Application of trans-aromatic folic acid

Abstract

The invention discloses application of trans-aromatic folic acid. The trans-aromatic folic acid is applied to cosmetics and has the effects of resisting skin aging and tightening skin. The skin anti-aging and firming composition has the advantage of being capable of achieving skin anti-aging and firming effects when applied to cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetic technology, and particularly relates to an application of trans-geranic acid. BACKGROUND

[0002] Skin, as the barrier structure of human body, its core functions include preventing water loss, blocking external stimuli, and maintaining microecological balance and immune response. Skin aging is affected by both endogenous and exogenous factors, among which ultraviolet light is the main factor. When the skin is exposed to ultraviolet light, a large number of free radicals are generated, which can destroy the extracellular matrix like a "molecular scissors", resulting in a significant decrease in collagen content, thereby causing wrinkles, skin relaxation, and pigment deposition.

[0003] In the "steel and iron" structure of the skin, type I collagen plays a core role. It is formed by three alpha chains of about 1000 amino acid residues winding around each other to form a stable triple helix structure, which provides strong support and elasticity to tissues such as skin and bones. However, matrix metalloproteinase 1 (MMP-1) acts as a "destroyer" and specifically degrades type I collagen during skin photoaging, accelerating the aging process of the skin.

[0004] The systematic name of trans-geranic acid is trans-3,7-dimethyl-2,6-octadienoic acid, which is a product of plant secondary metabolism, mainly existing in essential oils of aromatic plants such as citrus (orange, lemon), geranium, rose, etc.; or in the metabolic pathway of plants, which is usually generated by oxidation of monoterpene alcohols (such as geraniol) (the terminal hydroxyl group of geraniol is oxidized to carboxyl group, i.e. geranic acid, and cis-trans isomers are produced due to the difference in double bond configuration).

[0005] The existing research on geranic acid in cosmetics does not distinguish between cis-trans isomers, and it is a mixture of cis-geranic acid and trans-geranic acid. However, cis-geranic acid and trans-geranic acid may have significant differences in some effects, but no relevant research on trans-geranic acid alone in cosmetics has been found. SUMMARY

[0006] The purpose of the present application is to provide an application of trans-geranic acid. The present application has the advantages of achieving skin anti-aging and skin tightening effects in cosmetics.

[0007] The technical scheme of the present application is as follows: An application of trans-geranic acid as an active ingredient in the preparation of cosmetics with skin anti-aging and skin tightening effects.

[0008] In the aforementioned application of trans-geranic acid, the mass percentage of trans-geranic acid added in the cosmetics is 0.025%-0.05%.

[0009] In the aforementioned application of trans-germicidal acid, the mass percentage of trans-germicidal acid added to the cosmetic is 0.05%.

[0010] In the aforementioned applications of trans-germicidal acid, the dosage forms of the cosmetics include at least lotions, creams, serums, masks, and toners.

[0011] Compared with the prior art, the trans-geraniol of this application, when used as an active ingredient in cosmetics, can promote the synthesis of type I collagen in fibroblasts and inhibit the activity of matrix metalloproteinase 1 (MMP-1) in fibroblasts, thereby achieving the effects of skin anti-aging and firming.

[0012] Therefore, this invention has the advantage of achieving anti-aging and firming effects when applied in cosmetics. Attached Figure Description

[0013] Figure 1 This is the cell viability curve of trans-germicidal acid in Experiment 1; Figure 2 This is a cell morphology diagram of trans-germicidal acid in Experiment 1; Figure 3 This is a graph showing the results of Collagen I immunofluorescence in Experiment 2; Figure 4 This is a bar chart of Collagen I relative integrated optical density (IOD) / cell numerical values ​​in Experiment 2; Figure 5 This is a graph showing the concentration change of MMP-1 (matrix metalloproteinase-1) after treatment with trans-geranilic acid in Experiment 3. Detailed Implementation

[0014] 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.

[0015] Example 1. Application of trans-geranilic acid as an active ingredient in the preparation of cosmetics with skin anti-aging and skin firming effects.

[0016] The trans-germic acid is a monoterpene acid with the following structural formula: .

[0017] 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".

[0018] The trans-germicidal acid is added to the cosmetic at a mass percentage of 0.025% (cell experiment concentration is 0.0025%).

