Alligator oil anti-aging treatment

Abstract

The application discloses alligator oil anti-aging protection agent and belongs to the technical field of skin care products. The anti-aging protection agent is composed of the following components in mass parts: alligator oil 15-20 parts, adenosine 1-3 parts, safflower extract 1-3 parts, ginseng extract 1-3 parts, gynura extract 1-3 parts, salvia miltiorrhiza extract 0.5-2.5 parts, panax notoginseng extract 0.2-1.5 parts, spilanthes acmella extract 0.5-2.5 parts, polygonatum odoratum extract 1-5 parts, and deionized water, which is supplemented to 100 parts. The alligator oil is preferably alligator oil nanoliposomes prepared by an enzymatic extraction-nanoliposome continuous process. The application builds a multi-target anti-aging link of 'promoting synthesis-anti-degradation-promoting circulation-anti-oxidation-protecting barrier' through the compounding of alligator oil, adenosine and specific plant extracts such as gynura extract, the components have synergistic effect, have good anti-aging and skin protection effects, and can be used for preparing cosmetics or skin care products.

Description

Technical Field

[0001] This invention relates to the field of skin care products technology, and more specifically, to a crocodile oil anti-aging and repairing agent. Background Technology

[0002] Crocodile oil is an oil extracted from the fatty tissue of crocodiles. Its fatty acid composition is similar to that of human fat, and it is rich in unsaturated fatty acids (such as oleic acid, linoleic acid, and palmitoleic acid), squalene, vitamin E, and various natural active ingredients. It has excellent skin permeability, moisturizing and repairing properties, and anti-inflammatory effects. In recent years, with the deepening of research and development of cosmetic raw materials, the application potential of crocodile oil in the skincare field has received widespread attention.

[0003] Currently, crocodile oil has a certain research foundation in the field of anti-aging skincare. Existing technologies disclose various skincare compositions containing crocodile oil, such as combining crocodile oil with plant extracts like tocopherol, witch hazel, and purslane for stretch mark repair; or combining crocodile oil with niacinamide, retinol, and licorice root extract for whitening, spot removal, and anti-aging. Furthermore, adenosine, as an endogenous nucleoside, has been reported to promote skin energy metabolism and improve wrinkles, and has been applied in various anti-aging skincare products. The skincare benefits of plant extracts such as centella asiatica, ginseng, and safflower in terms of anti-inflammatory, antioxidant, and collagen synthesis-promoting effects are also widely recognized.

[0004] However, existing anti-aging skincare products containing crocodile oil still have the following shortcomings:

[0005] (1) Single mechanism of action and insufficient synergy: Existing formulations mostly use crocodile oil as the base oil component, lacking targeted synergistic design with other active ingredients, and failing to fully utilize the transdermal absorption properties of crocodile oil and the synergistic effect with other anti-aging active ingredients.

[0006] (2) Crocodile oil active ingredients are easily lost: Existing crocodile oil extraction methods mostly use conventional processes such as high-temperature boiling, organic solvent extraction or supercritical CO2 extraction. During the extraction process, heat-sensitive active ingredients such as unsaturated fatty acids are easily oxidized and degraded, and subsequent refining steps (such as degumming, deacidification, decolorization and deodorization) are often accompanied by further loss of active ingredients.

[0007] (3) Formulation affects efficacy: In conventional crocodile oil formulations, crocodile oil exists in the form of free oil, which makes it difficult to form a stable and uniform delivery system with the water-soluble active ingredients in the formulation, affecting transdermal absorption efficiency and compatibility stability.

[0008] Therefore, it is necessary to propose a new type of crocodile oil anti-aging and repair product. Summary of the Invention

[0009] To address the aforementioned issues, this invention provides a crocodile oil anti-aging and repairing agent. By combining crocodile oil nanoliposomes in a specific ratio with adenosine, safflower extract, ginseng extract, centella asiatica extract, salvia miltiorrhiza extract, notoginseng extract, purslane extract, and polygonatum odoratum extract, a multi-target synergistic anti-aging and skin repair effect is achieved.

[0010] To achieve the above-mentioned technical objectives, the specific technical solution of the present invention is as follows:

[0011] A crocodile oil anti-aging and repairing agent, comprising the following components in parts by weight: 15-20 parts crocodile oil, 1-3 parts adenosine, 1-3 parts safflower extract, 1-3 parts ginseng extract, 1-3 parts centella asiatica extract, 0.5-2.5 parts salvia miltiorrhiza extract, 0.2-1.5 parts notoginseng extract, 0.5-2.5 parts purslane extract, 1-5 parts polygonatum odoratum extract, and deionized water to make up to 100 parts.

