New application of iso15 fatty acid

Abstract

The invention discloses a new application of isopentadecanoic acid, and discloses an application of isopentadecanoic acid in preparation of skin care cosmetics, isopentadecanoic acid has the effects of up-regulating expression of hBD-2 and silk protein in human immortalized keratinocytes, reducing expression synthesis of ROS and TRPV1 in the human immortalized keratinocytes, inhibiting release of NO by mouse macrophages, and inhibiting expression of the human immortalized keratinocytes. And the secretion of sebaceous gland cell grease is reduced.

Description

Technical Field

[0001] The present invention relates to an application of isopentadecanoic acid, in particular to a new application of isopentadecanoic acid. Background Art

[0002] In traditional skincare and related fields, the most commonly used fatty acids are those with even carbon chains, such as stearic acid and oleic acid. Research and application of odd-numbered carbon chain fatty acids are relatively limited. Known odd-numbered carbon chain fatty acids are mostly used in food, industry, and other fields. For example, in the food sector, they promote the absorption of fat-soluble vitamins, provide emulsification, and lubricate. In the industrial sector, they serve as lubricants, promoters, stabilizers, and more. Branched-chain fatty acids (BCFAs) are primarily saturated fatty acids (SFAs) with monomethyl, dimethyl, or polymethyl branching. Food and dairy products are mostly composed of single-branched-chain fatty acids, which are volatile. In monomethyl BCFAs, the main branch is located at the terminal methyl group (iso) or next to the terminal methyl group (anteiso).

[0003] Iso 15:0 fatty acids are composed of repeating structural units represented by formula I: Formula I; its main chain is composed of a hydrocarbon chain with 14 carbon atoms, belonging to the tetradecanoic acid series, with a carboxyl group (-COOH) at one end and a methyl branch on the carbon chain, which is located at the 13th carbon atom. Its system name is 13-methyltetradecanoic acid, and its English name is 13-Methyltetradecanoic acid, which can also be called isopentadecanoic acid. Its molecular formula is C 15 H 30 O2, CAS number is 2485-71-4.

[0004] Prior art discloses the anti-cancer and neuroprotective effects of isopentadecanoic acid, such as inducing cancer cell apoptosis through mitochondrial-mediated pathways, inhibiting tumor growth by regulating signaling pathways, protecting neurons, inhibiting neuronal apoptosis, and repairing cerebral ischemia damage. However, there are no public reports on the effects and applications of isopentadecanoic acid in cosmetics and personal care for improving skin and scalp. Summary of the Invention

[0005] The purpose of the present invention is to provide a new application of isopentadecanoic acid and disclose the application of isopentadecanoic acid in the preparation of skin care cosmetics. Isopentadecanoic acid has the characteristics of being able to upregulate the expression of hBD-2 and filaggrin in human immortalized keratinocytes, reduce the expression and synthesis of ROS and TRPV1 in human immortalized keratinocytes, inhibit the release of NO by mouse macrophages, and reduce the secretion of oil by sebaceous gland cells.

[0006] The technical solution of the present invention is to use isopentadecanoic acid in the preparation of skin care cosmetics.

[0007] In the aforementioned application, isopentadecanoic acid is used in the preparation of skin care cosmetics for upregulating the expression of hBD-2 in human immortalized keratinocytes.

[0008] In the aforementioned application, isopentadecanoic acid is used in the preparation of skin care cosmetics for reducing ROS expression in human immortalized keratinocytes.

[0009] In the aforementioned application, isopentadecanoic acid is used in the preparation of skin care cosmetics for increasing the expression of filaggrin in human immortalized keratinocytes.

[0010] In the aforementioned application, isopentadecanoic acid is used in the preparation of skin care cosmetics for reducing TRPV1 expression in human immortalized keratinocytes.

[0011] In the aforementioned application, isopentadecanoic acid is used in the preparation of skin care cosmetics that inhibit the release of NO by mouse macrophages.

[0012] Among the aforementioned applications, isopentadecanoic acid is used in the preparation of skin care cosmetics that reduce the oil secretion of sebaceous gland cells.

[0013] In the aforementioned application, the cellular usage mass concentration of isopentadecanoic acid is 10ppm~50ppm.

[0014] In the aforementioned application, the cell usage concentration of isopentadecanoic acid was 25 ppm.

[0015] In the aforementioned application, the skin care cosmetics include skin and / or scalp care cosmetics.

[0016] The present invention also provides a skin care cosmetic comprising isopentadecanoic acid.

[0017] Compared with the prior art, the present invention has the following beneficial effects: Compared with conventional saturated fatty acids, isopentadecanoic acid can significantly upregulate the expression of hBD-2 in human immortalized keratinocytes, thereby increasing the activity of antimicrobial peptides, improving skin defense capabilities, effectively regulating the balance of skin microecology, providing a suitable living environment for resident bacteria on the skin, promoting the growth and reproduction of beneficial bacteria, and inhibiting the invasion and reproduction of harmful bacteria.

[0018] Isopentadeca fatty acid can reduce the expression of ROS in human immortalized keratinocytes, reduce free radicals, alleviate oxidative stress damage, maintain skin homeostasis, promote skin renewal, delay cell aging, and maintain cell vitality.

[0019] Isopentadeca fatty acid can increase the expression and synthesis of filaggrin (FLG) in human immortalized keratinocytes, has moisturizing and skin barrier repair effects, promotes the renewal and repair of skin cells, and brings more comprehensive maintenance effects to the skin.

