Application of Antarctic haematochrome in preparation of composition with skin tightening function

Abstract

The invention relates to application of Antarctic red in preparation of a composition with a skin tightening function, and belongs to the technical field of biology, the Antarctic red is derived from a fungus Geomyces sp.wnf-18C or a fermentation product of Geomyces sp.wnf-15A, the preservation number of the Geomyces sp.wnf-18C is CCTCC NO: M2019086, and the preservation number of the Geomyces sp.wnf-15A is CCTCC NO: M2012255. The Antarctic red has no toxicity to cells when the concentration is not higher than 250g / mL, and the Antarctic red can significantly improve the cell viability of human-derived fibroblasts and has the effect of increasing skin compactness.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of Antarctic red pigment in the preparation of compositions with skin-tightening functions. Background Technology

[0002] Increasing skin firmness refers to the physiological process of combating sagging and wrinkles by strengthening the skin's internal support structure, improving elasticity, and lifting contours. The core of this process lies in maintaining and repairing the extracellular matrix of the dermis, particularly the collagen, elastin, and hyaluronic acid network synthesized and regulated by fibroblasts. With age, photoaging, and oxidative stress, skin firmness gradually declines, primarily due to reduced activity of dermal fibroblasts, decreased collagen and elastin synthesis, and increased degradation. This leads to the collapse of the skin's "skeleton" and decreased elasticity, resulting in sagging contours and deepening of static wrinkles such as nasolabial folds. Currently, protecting fibroblasts and improving cell vitality are important directions for maintaining skin firmness.

[0003] Currently, protecting fibroblasts and maintaining their synthetic activity has become a major research direction for promoting skin firmness. In cell experiments studying skin firmness, fetal bovine serum (FBS), rich in various growth factors, hormones, and other bioactive substances, can effectively promote fibroblast proliferation and the synthesis of collagen and elastin, thus providing a clear and reliable efficacy benchmark for evaluating the efficacy of test substances. However, due to core issues such as safety (risk of animal-derived viruses), uncontrollable ingredients (large batch-to-batch variations), ethical controversies (animal welfare issues in the collection process), and high costs (unstable supply and large price fluctuations), it cannot be directly applied to the cosmetic field. Therefore, the current strategy still relies on classic approaches using antioxidants (such as vitamin E and coenzyme Q10) to neutralize free radicals, protect cells from damage, and maintain their normal function. However, these traditional antioxidants have significant limitations, mainly due to their relatively simple mechanisms of action, and the potential for irritation or cytotoxicity of some synthetic antioxidants at high concentrations, limiting their safe application. Therefore, developing safe, low-concentration effective, and naturally derived compositions to protect fibroblasts has become a key breakthrough in promoting skin firmness.

[0004] Patent No. 2015101171967, entitled "A Coloring Compound for Antarctic Fungi," discloses a fungus isolated from Antarctic soil. Geomyces sp. wnf - 15A (phy) is a strain capable of fermenting Antarctic Red, the safety of which has been proven. Antarctic Red, the raw material, is a natural product prepared through this fermentation process, which involves strain cultivation, fermenter optimization, and extraction and purification, offering advantages in terms of green sustainability. Summary of the Invention

[0005] The purpose of this invention is to provide a safe and efficient solution for increasing skin firmness by using Antarctic Red to protect fibroblasts and maintain their synthetic activity, thereby reducing the damage to fibroblasts caused by environmental stress and maintaining the youthful state of cells and related skin laxity caused by synthetic function.

[0006] This invention is achieved through the following technical solution: Application of Antarctic Red in the preparation of compositions with skin-tightening functions, wherein the Antarctic Red is derived from fungi. Geomyces sp. WNF-18C or Geomyces sp. The fermentation product of wnf-15A, said Geomyces sp. The accession number for wnf-18C is CCTCC NO:M2019086. Geomyces sp. The WNF-15A strain, with accession number CCTCC NO: M2012255, undergoes a specific process including microbial culture, fermentation tank optimization, extraction, and drying to obtain a powdered raw material, which is then stored refrigerated and protected from light. This method ensures the naturalness and consistency of the raw material.

[0007] Furthermore, applying Antarctic Red to the skin can enhance the cell vitality of human fibroblasts, thereby achieving the goal of skin tightening.

[0008] Furthermore, the application method involves applying Antarctic Red in the form of serum, lotion, cream, repair mask, or soothing spray to the skin surface.

[0009] Furthermore, in the aforementioned applications, the concentration of Antarctic Red is 7.81-250 μg / mL.

[0010] A liquid or ointment for increasing skin firmness, wherein the concentration of Antarctic Red in the liquid or ointment is 7.81-250 μg / mL; the liquid may be a moisturizing lotion, and the ointment may be a lotion or cream for use on the body or face.

[0011] A facial mask for increasing skin firmness, the mask containing Antarctic Red, wherein the concentration of Antarctic Red in the mask liquid is 7.81-250 μg / mL.

