Application of trans-aromatic folic acid in preparation of cosmetics with soothing effect

Abstract

The invention discloses an application of trans-aromatic folic acid in preparation of cosmetics with a soothing effect. The composition has the advantage of being capable of being applied to preparation of cosmetics with the soothing effect.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to the application of trans-geraniol in the preparation of cosmetics with soothing effects. Background Technology

[0002] As the largest barrier organ in the human body, the skin's homeostasis depends on the integrity of the epidermal structure, the precision of immune regulation, and the coordination of signal transduction. Keratinocytes, as the main constituent cells of the epidermis, not only maintain the physical barrier by synthesizing keratin proteins and lipids, but also play a central role in skin homeostasis by secreting cytokines and participating in the regulation of inflammatory signals. When the skin is subjected to external stimuli (such as ultraviolet radiation, chemicals, mechanical friction, etc.) or internal imbalances, keratinocyte function is easily disrupted, leading to a series of skin discomfort problems.

[0003] The core of skin soothing mechanisms lies in blocking or alleviating the generation and transmission of discomfort signals. Nuclear factor-κB (NF-κB), a key transcription factor in classic inflammatory signaling pathways, is activated upon stimulation and enters the cell nucleus, initiating the expression of pro-inflammatory factors such as IL-6 and TNF-α, triggering inflammatory responses such as erythema and swelling. It is a crucial upstream driver of skin discomfort. Simultaneously, external stimuli (such as UVB irradiation) induce excessive production of intracellular reactive oxygen species (ROS). ROS not only directly damage the structure and function of keratinocytes but can also further activate the NF-κB pathway through oxidative stress, exacerbating skin barrier damage.

[0004] In addition, transient receptor potential vanillic acid subtype 1 (TRPV1), as an important ion channel in the sensory nerve endings of the skin, is activated when exposed to inflammatory factors, temperature changes or chemical stimulation. It can transmit discomfort signals such as itching and burning, and works synergistically with NF-κB-mediated inflammatory response and ROS-induced oxidative stress to amplify the discomfort of the skin.

[0005] Currently, most skin-soothing ingredients focus on regulating a single target, but cannot simultaneously address inflammatory signals and neurosensory regulation, resulting in limited and unstable soothing effects.

[0006] Therefore, developing a multi-target soothing ingredient that can simultaneously act on keratinocyte protection, NF-κB pathway inhibition, ROS clearance, and TRPV1-related signal regulation has become an important direction for solving current skin discomfort problems.

[0007] The systematic name for trans-geranilic acid is trans-3,7-dimethyl-2,6-octadienoic acid. It is a product of plant secondary metabolism and is mainly found in the essential oils of aromatic plants, such as citrus (orange, lemon), geranium, rose, etc.; or in the metabolic pathways of plants. It is usually generated by the oxidation of monoterpenoid alcohols (such as geraniol) (the terminal hydroxyl group of geraniol is oxidized to a carboxyl group, which forms geranilic acid, and then cis-trans isomers are produced due to the difference in double bond configuration).

[0008] Existing research on geraniol in cosmetics does not distinguish between its cis and trans isomers; all studies present it as a mixture of cis and trans geraniol. While cis and trans geraniol may have significant differences in certain effects, no research has yet been found specifically targeting the soothing effects of trans geraniol in cosmetics. Summary of the Invention

[0009] The purpose of this invention is to provide an application of trans-geranilic acid in the preparation of cosmetics with soothing effects. This invention has the advantage of achieving soothing effects when applied in cosmetics.

[0010] The technical solution of the present invention: The application of trans-geranilic acid as an active ingredient in the preparation of cosmetics with skin-soothing effects.

[0011] In the aforementioned application of trans-germicidal acid in the preparation of cosmetics with soothing effects, the mass percentage of trans-germicidal acid added to the cosmetic is 0.001%-0.05%.

[0012] In the aforementioned application of trans-germicidal acid in the preparation of cosmetics with soothing effects, the mass percentage of trans-germicidal acid added to the cosmetic is 0.005%-0.025%.

[0013] In the aforementioned application of trans-germicidal acid in the preparation of cosmetics with soothing effects, the dosage forms of the cosmetics include at least lotions, creams, serums, masks, and toners.

[0014] Compared with the prior art, this application has the following beneficial effects: 1. Excellent safety: Trans geraniol at a concentration of ≤0.025% has a survival rate of ≥90% on HaCaT cells and no obvious cytotoxicity, which solves the problem of "effective concentration and toxic concentration being close" of some existing anti-inflammatory ingredients, making it suitable for sensitive skin and long-term use scenarios; 2. Comprehensive soothing mechanism: It can act on related signaling pathways, cell function regulation and multiple related factors at the same time. Compared with existing single-target soothing ingredients, it can relieve skin discomfort more systematically and the effect is more stable and lasting.