[0020] Isopentadeca fatty acid can reduce the expression and synthesis of TRPV1 in human immortalized keratinocytes, reduce skin inflammatory response, relieve skin itching, pain and other sensitive symptoms, and has a soothing effect.

[0021] Isopentadeca fatty acid can inhibit the release of NO by mouse macrophages, inhibit skin inflammatory response, reduce skin redness symptoms, and has a soothing effect.

[0022] Isopentadecanoic acid can reduce the secretion of oil in sebaceous gland cells, reduce lipid peroxidation reactions, reduce acne, inflammatory papules, etc., reduce symptoms such as redness, swelling, and sensitivity, and also has a certain auxiliary improvement effect on seborrheic alopecia. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the cytotoxicity of Iso 15:0 fatty acids on human immortalized keratinocytes.

[0024] Figure 2 This is a relative quantitative histogram of hBD-2 immunofluorescence of different fatty acids. In the figure, "*" indicates a significant level compared with the BC group, and two asterisks "**" indicate a significant level. P <0.01, three asterisks "***" indicate a significant level P <0.001.

[0025] Figure 3 Schematic diagram of the immunofluorescence staining results of hBD-2 for Iso 15:0 fatty acid (200×).

[0026] Figure 4 Schematic diagram of the results of immunofluorescence staining of Iso 15:0 fatty acid ROS (200×).

[0027] Figure 5 This is a relative quantitative bar graph of Iso 15:0 fatty acid ROS immunofluorescence; "#" in the figure indicates a significant level compared with the BC group, and "##" indicates a significant level P <0.01; “*” in the figure indicates the significant level compared with the NC group, and “*” indicates the significant level P <0.05, “**” indicates the significance level P <0.01.

[0028] Figure 6 Schematic diagram of the immunofluorescence staining results of Iso 15:0 fatty acid FLG (200×).

[0029] Figure 7 This is a relative quantitative histogram of Iso 15:0 fatty acid FLG immunofluorescence; "#" in the figure indicates a significant level compared with the BC group, and "#" indicates a significant level. P <0.05; “*” in the figure indicates the significant level compared with the NC group, and “*” indicates the significant level P <0.05, “***” indicates the significance level P <0.001.

[0030] Figure 8 Schematic diagram of the results of TRPV1 immunofluorescence staining with Iso 15:0 fatty acid (200×).

[0031] Figure 9 This is a relative quantitative bar graph of Iso 15:0 fatty acid TRPV1 immunofluorescence; "#" in the figure indicates a significant level compared with the BC group, and "##" indicates a significant level P <0.01; “*” in the figure indicates the significant level compared with the NC group, and “*” indicates the significant level P <0.05, “**” indicates the significance level P <0.01.

[0032] Figure 10 Schematic diagram of the cytotoxicity of Iso 15:0 fatty acids on mouse macrophages.

[0033] Figure 11 This is a bar graph of the NO content released by mouse macrophages; in the figure, "#" indicates a significant level compared with the BC group, and "####" indicates a significant level P <0.0001; in the figure, "*" indicates the significant level compared with the NC group, and "****" indicates the significant level P <0.0001.

[0034] Figure 12 Schematic diagram of Oil Red O staining and Nile Red staining results (400×).

[0035] Figure 13 This is a relative quantitative bar graph of the Nile red staining results; in the figure, "*" indicates the significant level compared with the BC group, and "***" indicates the significant level P <0.001, “****” indicates the significance level P <0.0001. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the examples, but they are not intended to limit the present invention.

[0037] Experimental Example 1: Immunofluorescence staining to evaluate the effect of isopentadecanoic acid (Iso 15:0 fatty acid) on antimicrobial peptides in human immortalized keratinocytes.

[0038] 1. Principle Defensin 2 (hBD-2) is a key regulatory molecule in the immune system, possessing direct bactericidal properties. It is a major antimicrobial peptide widely distributed in animals. Upregulating hBD-2 gene expression in immortalized human keratinocytes promotes defensin 2 synthesis, thereby exerting an immunomodulatory effect.

[0039] 2. Instruments and Equipment Microbalance, clean bench, carbon dioxide incubator, microplate reader, 96-well plate, fluorescence microscope.

[0040] 3. Reagents and Materials Cell material: human immortalized keratinocytes.

[0041] Reagents: DMEM medium, fetal bovine serum, double antibody, trypsin, PBS, Cell Counting Kit-8 reagent, trypan blue; DMEM complete medium: 89mL DMEM + 10mL FBS + 1mL P / S; hBD-2 rabbit antibody, DAPI staining solution, paraformaldehyde fixative, PBS, immunostaining blocking solution, immunostaining permeabilization solution.

[0042] 4. Operation process 4.1 CCK-8 cytotoxicity test 4.1.1 Pretreatment of test substance: Use a 0.22 μm filter to sterilize and filter the sample solution before pretreatment, and then use culture medium to prepare the sample to the required test concentration according to the table.

