Anti-aging composition containing collagen and preparation method and application thereof

Abstract

The present application relates to the technical field of collagen cosmetic, in particular to an anti-aging composition containing collagen, and a preparation method and application thereof. The anti-aging composition comprises recombinant human collagen type III and plant active ingredients, wherein the plant active ingredients at least include any one of fruiting body extract of NAEMATELIA AURANTIALBA and extract of GENTIANA TRIFLORA, and the anti-aging composition further comprises an auxiliary agent, wherein the auxiliary agent at least includes any one or more of trehalose, mannitol, carbomer, hydroxyethyl cellulose, glycerol, butanediol and pentanediol. The anti-aging composition can not only achieve a synergistic effect in terms of anti-aging effect, but also solve the problem of insufficient stability of recombinant human collagen type III to a certain extent through reasonable compounding of components.

Description

Technical Field

[0001] This invention relates to the field of collagen-based cosmetics technology, and more particularly to an anti-aging composition containing collagen, its preparation method, and its application. Background Technology

[0002] In today's society, with people paying close attention to beauty and health, anti-aging has become a research hotspot. Collagen, as an important biomaterial, has significant effects on skin anti-aging. Collagen is the main structural protein of human skin, accounting for 70%-80% of the total skin protein. It can maintain the elasticity and firmness of the skin, reduce the formation of wrinkles, and keep the skin youthful.

[0003] However, traditional single-component collagen has certain limitations in anti-aging applications. First, collagen itself is relatively unstable and easily degraded or denatured by external factors such as temperature, light, and enzymes, leading to reduced efficacy. Second, single-component collagen has relatively limited effects on promoting skin cell regeneration, anti-oxidation, and repairing damaged skin tissue, failing to comprehensively meet the demand for integrated anti-aging effects. Furthermore, using collagen alone is insufficient to form a complete anti-aging system, unable to address the multiple mechanisms of skin aging from multiple angles. Skin aging is a complex physiological process involving the combined effects of free radical damage, slowed cell metabolism, loss of collagen and elastin, and impaired skin microcirculation. Simply supplementing collagen quantity is unlikely to fundamentally reverse the aging process. Therefore, collagen-containing compositions have become a key focus of current research and development.

[0004] Currently, the main sources of collagen on the market include animal-derived and genetically engineered sources. Animal collagen has high bioactivity, is extracted from natural biological tissues, and possesses a complete and stable triple helix structure with high homology and compatibility with the human body. However, its disadvantages include high immunogenicity and a higher risk of viral infection. Recombinant collagen, on the other hand, is prepared by genetic engineering. It is obtained by constructing human collagen DNA into bacteria such as E. coli and yeast and then fermenting it. It features high water solubility, low allergenicity, and controllable mass production, reducing the occurrence of immune rejection and eliminating viral risks. However, the drawback of recombinant collagen is that its main functional triple helix structure is not stable enough, limiting its use in certain scenarios.

[0005] While existing technologies have explored various collagen-containing composite anti-aging / repair compositions, such as patent CN116617144B which combines collagen, modified sodium hyaluronate, and eucalyptus / cardamom extracts to promote the penetration of active ingredients and achieve safe, non-irritating moisturizing and repair; patent CN118787723B which uses fish collagen powder as the main component, combined with astaxanthin powder and amomum / lophatherum extracts, focusing on scavenging free radicals and improving skin elasticity; and patent CN119367238B which further combines deer bone collagen and tripeptide-1 extracts with active ingredients such as *Desmodium styracifolium* extract and *Vermicomyces cerevisiae* extract, emphasizing synergistic effects to promote collagen regeneration, these technologies, while addressing the synergistic effect of collagen to some extent, still have limitations. In particular, they do not specifically address the core issue of insufficient collagen stability. Therefore, more comprehensive and stable composite anti-aging compositions are still needed to meet market demands. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide an anti-aging composition containing collagen, its preparation method, and its application. This composition not only has a synergistic effect in anti-aging, but also, through the rational compounding of its components, can to a certain extent solve the problem of insufficient stability of recombinant type III human collagen.

