TGF-beta / Smad signaling pathway inhibitor and application thereof in anti-aging cosmetics

Abstract

The invention discloses a TGF-beta / Smad signal channel inhibitor. The TGF-beta / Smad signal channel inhibitor is prepared from the following components in parts by mass: 1000 parts of tea oil, 0.09 to 9.8 parts of bakuchiol and 0.01 to 0.2 part of acetyl hexapeptide-8, the invention also discloses a method for preparing the TGF-beta / Smad signal channel inhibitor as described above. The invention also discloses a cosmetic, which contains the TGF-beta / Smad signal channel inhibitor as an active component. The invention further discloses a method for evaluating the anti-aging effect of the cosmetic. According to the composition, by inhibiting abnormal activation of a TGF-beta / Smad signal channel, excessive deposition and crosslinking of an extracellular matrix are reduced, meanwhile, synthesis of collagen and elastin is promoted, aging characterization such as skin laxity and wrinkles is effectively improved, and deep repair and rejuvenation remodeling of skin aging damage are achieved.

Description

Technical Field

[0001] This invention belongs to the field of daily cosmetic technology, specifically relating to a TGF-β / Smad signaling pathway inhibitor and its application in anti-aging cosmetics. Background Technology

[0002] The extracellular matrix (ECM), as the core of structural and signaling regulation in aging, is a dynamic three-dimensional network composed of collagen, elastin, hyaluronic acid (HA), and other components between cells, and has been established as the 13th major biomarker of aging. Its contribution to anti-aging is mainly reflected in two aspects: First, the ECM provides crucial physical support for tissues. The degradation of its components (such as collagen breakage and HA loss) directly leads to aging characteristics such as skin laxity and wrinkle formation. Studies have found that supplementing with high-molecular-weight HA can effectively delay ECM degradation, extend the lifespan of model organisms, and reduce the risk of inflammation. Second, ECM components are not only structural units but also important signal carriers. For example, HA, by binding to CD44 receptors on the surface of fibroblasts, can activate collagen synthesis pathways, forming a bidirectional regulatory axis of "ECM-cell," thereby maintaining the tissue's regenerative and repair capabilities.

[0003] The TGF-β / Smad signaling pathway is one of the core pathways regulating cell fate and tissue homeostasis, playing a dual and crucial role in extracellular matrix homeostasis and the aging process. In the ECM, this pathway is a powerful "pro-fibrotic" engine: it strongly stimulates fibroblasts and other cells to synthesize ECM components such as collagen while inhibiting their degradation, thus dominating ECM deposition and remodeling. However, during aging, overactivation of the TGF-β / Smad pathway disrupts this balance, leading to excessive ECM deposition, cross-linking, and tissue fibrosis and hardening—a typical characteristic of organ dysfunction. More importantly, this pathway can directly induce cell cycle arrest, promote cellular senescence, and synergize with the senescence-associated secretory phenotype (SASP) to create a pro-inflammatory and pro-fibrotic microenvironment, thereby accelerating the overall aging process. Therefore, inhibiting the abnormal activation of the TGF-β / Smad pathway is considered an important anti-aging strategy, aiming to reverse the pathological hardening of the ECM, clear senescent cells, and restore tissue regeneration capacity, providing a key target for intervention in age-related diseases.

[0004] Bakuchiol's core anti-aging effect lies in its powerful stimulation of dermal collagen (type I and type III) and elastin synthesis, thereby improving skin firmness, reducing static wrinkles (such as nasolabial folds), and restoring plumpness and elasticity. Compared to traditional retinol, its advantages lie in significantly reduced irritation and superior photostability, providing a highly effective and gentle anti-aging option for sensitive skin. Acetyl hexapeptide-8 (commonly known as argirelin) is a bioactive peptide based on biomimetic design, known for its unique "botox-like" mechanism. Its effects are similar to Botox injections, but its action is gentle, reversible, and non-invasive, avoiding stiffness, making it suitable for daily care to smooth expression lines and improve facial smoothness. Some studies also suggest that it may help improve the skin matrix.

[0005] Therefore, the industry currently needs an innovative solution that aims to leverage the anti-aging mechanism of the TGF-β / Smad signaling pathway to penetrate deep into the cellular level, targeting and strengthening the structure and function of the extracellular matrix, thereby achieving deep repair and rejuvenation of skin aging damage. Summary of the Invention

[0006] To address the shortcomings of existing anti-aging cosmetics, such as insufficient regulation at the cellular level and the high irritation and poor stability of some ingredients, this invention proposes a TGF-β / Smad signaling pathway inhibitor and its application in anti-aging cosmetics.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a TGF-β / Smad signaling pathway inhibitor comprising the following components in parts by weight: 1000 parts of tea oil, 0.09-9.8 parts of psoralen, and 0.01-0.2 parts of acetyl hexapeptide-8.

[0008] Preferably, the components in the mass fractions further include: 1000 parts of tea oil, 0.99-1.99 parts of psoralen, and 0.01-0.1 parts of acetyl hexapeptide-8.

