Application of Antarctic red in preparation of composition for skin anti-inflammatory soothing

Abstract

The invention relates to an application of Antarctic red in preparation of a composition for skin anti-inflammatory soothing, and belongs to the field of biotechnology and cosmetics, the Antarctic red has a concentration of 7.81-62.50 g / mL, is non-toxic to cells, and can significantly inhibit excessive secretion of a macrophage inflammatory factor TNF-alpha caused by lipopolysaccharide stimulation, and the inhibition rate is 33.40%; the Antarctic red is derived from a fermentation product of a fungus Geomyces sp.wnf-18C (the preservation number is CCTCC NO: M2019086) or Geomyces sp.wnf-15A (the preservation number is CCTCC NO: M2012255), the Antarctic red is prepared by adopting a fermentation method, the components are natural, the potential irritation and safety risk of chemically synthesized anti-inflammatory components are avoided, and the Antarctic red is suitable for sensitive skin.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and cosmetics, specifically relating to the application of Antarctic Red in the preparation of anti-inflammatory and soothing compositions for the skin. Background Technology

[0002] Skin irritation, redness, sensitivity, and other discomfort are fundamentally linked to the skin's immune-inflammatory response. When the skin is exposed to external stimuli (such as physical friction, chemicals, or microbial components), immune cells (such as macrophages) are activated and release pro-inflammatory cytokines. These cytokines secrete inflammatory factors, such as tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-8 (IL-8), and interleukin-6 (IL-6), causing skin irritation. These inflammatory factors trigger and amplify local inflammatory responses, leading to vasodilation, nerve sensitivity, and impaired barrier function, clinically manifesting as redness, burning, stinging, and discomfort. Therefore, the soothing efficacy of a test sample can be evaluated by assessing changes in the levels of the inflammatory factor (TNF-α) secreted by cells.

[0003] Currently, most commercially available soothing cosmetics contain active ingredients derived from chemically synthesized anti-inflammatory drugs (such as glucocorticoid analogs) or some plant extracts. While chemically synthesized ingredients are highly effective, long-term use may pose safety risks such as skin dependence, thinning, and side effects. Many natural plant extracts, on the other hand, suffer from unstable efficacy, high effective concentrations, or complex compositions that can easily cause allergies. Therefore, developing a novel soothing ingredient that is naturally derived, highly safe, can clearly inhibit inflammatory pathways at low concentrations, and is supported by sufficient scientific data has become a crucial need in the cosmetics industry. Antarctic red is a natural product prepared through a specific microbial fermentation process, a green and sustainable process. This ingredient has previously been shown to possess various biological activities, but its precise efficacy in skin soothing, particularly based on cellular inflammation models, has not yet been systematically scientifically validated.

[0004] Patent No. 2015101171967, entitled "A Coloring Compound for Antarctic Fungi," discloses a fungus isolated from Antarctic soil. Geomyces sp. WNF-15A (Phy), this strain can ferment and produce Antarctic Red, and its safety has been proven. Antarctic Red, the raw material, is a natural product prepared through the fermentation process of this strain. The preparation process involves strain cultivation, fermenter optimization, and extraction and purification, offering advantages of being green and sustainable. However, its anti-inflammatory and soothing potential has not been previously verified. Summary of the Invention

[0005] The purpose of this invention is to provide a safe, effective, and naturally derived skin soothing solution that uses Antarctic Red to inhibit the release of TNF-α, thereby preventing and improving skin irritation, redness, and sensitivity.

[0006] The principle of this invention is based on the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage model. The soothing efficacy of the sample is evaluated by detecting changes in the levels of the inflammatory cytokine (TNF-α) secreted by the cells. Test data show that this component, at low concentrations (7.81-62.50 µg / mL, m / V), significantly inhibited the excessive secretion of the inflammatory cytokine TNF-α induced by LPS stimulation in macrophages, with an inhibition rate of 33.40%, and the results were statistically significant. p The concentration of Antarctic Red (<0.01) demonstrates its clear anti-inflammatory and soothing activity. This result not only fills a gap in the application of Antarctic Red in soothing cosmetic products but also provides the cosmetics industry with a natural solution based on empirical data.

[0007] This invention is achieved through the following technical solution: The use of Antarctic Red in the preparation of anti-inflammatory and soothing compositions for the skin, wherein the Antarctic Red is derived from fungi. Geomyces sp. wnf-18C (accession number CCTCC NO:M2019086) or Geomyces sp. The fermentation product of wnf-15A (accession number CCTCC NO: M2012255) is processed through a process including microbial culture, fermentation tank optimization, extraction, and drying to obtain a powdered raw material, which is then stored under cold and light-proof conditions. This method ensures the naturalness and consistency of the raw material.