[0012] This crocodile oil anti-aging repair product is specifically designed to address the main layers of skin aging (decreased epidermal barrier function, collagen loss in the dermis, microcirculation disorders, and oxidative stress damage). Crocodile oil, as the core active ingredient and natural penetration enhancer, has a fatty acid composition similar to human sebum. It not only replenishes essential unsaturated fatty acids for the skin (especially linoleic acid, which the body cannot synthesize on its own) and repairs the skin barrier, but also promotes the transdermal absorption of other active ingredients in the formula. Building upon this foundation, the compound adenosine stimulates dermal fibroblasts to synthesize type I and type III collagen through the A2A receptor pathway, improving skin elasticity and reducing wrinkles from a deep level. Centella asiatica extract reduces collagen degradation by inhibiting matrix metalloproteinase-1 (MMP-1) activity, forming a bidirectional regulatory mechanism of "promoting synthesis and resisting degradation" with adenosine. Ginseng extract activates the Nrf2 antioxidant pathway and scavenge free radicals; safflower and Panax notoginseng extracts improve skin microcirculation; Salvia miltiorrhiza extract has anti-inflammatory and synergistic antioxidant effects; Portulaca oleracea extract soothes and reduces inflammation to neutralize potential irritation; and Polygonatum odoratum extract provides moisturizing and nourishing effects, complementing the occlusive moisturizing effect of crocodile oil. Each component acts on different levels, including the epidermis, dermis, microcirculation, and antioxidants, forming a complete anti-aging chain of "promoting synthesis, resisting degradation, promoting circulation, resisting oxidation, and protecting the skin barrier," achieving synergistic effects across multiple targets.

[0013] Preferably, the product is composed of the following components in parts by weight: 20 parts crocodile oil, 2 parts adenosine, 2 parts safflower extract, 2 parts ginseng extract, 2 parts centella asiatica extract, 1.5 parts salvia miltiorrhiza extract, 0.8 parts notoginseng extract, 1.5 parts purslane extract, 3 parts Solomon's seal extract, and deionized water to make up to 100 parts.

[0014] Preferably, the crocodile oil is prepared by the following method: crocodile abdominal fat is taken, cleaned to remove impurities, cut into pieces, and minced into a fat paste; neutral protease is added to the fat paste for enzymatic hydrolysis, and the supernatant is collected by centrifugation to obtain crude crocodile oil; the crude crocodile oil is mixed with soybean lecithin, cholesterol, and Tween-80 at a mass ratio of 1:5:1:1.5, dissolved in ethanol, and evaporated at 36°C to form a film. After hydration, the film is sonicated at 400W for 10 minutes to obtain crocodile oil nanoliposomes.

[0015] Preferably, a neutral protease is added to the chyme, and the enzymatic hydrolysis temperature is controlled at 48-52°C, the pH at 7.0-7.8, and the hydrolysis time at 3.5-4.5 hours, so that the degree of hydrolysis reaches 15%-20%. The supernatant is then collected by centrifugation to obtain crocodile crude oil.

[0016] In this invention, crocodile oil is prepared using a continuous process of "enzymatic extraction-nanoliposome formation," aiming to solve the technical problems of easy oxidation and degradation of unsaturated fatty acids, severe loss of active ingredients, and difficulty in stable compatibility between free oil and water-soluble active ingredients in the formulation in existing high-temperature extraction processes. Specifically, crocodile abdominal fat is minced into a paste and enzymatically hydrolyzed with neutral protease at a mild temperature of 48–52°C and pH 7.0–7.8 for 3.5–4.5 hours, achieving a degree of hydrolysis of 15%–20%. The resulting crude crocodile oil is obtained by centrifugation. The crude crocodile oil is then mixed with soybean lecithin, cholesterol, and Tween-80 in a mass ratio of 1:5:1:1.5, dissolved in ethanol, formed by rotary evaporation, hydrated, and ultrasonically treated to obtain crocodile oil nanoliposomes. Enzymatic hydrolysis replaces traditional high-temperature boiling or organic solvent extraction, avoiding damage to heat-sensitive unsaturated fatty acids such as oleic acid and linoleic acid. Nanoliposome formation encapsulates crocodile oil through a phospholipid bilayer, transforming it into a nanoscale delivery carrier (with a particle size capable of penetrating the stratum corneum barrier), effectively enhancing the transdermal absorption efficiency of active ingredients. This allows crocodile oil to not only act on the epidermis but also penetrate deep into the dermis to exert its repairing effects. Furthermore, the liposome structure delays the oxidative rancidity of unsaturated fatty acids and allows crocodile oil to form a stable co-delivery system with water-soluble active ingredients (such as adenosine and plant extracts), preventing oil-water separation. Testing showed that the repair agent prepared by this formula exhibited DPPH and hydroxyl radical scavenging rates of 82.67% and 78.92%, respectively, demonstrating both highly efficient delivery and synergistic antioxidant functions.

[0017] Preferably, the safflower extract, ginseng extract, centella asiatica extract, salvia miltiorrhiza extract, notoginseng extract, purslane extract, and polygonatum odoratum extract are water extracts or alcohol extracts of the corresponding plants.

[0018] This invention provides the application of the described crocodile oil anti-aging and repairing agent in the preparation of anti-aging or skin repair products.

[0019] This invention provides a product containing the aforementioned crocodile oil anti-aging and repairing agent.