[0007] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an anti-aging composition containing collagen, comprising:

[0009] Recombinant type III human collagen and plant active ingredients, wherein the plant active ingredients include at least one of the following: extract of the fruiting body of *Naemonella aurantilia* and extract of *Gentiana trifoliata*.

[0010] Preferably, the plant active ingredient is a combination of extracts from the fruiting body of *Auricularia aurantii* and extracts from *Gentiana trifoliata*.

[0011] Preferably, the composition further includes an adjuvant, and the adjuvant includes at least one or more of trehalose, mannitol, carbomer, hydroxyethyl cellulose, glycerol, butylene glycol, and pentanediol.

[0012] Preferably, the collagen-containing anti-aging composition comprises, by weight parts:

[0013] 1-10 parts of recombinant type III human collagen, 10-20 parts of Naematelia aurantialba fruiting body extract, 10-20 parts of Gentiana triflora extract, and 5-20 parts of adjuvants.

[0014] Preferably, the extract of Auricularia auricula-judae fruiting body is prepared by the following method: after pulverizing the fruiting body of Auricularia auricula-judae, it is extracted with 5-10 times the amount of 70% ethanol solution at 55-65℃ for 2-3 times, each time for 2 hours. The extracts are combined, concentrated under reduced pressure, and then freeze-dried to obtain the extract of Auricularia auricula-judae fruiting body.

[0015] Preferably, the preparation method of the Gentiana triflora extract is as follows:

[0016] After pulverizing the dried whole herb of Gentiana triflora, add 60% methanol solution at a material-to-liquid ratio of 1:10-15 (g / mL), and extract under ultrasonic power of 300-500W for 30-45 minutes. Filter and collect the extract, purify it with HPD-600 macroporous resin, use 30% ethanol as eluent, collect the target fraction, and dry it to obtain Gentiana triflora extract.

[0017] Furthermore, in the preparation of the Gentiana triflora extract, the collection method for this target fraction is as follows: elution is first performed using 1-2 times the volume of pure water in a macroporous resin column, with 30% ethanol as the eluent. The ethanol eluent is collected, concentrated under reduced pressure, and then freeze-dried to obtain the Gentiana triflora extract. In this preparation method, the anti-aging effect of the extract is ensured by efficiently enriching active substances such as iridoids (represented by gentiopicrin) and flavonoids.

[0018] Preferably, in the collagen-containing anti-aging composition, the adjuvant is composed of the following components in parts by weight: 2-8 parts trehalose, 1-6 parts mannitol, 1-4 parts carbomer, and 3-10 parts glycerol. In the adjuvant, trehalose and mannitol, as small molecule sugar alcohols, can protect the molecular structure of collagen and plant active ingredients through hydrogen bonding.

[0019] Secondly, the present invention also provides a method for preparing the aforementioned collagen-containing anti-aging composition, characterized by comprising the following steps:

[0020] S1: Preparation of premixed solvent: Add 50-70% by mass of deionized water to the reaction vessel, heat to 35-45°C, and control the speed at 200-300 rpm while stirring; slowly add the auxiliary agent and continue stirring for 20-30 minutes until completely dissolved to obtain the basic solution;

[0021] S2: Plant extract dispersion: Add the plant active ingredients to the base solution of step S1, reduce the stirring speed to 150-200 rpm, and continue stirring at 35-40℃ for 15-25 minutes to fully disperse the plant extract and make it compatible with the base solution, thus obtaining a mixed system;

[0022] S3: Adding recombinant collagen: Lower the temperature of the mixture to 25-30℃, add recombinant type III human collagen and replenish deionized water, adjust the stirring speed to 100-150 rpm, and stir at low speed for 10-15 minutes until completely dispersed;

[0023] S4: Adjust the pH to 5.5-6.8 and perform sterile filtration to obtain the collagen-containing anti-aging composition.

[0024] Thirdly, this aspect also provides the application of the collagen-containing anti-aging composition provided in the first aspect above in cosmetic processing.