[0009] Preferably, the components in the mass fractions further include: 1000 parts of tea oil, 1.99 parts of psoralen, and 0.01 parts of acetyl hexapeptide-8.

[0010] A second aspect of the present invention provides a method for preparing a TGF-β / Smad signaling pathway inhibitor as described in any one of claims 1 to 3, comprising the following steps:

[0011] (1) Weigh each component according to its mass fraction;

[0012] (2) Heat the tea oil slowly;

[0013] (3) Add psoralen and acetyl hexapeptide-8 to the tea oil under stirring conditions, and stir until completely dissolved;

[0014] (4) Allow the system to stand and cool to obtain the inhibitor.

[0015] Preferably, the heating temperature in step (2) is 40°C.

[0016] Preferably, the stirring speed in step (3) is 200 rpm.

[0017] Preferably, the cooling temperature in step (4) is room temperature.

[0018] In a third aspect, the present invention provides a cosmetic product, wherein the dosage form of the cosmetic product is a skin care oil, lotion, cream, serum, freeze-dried powder, or skin care water.

[0019] A fourth aspect of the present invention provides a method for evaluating the anti-aging efficacy of the cosmetic as described in claim 8 or 9, characterized in that the method comprises one or more of the following:

[0020] Method 1: By establishing an H2O2-induced HaCaT cell senescence model, the expression levels of TGF-β1 and / or Smad2 mRNA were detected by qPCR.

[0021] Method 2: By establishing an H2O2-induced HaCaT cell senescence model, the expression level of COL1a1b mRNA was detected by qPCR.

[0022] Method 3: By establishing an H2O2-induced HaCaT cell senescence model, the activity of matrix metalloproteinases (MMPs) was detected using the ELISA method;

[0023] Method 4: By establishing an H2O2-induced HaCaT cell senescence model, the activity of senescence-related β-galactosidase SA-β-gal was detected by staining.

[0024] The present invention has the following beneficial effects:

[0025] 1. The composition of the present invention reduces excessive deposition and cross-linking of the extracellular matrix by inhibiting the abnormal activation of the TGF-β / Smad signaling pathway, while promoting the synthesis of collagen and elastin, effectively improving aging characteristics such as skin laxity and wrinkles, and achieving deep repair and rejuvenation of aging-related skin damage.

[0026] 2. In a 1000g tea oil system, when the amount of psoralen is 0.99g to 1.99g and the amount of acetyl hexapeptide-8 is 0.01g to 0.1g, the composition can produce a synergistic effect, showing significant effects in inhibiting the TGF-β / Smad signaling pathway, promoting extracellular matrix synthesis and clearing senescent cells, and the anti-aging effect is better than that of using a single component. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the effect of the composition treatments of each embodiment on TGF-β1 mRNA expression in HaCaT cells.

[0028] Figure 2 This is a schematic diagram illustrating the effect of the composition treatments of each embodiment on Smad2 mRNA expression in HaCaT cells.

[0029] Figure 3 This is a schematic diagram illustrating the effect of the composition treatments of each embodiment on the expression of COL1a1b mRNA in HaCaT cells.

[0030] Figure 4 This is a schematic diagram illustrating the results of MMP activity assays in HaCaT cells using the compositions of each embodiment.

[0031] Figure 5 This is a schematic diagram illustrating the results of the composition treatments of various embodiments on the detection of β-galactosidase activity in HaCaT cells. Detailed Implementation

[0032] The present invention will be described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are all available through conventional commercial channels.

[0034] This invention uses skin-care tea oil as a typical dosage form for illustration, but the application of the TGF-β / Smad signaling pathway inhibitor is not limited to this, and can also be applied to other types of cosmetic dosage forms such as lotions, creams, serums, freeze-dried powders, and skin-care toners.

[0035] Examples 1-8: Preparation of TGF-β1 / Smad2 signaling pathway inhibitor compositions

[0036] Examples 1-8 provide a TGF-β1 / Smad2 signaling pathway inhibitor, which is based on tea oil and contains a specific ratio of psoralen and acetyl hexapeptide-8. The specific preparation process is as follows:

[0037] The first step is to select refined tea oil as the base oil for cosmetics;

[0038] The second step is to weigh 1000 g of tea oil, place it in a clean glass reaction vessel, and slowly heat it to 40°C. Then, add psoralen and acetyl hexapeptide-8 in the proportion shown in Table 1, and stir continuously at 200 rpm until completely dissolved.

[0039] Third, after complete dissolution, stop heating and allow to cool to room temperature (approximately 25°C) to obtain the inhibitor composition, which should be sealed and stored in a light-proof container for later use.

[0040] Table 1. Composition of the compositions described in Examples 1-8

[0041]

[0042] To verify the anti-aging efficacy of the compositions prepared in the above embodiments, the following cellular level experiments were conducted.

[0043] Immortalized human keratinocytes were used in the experiments after being revived and passaged three times. The experimental groups were as follows:

[0044] (1) Blank control group: No test composition was added.