[0043] 4.1.2 Cell digestion: Cells that have reached the logarithmic growth phase are plated in a 96-well plate for detection. When the keratinocytes in the cell culture flask are more than 50-70% confluent, the culture medium is removed and the cells are washed twice with 5-10 mL of sterile PBS. Gently shake to ensure that no obvious foam is generated, and then remove the washing solution. Add trypsin / EDTA solution to the culture flask (1 mL for T25 cm2 culture flasks and 1 mL for T75 cm2 culture flasks). 2 Use 2 mL of culture flask), aspirate after 30 seconds, incubate at room temperature for 3-7 minutes, and observe the cell status from time to time. When more than 50% of the cells are floating, gently tap the culture flask to detach the cells. When most of the cells are detached, add 2 times the volume of trypsin solution and add room temperature complete medium containing FBS to gently pipette the cells. Transfer the cell suspension to a centrifuge tube; centrifuge at about 1000 rpm for 5 minutes and remove the supernatant.

[0044] 4.1.3 Cell Counting and Plating: Resuspend the cell pellet gently with complete culture medium to form a uniformly distributed single-cell suspension. Count the cell concentration using a cell counter and calculate the number of viable cells and cell survival rate. After cell counting, use a multichannel pipette to plate the cells so that the culture medium volume is 100 μL / well and the number of viable cells is 1-2 × 10 4 cell / well; set up a group of zero wells without cells as solvent control.

[0045] 4.1.4 Sample Addition: 24 hours after plating, when the cell confluence is 40-60%, add the test substance at varying concentrations to achieve the final concentration of the test concentration. Set up a test well with n ≥ 3 replicates. The test groups are set as a blank control group and a sample group. The specific groupings are shown in Table 1.

[0046] Table 1. Grouping table 4.1.5 CCK-8 Assay: After 24 hours of incubation, discard the supernatant and add 100 μl of 10% CCK-8 solution to each well. Incubate at 37°C in the dark for 1 hour. After incubation, read the OD value at 490 nm. Calculate cell viability according to the formula.

[0047] Cell viability (%) = (OD of sample well - OD of zero-adjustment well) / (OD of blank well - OD of zero-adjustment well) × 100%.

[0048] 4.2 Effects of antimicrobial peptides 4.2.1 Cell plating After counting the cells, dilute to the desired concentration and add the cell suspension to the cell culture dish so that the culture medium volume is 1 mL / well and the number of viable cells is 5 × 10 4 cell / well.

[0049] 4.2.2 Sample addition 24 hours after plating, when the cell confluence rate was 40-60%, the test substance was added at various concentrations to achieve the test concentration, with three parallel wells. The test groups were set up as a blank control group and a sample group. The specific groupings are shown in Table 2.

[0050] Table 2. Grouping of hBD-2 by different fatty acids 4.2.3 Immunofluorescence staining After 24 hours of sample addition, the culture medium was removed and the cells were washed three times with PBS.

[0051] Add fixative and fix at 37°C for 30 minutes. Aspirate the fixative and wash three times with washing solution, 3-5 minutes each time.

[0052] Add immunostaining permeabilization solution and permeabilize for 10 minutes at room temperature. Aspirate the permeabilization solution and wash three times with washing solution for 3-5 minutes each time.

[0053] Add immunostaining blocking solution and block at room temperature for 60 minutes. Use enough blocking solution to cover the sample. For samples in a 24-well plate, typically add 0.5 mL of blocking solution.

[0054] Aspirate the immunostaining blocking buffer and add hBD-2 rabbit monoclonal antibody. Incubate overnight at 4°C in the dark. Use only enough hBD-2 rabbit monoclonal antibody to cover the sample. Carefully aspirate the hBD-2 rabbit monoclonal antibody into an appropriate container. Wash three times with washing buffer for 5-10 minutes each.

[0055] Add anti-rabbit secondary antibody and incubate at 37°C for 1 hour. Use just enough anti-rabbit secondary antibody to cover the sample. Carefully aspirate the anti-rabbit secondary antibody into an appropriate container. Wash three times with washing buffer for 5-10 minutes each.

[0056] Add DAPI nuclear staining solution and stain at room temperature for 10 minutes. Aspirate the nuclear staining solution and wash three times with washing solution, each time for 3-5 minutes.

[0057] Observe under a fluorescence microscope. hBD-2 staining shows green fluorescence, and cell nuclei staining shows blue fluorescence.

[0058] V. Judgment Basis If the cell viability is >90%, the concentration at this cell viability is considered to be non-toxic to the cells.

[0059] If the expression level of hBD-2 in human immortalized keratinocytes under the action of the sample group is significantly higher than that in the blank control group BC, it is determined that the test substance in the sample group can effectively upregulate the expression of hBD-2 in human immortalized keratinocytes.

[0060] 6. Test Results Toxicity test results are shown in Figure 1 and as shown in Table 3.

[0061] Table 3. Cytotoxicity of Iso 15:0 fatty acids on human immortalized keratinocytes The results of toxicity experiments demonstrated that Iso 15:0 fatty acid was non-toxic to human immortalized keratinocytes at concentrations ranging from 0.5 to 50 μg / ml.

[0062] The test results are shown in Figure 2 、 Figure 3 and as shown in Table 4.

[0063] Table 4. Relative quantitative results of hBD-2 immunofluorescence for different fatty acids from Figure 2 、 Figure 3 As shown in Table 3, not all commercially available straight-chain fatty acids with varying carbon chains and degrees of saturation promote the expression and synthesis of hBD-2 in keratinocytes. Some even significantly reduce hBD-2 synthesis in keratinocytes, downregulating hBD-2 expression in immortalized human keratinocytes. However, Iso 15:0 fatty acids significantly upregulated hBD-2 expression and synthesis in immortalized human keratinocytes at all tested concentrations.