[0025] Preferably, in the application, the cosmetic is a leave-on cosmetic, and the amount of the collagen-containing anti-aging composition added to the cosmetic is 0.1%-5% wt.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention provides an anti-aging composition containing collagen. Regarding the plant-based active ingredients, the preferred combination is a synergistic blend of *Naematelia aurantialba* fruiting body extract and *Gentiana triflora* extract. The *Naematelia aurantialba* fruiting body extract is obtained through a specific concentration of ethanol, temperature, and multiple extraction processes, ensuring efficient extraction of the active ingredients. The *Gentiana triflora* extract is purified using ultrasound-assisted macroporous resin and a precise fraction collection method based on the characteristic peak of gentianin, ensuring high purity and high activity of the extract, providing a solid guarantee for the anti-aging efficacy of the composition. In this combination, the *Naematelia aurantialba* fruiting body extract is rich in polysaccharides and flavonoids, possessing strong antioxidant capabilities, scavenging free radicals, and delaying cell aging. The gentianin in the *Gentiana triflora* extract has anti-inflammatory and repairing properties, accelerating skin cell metabolism. The combination of these three ingredients works from multiple dimensions, including nutritional supplementation, antioxidant effects, and anti-inflammatory repair, achieving highly effective anti-aging.

[0028] Meanwhile, the plant active ingredients and adjuvants provided by this invention also play a key stabilizing role in the composition. The preferred composition of the adjuvant is: the adjuvant is composed of 2-8 parts of trehalose, 1-6 parts of mannitol, 1-4 parts of carbomer and 3-10 parts of glycerol. The components of the adjuvant can effectively enhance the synergistic stability between the components, extend the shelf life of the product, and ensure the quality stability of the product during storage and use.

[0029] Finally, the collagen-containing anti-aging composition provided by this invention has broad application prospects in the cosmetics processing field, especially suitable for the development of leave-on cosmetics. In leave-on cosmetics such as lotions, creams, and serums, the recommended addition amount of this anti-aging composition is 0.1%-5% (wt%). In practical applications, the addition amount can be reasonably adjusted within this range according to the specific formulation system and expected efficacy of the cosmetic, so that it can effectively exert its anti-aging effects while being compatible with various cosmetic systems. It can be used in various products such as creams, serums, and masks to meet the needs of different consumers, and has great market application potential. Detailed Implementation

[0030] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are commercially available conventional products.

[0031] To better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art will understand that the present invention can be practiced without certain specific details. In other embodiments, methods, means, apparatus, and steps well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include unavoidable errors in industrial production.

[0033] Example 1

[0034] This embodiment provides an anti-aging composition containing collagen, which, by weight parts, comprises:

[0035] Three portions of recombinant type III human collagen, ten portions of extract from the fruiting body of Auricularia auricula-judae, and ten portions of extract from Gentiana triflora;

[0036] Meanwhile, this embodiment also provides a method for preparing the aforementioned collagen-containing anti-aging composition, wherein the anti-aging composition uses 3 parts of recombinant type III human collagen, 10 parts of *Auricularia auricula-judae* fruiting body extract, and 10 parts of *Gentiana triflora* extract as active ingredients, and the total concentration of active ingredients is 3 mg / mL, including the following steps:

[0037] S1: Preparation of premixed solvent:

[0038] Add 50-70% by mass of deionized water to the reactor, heat to 40°C, and control the rotation speed at 300 rpm while stirring.

[0039] S2: Plant extract dispersion:

[0040] (1) Preparation of extract from fruiting body of Auricularia auricula-judae:

[0041] Take dried fruiting bodies of *Auricularia auricula-judae*, pulverize them to 40-60 mesh, add 70% ethanol solution at a material-to-liquid ratio of 1:10 (g / mL), control the temperature at 60℃, stir and extract twice, each time for 2 hours, and collect the extracts from the two extractions; combine the extracts, concentrate them under reduced pressure using a rotary evaporator, and then transfer the concentrate to a freeze dryer for freeze drying to obtain the fruiting body extract of *Auricularia auricula-judae*.