[0019] Example 2. An application of trans-germicidal acid, similar to Example 1, except that the mass percentage of trans-germicidal acid added to the cosmetic is 0.05% (cell experiment concentration is 0.005%).

[0020] Trans geraniol efficacy verification I. Experiment 1: Detection of fibroblast toxicity (MTT method) Experimental Groups: The experiment included a zeroing group, a solvent control group, a positive control group, and a sample group. Each sample in the sample group had eight concentrations, with three replicate wells for each concentration.

[0021] Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table (Table 1);

[0022] Note: Human skin is more tolerant, but has a low transdermal absorption rate. Therefore, the concentration added to the formula must be higher than the concentration at which the cell takes effect in order to compensate for the loss and ensure that the active ingredients can penetrate deep into the skin to take effect. Due to the presence of the human skin barrier and transdermal loss, the actual concentration added in the formulation is often higher than the concentration for cell administration (e.g., 5 to 10 times) to compensate for the loss and ensure the effectiveness of the active ingredient; therefore, the concentration of transgerminic acid in the experimental verification is one-tenth of the actual concentration added to the cosmetic.

[0023] After reviving the cells, when the cell plating rate reached about 60%, they were seeded into 96-well plates and incubated overnight in a CO2 incubator at 37°C and 5% CO2. When the cell seeding rate in the 96-well plate reaches 50%-60%, the drug is administered. For the solvent control group, 200 μL of culture medium is added to each well; for the positive control group, 200 μL of culture medium containing 10% DMSO is added to each well; for the sample group, 200 μL of culture medium containing the corresponding concentration of the sample is added to each well; and for the zeroing group, no cells are seeded, only 200 μL of culture medium is added. After administration, the 96-well plate was returned to the incubator under the same conditions and incubated for 24 hours. Then the supernatant was discarded, and 0.5 mg / mL MTT working solution was added and incubated in the dark for 4 hours. After incubation, discard the supernatant, add 150 µL of DMSO to each well, and finally measure the OD value at 490 nm.

[0024] Relative cell viability calculation: Calculated according to the formula. .

[0025] Based on MTT and morphological results (see appendix) Figure 1 and 2It was found that transgermicic acid, based on fibroblasts, did not show significant cytotoxicity at a concentration of 0.005%, and subsequent experiments were conducted at a concentration of 0.005%.

[0026] II. Experiment 2: Detection of Type I Collagen (Collagen I) Content in Fibroblasts Irradiated by UVA Cell seeding: After cell resuscitation, when the cell plating rate reaches about 60%, seed cells into 24-well plates and incubate overnight in a CO2 incubator (37 ℃, 5% CO2).

[0027] Solution preparation: Prepare working solutions of the test substances according to the test groups (Table 2).

[0028]

[0029] UVA irradiation: According to the test groups, when the cell deposition rate in the 24-well plates reached 30%-50%, all groups except the blank control group were subjected to UVA irradiation at a dose of 30 J / cm². 2 ; After irradiation, the drugs were administered in groups, with each group having 3 replicates. 1 mL of culture medium was added to each well of the blank control group and negative control group, 1 mL of culture medium containing TGF-β1 was added to each well of the positive control group, and 1 mL of culture medium containing the corresponding concentration of the sample was added to each well of the sample group. After drug administration, the 24-well plate was placed in a CO2 incubator (37 ℃, 5% CO2) and incubated for 24 h. Cells were fixed with 4% paraformaldehyde for 30 min, and then immunofluorescence was performed. The staining results were observed and photographed under a fluorescence microscope. Figure 3 , attached Figure 3 The analysis was performed using Image-Pro Plus software.

[0030] Lift rate calculation: .

[0031] like Figure 4 As shown, in fibroblasts irradiated with UVA, compared with the negative control group, the content of type I collagen (Collagen I) in the sample group was significantly increased after 24 hours of treatment with monoterpene acid at a concentration of 0.005% (m / m), with an increase rate of 114.29%. This indicates that the sample can increase the content of type I collagen (Collagen I) at this concentration and has a skin-firming effect.