[0064] Experimental Example 2: Immunofluorescence staining was used to evaluate the effect of isopentadecanoic acid (Iso 15:0 fatty acid) on ROS expression in human immortalized keratinocytes.

[0065] 1. Principle The intracellular redox state is a key factor in maintaining normal cellular physiological function. Reactive oxygen species (ROS), as key players in intracellular redox reactions, changes in their content directly reflect the cellular redox balance. By measuring ROS levels, we can determine whether cells are in a state of oxidative stress or a relatively stable reduced state, and thus assess their overall health. For example, under normal physiological conditions, intracellular ROS production and clearance are in a dynamic equilibrium. When cells are exposed to external stimuli such as ultraviolet radiation or chemical toxins, ROS production increases. If ROS production exceeds the cell's clearance capacity, this imbalance can be disrupted, leading to oxidative stress and potentially causing cell damage or even death. Fluorescent dyes that specifically bind to ROS are then used to assess ROS levels in keratinocytes by observing the intensity of the fluorescence signal under a fluorescence microscope. Commonly used fluorescent dyes, such as DCFH-DA (dichlorodihydrofluorescein diacetate), are non-fluorescent. Upon entry into cells, they are hydrolyzed by intracellular esterases to produce DCFH, which is then oxidized by ROS to the fluorescent DCF (dichlorofluorescein). The fluorescence intensity is proportional to the intracellular ROS content.

[0066] 2. Instruments and Equipment Microbalance, clean bench, carbon dioxide incubator, fluorescence microscope, centrifuge, cell counting chamber.

[0067] 3. Reagents and Materials Cell material: human immortalized keratinocytes.

[0068] Reagents: trypsin / EDTA solution, phosphate-buffered saline (PBS), DMEM medium (containing 10% newborn calf serum), DCFH-DA.

[0069] 4. Operation process 4.1 Test Substance Pretreatment and Test Grouping 4.1.1 Cell plating After counting the cells, dilute to the desired concentration and add the cell suspension to the cell culture plate so that the culture medium volume is 1 mL / well and the number of viable cells is 5 × 10 4 cell / well.

[0070] 4.1.2 Sample processing After 24 hours of plating, when the cell confluence rate is 40-60%, add the test substance at various concentrations to achieve the test concentration. Set up three parallel test wells. Test groups are: blank control, negative control, positive control, and sample group. Vitamin E (VE) is used in the positive control group.

[0071] The positive control group and the sample group were protected with the test substance for 4 hours, and then the negative control group, the positive control group, and the sample group were stimulated with the stimulant for 20 hours. The specific grouping is shown in Table 5.

[0072] Table 5. Test group grouping 4.2 Immunofluorescence staining After 20 h of SDS stimulation, the culture medium was removed and the cells were washed three times with PBS.

[0073] DCFH-DA was diluted with PBS at a dilution ratio of 1:500.

[0074] Add DCFH-DA working solution and incubate at 37°C for 30 minutes. Aspirate the liquid and wash three times with washing solution for 3-5 minutes each time.

[0075] Observe under a fluorescence microscope. ROS staining is green fluorescence.

[0076] V. Judgment Basis If, under the action of the sample group, the expression level of ROS in the human immortalized keratinocytes is significantly lower than that in the negative control group, it is determined that the test substance in the sample group can effectively downregulate the expression of ROS in the human immortalized keratinocytes.

[0077] 6. Test Results The results of ROS immunofluorescence assay are shown in Figure 4 and Figure 5 shown.

[0078] Depend on Figure 3 It can be seen from the above that Iso 15:0 fatty acid can reduce the expression of ROS in human immortalized keratinocytes at a test concentration of 25 ppm.

[0079] Experimental Example 3: Immunofluorescence staining to evaluate the effect of isopentadecanoic acid (Iso 15:0 fatty acid) on filaggrin in human immortalized keratinocytes.

[0080] 1. Principle Filaggrin (FLG) is a protein that plays a vital role in the stratum corneum and plays a central role in the skin's barrier function. By binding to keratin fibers, it helps stratum corneum cells form a dense structure, reducing water loss from the skin and preventing the invasion of harmful external substances such as bacteria, viruses, and chemicals. Furthermore, it can regulate the release of skin cytokines and influence the recruitment and activation of immune cells, thereby playing a role in immune regulation in the skin's defense against external pathogens and allergens. Increased FLG expression in immortalized human keratinocytes can make the stratum corneum more compact, helping to maintain the integrity of the cell layer, reducing transepidermal water loss, and effectively blocking the invasion of harmful external substances such as bacteria, viruses, and allergens, thereby enhancing the skin's barrier function.

[0081] 2. Instruments and Equipment Microbalance, clean bench, carbon dioxide incubator, fluorescence microscope.

[0082] 3. Reagents and Materials Cell material: human immortalized keratinocytes.

[0083] Reagents: FLG rabbit antibody, DAPI staining solution, paraformaldehyde fixative, PBS, immunostaining blocking solution, immunostaining permeabilization solution.

[0084] 4. Operation process 4.1 Test Substance Pretreatment and Test Grouping 4.1.1 Cell plating After counting the cells, dilute to the desired concentration and add the cell suspension to the cell culture plate so that the culture medium volume is 1 mL / well and the number of viable cells is 5 × 10 4 cell / well.

