxidant Performance Test The test method is as follows: DPPH methanol solution is violet and has a strong absorbance at 517nm. If it combines with the sample, the absorbance value at 517nm will be reduced, thereby judging the ability of the sample to scavenge DPPH free radicals.

[0036] The specific method is as follows: (1) Take 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 -4 mol / L DPPH solution was mixed (A1); (2) Take an equal volume of anhydrous ethanol (solvent for the test substance) and 2×10 -4 mol / L DPPH solution and mix well (A2); (3) Take an equal volume of anhydrous ethanol and mix it with the test solution (A3); (4) After 40 minutes of reaction, measure the absorbance of tubes A1, A2, and A3 at 517 nm.

[0037] The formula for calculating the clearance rate is: Clearance rate (%) = [1-(A1-A3) / A2] × 100% The test results are shown in Table 3.

[0038] Table 3 Antioxidant test results

[0039] The blueberry seed oil samples in Table 3 contain a certain amount of emulsifier (Tween 40); the unfermented artichoke leaves are from the sample prepared in the "unfermented artichoke leaves" in Example 1.

[0040] Example 4 The preparation of the anti-aging composition comprises the following steps: Weigh each raw material according to the mass parts recorded in Table 4 below; mix well and seal for storage.

[0041] Table 4 Anti-aging composition ratio

[0042] The performance test of the anti-aging composition prepared in Example 4 was carried out, specifically: (1) Allergy test The 10 groups of products obtained were subjected to human skin patch tests in accordance with the 2022 Cosmetic Safety Technical Specifications; The method of skin occlusion patch test is: select 50 people aged 18-60 years old, and select an area not exceeding 50mm 2 , a qualified patch test device with a depth of approximately 1mm. Place 0.020 mL of the finished product prepared in Example (diluted to a 10% concentration) into the patch test chamber. The control well serves as a blank control (no substance). Apply the patch test device containing the finished product to the flexed side of the subject's forearm using hypoallergenic tape. Apply light pressure with the palm of your hand to evenly adhere the patch to the skin for 24 hours. Observe the skin for standard reactions 30 minutes after removal of the finished product (after the indentation disappears), 24 hours, and 48 hours afterward, and record the results.

[0043] The reaction level of all subjects was "-", level 0 reaction; that is, negative reaction, no allergic reaction occurred.

[0044] (2) Antioxidant test The test method is the same as that of Example 3, and the results are shown in Table 5 below.

[0045] Table 5 Antioxidant test results

[0046] (3) Promoting Type I Collagen Test The test method is as follows: Elastic fibers, composed of elastin and microfibrils, are distributed throughout the dermis and subcutaneous tissue, contributing to the skin's elasticity. Environmental factors such as UV radiation, stress, and pollution stimulate the production of elastase. Elastase, a member of the chymotrypsin family, degrades elastin, leading to the loss of connective tissue in the epidermis and contributing to skin aging, wrinkles, and photoaging. This study investigated porcine pancreatic elastase, using N-succinyl-alanine-alanine-alanine-p-nitroanilide (AAAPVN) as a substrate. Porcine pancreatic elastase hydrolyzes AAAPVN, and the resulting hydrolysis products increase absorbance at 420 nm. This absorbance is measured using a microplate reader to assess the firming and anti-wrinkle efficacy of the test samples.

[0047] 1. Solution Preparation (1) Sample concentration setting: The anti-aging composition prepared in Example 4 was selected, and the test concentration was 1%.

[0048] (2) Prepare Tris-HCl buffer (0.1 M pH = 8.0): Weigh 2.42 g of Tris into a beaker, add 200 mL of ultrapure water, and adjust the pH to 8.0 with concentrated HCl after complete dissolution.

[0049] (3) Preparation of positive control tea polyphenol solution (1 mg / ml, 0.1%): Weigh 5 mg of tea polyphenols and dissolve it in 5 mL of Tris-HCl buffer.

[0050] (4) Prepare substrate solution AAAPVN (2 mM): Weigh 4.51 mg of N-succinyl-alanine-alanine-alanine-p-nitroaniline and dissolve it in 5 ml of Tris-HCl buffer.

[0051] (5) Prepare porcine pancreatic elastase solution (0.171 U / mL): Dissolve 280 μl in 10 ml Tris-HCl buffer.