[0020] The product provided by this invention is a cosmetic or skin care product.

[0021] This application has the following beneficial effects:

[0022] (1) By forming a core triangular combination of "promoting collagen synthesis - resisting collagen degradation - promoting penetration" through crocodile oil nanoliposomes, adenosine, and centella asiatica extract, multi-target synergistic anti-aging is achieved. Each component is indispensable, and the overall effect is significantly better than that of a single component or simple superposition.

[0023] (2) Crocodile oil is prepared by a continuous process of enzymatic extraction-nanopeptidation. The entire process is gentle and avoids the oxidation and degradation of unsaturated fatty acids. The liposome structure upgrades the crocodile oil from epidermal function to dermal repair. At the same time, it achieves stable co-delivery with water-soluble active ingredients. The DPPH clearance rate is 82.67% and the transdermal depth reaches 108μm.

[0024] (3) The contribution of extracts of blood-activating and stasis-removing plants such as safflower, Panax notoginseng, and Salvia miltiorrhiza to fading stretch marks, reducing their area and restoring skin color decreased as the number of removed components increased. The total score of the complete formula in human trials reached 18.30 points, indicating that the present invention covers the main links of skin aging from multiple levels such as epidermis, dermis, microcirculation and anti-oxidation, and has comprehensive and significant anti-aging and skin repair effects. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments and comparative examples, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0026] I. Preparation of Crocodile Oil Nanoliposomes

[0027] The crocodile oil used in this invention is prepared using a continuous process of "enzymatic extraction-nanoliposome formation," and the specific steps are as follows:

[0028] (1) Take the fat from the belly of the crocodile, clean it to remove impurities, cut it into pieces and mince it into fat paste;

[0029] (2) Neutral protease was added to the fat chyme for enzymatic hydrolysis. The hydrolysis temperature was controlled at 48-52℃, the pH at 7.0-7.8, and the hydrolysis time at 3.5-4.5 hours, so that the degree of hydrolysis reached 15%-20%. The supernatant was collected by centrifugation to obtain crocodile crude oil.

[0030] (3) Mix crocodile crude oil with soybean lecithin, cholesterol and Tween-80 in a mass ratio of 1:5:1:1.5, add ethanol to dissolve, evaporate at 36℃ to form a film, hydrate and sonicate at 400W power for 10min to obtain crocodile oil nanoliposomes.

[0031] II. Source of Raw Materials

[0032] All components used in the embodiments of this invention can be obtained through commercial channels. Information on the sources of some raw materials is as follows:

[0033] Table 1. Component Sources

[0034] Components Specification Manufacturers adenosine Purity ≥ 99%, CAS: 58-61-7 Nanjing Youchun Biotechnology Co., Ltd. Safflower extract 10:1 water extract, CAS: 42553-65-1 Guangdong Mingtong Biotechnology Co., Ltd. Ginseng extract 10:1 alcohol extract, CAS: 90045-38-8 Guangdong Fangxin Biotechnology Co., Ltd. Centella Asiatica extract 70%–80% total saponins, CAS: 84696-21-9 Changsha Hepu Biotechnology Co., Ltd. Salvia miltiorrhiza extract 10:1 alcohol extract, CAS: 90106-50-6 Jiangsu Huace Biotechnology Co., Ltd. Panax notoginseng extract 10:1 alcohol extract, CAS: 80418-29-7 Shaanxi Xintianyu Biotechnology Co., Ltd. Purslane extract 10:1 water extract, CAS: 90083-07-1 Shaanxi Xintianyu Biotechnology Co., Ltd. Polygonatum odoratum extract 10:1 water extract, CAS: 100-00-4 Shaanxi Xintianyu Biotechnology Co., Ltd. Soy lecithin CAS: 8002-43-5 Xi'an Zebang Biotechnology Co., Ltd. cholesterol CAS: 57-88-5 Changsha Yuteng New Materials Co., Ltd. Twain-80 CAS: 9005-65-6 Tianjin Xiens Biochemical Technology Co., Ltd. neutral protease CAS: 9068-59-1 Shanghai Yuanye Biotechnology Co., Ltd.

[0035] III. Examples and Comparative Examples

[0036] (a) Examples 1-7

[0037] The anti-aging repair agent was prepared according to the formula shown in Table 1 (by weight, deionized water was added to bring the total to 100 parts). The crocodile oil was prepared using the aforementioned method for "crocodile oil nanoliposomes". The preparation method is as follows: crocodile oil nanoliposomes, adenosine, and various plant extracts were mixed in proportion, stirred at 40–50°C until homogeneous, and then cooled to obtain the final product.