[0085] 4.1.2 Sample processing 24 hours after plating, when the cell confluence reached 40-60%, test substances were added at varying concentrations to achieve the test concentrations, with three replicate wells. Test groups included a blank control, a negative control, a positive control, and a sample group. The positive control group was treated with dexamethasone (DEX). The positive and sample groups were protected with the test substances for 4 hours. Subsequently, the negative, positive, and sample groups were stimulated with the stimulator for 20 hours. Grouping is shown in Table 6.

[0086] Table 6. Test group grouping 4.2 Immunofluorescence staining After 20 h of stimulation, the culture medium was removed and the cells were washed three times with PBS.

[0087] Add fixative and fix at 37°C for 30 minutes. Aspirate the fixative and wash three times with washing solution, 3-5 minutes each time.

[0088] Add immunostaining permeabilization solution and permeabilize for 10 minutes at room temperature. Aspirate the permeabilization solution and wash three times with washing solution for 3-5 minutes each time.

[0089] Add immunostaining blocking solution and block at room temperature for 60 minutes. Use enough blocking solution to cover the sample. For samples in a 24-well plate, typically add 0.5 mL of blocking solution.

[0090] Aspirate the immunostaining blocking solution and add FLG rabbit monoclonal antibody. Incubate overnight at 4°C in the dark. Use only enough FLG rabbit monoclonal antibody to cover the sample. Carefully aspirate the FLG rabbit monoclonal antibody into an appropriate container. Wash three times with washing buffer for 5-10 minutes each.

[0091] Add anti-rabbit secondary antibody and incubate at 37°C for 1 hour. Use just enough anti-rabbit secondary antibody to cover the sample. Carefully aspirate the anti-rabbit secondary antibody into an appropriate container. Wash three times with washing buffer for 5-10 minutes each.

[0092] Add DAPI nuclear staining solution and stain at room temperature for 10 minutes. Aspirate the nuclear staining solution and wash three times with washing solution, each time for 3-5 minutes.

[0093] Observe under a fluorescence microscope. FLG staining shows green fluorescence, and cell nuclei staining shows blue fluorescence.

[0094] V. Judgment Basis If, under the action of the sample group, the expression level of FLG in the human immortalized keratinocytes is significantly higher than that in the negative control group, it is determined that the test substance in the sample group can effectively upregulate the expression of FLG in the human immortalized keratinocytes.

[0095] 6. Test Results The results of FLG immunofluorescence test are shown in Figure 6 and Figure 7 shown.

[0096] From the above, it can be seen that Iso 15:0 fatty acid can enhance the expression and synthesis of FLG in human immortalized keratinocytes at the tested concentrations of 50 ppm, 25 ppm and 10 ppm.

[0097] Experimental Example 4: Immunofluorescence staining to evaluate the effect of isopentadecanoic acid (Iso 15:0 fatty acid) on TRPV1 expression in human immortalized keratinocytes.

[0098] 1. Principle TRPV1 (Transient Receptor Potential Vanilloid 1) is an ion channel protein widely expressed in sensory neurons. It plays a key role in physiological processes such as pain perception, temperature regulation, and inflammatory responses. During inflammation, damaged tissue releases a variety of inflammatory mediators, such as prostaglandins and bradykinin. These substances can indirectly activate TRPV1, leading to the release of neuropeptides such as substance P and calcitonin gene-related peptide from sensory nerve endings, further exacerbating the inflammatory response and pain perception.

[0099] 2. Instruments and Equipment Microbalance, clean bench, carbon dioxide incubator, fluorescence microscope.

[0100] 3. Reagents and Materials Cell material: human immortalized keratinocytes.

[0101] Reagents: TRPV1 rabbit antibody, DAPI staining solution, paraformaldehyde fixative, PBS, immunostaining blocking solution, immunostaining permeabilization solution.

[0102] 4. Operation process 4.1 Test Substance Pretreatment and Test Grouping 4.1.1 Cell plating After counting the cells, dilute to the desired concentration and add the cell suspension to the cell culture plate so that the culture medium volume is 1 mL / well and the number of viable cells is 5 × 10 4 cell / well.

[0103] 4.1.2 Sample processing After 24 hours of plating, when the cell confluence rate reached 40-60%, the test substance was added at varying concentrations to achieve the test concentration, with three parallel wells. The test groups were: blank control, negative control, positive control, and sample group. The positive control and sample groups were incubated with the test substance for 4 hours, followed by stimulation with the stimulant for 20 hours. The specific groupings are shown in Table 7.

[0104] Table 7. Test group grouping 4.2 Immunofluorescence staining After 20 h of stimulation, the culture medium was removed and the cells were washed three times with PBS.

[0105] Add fixative and fix at 37°C for 30 minutes. Aspirate the fixative and wash three times with washing solution, 3-5 minutes each time.

[0106] Add immunostaining permeabilization solution and permeabilize for 10 minutes at room temperature. Aspirate the permeabilization solution and wash three times with washing solution for 3-5 minutes each time.

[0107] Add immunostaining blocking solution and block at room temperature for 60 minutes. Use enough blocking solution to cover the sample. For samples in a 24-well plate, typically add 0.5 mL of blocking solution.

[0108] Aspirate the immunostaining blocking solution and add TRPV1 rabbit monoclonal antibody. Incubate overnight at 4°C in the dark. Use enough TRPV1 rabbit monoclonal antibody to cover the sample. Carefully aspirate the TRPV1 rabbit monoclonal antibody into an appropriate container. Wash three times with washing buffer for 5-10 minutes each.