[0015] Therefore, this invention has the advantage of achieving a soothing effect when applied in the preparation of cosmetics. Attached Figure Description

[0016] Figure 1 This is a graph showing the effect of transgermicic acid on HaCaT cell toxicity in Experiment 1; Figure 2 This is a diagram illustrating the protective effect of trans-germicidal acid against UVB-induced oxidative damage (ROS) in Experiment 2. Figure 3 This is a fluorescence staining image showing the inhibition of UVB-induced fluorescence by trans-geranilic acid in Experiment 3. Figure 4 This is a graph showing the results of immunofluorescence staining quantitative analysis of NF-κB induced by UVB inhibition by trans-geranilic acid in Experiment 3. Figure 5 This is a graph showing the results of immunofluorescence staining quantitative analysis of TRPV1 induced by UVB inhibition with trans-geranilic acid in Experiment 3. Figure 6 This is an immunofluorescence staining image showing the inhibition of SDS-induced immunofluorescence by trans-germicidal acid in Experiment 3. Figure 7 This is a graph showing the results of immunofluorescence staining quantitative analysis of NF-κB induced by SDS inhibition by trans-germicic acid in Experiment 3. Figure 8 This is a graph showing the results of immunofluorescence staining quantitative analysis of TRPV1 induced by SDS under the inhibition of trans-germicic acid in Experiment 3. Figure 9 This is a typical image of the soothing effect in Experiment 4, where the yellow dashed line represents the quantitative region; Figure 10 This is a bar chart showing the number of neutrophils in zebrafish during Experiment 4; compared with the model control group, ***p<0.001. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0018] Example. The application of trans-geranilic acid as an active ingredient in the preparation of cosmetics with soothing effects.

[0019] The trans-germic acid is a monoterpene acid with the following structural formula: .

[0020] The trans-germicolic acid is prepared using existing methods, such as the method described in Chinese invention patent application CN119823954A, "A Germicidal Dehydrogenase and Its Preparation and Application Methods".

[0021] The trans-germicidal acid is added to the cosmetic at a mass percentage of 0.025%.

[0022] Trans geraniol efficacy verification Experiment 1: Detection of cell viability using the CCK8 assay Set up an experimental group, a blank control group, and a zero calibration group (PBS only), with 3 replicates for each group; seed HaCaT cells at an appropriate density into a 96-well plate (100 μL / well), and add 100 μL of PBS to the zero calibration group and the outer wells to prevent evaporation; Incubate overnight at 37℃ and 5% CO2 (16-18h). When the cell confluence reaches 40%-60%, the experimental group is replaced with fresh culture medium containing different concentrations of the sample, and the blank control group is replaced with fresh culture medium without the sample. After incubation, cell morphology was observed, CCK-8 reagent was added, and the OD value at 450 nm was measured using an ELISA reader after 2 hours.

[0023] like Figure 1 The results showed that when the sample concentration was ≤0.025%, the survival rate of HaCaT cells was ≥90%, and this concentration was determined to be the safe concentration for subsequent experiments.

[0024] Experiment 2: Flow cytometry detection of reactive oxygen species (ROS) in HaCaT cells using DCFH-DA. The experiment was divided into a blank control group (BC, cells without UVB irradiation), a model control group (NC, cells irradiated with UVB), a positive control group (PC, cells irradiated with UVB and treated with 0.001% tocopherol before and after), and a sample group (cells irradiated with UVB and treated with the test substance before and after). Each group had at least 3 replicates. Resuscitate cells at passage P < 25, culture in DMEM containing 10% FBS, passage to 90% confluence, digest with 0.25% trypsin, centrifuge at 1200 rpm for 5 min, resuspend, and culture at 3.0 × 10⁻⁶ cells / mL. 5 Inoculate 1 mL per well into a 12-well plate and incubate at 37°C with 5% CO2. 2 Incubate overnight; When the cell confluence reaches 40-60%, change the medium and administer the drug: add 1 mL of culture medium to the BC and NC groups, add 1 mL of culture medium containing tocopherol to the PC group, and add 1 mL of culture medium containing the corresponding concentration of the test substance to the sample group; After 4 hours of incubation, except for group BC, the other groups were replaced with 500 μL of HBSS at 100 mJ / cm². 2 Dosage (Bio-SUN irradiator) UVB irradiation; After irradiation, the medium was changed. 1 mL of DMEM containing 10% FBS was added to the NC group, while culture medium containing tocopherol and the test substance was added to the PC and sample groups, respectively. The DMEM for the BC group was replaced and incubated overnight. The solution was then discarded, washed once with HBSS, and 500 μL of diluted DCFH-DA probe was added to each well. The solution was then incubated at 37°C and 5% CO2 for 30 min. After probe incubation, the cells were washed twice with HBSS, digested with trypsin, centrifuged at 1200 rpm for 5 min, washed twice with HBSS / PBS, resuspended in 300 μL of HBSS / PBS, and analyzed at medium speed in the 488nm channel of a flow cytometer.

[0025] The mean fluorescence values ​​(GE) of each group were obtained using FlowJo software, and plotted using GraphPad Prism 8 (GE on the ordinate, concentration and category on the abscissa). The data are expressed as mean values, and comparisons were made by t-test or one-way ANOVA. P < 0.05 was considered statistically significant.