[0038] Table 1. Formulation composition (parts by weight) of Examples 1-7

[0039] Components Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Crocodile oil nanoliposomes 15 20 18 16 22 25 / Commercially available crocodile oil (non-nano) / / / / / 20 adenosine 1.5 2 1 3 2.5 1 2 Safflower extract 1 2 2.5 1.5 3 1 2 Ginseng extract 3 2 1 2.5 1.5 1 2 Centella Asiatica extract 1.5 2 3 1 2.5 3 2 Salvia miltiorrhiza extract 2.5 1.5 0.5 2 1 2.5 1.5 Panax notoginseng extract 0.2 0.8 1.5 0.5 1.2 0.2 0.8 Purslane extract 2.5 1.5 0.5 2 1 2.5 1.5 Polygonatum odoratum extract 2 3 1 5 4 1 3

[0040] (ii) Comparative Examples 1-10

[0041] To verify the synergistic effect of the components of this invention and the advantages of the nanoliposome process, the following comparative examples were set up (by mass, deionized water was added to make up to 100 parts):

[0042] Comparative Example 1: Except for the absence of crocodile oil, the other components are the same as in Example 2, used to verify the core role of crocodile oil in the formulation.

[0043] Comparative Example 2: Except for the absence of adenosine, the other components were the same as in Example 2, used to verify the role of adenosine in the formulation.

[0044] Comparative Example 3: Except for the absence of Centella Asiatica extract, the other components were the same as in Example 2, used to verify the effect of Centella Asiatica extract.

[0045] Comparative Example 4: Contains only 20 parts of crocodile oil nanoliposomes, 2 parts of adenosine, 2 parts of ginseng extract, 2 parts of centella asiatica extract, and 74 parts of deionized water, without the addition of five plant extracts: safflower, notoginseng, salvia miltiorrhiza, purslane, and polygonatum odoratum.

[0046] Comparative Example 5: Contains only 20 parts of crocodile oil nanoliposomes, 2 parts of adenosine, 2 parts of ginseng extract, 2 parts of centella asiatica extract, 3 parts of Solomon's seal extract, and 71 parts of deionized water, without the addition of four plant extracts: safflower, Panax notoginseng, Salvia miltiorrhiza, and Portulaca oleracea.

[0047] Comparative Example 6: Contains only 20 parts of crocodile oil nanoliposomes, 2 parts of adenosine, 2 parts of ginseng extract, 2 parts of centella asiatica extract, 1.5 parts of purslane extract, 3 parts of Solomon's seal extract, and 69.5 parts of deionized water, without the addition of safflower, Panax notoginseng, and Salvia miltiorrhiza extracts.

[0048] Comparative Example 7: Contains only 20 parts of crocodile oil nanoliposomes, 2 parts of adenosine, 2 parts of ginseng extract, 2 parts of centella asiatica extract, 1.5 parts of salvia miltiorrhiza extract, 1.5 parts of purslane extract, 3 parts of Solomon's seal extract, and 68 parts of deionized water, without the addition of safflower and Panax notoginseng extracts.

[0049] Comparative Example 8: Contains only 20 parts of crocodile oil nanoliposomes, 2 parts of adenosine, and 78 parts of deionized water, without any added plant extracts.

[0050] Comparative Example 9: Contains only 20 parts of crocodile oil nanoliposomes and 80 parts of deionized water, without any other active ingredients.

[0051] Comparative Example 10: The formula of Example 2 was used, but the crocodile oil was directly added using free crocodile oil extracted by conventional high-temperature boiling (without nanoliposome treatment). The other components and their amounts remained unchanged. This was used to compare with the enzymatic hydrolysis + nanoliposome process of the present invention.

[0052] IV. Test Items and Test Methods

[0053] Test 1: In vitro antioxidant activity assay (DPPH free radical scavenging rate)

[0054] Using vitamin E as a positive control, the scavenging abilities of each example and comparative sample against DPPH and hydroxyl radicals were determined. The specific method was as follows: A solution with a concentration of 1 mg / mL was prepared for each sample. 2 mL of the sample solution was mixed with 2 mL of DPPH ethanol solution (0.1 mmol / L), and the mixture was reacted at room temperature in the dark for 30 min. The absorbance was measured at 517 nm, and the scavenging rate was calculated.

[0055] Clearance rate (%) = [1 - (Sample A - Control A) / Blank A] × 100%

[0056] Test 2: Human skin fibroblast proliferation assay (CCK-8 assay)

[0057] Human skin fibroblasts (HSF) in logarithmic growth phase were collected and seeded at a density of 4 × 10³ cells / well in 96-well plates, 100 μL per well, and cultured for 24 h at 37 °C with 5% CO2. Cells were then divided into a blank control group and a drug-treated group. The blank control group was treated with 100 μL of DMEM complete medium, while the drug-treated groups were treated with 100 μL of medium containing the samples from each example and comparative example (concentration 50 μg / mL), with three replicates per group. Cells were cultured for another 48 h, and then 10 μL of CCK-8 solution was added to each well, followed by 2 h of further culture. The absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0058] Cell viability (%) = (Average absorbance of the drug-treated group / Average absorbance of the blank control group) × 100%

[0059] Test 3: Evaluation of Anti-wrinkle Efficacy in Mouse Skin

[0060] A photoaging model was established using female BALB / c mice (18–20 g). After shaving the back hair (2 × 3 cm area), the mice were irradiated with UVB lamps (wavelength 280–320 nm, dose 180 mJ / cm²) every other day for 4 weeks. After successful model validation, the mice were divided into the following groups: model group (0.05% saline applied topically), Examples 1–7, Comparative Examples 1–10, positive control group (0.05% retinoic acid cream applied topically), and normal control group (no irradiation + 0.05% saline applied topically). The treatment was applied twice daily, 6 hours apart, for 4 consecutive weeks. The measured indicators included skin wrinkle score (0–4 points), epidermal thickness (HE staining), and collagen area ratio (Masson staining).