[0109] Add anti-rabbit secondary antibody and incubate at 37°C for 1 hour. Use just enough anti-rabbit secondary antibody to cover the sample. Carefully aspirate the anti-rabbit secondary antibody into an appropriate container. Wash three times with washing buffer for 5-10 minutes each.

[0110] Add DAPI nuclear staining solution and stain at room temperature for 10 minutes. Aspirate the nuclear staining solution and wash three times with washing solution, each time for 3-5 minutes.

[0111] Observe under a fluorescence microscope. TRPV1 staining shows green fluorescence, and the cell nucleus staining shows blue fluorescence.

[0112] V. Judgment Basis If, under the action of the sample group, the expression level of TRPV1 in the human immortalized keratinocytes is significantly lower than that in the negative control group, it is determined that the test substance in the sample group can effectively downregulate the expression of TRPV1 in the human immortalized keratinocytes.

[0113] 6. Test Results The results of TRPV1 immunofluorescence assay are shown in Figure 8 and Figure 9 shown.

[0114] Depend on Figure 8 and Figure 9 It can be seen that Iso 15:0 fatty acid can increase the expression and synthesis of TRPV1 in human immortalized keratinocytes at a test concentration of 25 ppm.

[0115] Experimental Example 5: Determination of nitric oxide content in a macrophage inflammatory cell model.

[0116] 1. Principle Cosmetics with soothing properties can help alleviate skin irritation. The occurrence of skin irritation primarily involves the physiological process of the skin barrier, neurovascular system, and immune inflammation. Alleviating skin inflammation can alleviate skin irritation. Nitric oxide (NO), as an immunomodulator, participates in the physiological process of skin inflammation. By inhibiting the release of excessive NO by activated immune cells, it can reduce the skin's immune response, inhibit inflammation, and thus improve skin irritation.

[0117] Methods Bacterial lipopolysaccharide (LPS) was used to induce the release of NO from the mouse mononuclear macrophage leukemia cell line RAW264.7. The NO concentration in the cell culture supernatant was determined by the Griess method. The inhibition rate of the test substances on the release of NO from mouse macrophages was calculated, and the soothing efficacy of the test substances was evaluated.

[0118] 2. Instruments and Equipment Microbalance, clean bench, carbon dioxide incubator, microplate reader, cell counter, inverted microscope, low-speed centrifuge.

[0119] 3. Reagents and Materials Cell material: Mouse mononuclear macrophage leukemia cell line RAW 264.7.

[0120] Reagents: high-glucose DMEM culture medium, fetal bovine serum, double-stranded antibodies, Cell Counting Kit-8 reagent, trypan blue, phosphate buffered saline (1xPBS), bacterial lipopolysaccharide (LPS), Beyotime nitric oxide (NO) detection kit (with standard); DMEM complete culture medium: 89mL DMEM + 10mL FBS + 1mL P / S.

[0121] 4. Operation process 4.1 CCK-8 cytotoxicity test 4.1.1 Pretreatment of test substance: Use a 0.22 μm filter to sterilize and filter the sample solution before pretreatment, and then use culture medium to prepare the sample to the required test concentration according to the table.

[0122] 4.1.2 Cell perfusion: Cells that have reached the logarithmic growth phase are plated in a 96-well plate for detection. When the macrophages in the cell culture flask are more than 50-70% confluent, gently tap the flask to detach the cells. When most of the cells are detached, gently perfuse the cells. Transfer the cell suspension to a centrifuge tube and centrifuge at approximately 1000 rpm for 5 minutes. Remove the supernatant.

[0123] 4.1.3 Cell Counting and Plating: Resuspend the cell pellet gently with complete culture medium to form a uniformly distributed single-cell suspension. Count the cell concentration using a cell counter and calculate the number of viable cells and cell survival rate. After cell counting, use a multichannel pipette to plate the cells so that the culture medium volume is 100 μL / well and the number of viable cells is 1-2 × 10 4 cell / well; set up a group of zero wells without cells as solvent control.

[0124] 4.1.4 Sample Addition: 24 hours after plating, when the cell confluence rate is 40-60%, add the test substance at various concentrations to achieve the final concentration of the test concentration. Set up a test well with n ≥ 3 replicates. The test groups are set as: blank control group and sample group. The specific grouping is shown in Table 8.

[0125] Table 8. Grouping situation 4.1.5 CCK-8 Assay: After 24 hours of incubation, discard the supernatant and add 100 μl of 10% CCK-8 solution to each well. Incubate at 37°C in the dark for 1 hour. After incubation, read the OD value at 490 nm. Calculate cell viability according to the formula.

[0126] Cell viability (%) = (OD of sample well - OD of zero-adjustment well) / (OD of blank well - OD of zero-adjustment well) × 100%.

[0127] 4.2 Nitric oxide content determination test 4.2.1 Cell plating After counting the cells, dilute to the desired concentration and add the cell suspension to the cell culture plate so that the culture medium volume is 1 mL / well and the number of viable cells is 1.5 × 10 5 cell / well.