[0012] A method for increasing skin firmness involves applying Antarctic Red to the skin at a concentration of 7.81-250 μg / mL. This method aims to tighten the skin by enhancing the cell viability of human fibroblasts. The Antarctic Red is derived from fungi. Geomyces sp. WNF-18C or Geomyces sp. The fermentation product of wnf-15A, said Geomyces sp.The accession number for wnf-18C is CCTCC NO:M2019086. Geomyces sp. The accession number for wnf-15A is CCTCC NO:M2012255.

[0013] Furthermore, the method of use involves preparing Antarctic Red into formulations such as serums, lotions, creams, repairing masks, or soothing sprays.

[0014] The beneficial effects of this invention compared to the prior art are as follows: This invention is the first to demonstrate the skin-tightening effects of Antarctic Red through standardized experiments. Test data showed that after treatment at a concentration of 250 (μg / mL, m / V) for 48 hours, the cell viability of human fibroblasts significantly increased, with an increase rate of 19.76%, and this difference was statistically significant compared to the control group. p <0.01). Furthermore, it remained effective at a concentration of 7.81 (μg / mL, m / V) (increase rate of 15.65%). p The result (<0.01) indicates that the sample has the effect of increasing skin firmness. This result not only fills the gap in the application of Antarctic Red in the preparation of compositions with firming function, but also provides the cosmetics industry with a natural solution based on empirical data.

[0015] Antarctic red, a natural active ingredient extracted from Antarctic fungi, possesses superior safety and increases the cell vitality of fibroblasts. Therefore, this ingredient increases skin firmness and has application value in improving skin aging and sagging. Furthermore, although Antarctic red is a red pigment, the effective dose for increasing skin firmness is only 7.81-250 µg / mL, so it will not affect the appearance of the skin after use. Attached Figure Description

[0016] Figure 1 This is a graph showing the toxicity of Antarctic red samples to human fibroblasts. Figure 2 The bar chart shows the effect of Antarctic Red on the viability of human fibroblasts. Detailed Implementation

[0017] The technical solution of the present invention will be further explained below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0018] The Antarctic red pigment used in the following examples was prepared according to the method disclosed in patent number 2019101866038, entitled "A Mutant Strain of *Gastromyxoides* (accession number CCTCC NO: M2019086) and its Applications." This mutant strain is a mutation of the original strain disclosed in patent number 2015101171967, entitled "A Colorimetric Compound of Antarctic Fungi." The fermentation broth contained 10 g / L glucose and 200 g / L potato, the fermentation temperature was 15°C, and the fermentation time was 12 days. The fermentation broth obtained from the above 150 L fermenter was transferred to a centrifuge bottle and centrifuged at 7500 r / min for 15 min. The supernatant was then filtered through a 200-mesh sieve to remove bacterial cells, and the resulting filtrate was the crude pigment solution. The crude pigment solution was adsorbed onto DPH-722 macroporous adsorption resin until saturation. The resin was then packed into a chromatography column, and two column volumes were washed with water at a rate of 15 mL / min. The pigment was then eluted with 70% ethanol at the same rate, and the eluent was collected. The ethanol was removed by rotary evaporation, and the resulting solid red pigment powder was obtained by vacuum freeze-drying and named Antarctic Red.

[0019] Example 1: Antarctic Erythrocyte Toxicity Experiment 1. Experimental reagents and equipment; Experimental reagents: low-glucose DMEM culture medium (Pronosai), fetal bovine serum (Gibco), PBS (VivaCell), MTT (Sigma), DMSO (Sinopharm), trypsin (Gibco), Antarctic red (Linyi University, red powder, stored at 4~8℃, protected from light).

[0020] Experimental equipment: CO2 incubator (Thermo, 160i), biosafety cabinet (ESCO, LA2-6A1), inverted fluorescence microscope (Keyence BZ-X1000), microplate reader (Tecan, Spark).

[0021] 2. Testing methods; Cell seeding: at 8 × 10 3 Human fibroblasts were seeded into 96-well plates at a seeding density of cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0022] Experimental Groups: The experiment included a zeroing group, a control group, a positive control group, and a sample group. Within the sample group, each sample had eight concentration gradients, with three replicate wells for each gradient.

[0023] Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table 1; Table 1 Test Concentration Setting Table .

[0024] Sample loading: Load the cells into the 96-well plate when the cell seeding rate reaches 40%–60%. Add 200 μL of culture medium containing 10% PBS to each well of the control group; add 200 μL of culture medium containing 10% DMSO to each well of the positive control group; add 200 μL of culture medium containing the corresponding concentration of the sample to each well of the sample group; add 200 μL of cell culture medium to each well of the zeroing group without cell seeding. After loading, place the 96-well plate in an incubator (37 ℃, 5% CO2) for incubation.

[0025] Detection: After culturing cells for 24 h, the supernatant was discarded, and MTT working solution (0.5 mg / mL) was added. The cells were incubated at 37 ℃ in the dark for 4 h. After incubation, the supernatant was discarded, and 100 μL of DMSO was added to each well. The OD value was read at 490 nm.

[0026] Cell viability calculation: Calculated according to Formula 1 Formula 1.

[0027] 3. Experimental results; Eight sample loading concentrations were set, and cytotoxicity assays were performed. The MTT assay results are shown in Table 2. Figure 1 According to MTT results, the samples did not show human fibroblast toxicity within the Antarctic Red 250 concentration range (μg / mL, m / V); Table 2. Results of MTT assay for cytotoxicity .