[0042] (2) Preparation of Gentiana triflora extract:

[0043] Raw material preparation: The dried whole herb of Gentiana triflora was pulverized and passed through an 80-mesh sieve. 60% methanol solution was added at a material-to-liquid ratio of 1:12 (g / mL), and the mixture was extracted for 40 minutes at an ultrasonic power of 400W and an ultrasonic frequency of 20kHz. The extract was then filtered, collected, and concentrated to 1 / 3 of the original extract volume. Then, it was purified using HPD-600 macroporous resin. The purification process involved eluting with twice the volume of pure water of the macroporous resin column to remove impurities, followed by elution with 30% ethanol. The ethanol eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain the Gentiana triflora extract.

[0044] (3) Weigh the plant active ingredients according to the mass ratio in the formula. In this embodiment, the plant active ingredients are composed of extracts of Auricularia auricula-judae fruit and extracts of Gentiana triflora in a 1:1 ratio. After mixing the two, add them to the deionized water in step S1, reduce the stirring speed to 200 rpm, and continue stirring at 40°C for 20 minutes to fully disperse the plant extracts and make them compatible with the deionized water, thus obtaining a mixed system.

[0045] S3: Added recombinant collagen:

[0046] Lower the temperature of the mixture to 30°C, add recombinant type III human collagen according to the mass fraction, and add deionized water. Adjust the stirring speed to 100-150 rpm and stir at low speed for 10-15 minutes until completely dispersed.

[0047] S4: Adjust the pH to 5.5-6.8, perform sterile filtration, and obtain the collagen-containing anti-aging composition, which is recorded as experimental group 1.

[0048] Experimental Example 1

[0049] This experimental example, based on the aforementioned Example 1, sets up several different anti-aging compositions containing collagen and tests their efficacy, specifically including:

[0050] 1.1 Experimental Grouping

[0051] This experiment set up multiple experimental groups, and the composition of the effective components in each experimental group is shown in Table 1:

[0052] Table 1. Composition of effective components in the compositions of each experimental group

[0053]

[0054] 1.2 Efficacy Test and Test Results

[0055] The test samples were prepared according to the composition of each experimental group above. The specific method was as follows: each component was weighed according to the mass ratio of the formula composition, and dissolved in water to prepare a test sample with a total effective ingredient concentration of 3 mg / mL for testing.

[0056] Experimental results were analyzed using SPSS, and all data are expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons between groups. If the ANOVA results showed a statistically significant difference (P < 0.05), Tukey's test was used for further pairwise comparisons between groups.

[0057] (1) In vitro antioxidant test

[0058] DPPH free radical scavenging rate test: 6×10 -5 For a 3 mg / mL DPPH ethanol solution, mix 2 mL of the sample solution with 2 mL of DPPH solution and incubate at 25 ± 1 °C in the dark for 30 min. Measure the absorbance at 517 nm using a microplate reader, and record this as A1. Similarly, mix 2 mL of the sample solution with 2 mL of anhydrous ethanol and measure the absorbance, recording this as A2. Finally, mix 2 mL of DPPH solution with 2 mL of anhydrous ethanol and measure the absorbance, recording this as A0. The DPPH free radical scavenging rate is calculated using the following formula:

[0059] DPPH free radical scavenging rate (%) = [(A0-A1) / A0] × 100%. Each sample was measured in triplicate, and the average value was taken.

[0060] SOD activity assay (xanthine oxidase method): Prepare 0.2 mmol / L xanthine substrate solution and 0.1 U / mL xanthine oxidase solution. Take 0.1 mL of sample solution, add 2.8 mL of Tris-HCl buffer (pH 8.2) and 0.1 mL of xanthine substrate solution, preheat at 37℃ for 5 min, then add 0.1 mL of xanthine oxidase solution to start the reaction. After reacting for 10 min, terminate the reaction and measure the absorbance at 560 nm. Define the amount of sample required to inhibit xanthine oxidase activity by 50% as one unit of SOD activity (U). Calculate the SOD activity (U / mL) of the sample. Each sample is measured in triplicate, and the average value is taken.