[0061] Mouse skin wrinkle scoring criteria (0-4 points): 0 points: smooth skin surface, no visible fine lines or wrinkles; 1 point: very slight fine lines, few in number and shallow; 2 points: obvious fine lines, increased in number and widely distributed; 3 points: moderate to deep wrinkles, uneven skin surface; 4 points: deep groove wrinkles, dense wrinkles with a strong three-dimensional effect, poor skin elasticity.

[0062] Test 4: In vitro transdermal depth test

[0063] After labeling the samples of each example and comparative example with fluorescein isothiocyanate (FITC), fresh pig skin was washed with PBS, and fixed with the stratum corneum facing upward in a transdermal diffusion apparatus and incubated at 37°C. 0.5 mL of a 1 mg / mL fluorescently labeled sample solution was dropped onto the pig skin tissue, and after 24 hours, it was frozen sectioned and the penetration depth was observed under a confocal microscope.

[0064] Test 5: Human Trial Evaluation

[0065] One hundred healthy women aged 25-42 years were selected on a voluntary basis. All participants were more than 3 months postpartum, had noticeable stretch marks around their abdomen, and had no serious systemic diseases, immunodeficiency, autoimmune diseases, or active allergic diseases. During the trial, participants were prohibited from applying any medications or cosmetics unrelated to the experiment. Participants were randomly divided into 20 groups (Example 1-7, Comparative Examples 1-10, Blank Control Group, Positive Control Group, and Model Group), with 5 participants in each group. After cleansing the abdominal skin morning and evening, the corresponding sample (consistent concentration) was applied to the stretch mark areas at a dosage of 5-10g per application, followed by 5 minutes of massage, for 3 consecutive months.

[0066] Evaluation criteria: The evaluation will assess five aspects: the degree of fading of stretch marks, the degree of reduction in the area of ​​stretch marks, the degree of restoration of normal skin tone, and nourishing and moisturizing effects. Each aspect will be scored from 0 to 5 points, for a total of 20 points. Detailed evaluation rules are as follows:

[0067] 5 points: Completely satisfied; 4-5 points: Very satisfied; 3-4 points: Acceptable; Below 3 points: Unacceptable.

[0068] Total score of 16 or above: Subjects are relatively satisfied and the repair effect is good; Total score of 12-16: Subjects can accept it and there is a certain repair effect; Total score of 0-12: Subjects cannot accept it and there is no obvious repair effect.

[0069] V. Test Results

[0070] (I) Results of in vitro antioxidant activity assay

[0071] The scavenging rates of DPPH and hydroxyl radicals for each example and comparative sample are shown in Table 2.

[0072] Table 2. Scavenging rates of DPPH and hydroxyl radicals for each sample

[0073] Group DPPH clearance rate (%) Hydroxyl radical scavenging rate (%) Vitamin E (positive control) 85.32±2.15 80.56±3.21 Example 1 76.54±3.21 72.38±2.89 Example 2 82.67±2.56 78.92±3.02 Example 3 74.23±3.65 70.15±2.76 Example 4 79.86±2.98 75.63±3.11 Example 5 80.12±3.02 76.48±2.95 Example 6 72.56±3.54 68.23±2.88 Example 7 65.23±4.12 58.67±3.56 Comparative Example 1 56.32±3.89 48.56±4.02 Comparative Example 2 68.54±3.22 62.38±3.45 Comparative Example 3 62.15±3.98 55.69±3.87 Comparative Example 4 54.23±4.12 46.32±3.98 Comparative Example 5 56.78±3.89 48.96±3.76 Comparative Example 6 58.92±3.56 50.23±3.65 Comparative Example 7 60.34±3.42 52.18±3.58 Comparative Example 8 50.12±4.23 42.36±4.11 Comparative Example 9 42.56±4.56 35.28±4.32 Comparative Example 10 60.85±3.78 53.42±3.69

[0074] The results showed that the DPPH scavenging rate of Examples 1-6 was between 72% and 83%, and the hydroxyl radical scavenging rate was between 68% and 79%, which was superior to the comparative examples (P<0.05). Among them, Example 2 showed the best effect, approaching the level of the positive control vitamin E. Example 7 used commercially available crocodile oil, and the scavenging rate was lower than that of Example 2, indicating that the crocodile oil prepared by the enzymatic hydrolysis + nanoliposome process of this invention has higher antioxidant activity. Comparative Example 9 (containing only crocodile oil) had the lowest scavenging rate, proving that the combination of various plant extracts contributed significantly to the overall antioxidant activity.