[0028] Example 2: Antarctic Red Cell Proliferation Test Experiment 1. Experimental reagents and equipment; Experimental reagents: low-glucose DMEM culture medium (Pronosai), fetal bovine serum (Gibco), PBS (VivaCell), MTT (Sigma), DMSO (Sinopharm), trypsin (Gibco), Antarctic red (Linyi University, red powder, stored at 4~8℃, protected from light).

[0029] Experimental equipment: CO2 incubator (Thermo, 160i), biosafety cabinet (ESCO, LA2-6A1), inverted fluorescence microscope (Keyence BZ-X1000), microplate reader (Tecan, Spark).

[0030] 2. Testing methods; Cell seeding: Collect human fibroblasts in logarithmic growth phase and seed them at a cell density of 8 × 10⁶ cells / year. 4 / well inoculated into a 24-well culture plate.

[0031] Sample loading: After incubation in an incubator (37 ℃, 5% CO2) for 24 h, the specific settings for experimental groups are shown in Table 3; Table 3 Experimental Design .

[0032] Detection: 48 h after sample loading, the supernatant was discarded, and 500 μL of culture medium containing 0.5 mg / mL MTT was added. The mixture was incubated at 37°C in the dark for 2 h. After incubation, the supernatant was discarded, and 300 µL of DMSO was added to each well. The OD value was read at 490 nm.

[0033] Cell viability calculation: Calculated according to Formula 1.

[0034] 3. Experimental results; The test results are shown in Table 4 and Figure 2 As shown, compared with the BC group, the PC group with 10% FBS medium significantly improved the cell viability of human fibroblasts. p <0.01 indicates that the positive control assay was effective. Compared with the BC group, the cell viability of human fibroblasts was significantly increased at a sample concentration of 250 (μg / mL, m / V). p <0.01%, with an improvement rate of 19.76%, and it still maintained efficacy at a concentration of 7.81 (μg / mL, m / V) (improvement rate of 15.65%). p The value <0.01 indicates that the Antarctic Red sample has a firming effect.

[0035] In summary, based on the human fibroblast model, treatment with Antarctic Red at concentrations of 7.81-250 (μg / mL, m / V) for 48 hours significantly increased the cell viability of human fibroblasts, with a maximum increase rate of 19.76%, which was statistically different from that of the BC group. p <0.01 indicates that the sample has a firming effect.

[0036] Table 4 Summary of Cell Viability Results ; ; Note: Compared with group BC, the significance of group PC and sample group is as follows: express, p -value < 0.05 means , p -value < 0.01 means .

Claims

1. Use of Antarctic red in the preparation of a composition having skin tightening function, characterized in that, The Antarctic red is derived from a fungus Geomyces sp. wnf-18C or Geomyces sp. wnf-15A fermentation product, the Geomyces sp. wnf-18C has a CCTCC NO: M2019086, Geomyces sp. wnf-15A has a CCTCC NO: M2012255.

2. Use according to claim 1, characterized in that, Antarctic red is applied to the skin to tighten the skin by increasing the cell viability of human fibroblasts.

3. Use according to claim 2, characterized in that, The application method is to apply the Antarctic red to the skin surface in the form of an essence, a cream, a cream, a repair mask or a soothing spray.

4. Use according to claim 3, characterized in that, The concentration of Antarctic red in the application is 7.81-250 μg / mL.

5. A liquid or paste for increasing skin firmness, characterized by, The concentration of Antarctic red in the liquid or paste is 7.81-250 μg / mL; the liquid is skin lotion, the paste is body or face emulsion or cream, and the Antarctic red is derived from fungi Geomyces sp. wnf-18C or Geomyces sp.wnf-15A fermentation product, the Geomyces sp. wnf-18C has a preservation number of CCTCC NO: M2019086, Geomyces sp. wnf-15A has a preservation number of CCTCC NO: M2012255.

6. A mask for increasing skin firmness, characterized by, The mask contains the Antarctic red, the concentration of Antarctic red in the mask liquid is 7.81-250 μg / mL, and the Antarctic red is derived from fungi Geomyces sp. wnf-18C or Geomyces sp. wnf-15A fermentation product, the Geomyces sp. wnf-18C has a preservation number of CCTCC NO: M2019086, Geomyces sp. wnf-15A has a preservation number of CCTCC NO: M2012255.

7. A method of increasing skin firmness, characterized by, The method is to apply the Antarctic red on the skin, the use concentration of the Antarctic red is 7.81-250 mu g / mL, the cell viability of human fibroblasts is improved, so that the skin is tightened; the Antarctic red is derived from fungi Geomyces sp. wnf-18C or Geomyces sp.wnf-15A fermentation product, the Geomyces sp. The preservation number of wnf-18C is CCTCC NO: M2019086, Geomyces The preservation number of sp.wnf-15A is CCTCC NO: M2012255.

8. The method of claim 7, wherein, The Antarctic red is used in the form of an essence, a cream, a cream, a repair mask or a soothing spray.

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