[0008] Furthermore, the composition contains an effective amount of Antarctic Red at a concentration of 7.81-62.50 µg / mL. The composition can be formulated into dosage forms such as serums, lotions, creams, repair masks, and soothing sprays.

[0009] The use of Antarctic Red in anti-inflammatory and soothing effects on the skin: Antarctic Red at concentrations of 7.81-62.50 µg / mL can significantly inhibit the excessive secretion of macrophage inflammatory factor TNF-α induced by LPS stimulation, thereby having an anti-inflammatory and soothing effect on the skin.

[0010] A method for skin anti-inflammatory and soothing, wherein the method comprises applying Antarctic Red at a concentration not exceeding 62.50 µg / mL to the skin.

[0011] Furthermore, the aforementioned method of use allows for the direct application of Antarctic Red solution, or the Antarctic Red can be formulated into serums, lotions, creams, repair masks, soothing sprays, and other dosage forms.

[0012] The advantages of this invention compared to existing technologies are: It is naturally sourced, has good compatibility, is produced using bio-fermentation technology, and its ingredients are natural, stable, and easily combined with other skincare ingredients, making it widely applicable in various soothing, repairing, and barrier-strengthening cosmetic formulations. It has a low effective concentration and high safety; the ingredient exhibits significant efficacy at microgram concentrations (7.81-62.50 µg / mL), and MTT assays have verified its non-toxicity to cells at this concentration and within a wider range. As a natural fermentation product, it avoids the potential irritation and safety risks of chemically synthesized anti-inflammatory ingredients, making it suitable for sensitive skin. Furthermore, although Antarctic Red is a red pigment, its effective anti-inflammatory and soothing dose is only 7.81-62.50 µg / mL; therefore, it will not affect the skin's appearance after use. Attached Figure Description

[0013] Figure 1 Image showing the viability of RAW264.7 macrophages; Figure 2 This is a bar chart showing TNF-α content. Detailed Implementation

[0014] 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.

[0015] 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 *Geotrichum candida* (accession number CCTCCNO: 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 a 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 collected pigment eluent was then subjected to rotary evaporation to remove the ethanol, followed by vacuum freeze-drying to obtain a red pigment solid powder, named Antarctic Red.

[0016] Example 1: Antarctic Erythrocyte Toxicity Experiment 1. Experimental reagents and equipment; Experimental reagents: High glucose DMEM culture medium (Gibco), fetal bovine serum (Gibco), PBS (VivaCell), MTT (Sigma), DMSO (Sinopharm), trypsin (Gibco), LPS (Sigma), dexamethasone (Sigma), Mouse TNF-α ELISA kit (Shanghai Enzyme-Linked Biotechnology).

[0017] Experimental equipment: CO2 incubator (Thermo, 160i), biosafety cabinet (Sujing Antai, BSC-1604ⅡA2), microplate reader (Tecan, Spark).

[0018] 2. Testing Method: Cell seeding: 1×10 4 Macrophages were seeded at a density of cells / well in RAW264.7 to 96-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0019] 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.

[0020] Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table 1; Table 1 Test Concentration Setting Table .

[0021] Sample loading: Load samples when the cell seeding rate in the 96-well plate 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 sample loading, place the 96-well plate in an incubator (37 ℃, 5% CO2) for incubation.

[0022] 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°C 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.

[0023] Cell viability calculation: Calculated according to Formula 1 (Formula 1) 3. Experimental Results: Table 2 MTT test results .

[0024] The cell viability test results for different experimental groups are shown in Table 2. A cell viability graph was plotted with the eight selected concentrations of the samples as the x-axis and the cell viability values ​​as the y-axis (see Table 2). Figure 1 Therefore, according to the MTT results, Antarctic Red samples did not exhibit macrophage RAW264.7 cytotoxicity within the concentration range of 62.50 (µg / mL, m / V).

[0025] Example 2: Inflammatory factor content test 1. Experimental reagents and equipment: Experimental reagents: BSA (Abixin), glucose (Sinopharm), PBS (VivaCell), aminoguanidine hydrochloride (Yuanye Biotechnology), Antarctic red (Linyi University, red powder, stored at 4~8℃, protected from light).