[0052] 2. Add sample The experiment was divided into four groups: sample group, positive control group, blank control group, and model control group. Four replicate wells were set up for each group at the same concentration. The amount of each solution added is shown in Table 6.

[0053] Table 6

[0054] 3. Determination The reaction was allowed to proceed at room temperature for 15 min, and the absorbance was measured at 420 nm using a microplate reader.

[0055] 4. Calculation of results

[0056] Where: A0-average absorbance of blank control wells; A1-average absorbance of sample wells; A2 - average absorbance of model control wells.

[0057] 5. Statistics Statistical analysis was performed using the SPSS 22.0 statistical software package. Descriptive statistics were compiled for the measured values ​​in the test areas. Changes in values ​​and differences between the control and sample groups were calculated and analyzed. If the test data showed a normal distribution, the independent T-test was used for statistical analysis; if the test data showed a non-normal distribution, the rank sum test was used for statistical analysis. Two-tailed tests were used for all statistical analysis, with an α level of 0.05.

[0058] 6. Result determination Compared with the model control group, the absorbance of the sample group showed a significant positive difference, indicating that the tested sample had the effect of inhibiting porcine pancreatic elastase. Otherwise, there was no such effect.

[0059] 7. Experimental results are shown in Table 7.

[0060] Table 7 Results of porcine pancreatic elastase inhibition rate test

[0061] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. An anti-aging repair composition containing collagen, characterized in that: Contains the following ingredients by mass: Hydrolyzed collagen 2-5 Recombinant human collagen type III 1-3 Artichoke Leaf Extract 5-10 Fermented bilberry seed oil 25-50 The artichoke leaf extract is obtained by enzymatically hydrolyzing the artichoke leaves, fermenting them with plant lactic acid bacteria, and then purifying them. The fermented bilberry seed oil is obtained by purifying bilberry seed oil fermented with bumblebee Candida.

2. The anti-aging repair composition containing collagen according to claim 1, characterized in that: The preparation method of the artichoke leaf extract comprises the following steps: taking fresh artichoke leaf parts, washing and crushing them; performing enzymatic hydrolysis using pectinase and cellulase; inactivating the enzymes after enzymatic hydrolysis, and filtering and collecting the filtrate; The filtrate is inoculated with activated Lactobacillus plantarum and anaerobically fermented at 36-38°C and pH 5.5-6.0 for at least 48 hours; After fermentation, the mixture is sterilized and centrifuged to obtain the supernatant, ethanol is added to the supernatant, and then the supernatant is passed through an 800Da filter membrane. The filtered solution is then dried to form a solid.

3. The anti-aging repair composition containing collagen according to claim 2, characterized in that: When using pectinase and cellulase for enzymatic hydrolysis, the temperature is controlled at 45-50°C, pH 4.5-5.0, and the enzymatic hydrolysis time is at least 2 hours.

4. The anti-aging and repairing composition containing collagen according to claim 2, characterized in that: 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.

5. The anti-aging and repairing composition containing collagen according to claim 1, characterized in that: The fermented bilberry seed oil preparation method is: take bilberry seed oil, mixed with an aqueous medium emulsified to prepare a fermentation substrate and inoculated with activated bumblebee Candida; then fermented at 28-32 ° C, pH 5.0-6.0, OD value of at least 40% for at least 72h; After fermentation, the mixture is sterilized and centrifuged to obtain the upper oil phase; the oil phase is dehydrated and then molecularly distilled to collect the main fraction at 160-180°C.

6. The anti-aging and repairing composition containing collagen according to claim 5, characterized in that: The aqueous culture medium contains at least the following components in percentage by mass: At least 1% yeast extract; At least 2% glucose At least 1% peptone At least 0.1% magnesium sulfate At least 2.5% non-ionic lipophilic emulsifier.

7. The anti-aging and repairing composition containing collagen according to claim 1, characterized in that: It also includes a moisturizing agent; the moisturizing agent includes at least one of glycerin, β-glucan, sodium hyaluronate, and polyol.

8. Use of the anti-aging and repairing composition containing collagen according to any one of claims 1 to 7 in the preparation of cosmetics.

9. The use in preparing cosmetics according to claim 8, characterized in that: The minimum addition amount is 1wt%.

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