[0061] The experimental results are shown in Table 2:

[0062] Table 2 Results of in vitro antioxidant tests for each composition

[0063]

[0064]

[0065] *: indicates a significant difference compared to experimental group 9 (P<0.05); **: indicates an extremely significant difference compared to experimental group 9 (P<0.01);

[0066] The above experimental results show that the antioxidant activity (DPPH 84.2%, SOD 78.5U / mL) of experimental group 7 is significantly higher than that of aurantiacus or gentian alone, and exceeds the sum of the effects of the two alone, indicating that the two extracts can play a synergistic role in antioxidant effect.

[0067] Meanwhile, the antioxidant activity of experimental group 1 was significantly higher than that of experimental groups 4, 5, and 7. The experimental data of this group demonstrates that the combination of collagen with *Auricularia auricula-judae* fruiting body extract and *Gentiana triflora* extract can have a synergistic effect. Furthermore, experimental group 1 showed the best antioxidant effect compared to experimental groups 2 and 3, with a significantly higher DPPH scavenging rate. This indicates that the optimal ratio of recombinant type III human collagen, *Auricularia auricula-judae* fruiting body extract, and *Gentiana triflora* extract (by mass) of 3:10:10 exhibits the best antioxidant activity.

[0068] (2) Experimental cell culture of human fibroblasts:

[0069] Based on the aforementioned antioxidant effect tests, only experimental group 1 was tested below, while experimental groups 2 and 3 were not tested; similarly, only experimental group 7 was tested, while experimental groups 8 and 9 were not tested. The specific testing methods are as follows:

[0070] Cell culture and treatment: Human fibroblasts were seeded in 96-well plates at a density of 5 × 10⁶ cells / well. 4 Cells / well were placed in a 37°C, 5% CO2 incubator for culture. After the cells adhered, 20 μL of each experimental group sample was added, with 6 replicates per group. After culturing for 72 h, the culture medium was discarded, and the cells were washed 3 times with PBS, with each wash requiring thorough aspiration of any residual liquid.

[0071] Collagen production assay: Add 100 μL of RIPA lysis buffer containing protease inhibitor to each well, lyse on ice for 30 min, centrifuge at 12000 rpm for 15 min at 4 °C, and collect the supernatant. Perform the procedure according to the instructions of the Human Collagen Type III ELISA kit, plot a standard curve, and determine the collagen production in the sample (μg / mL).

[0072] The experimental results are shown in Table 3:

[0073] Table 3. Results of cell culture experiments using human fibroblasts in each experimental group.

[0074]

[0075] *: indicates a significant difference compared to experimental group 7 (P<0.05), **: indicates an extremely significant difference compared to experimental group 7 (P<0.01);

[0076] The above experimental results show that:

[0077] The collagen production in experimental group 1 reached 39.8 ± 1.5 μg / mL, an increase of 213.40% compared to experimental group 7. This fully demonstrates that the combination of recombinant type III human collagen with extracts of *Auricularia auricula-judae* fruit and *Gentiana triflora* has a significant synergistic effect in promoting collagen production in human fibroblasts. The collagen production in experimental groups 4 and 5 was also higher than that in experimental groups 7 and 6, indicating that individual plant extracts combined with collagen can also promote collagen production, but the effect is not as good as the combined effect of all three components. The data differences show that extracts of *Auricularia auricula-judae* fruit and *Gentiana triflora* extract play irreplaceable roles in the synergistic promotion of collagen production with collagen, and their combined effect achieves better results.

[0078] (3) Evaluation of human efficacy

[0079] (1) Subject screening

[0080] Basic requirements: 100 healthy female subjects aged 35-55 years were recruited and divided into 5 groups (n=20). The subjects had no obvious inflammation, damage, or history of allergies on their facial skin, and no serious systemic diseases (such as diabetes, thyroid dysfunction, or other diseases that may affect skin metabolism). They had not received any medical aesthetic treatments (such as laser, radiofrequency, or injectable fillers) in the past 3 months, and had not used any cosmetics or skin care products with special effects such as anti-aging, whitening, or acne removal in the past month.