[0128] 4.2.2 Sample addition 24 hours after plating, when the cell confluence rate was 40-60%, the culture medium in the cell culture plate was discarded. The test groups were set up as: blank control group, negative control group, positive control group, and sample group. The sample group was treated with culture medium containing different concentrations of sample and LPS, the negative control group was treated with cell culture medium containing LPS, the positive control group was treated with working solution containing DEX, and the blank control group was treated with only cell culture medium. The final concentration of the samples in different groups was ensured to be the test concentration. Three parallel test wells were set up. The specific grouping is shown in Table 9 below.

[0129] Table 9. Test group grouping 4.2.3 Collection of cell supernatant After the incubation, 1 mL of cell culture supernatant was collected from each well into a 1.5 mL sterile centrifuge tube and the NO content was immediately tested.

[0130] 4.2.4 NO content detection The nitric oxide (NO) detection kit was used for detection.

[0131] 4.2.5 Calculation of NO content Use professional curve creation software to draw a standard curve with the concentration of the standard included in the nitric oxide (NO) detection kit as the ordinate and the corrected OD540 value of the standard as the abscissa. Obtain a regression equation, substitute the corrected OD540 value of the test substance into the equation, calculate the NO content, and take the average of the three replicate wells as the final NO content result.

[0132] V. Judgment Basis If the cell viability is >90%, the concentration at this cell viability is considered to be non-toxic to the cells.

[0133] If the content of NO released by mouse macrophages under the action of the sample group is significantly lower than that of the negative control group, it means that the test substance in the sample group has an inhibitory effect on the release of NO by mouse macrophages, has a soothing effect, and can reduce skin redness.

[0134] 6. Test Results The results of the mouse macrophage cytotoxicity test are shown in Figure 10 and shown in Table 10.

[0135] Table 10. Cytotoxicity of Iso 15:0 fatty acids on mouse macrophages The results of toxicity experiments showed that Iso 15:0 fatty acid was non-toxic to mouse macrophages at concentrations of 10-50 μg / ml.

[0136] The results of the NO content determination test are shown in Figure 11 shown.

[0137] Depend on Figure 11 It can be seen that Iso 15:0 fatty acid can significantly inhibit the release of NO by mouse macrophages at the test concentrations of 50ppm, 25ppm, and 10ppm, and has a soothing effect.

[0138] Experimental Example 6: Evaluation of the oil-control effect of 15:0 fatty acids (Iso 15:0 fatty acids) in sebaceous gland cells by Oil Red staining.

[0139] 1. Principle Oil Red O is an azo dye, a fat-soluble, non-polar dye. Sebaceous gland cells are rich in lipids, primarily triglycerides and fatty acids. Based on the principle of like dissolves like, Oil Red O dissolves in lipids and binds to the non-polar portions of the lipids through interactions such as van der Waals forces, giving the lipids a red color. This allows for specific staining of lipids within sebaceous gland cells, allowing for clear microscopic observation of their distribution and content.

[0140] 2. Instruments and Equipment Clean bench, carbon dioxide incubator, fluorescence microscope, centrifuge, cell counting chamber, cell culture dish.

[0141] 3. Reagents and Materials Cell material: human sebaceous gland cells SZ95.

[0142] Reagents: phosphate-buffered saline (PBS), DMEM medium (containing 10% newborn calf serum), 4% paraformaldehyde fixative, Oil Red O powder, isopropanol, and hematoxylin staining solution.

[0143] 4. Operation process 4.1 Test Substance Pretreatment and Test Grouping 4.1.1 Cell plating After counting the cells, dilute to the required concentration and add the cell suspension to the cell culture plate so that the culture medium volume is 1 mL / well and the number of viable cells is 8 × 10 4 cell / well.

[0144] 4.1.2 Sample processing After 24 hours of plating, when the cell confluence rate reached 40-60%, the test substance was added at varying concentrations to achieve the test concentration. Three parallel wells were set up and cultured for another 24 hours. The test groups were: blank control, negative control, positive control, and sample group. The test group groups are shown in Table 11.

[0145] Table 11. Test group grouping 4.2 Oil red staining Prepare 60% isopropyl alcohol (30 mL 100% isopropyl alcohol + 20 mL water); Prepare 60% Oil Red (6 mL Oil Red + 4 mL water, preheat to 60°C, cool naturally, filter with a 0.45 μM filter, and prepare immediately before use); The culture medium was discarded, and the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min.

[0146] The fixative was discarded, the cells were washed three times with PBS, and 60% isopropanol was added to infiltrate the cells for 15-20 seconds.

[0147] Discard 60% isopropanol, add the prepared oil red staining solution, and stain in the dark for 1 hour.

[0148] To remove the oil red, rinse with 60% isopropyl alcohol for 3-5 seconds.

[0149] Wash with PBS three times; add hematoxylin to stain for 5 seconds.

[0150] V. Judgment Basis Oil Red staining primarily stains intracellular lipids. Normally, lipids stain bright red with Oil Red O. Distinct red areas within cells indicate the presence of lipids, and darker red areas generally indicate higher lipid content. Counterstaining with hematoxylin results in a blue nucleus, creating a sharp contrast and facilitating observation of cell morphology and the distribution of lipids within the cell.

[0151] 6. Test Results The results of the oil red staining test are shown in Figure 12 As shown. Figure 12 It can be seen that Iso 15:0 fatty acid can reduce the secretion of oil in sebaceous gland cells at the test concentrations of 50ppm, 25ppm and 10ppm.

[0152] Test Example 7: Nile red staining to evaluate the oil-control effect of isopentadecanoic acid (Iso 15:0 fatty acid) in sebaceous gland cells.