[0075] (II) Results of human skin fibroblast proliferation experiment

[0076] The effects of each embodiment and comparative sample on the survival rate of human skin fibroblasts are shown in Table 3.

[0077] Table 3 Effects of each sample on fibroblast survival rate

[0078] Group Cell viability (%) Blank control group 100.00 Example 1 106.32±4.25 Example 2 118.56±3.86 Example 3 109.86±4.02 Example 4 115.62±3.98 Example 5 112.38±4.12 Example 6 104.23±4.56 Example 7 103.65±4.23 Comparative Example 1 101.21±3.65 Comparative Example 2 99.68±4.02 Comparative Example 3 97.32±4.56 Comparative Example 4 96.32±4.15 Comparative Example 5 97.21±4.23 Comparative Example 6 98.56±3.89 Comparative Example 7 99.23±3.76 Comparative Example 8 92.65±4.56 Comparative Example 9 88.32±5.02 Comparative Example 10 102.36±4.12

[0079] The results showed that the fibroblast survival rate of Examples 1-6 was greater than 104%, higher than that of the blank control group, indicating that the composition of the present invention can promote fibroblast proliferation, with Example 2 showing the best effect. Although the cell survival rate of Comparative Example 1 without crocodile oil was slightly higher than that of the blank control group, it was much lower than that of Example 2, indicating that crocodile oil is not only a basic oil component in the formulation, but its role in promoting transdermal absorption of active ingredients and synergistically promoting proliferation is irreplaceable. The cell survival rate of Comparative Example 2 without adenosine was basically the same as that of the blank control group, confirming the key role of adenosine in stimulating collagen synthesis in fibroblasts through the A2A receptor pathway. The cell survival rate of Comparative Example 3 without Centella asiatica extract was even lower than that of Comparative Example 2, indicating that the lack of Centella asiatica extract resulted in the loss of MMP-1-mediated collagen degradation inhibition, leading to a decrease in the overall proliferation-promoting effect. In summary, the core triangular combination of crocodile oil, adenosine, and Centella asiatica extract—"promoting collagen synthesis - resisting collagen degradation - promoting penetration"—has a synergistic effect and none of them can be omitted.

[0080] (III) Evaluation results of anti-wrinkle efficacy of mouse skin

[0081] The skin repair effects of each embodiment and comparative example on photo-aged mouse models are shown in Table 4.

[0082] Table 4. Skin repair effects of each sample on photo-aged mouse model

[0083] Group Wrinkle score (0-4 points) Skin thickness (μm) Collagen area ratio (%) normal control group 1.0±0.2 17.6±2.1 47.5±3.2 Model group 3.6±0.3 65.8±4.5 43.3±3.8 Positive control group 1.1±0.3 18.3±2.8 60.3±4.1 Example 1 1.4±0.4 35.2±3.6 62.5±3.8 Example 2 1.0±0.3 28.6±3.2 68.2±3.5 Example 3 1.5±0.4 38.5±3.8 60.8±4.0 Example 4 1.2±0.3 32.4±3.5 65.6±3.6 Example 5 1.3±0.4 34.8±3.7 63.8±3.9 Example 6 1.6±0.4 40.2±4.0 58.6±4.2 Example 7 2.0±0.5 48.5±4.2 52.3±4.0 Comparative Example 1 2.8±0.5 58.6±4.5 46.8±4.2 Comparative Example 2 2.5±0.5 52.3±4.0 50.5±3.9 Comparative Example 3 2.3±0.5 49.6±4.1 52.1±4.0 Comparative Example 4 2.6±0.5 55.2±4.3 48.2±4.1 Comparative Example 5 2.5±0.4 53.8±4.2 49.5±4.0 Comparative Example 6 2.4±0.5 51.6±4.1 50.8±3.9 Comparative Example 7 2.3±0.5 50.2±4.0 51.5±4.0 Comparative Example 8 2.8±0.5 57.3±4.5 47.2±4.1 Comparative Example 9 3.1±0.6 60.5±4.8 44.6±4.3 Comparative Example 10 2.2±0.5 46.8±4.0 53.8±3.9

[0084] The results showed that after UVB irradiation, the wrinkle score of mice in the model group increased to 3.6 points, the epidermal thickness increased to 65.8 μm, and the collagen area ratio decreased to 43.3%, indicating that the photoaging model was successfully established. The wrinkle scores, epidermal thickness, and collagen area ratio of mice in treatment groups 1-6 were all superior to those in the model group (P<0.01), with Example 2 showing the best results. The wrinkle score of 1.0 point was comparable to the normal control group, and the epidermal thickness of 28.6 μm and collagen area ratio of 68.2% were both superior to the positive control group. This is consistent with the bidirectional regulatory mechanism of "promoting synthesis-anti-degradation" between adenosine promoting collagen synthesis and Centella asiatica extract inhibiting collagen degradation. Example 7, using commercially available non-nano crocodile oil, showed worse wrinkle score and epidermal thickness results than Example 2, verifying the superiority of the nanoliposome process of this invention in improving the bioavailability of active ingredients. Comparative Example 9, containing only crocodile oil, showed the worst repair effect, almost identical to the model group. This demonstrates that when crocodile oil is used alone, it only acts on the epidermis and is difficult to achieve deep repair of the dermis. It also shows that the combination of various plant extracts is the key to achieving multi-target anti-aging.