[0026] like Figure 2 As shown, UVB irradiation significantly increases intracellular ROS levels (oxidative damage), while trans-geraniol can significantly reduce UVB-induced ROS production, suggesting its potential to have antioxidant properties and protect cells from UVB oxidative damage.

[0027] Experiment 3: Cell Immunofluorescence Assay HaCaT cells in logarithmic growth phase were used at a rate of 3 × 10⁻⁶. 4 Cells were seeded at a density of cells / well in 8-well cell culture slides and cultured overnight in a 37 ℃, 5% CO2 incubator. After each group of cells was treated with the drug for 24 h, the cells were seeded. Discard the culture medium, wash the cells three times with PBS buffer for 5 min each time; add 100 μL of immunostaining fixative to each well and fix at room temperature for 15 min; wash three times again with PBS. Add 100 μL of immunostaining blocking solution to each well and block at room temperature for 30 min to block nonspecific binding. Discard the blocking solution, add 100 μL of primary antibody diluted with PBS at a ratio of 1:200 to each well, incubate at room temperature for 2 h, and wash 3 times with PBS; Add Alexa Fluor 594 goat anti-rabbit fluorescently labeled secondary antibody diluted 1:500, incubate at room temperature in the dark for 1 hour, and wash 3 times with PBS; Add 1 μg / mL DAPI staining solution and incubate at room temperature in the dark for 5 min to label cell nuclei. Wash three times with PBS. The coverslip was attached to the slide (cell side down) using an anti-fluorescence quenching mounting medium and observed in a Keyence BZX800. Images were acquired using the same exposure time, gain, and other parameters, and the average optical density of the fluorescent region was analyzed using ImageJ software.

[0028] like Figure 3-8The results showed that treatment of HaCaT cells with trans geraniol (0.025%) for 24 h could significantly inhibit the activation / expression of NF-κB, indicating that trans geraniol may be involved in soothing the skin and regulating cell function. Treatment of HaCaT cells with trans geraniol (0.025%) for 24 h significantly inhibited the activation / expression of TRPV1, suggesting that trans geraniol may be involved in soothing the skin and regulating cell function.

[0029] Figure 3 The medium NC is 20 mJ / cm 2 Model group cultured for 24 hours after UVB irradiation; Figure 4 BC represents the solvent control group, and NC represents the solvent obtained after treatment with 20 mJ / cm². 2 The model group was cultured for 24 hours after UVB irradiation. The sample contained 0.025% trans-germicolic acid, and the sample of *Panax senticosus* (PC) contained 100 μM bisabolol. Figure 5 BC represents the solvent control group; NC represents the solvent control group after treatment with 20 mJ / cm². 2 The model group was cultured for 24 hours after UVB irradiation; the sample contained 0.025% trans-germicolic acid; the ginseng (PC) contained 100 μM bisabolol. Figure 6 The NC group was the model group cultured with 10 μg / mL sodium dodecyl sulfate (SDS) for 24 h. Figure 7 BC was the solvent control group; NC was the model group cultured in 10 μg / mL sodium dodecyl sulfate (SDS) for 24 h; the sample was 0.025% trans-germicidal acid; and the ginseng (PC) was 100 μM bisabolol. Figure 8 BC was the solvent control group; NC was the model group cultured in 10 μg / mL sodium dodecyl sulfate (SDS) for 24 h; the sample was 0.025% transgermicic acid; and the ginseng (PC) was 100 μM bisabolol.

[0030] Experiment 4: Neutrophil Inhibition Rate Method in Zebrafish Juveniles Transgenic neutrophil-positive green fluorescent zebrafish (MPX) were randomly selected and placed in 6-well plates, with 15 fish per well; First, a zebrafish skin discomfort model was established by water-soluble administration of sodium dodecyl sulfate (SLS); Secondly, the sample was given in water, and a normal control group and a model control group were set up. The volume of each well was 3 mL, and the sample was incubated at 28°C in the dark for 18 h. Finally, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Advanced image processing software was used to analyze and collect data, and the number of neutrophils (N) in the zebrafish skin was analyzed. The formula was used to calculate and determine whether the sample had a soothing effect.

[0031] like Figure 9 and 10 (The attached images were taken using a Zeiss motorized fluorescence zoom microscope (instrument model: AXIO Zoom.V16) at a magnification of 32×. The green fluorescent dots represent neutrophils, and the more fluorescent dots, the more neutrophils there are.) Compared with the model control group, the number of neutrophils was significantly reduced at a concentration of 0.0005% (m / m), and the soothing effect was 36%, indicating that the sample has a soothing effect.

Claims

1. The application of trans-germicidal acid as an active ingredient in the preparation of cosmetics with soothing effects.

2. The application of trans-geranilic acid according to claim 1 in the preparation of cosmetics with soothing effects, characterized in that: The trans-germicidal acid is added to the cosmetic at a mass percentage of 0.001%-0.05%.

3. The application of trans-geranilic acid according to claim 2 in the preparation of cosmetics with soothing effects, characterized in that: The trans-germicidal acid is added to the cosmetic at a mass percentage of 0.005%-0.025%.

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