[0153] 1. Principle Nile Red staining is a fluorescent staining technique commonly used to assess oil content and has important applications in evaluating the oil-control effectiveness of cosmetics, such as skincare products. Leveraging its specific binding to oils, Nile Red can reflect changes in oil content through fluorescence signal intensity, thereby assessing the effectiveness of oil-control products. Nile Red is a lipophilic fluorescent dye with a highly lipid-soluble molecular structure. In oils, Nile Red emits strong fluorescence (maximum excitation wavelength approximately 550 nm, emission wavelength approximately 630 nm, resulting in red fluorescence). Its fluorescence intensity decreases significantly (to almost zero) in water or polar solvents. Nile Red embeds into the hydrophobic structure of oils (such as triglycerides, fatty acids, and sebum) through hydrophobic interactions, forming a stable bond, resulting in a distinct fluorescence signal under a fluorescence microscope or fluorescence spectrophotometer.

[0154] 2. Instruments and Equipment Clean bench, carbon dioxide incubator, fluorescence microscope, centrifuge, cell counting chamber, cell culture dish.

[0155] 3. Reagents and Materials Cell material: human sebaceous gland cells SZ95.

[0156] Reagents: DMEM high-glucose culture medium (without pyruvate), fetal bovine serum, double-antibody, PBS, trypsin, isotretinoin, finasteride, Nile red staining solution, and FDA staining solution.

[0157] 4. Operation process 4.1 Test Substance Pretreatment and Test Grouping 4.1.1 Cell plating After counting the cells, dilute to the desired concentration and add the cell suspension to the cell culture plate so that the culture medium volume is 1 mL / well and the number of viable cells is 5 × 10 4 cell / well.

[0158] 4.1.2 Sample processing After 24 hours of plating, when the cell confluence rate reached 40-60%, the test substance was added at varying concentrations to achieve the test concentration. Three parallel wells were set up and cultured for another 24 hours. The test groups were: blank control, negative control, positive control, and sample group. The specific groupings are shown in Table 12.

[0159] Table 12. Test group grouping 4.2 Nile red staining Rinse: discard the culture medium and wash with PBS three times; Fixation: Add 4% paraformaldehyde (300 μL) for 30 min; Staining: Prepare Nile red stain (working concentration: 10 μg / mL), discard the fixative, rinse the cells three times with PBS, add Nile red stain (300 μL) and stain for 15 min; Photography: Observe the staining of cells in each group under an inverted fluorescence microscope and take photos; Result analysis: Image Pro Plus software was used to quantitatively analyze the fluorescence intensity.

[0160] V. Judgment Basis The fluorescence intensity emitted by Nile Red after binding to lipids is positively correlated with the lipid content in the sample. A distinct red area within the cell indicates the presence of lipids, and darker red areas generally indicate a higher lipid content.

[0161] 6. Test Results The test results are shown in Figure 12 and Figure 13 As shown. Figure 12 and Figure 13 It can be seen that compared with the blank control group BC, Iso 15:0 fatty acid at the test concentrations of 50ppm, 25ppm, and 10ppm can reduce the secretion of oil in sebaceous gland cells.

[0162] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Those skilled in the art may modify the technical solutions described in the above embodiments, or make equivalent replacements for some of the technical features therein; and all these modifications and replacements should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Application of isopentadecanoic acid in the preparation of skin care cosmetics.

2. The use of the isopentadecanoic acid according to claim 1 in preparing skin care cosmetics, characterized in that: Application of isopentadecanoic acid in the preparation of skin care cosmetics for upregulating hBD-2 expression in human immortalized keratinocytes.

3. The use of the isopentadecanoic acid according to claim 1 in preparing skin care cosmetics, characterized in that: Application of isopentadecanoic acid in the preparation of skin care cosmetics for reducing ROS expression in human immortalized keratinocytes.

4. The use of the isopentadecanoic acid according to claim 1 in preparing skin care cosmetics, characterized in that: Application of isopentadecanoic acid in the preparation of skin care cosmetics for increasing filaggrin expression in human immortalized keratinocytes.

5. The use of the isopentadecanoic acid according to claim 1 in preparing skin care cosmetics, characterized in that: Application of isopentadecanoic acid in the preparation of skin care cosmetics for reducing TRPV1 expression in human immortalized keratinocytes.

6. The use of the isopentadecanoic acid according to claim 1 in preparing skin care cosmetics, characterized in that: Application of isopentadecanoic acid in the preparation of skin care cosmetics for inhibiting the release of NO from mouse macrophages.

7. The use of the isopentadecanoic acid according to claim 1 in preparing skin care cosmetics, characterized in that: Application of isopentadecanoic acid in the preparation of skin care cosmetics for reducing oil secretion of sebaceous gland cells.

8. Use of the isopentadecanoic acid according to claim 2, 4, 6 or 7 in preparing skin care cosmetics, characterized in that: The cell usage mass concentration of isopentadecanoic acid is 10ppm~50ppm.

9. Use of the isopentadecanoic acid according to claim 3 or 5 in preparing skin care cosmetics, characterized in that: The cell mass concentration of isopentadecanoic acid was 25 ppm.

10. The use of the isopentadecanoic acid according to claim 1 in preparing skin care cosmetics, characterized in that: The skin care cosmetics include skin and / or scalp care cosmetics.

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