[0081] Test method: Subjects were required to use the product containing the corresponding experimental group composition twice daily, morning and evening (the composition of the active ingredients is shown in Table 1, and the total concentration of active ingredients was kept consistent at 3 mg / mL. Phenoxyethanol 0.4% wt. was added to the system to ensure preservation requirements). The method of use was as follows: apply 2 mL of the product evenly to the face twice daily, morning and evening, and gently massage until absorbed. The product was used continuously for 28 days. During the use, the product was refrigerated at 2-4℃.

[0082] (2) Testing process

[0083] Test preparation: Subjects will be tested on day 0 and day 28. Facial cleanliness is required during testing. The tests include:

[0084] (01) Skin hydration measurement: Using a Corneometer skin hydration meter (probe model CM 825), five measurement points were selected on the face: the center of the forehead, the apple cheek area on both sides, and the center of the jaw. During measurement, the probe was pressed vertically and gently onto the skin surface. After the instrument data stabilized, the value was read. Each measurement point was measured three times, and the average value was taken as the skin hydration data for that point. Finally, the average value of the five points was taken as the result of the subject's facial skin hydration.

[0085] (02) Skin elasticity measurement: A Cutometer skin elasticity tester (model MPA580) was used, and the R0-R7 parameter measurement mode was selected. One measurement point was selected on each cheek apple area. During the measurement, the probe was placed tightly against the skin to ensure that there was no air residue. The instrument was started to measure. Each measurement point was measured three times. The elasticity parameters (such as R2, R7, etc.) of each measurement were recorded and saved. The average value was used for subsequent analysis.

[0086] (03) Wrinkle depth measurement: The VISIA skin analyzer was used to ensure consistent shooting conditions. After the instrument automatically captured facial images, its matching analysis software was used to process the images, calculate parameters such as the average depth and length of wrinkles, compare the changes of each indicator before and after the test, and calculate the improvement rate of each indicator for each subject. The experimental results are shown in Table 4.

[0087] Table 4 Comparison of experimental results for each test group (n=20)

[0088]

[0089] *: indicates a significant difference compared to experimental group 7 (P<0.05), **: indicates an extremely significant difference compared to experimental group 7 (P<0.01);

[0090] The above experimental results show that after 28 days of product use, the subjects' skin hydration, elasticity, and wrinkle depth all showed significant improvement. Experimental group 1 performed best in all three indicators: skin hydration increased by 42.3±3.1%, elasticity increased by 28.7±2.5%, and wrinkle depth decreased by 36.5±4.2%, significantly better than other experimental groups. This indicates that the composition formula not only effectively promotes collagen production in in vitro experiments but also tangibly improves skin condition and achieves anti-aging effects in actual human use. Experimental groups 4 and 5 showed the next best improvement in skin condition, further validating the importance of the synergistic effect between the two plant extracts and collagen. Experimental group 6, containing only collagen, and experimental group 7, containing only plant extracts, showed relatively weaker improvement effects, again confirming the advantages of using the three components in combination.

[0091] Experimental Example 2

[0092] Based on the aforementioned experimental example 1, this embodiment provides a stability testing method for a collagen-containing anti-aging composition system, used to test the stability of macromolecular collagen in different composition systems. The testing method is as follows:

[0093] S1: Sample Preparation

[0094] Sample 1: According to the formula of Experimental Group 1 in Example 1, the total effective ingredients are calculated by mass ratio and the composition is: recombinant type III human collagen: extract of Auricularia auricula-judae fruit: extract of Gentiana triflora = 3:10:10. The anti-aging composition containing collagen is prepared according to the total effective ingredients and the concentration of the total effective ingredients in the composition is 3 mg / mL.