[0026] Experimental equipment: Biosafety cabinet (Sujing Antai, BSC-1604ⅡA2), microplate reader (Tecan, Spark), high-speed constant temperature incubator (Yiheng, THZ).

[0027] 2. Test method: The specific settings for experimental grouping are shown in Table 3; Table 3 Experimental Design .

[0028] Cell seeding: 1×10 5 Cells were seeded into 24-well plates at a seeding density of cells / well and incubated overnight in an incubator (37 °C, 5% CO2).

[0029] Solution preparation: Prepare working solutions of different concentrations of the test substance according to Table 3 of the experimental design.

[0030] Induction and loading: When the cell deposition rate in the 24-well plates reached 40%–60%, 100 µL of 10×LPS working solution was added to each well according to the experimental design. The wells were shaken left and right to mix the drug. Simultaneously, the sample was added. The final LPS concentration was 1 µg / mL. Each group had 3 replicates. After loading, the plates were placed in an incubator (37℃, 5% CO2) and cultured for another 24 h.

[0031] Sample collection: After incubation, collect the cell culture supernatant into EP tubes, and then freeze the samples at -80°C.

[0032] 3. Experimental Results: Based on the experimental method, cell supernatant was collected, and TNF-α content was detected. The results are shown in Table 4, and the trend is shown in Figure 2. Compared with the BC group, the secretion of the inflammatory factor TNF-α in RAW264.7 macrophages in the NC group was significantly increased ( p <0.01 indicates that LPS stimulation modeling was successful in this experiment. Compared with the NC group, the PC group showed a significant decrease in the secretion of the inflammatory factor TNF-α in RAW264.7 macrophages at a loading concentration of 100 µg / mL dexamethasone. p <0.01 indicates that the positive control test was effective. Compared with the NC group, the secretion of the inflammatory factor TNF-α by macrophages was significantly reduced when the Antarctic Red sample was at a concentration of 62.50 (µg / mL, m / V). p <0.01%, the inhibition rate was 33.40%. Furthermore, it still exhibited some inhibitory effect at a concentration of 7.81 (µg / mL, m / V). p <0.05), showing a dose-dependent effect.

[0033] Therefore, based on the LPS-stimulated RAW264.7 macrophage model, Antarctic Red at concentrations of 7.81-62.50 (µg / mL, m / V) can inhibit the secretion of the inflammatory factor TNF-α in RAW264.7 macrophages, exhibiting anti-inflammatory and soothing effects. Table 4 Summary of TNF-α data ; Note: Significance between the NC group and the BC group is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##. Significance between the sample group, PC group, and NC group is indicated by... This means that a p-value < 0.05 is considered... A p-value < 0.01 indicates that... .

[0034] In summary, the compositions described, such as face creams, serums, lotions, repair masks, and soothing sprays, contain an effective amount of Antarctic Red, which has the effect of inhibiting the secretion of the inflammatory factor TNF-α from macrophage RAW264.7. Therefore, this indicates that Antarctic Red has anti-inflammatory and soothing effects on the skin.

Claims

1. The application of Antarctic Red in the preparation of anti-inflammatory and soothing compositions for the skin, characterized in that, The Antarctic Red originates 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.

2. The application according to claim 1, characterized in that, The composition contains an effective amount of Antarctic Red at a concentration of 7.81-62.50 µg / mL.

3. The application according to claim 1, characterized in that, The composition is a serum, lotion, cream, repairing mask, or soothing spray.

4. The use of Antarctic Red in anti-inflammatory and soothing skin treatments, characterized in that... Antarctic red, at concentrations of 7.81-62.50 µg / mL, can inhibit the excessive secretion of the macrophage inflammatory factor TNF-α induced by lipopolysaccharide stimulation, thereby exhibiting anti-inflammatory and soothing effects on the skin; 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.

5. A method for skin anti-inflammatory and soothing, characterized in that, The method involves applying Antarctic Red at a concentration of 7.81-62.50 µg / mL to the skin; the Antarctic Red is derived from fungi. Geomyces sp. WNF-18C or Geomyces sp. The fermentation product of wnf-15A, said Geomyces sp. wnf-18C has the accession number CCTCC NO:M2019086. Geomyces sp. The accession number for wnf-15A is CCTCC NO:M2012255.

6. The method according to claim 5, characterized in that, The method of use involves preparing Antarctic Red into formulations such as serums, lotions, creams, repairing masks, or soothing sprays.

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