[0085] (iv) Results of in vitro transdermal penetration test

[0086] The 24-hour penetration depth of each embodiment and comparative sample in pigskin is shown in Table 5.

[0087] Table 5 Transdermal penetration depth of each sample after 24 hours

[0088] Group Penetration depth (μm) Example 1 95±5 Example 2 108±6 Example 3 98±5 Example 4 102±4 Example 5 100±5 Example 6 92±5 Example 7 75±6 Comparative Example 1 78±5 Comparative Example 2 82±5 Comparative Example 3 80±4 Comparative Example 4 65±6 Comparative Example 5 62±5 Comparative Example 6 68±6 Comparative Example 7 70±5 Comparative Example 8 58±6 Comparative Example 9 55±7 Comparative Example 10 72±6

[0089] The results showed that the penetration depth of Examples 1-6 was concentrated in the range of 92-108 μm, while the penetration depth of each comparative example was lower than that of the Example group (P<0.05). Example 2 had the largest penetration depth, while the penetration depth of Example 7 was significantly lower than that of Example 2, verifying the advantage of nanoliposomes in significantly improving transdermal absorption efficiency. Comparative Example 9 had the lowest penetration depth, indicating that crocodile oil mainly remained in the epidermal layer when used alone.

[0090] The penetration depth of Comparative Example 1 was 78±5 μm, higher than Comparative Example 9 but lower than Examples 1-6. This indicates that without crocodile oil, although other active ingredients in the formulation have some penetration ability, the lack of the synergistic effect of crocodile oil significantly reduces penetration efficiency. The penetration depth of Comparative Example 2 was 82±5 μm, higher than Comparative Example 1, indicating that the absence of adenosine has a relatively small impact on the overall permeability of liposomes, and the decrease in penetration depth is mainly attributed to changes in the combination of active ingredients. The penetration depth of Comparative Example 3 was 80±4 μm, similar to Comparative Example 2, indicating that the absence of Centella asiatica extract has a limited impact on the physical permeability of liposomes.

[0091] Overall, based on the penetration depth data of Comparative Examples 1–10, nanoliposome formation is the key to achieving deep penetration (Examples 1–6 containing nanoliposomes are superior to Example 7 and Comparative Example 10 without nanoliposomes). The presence or absence of each active ingredient has a relatively small impact on the physical permeability of liposomes. However, due to the different bioactivities of crocodile oil, adenosine, and plant extracts after reaching the target site, the differences in their actual efficacy are more obvious (as shown in Tests 2–3).

[0092] (v) Results of human trial evaluation

[0093] The comprehensive scores of each embodiment and comparative sample in postpartum stretch mark repair are shown in Table 6.

[0094] Table 6. Overall Score of Stretch Mark Repair Efficacy

[0095] Group Stretch marks fade Narrowing the scope Skin tone restoration Nourishing Moisturizing Total Score Example 1 4.18±0.22 4.22±0.20 4.24±0.23 2.32±0.40 2.60±0.43 17.56 Example 2 4.42±0.21 4.38±0.20 4.36±0.20 2.52±0.22 2.62±0.24 18.30 Example 3 4.20±0.22 4.40±0.32 4.30±0.20 2.30±0.27 2.80±0.28 18.00 Example 4 4.28±0.24 4.18±0.42 4.22±0.24 2.38±0.24 2.58±0.22 17.64 Example 5 4.16±0.24 4.30±0.22 4.32±0.22 2.48±0.30 2.60±0.22 17.86 Example 6 4.10±0.24 4.16±0.24 4.18±0.24 2.28±0.24 2.52±0.24 17.24 Example 7 3.80±0.28 3.92±0.30 3.98±0.28 2.20±0.28 2.38±0.28 16.28 Comparative Example 1 2.56±0.20 3.16±0.47 3.26±0.21 2.02±0.24 1.85±0.14 12.85 Comparative Example 2 2.36±0.28 3.43±0.36 3.21±0.26 2.14±0.27 1.86±0.23 12.88 Comparative Example 3 2.12±0.27 3.13±0.43 3.23±0.27 1.95±0.25 1.02±0.12 11.45 Comparative Example 4 2.32±0.24 2.28±0.56 3.28±0.29 1.92±0.23 1.12±0.42 10.92 Comparative Example 5 2.42±0.24 2.38±0.52 3.32±0.28 1.96±0.24 1.18±0.40 11.26 Comparative Example 6 2.48±0.24 2.42±0.48 3.36±0.28 2.00±0.24 1.22±0.38 11.48 Comparative Example 7 2.52±0.22 2.48±0.46 3.40±0.26 2.04±0.24 1.28±0.36 11.72 Comparative Example 8 2.16±0.28 2.12±0.48 3.12±0.30 1.82±0.24 1.08±0.36 10.30 Comparative Example 9 1.92±0.32 1.88±0.52 2.98±0.32 1.68±0.28 1.02±0.32 9.48 Comparative Example 10 3.52±0.30 3.62±0.32 3.68±0.30 2.06±0.30 2.12±0.28 15.00