[0032] III. Experiment 3: Detection of matrix metalloproteinase-1 (MMP-1) content in UVA-irradiated fibroblasts This experiment first involves reasonable grouping and replication settings, including blank control group (BC), model control group (NC), positive control group (PC) and sample group, with at least 3 replicates in each group; The cell culture and seeding steps are as follows: Culture cells to 90% confluence, digest with 0.25% trypsin, centrifuge at 1000 rpm / min for 5 min, resuspend in low-glucose DMEM medium containing 10% FBS, and then seed at 5.0 × 10⁻⁶ cells / year. 6 Inoculate 2 mL of the sample into each well of a 6-well plate at a rate of 2 mL per well and incubate at 37 °C in a 5% CO2 incubator for 24 h. During UVA irradiation, first aspirate the original culture medium from the wells, then add 1 mL of HBSS solution to each well, at a concentration of 18 J / cm². 2 Irradiate for one and a half hours; after irradiation, change the culture medium. For the NC group, add 2 mL of low-glucose DMEM culture medium containing 10% FBS, for the sample group, add 2 mL of culture medium containing the corresponding concentration of the test substance, for the PC group, add 2 mL of culture medium containing 100 ng / mL TGF-β, and for the BC group, replace with new low-glucose DMEM culture medium. Then continue to incubate overnight in the incubator. After the culture is completed, collect the supernatant, centrifuge at 4 ℃ and 1000×g for 20 min to remove impurities, and take the supernatant for detection; during detection, add 100 μL of standard working solution or sample (standard is replicated) to each reaction well, and incubate at 37 ℃ for 90 min; after discarding the solution, add 100 μL of biotin-labeled matrix metalloproteinase 1 antibody working solution, and incubate at 37 ℃ for 60 min; Next, wash: add 350 μL of washing solution to each well, soak for 1-2 min, and then spin dry. Repeat 4 times. Then add 100 μL of HRP-labeled streptavidin working solution, incubate at 37 ℃ for 30 min, and then wash with 300 μL of washing solution every 30 s and spin dry. Repeat 4 times. Then add 90 μL of chromogenic reagent and develop color in the dark for about 15 min. Add 50 μL of stop solution and measure the OD value at 450 nm wavelength using an ELISA reader within 5 min. Finally, the average OD value of the standard and the sample (minus the OD value of the zero well) is calculated, and a standard curve is plotted using a "four-parameter logistic model" with the standard concentration as the x-axis and the OD value as the y-axis.

[0033] like Figure 5 As shown, the results indicated that, compared with the negative control group (NC), the MMP-1 concentration in the groups treated with transgeranic acid (whether at a concentration of 0.0025% or 0.005%) was reduced, and this reduction was statistically significant. This suggests that trans-geraniol may have the effect of inhibiting MMP-1 expression (skin anti-aging), and this effect is more significant at a concentration of 0.005%.

[0034] Application Example 1: Moisturizing Serum Manufacturing process: (All equipment and containers are clean and dry) A. Add all raw materials of phase A into the operating container, heat to 80°C, and stir until completely homogeneous; B. Add all raw materials of phase B into the operating container, heat to 78°C, and stir until completely homogeneous; C. Add phase B to phase A and mix thoroughly. D. Add the C phase raw material at 38°C and stir until completely homogeneous; if a filter is required, a dry 100-mesh filter must be used.

[0035]

[0036] Application Example 2: Moisturizing Cream Manufacturing process: (All equipment and containers are clean and dry) A. Add all raw materials of phase A into the operating container, heat to 80°C, and stir until completely homogeneous; B. Add all raw materials of phase B into the operating container, heat to 78°C, and stir until completely homogeneous; C. Add phase B to phase A and mix thoroughly. D. Add the C phase raw material at 60℃ and stir until completely homogeneous; E. Add the D phase raw material at 38℃ and stir until completely homogeneous; if a filter is required, a dry 100-mesh filter must be used.

[0037]

Claims

1. The application of trans-geranilic acid as an active ingredient in the preparation of cosmetics with anti-aging and skin-firming effects.

2. The application of trans-germicidal acid according to claim 1, characterized in that: The trans-germicidal acid is added to the cosmetic at a mass percentage of 0.025%-0.05%.

3. The application of trans-germicidal acid according to claim 2, characterized in that: The trans-germicidal acid is added to the cosmetic at a mass percentage of 0.05%.

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