[0095] Sample 2: The difference from Sample 1 is that the total effective ingredients, calculated by mass ratio, are: recombinant type III human collagen: Gentiana triflora extract = 3:20;

[0096] Sample 3: The difference from Sample 1 is that the total effective ingredients, calculated by mass ratio, consist of: recombinant type III human collagen: extract of Auricularia auricula-judae fruiting body = 3:20;

[0097] Sample 4: Based on Sample 1, add adjuvants, and according to the mass ratio, recombinant type III human collagen: Auricularia auricula-judae fruit extract: Gentiana triflora extract: adjuvants = 3:10:10:20, wherein the adjuvants are composed of the following: trehalose 6 parts, mannitol 2 parts, carbomer 2 parts and glycerin 10 parts;

[0098] Sample 5: Based on Sample 1, add adjuvants, and according to the mass ratio, recombinant type III human collagen: Auricularia auricula-judae fruit extract: Gentiana triflora extract: adjuvants = 3:10:10:20, wherein the adjuvant composition is as follows (by mass parts): trehalose 8 parts, carbomer 2 parts and glycerin 10 parts;

[0099] Sample 6: Based on Sample 1, add adjuvants, and according to the mass ratio, recombinant type III human collagen: Auricularia auricula-judae fruit extract: Gentiana triflora extract: adjuvants = 3:10:10:20, wherein the adjuvant composition is as follows (by mass parts): mannitol 8 parts, carbomer 2 parts and glycerin 10 parts;

[0100] Control group: Similar to sample 1, but without the addition of Auricularia auricula-judae fruit extract, Gentiana triflora extract, and adjuvants; the active ingredient was calculated based solely on recombinant type III human collagen, which was approximately 0.391 mg / mL.

[0101] During the experiment, except for the control group, the concentration of total effective components in all samples was kept consistent at 3 mg / mL (only recombinant type III human collagen, extract of *Auricularia auricula-judae* fruit, and extract of *Gentiana triflora* were considered as effective components for calculation). After preparation, each sample was aliquoted and placed in environments of 4℃ (refrigeration), 25℃ (room temperature), and 40℃ (accelerated degradation), respectively. Samples were collected after 30 days, with three samples per group. SDS-PAGE electrophoresis was performed (using a 12% separating gel and a 5% stacking gel, with 10 μL loaded per well). After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 solution with shaking for 2 hours. Then, they were transferred to destaining solution (acetic acid:methanol:water = 1:4:5) with shaking, changing the destaining solution every 30 minutes until the background was transparent and the bands were clearly visible. Gel images were acquired using a gel imaging system (Typhoon FLA 9500), and the images were quantitatively analyzed using ImageQuantTL software to calculate the collagen retention rate.

[0102] Retention rate (%) = IOD value of the main band after storage / IOD value of the main band of the control sample at day 0 × 100%;

[0103] The experimental results are shown in Table 5:

[0104] Table 5. Collagen retention rate (%) of each sample under different storage conditions

[0105]

[0106]

[0107] The above experimental results show that:

[0108] Sample 1 exhibited significantly improved stability under all temperature conditions. Under accelerated conditions at 40℃, its collagen retention rate reached 82.7%, a 46.6% increase compared to the control group (56.4%). This result confirms a clear synergistic protective mechanism between the extract of *Auricularia auricula-judae* fruit and the extract of *Gentiana triflora*.

[0109] The experimental results of samples 2 and 3 showed that collagen stability was significantly deteriorated when only one active ingredient was retained. Sample 2, with only Gentiana triflora extract added, had a retention rate of only 42.1% at 40°C, 25.4% lower than the control group, indicating that Gentiana triflora extract alone may promote collagen hydrolysis; this may be related to the organic acids (such as gentianic acid) or small amounts of free amino acids present. Sample 3, with a retention rate of 55.2% at 40°C, was slightly higher than the control group but 27.5% lower than sample 1, demonstrating that the physical protective effect of the *Auricularia auricula-judae* extract depends on the synergistic antioxidant effect of Gentiana triflora.