[0096] Human trial evaluation results showed that the total score of all seven groups (Examples 1-7) exceeded 16 points (subject satisfaction), with Example 2 achieving the highest total score, and scores exceeding 4.3 points in three areas: fading of stretch marks, reduction in affected area, and restoration of skin tone. Comparative Example 1 scored 12.85 points, only at an "acceptable" level, demonstrating the irreplaceable role of crocodile oil as a core active ingredient and penetration enhancer in the formula. Comparative Example 2 scored 12.88 points, indicating the key role of adenosine in combating deep wrinkles by promoting collagen synthesis. Comparative Example 3 scored only 11.45 points, below the lower limit of "acceptable," indicating that the lack of Centella Asiatica extract resulted in the loss of the protective mechanism of "inhibiting collagen degradation," significantly reducing the overall anti-aging effect. It is worth noting that from Comparative Example 4 to Comparative Example 9, as extracts of blood-activating and stasis-removing plants such as safflower, Panax notoginseng, and Salvia miltiorrhiza were gradually removed, the total score decreased progressively. This demonstrates the contribution of safflower, Panax notoginseng, and Salvia miltiorrhiza to the overall repair effect in improving skin microcirculation. Among them, safflower and Panax notoginseng made particularly significant contributions to fading and reducing the area of ​​stretch marks (observable in comparison of the differences between Example 2 and Comparative Examples 6-7). The total score of the free crocodile oil prepared by the conventional high-temperature method in Comparative Example 10 was 15.00 points, which is between "acceptable" and "satisfactory," but significantly lower than the 18.30 points of Example 2. This proves that the enzymatic hydrolysis + nanoliposome process of this invention has substantial advantages in improving transdermal absorption efficiency and bioavailability.

[0097] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A crocodile oil anti-aging and repairing agent, characterized in that, It is composed of the following components in parts by weight: 15-20 parts crocodile oil, 1-3 parts adenosine, 1-3 parts safflower extract, 1-3 parts ginseng extract, 1-3 parts centella asiatica extract, 0.5-2.5 parts salvia miltiorrhiza extract, 0.2-1.5 parts notoginseng extract, 0.5-2.5 parts purslane extract, 1-5 parts polygonatum odoratum extract, and deionized water to make up to 100 parts.

2. The crocodile oil anti-aging and repairing agent according to claim 1, characterized in that, It consists of the following components in parts by weight: 20 parts crocodile oil, 2 parts adenosine, 2 parts safflower extract, 2 parts ginseng extract, 2 parts centella asiatica extract, 1.5 parts salvia miltiorrhiza extract, 0.8 parts notoginseng extract, 1.5 parts purslane extract, 3 parts polygonatum odoratum extract, and deionized water to make up to 100 parts.

3. The crocodile oil anti-aging and repairing agent according to claim 1, characterized in that, The crocodile oil is prepared by the following method: crocodile abdominal fat is taken, cleaned to remove impurities, cut into pieces, and minced into a fat paste; neutral protease is added to the fat paste for enzymatic hydrolysis, and the supernatant is collected by centrifugation to obtain crude crocodile oil; the crude crocodile oil is mixed with soybean lecithin, cholesterol, and Tween-80 at a mass ratio of 1:5:1:1.5, dissolved in ethanol, and evaporated at 36°C to form a film. After hydration, it is sonicated at 400W for 10 minutes to obtain crocodile oil nanoliposomes.

4. The crocodile oil anti-aging and repairing agent according to claim 3, characterized in that, Neutral protease was added to the chyme, and the enzymatic hydrolysis temperature was controlled at 48–52°C, the pH at 7.0–7.8, and the hydrolysis time at 3.5–4.5 hours, so that the degree of hydrolysis reached 15%–20%. The supernatant was collected by centrifugation to obtain crocodile crude oil.

5. The crocodile oil anti-aging and repairing agent according to claim 1, characterized in that, The safflower extract, ginseng extract, centella asiatica extract, salvia miltiorrhiza extract, notoginseng extract, purslane extract, and polygonatum odoratum extract are water or alcohol extracts of the corresponding plants.

6. The use of the crocodile oil anti-aging repair agent according to any one of claims 1 to 5 in the preparation of anti-aging or skin repair products.

7. A product characterized in that, Contains the crocodile oil anti-aging and repairing agent as described in any one of claims 1 to 5.

8. The product according to claim 7, characterized in that, The product in question is a cosmetic or skin care product.