[0110] Meanwhile, after adding the adjuvants, samples 4-6 were further optimized based on sample 1, with the composite adjuvant (sample 4) showing the most outstanding effect, indicating that the combination of trehalose and mannitol produces a synergistic effect:

[0111] The aforementioned studies have demonstrated that the formulation provided by this invention can significantly reduce the dependence on cold chain transportation and low-temperature storage for recombinant type III human collagen, and is suitable for heat-sensitive product systems such as skin care essences and freeze-dried preparations.

[0112] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. An anti-aging composition comprising collagen, characterized in that, It comprises recombinant human collagen type III, extract of Tremella aurantialba and extract of Gentiana triflora, and an auxiliary agent; wherein, by mass fraction, it comprises 1-10 parts of recombinant human collagen type III, 10-20 parts of extract of Tremella aurantialba, 10-20 parts of extract of Gentiana triflora, and 5-20 parts of auxiliary agent; the auxiliary agent at least comprises any one or more of trehalose, mannitol, carbomer, hydroxyethyl cellulose, glycerol, butanediol, and pentanediol; The extract of Tremella aurantialba is prepared by the following method: after the fruiting body of Tremella aurantialba is crushed, it is extracted with 5-10 times the amount of 70% ethanol solution at 55-65°C for 2-3 times, each time for 2 hours, the extract is combined, concentrated under reduced pressure, and then freeze-dried to obtain the extract of Tremella aurantialba; The preparation method of the extract of Gentiana triflora is as follows: The whole plant of Gentiana triflora is crushed, 60% methanol solution is added at a solid-liquid ratio of 1:10-15 g / mL, and the mixture is extracted under ultrasonic power of 300-500 W for 30-45 minutes; the extract is collected by filtration, purified by HPD-600 macroporous resin, eluted with 30% ethanol as eluent, and the target fraction is collected to obtain the extract of Gentiana triflora after drying. The collection method of the target fraction is as follows: first, elute with 1-2 times the volume of pure water, then elute with 30% ethanol as eluent, collect the ethanol eluate, and freeze-dry the eluate after concentration under reduced pressure to obtain the extract of Gentiana triflora.

2. The anti-aging composition comprising collagen according to claim 1, wherein: The auxiliary agent is composed of the following components by mass fraction: trehalose 2-8 parts, mannitol 1-6 parts, carbomer 1-4 parts, and glycerol 3-10 parts.

3. The anti-aging composition containing collagen according to claim 1, wherein It comprises the following steps:

4. The method of claim 1, wherein the collagen-containing anti-aging composition is prepared by mixing collagen with a pharmaceutically acceptable carrier, and then adding a cross-linking agent to the mixture. S1: Prepare a premixed solvent: add deionized water in an amount of 50-70% of the total mass of the composition to a reaction kettle, heat to 35-45°C, and control the stirring speed at 200-300 rpm under stirring; slowly add the auxiliary agent and continue stirring for 20-30 minutes until completely dissolved to obtain a base solution; S2: Disperse the extract of Tremella aurantialba and the extract of Gentiana triflora: add the extract of Tremella aurantialba and the extract of Gentiana triflora to the base solution of step S1, reduce the stirring speed to 150-200 rpm, and continue stirring at 35-40°C for 15-25 minutes to fully disperse the extract of Tremella aurantialba and the extract of Gentiana triflora and make them compatible with the base solution to obtain a mixed system; S3: Add recombinant collagen: reduce the temperature of the mixed system to 25-30°C, add recombinant human collagen type III, and adjust the stirring speed to 100-150 rpm by adding deionized water; low-speed stirring for 10-15 minutes until completely dispersed; S4: Adjust the pH to 5.5-6.8, and perform sterile filtration to obtain the collagen-containing anti-aging composition.

5. Use of the collagen-containing anti-aging composition according to any one of claims 1-3 in the preparation of a cosmetic product. The cosmetic product is a resident cosmetic product, and the amount of the collagen-containing anti-aging composition added in the cosmetic product is 0.1-5 wt%.

6. The use according to claim 5, characterized in that: ​

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