[0045] (2) Model group: Cell senescence was induced for 28 h using 200 μM H2O2.

[0046] (3) Sample group: After inducing cells with 200 μM H2O2 for 4 h, the culture medium was replaced with 200 μM H2O2 and samples from each example, and the treatment continued for 24 h.

[0047] Example 9: Detection of TGF-β1, Smad2 and COL1a1b gene expression levels

[0048] (1) Sample preparation: After treating HaCaT cells according to the above grouping, total RNA was extracted from the cells using TRIzol reagent.

[0049] Specific steps: Discard the culture medium, wash the cells twice with PBS, add 1 mL TRIzol to each well, let stand for 5 min, scrape the cells with a cell scraper, and transfer them to an enzyme-free centrifuge tube.

[0050] (2) RNA extraction: After lysing the sample with TRIzol solution, add 200 μL of chloroform, vortex for 15 sec, and let stand for 3 min. Centrifuge at 12000 g for 15 min at 4 ℃, and carefully aspirate the upper aqueous phase into a new tube. Add an equal volume of isopropanol pre-cooled to -20 ℃, gently invert to mix, and let stand at -20 ℃ for 15 min. Centrifuge at 12000 g for 10 min at 4 ℃, and discard the supernatant. Add 1 mL of 75% ethanol prepared with enzyme-free water, vortex to mix, centrifuge at 10000 g for 5 min at 4 ℃, and discard the supernatant. Dry in a fume hood for 3 min, add 20 μL of enzyme-free water to dissolve the RNA, and determine the concentration and purity of the RNA using a Nanodrop 2000 instrument.

[0051] (3) cDNA synthesis: Reverse transcription was performed using the PrimeScript RT reagent Kit (purchased from Takara, Japan). The reaction volume was 20 μL, and the specific system was as follows:

[0052]

[0053] The reaction procedure was as follows: react at 25°C for 5 minutes for primer annealing, then react at 42°C for 30 minutes for reverse transcription, and finally treat at 85°C for 5 minutes to inactivate the enzyme.

[0054] (4) qPCR reaction system: TB Green Premix Ex Taq II (purchased from Takara, Japan) was used for real-time quantitative PCR reaction. The reaction system was 20 μL, and the specific system is as follows:

[0055]

[0056] qPCR run procedure (using Applied Biosystems 7500 Real-Time PCR System):

[0057]

[0058] The primer sequences used for qPCR are shown in the table below.

[0059]

[0060] To evaluate the effect of the composition on the TGF-β / Smad signaling pathway, the expression levels of key molecules in this pathway in HaCaT cells were detected by qPCR.

[0061] Figure 1 This diagram illustrates the effect of the compositions from each embodiment on TGF-β1 mRNA expression in HaCaT cells. Figure 1 As shown, compared with the blank control group, the mRNA expression level of TGF-β1 in the H2O2-induced aging model group was significantly upregulated. After intervention with the compositions of Examples 1 to 8 of this invention, the mRNA expression level of TGF-β1 showed varying degrees of downregulation.

[0062] Figure 2 This diagram illustrates the effect of the compositions from each embodiment on Smad2 mRNA expression in HaCaT cells. Figure 2 As shown, the mRNA expression level of Smad2 in the model group was significantly increased, while its expression level was effectively inhibited after treatment with the composition of the present invention.

[0063] Figure 3This diagram illustrates the effect of the treatments with the compositions of each embodiment on COL1a1b mRNA expression in HaCaT cells. Figure 3 As shown, the expression level of COL1a1b mRNA in the model group was significantly lower than that in the blank control group. After treatment with the composition of the present invention, the expression level of COL1a1b mRNA significantly rebounded.

[0064] Comprehensive analysis showed that in a 1000g tea oil system, when the amount of psoralen was 0.99g to 1.99g and the amount of acetyl hexapeptide-8 was 0.01g to 0.1g, its inhibitory effect on the TGF-β / Smad signaling pathway and its promoting effect on collagen synthesis were particularly significant. This range corresponds to Examples 2 to 5, among which Example 4, based on 1000g tea oil, showed the most outstanding effect with a ratio of 1.99g psoralen to 0.01g acetyl hexapeptide-8.

[0065] Example 10: Detection of matrix metalloproteinase (MMP) activity

[0066] The activity of MMPs in cell culture medium was detected using an enzyme-linked immunosorbent assay (ELISA). The Human MMP-1 ELISA Kit (purchased from R&D Systems, USA) was used, and the specific steps are as follows:

[0067] (1) Sample pretreatment: Collect cell culture medium, centrifuge at 3000 g for 10 min at 4 ℃, take the supernatant and store at -80 ℃ for testing.

[0068] (2) Preparation of standard: The standard powder was reconstituted with 1 mL of standard diluent, allowed to stand for 10 min, vortexed and mixed, and serially diluted to a concentration range of 1000 pg / mL to 7.8 pg / mL, for a total of 8 concentration points.

[0069] (3) Adding and incubating: Add 100 μL of standard or sample to a 96-well plate pre-coated with antibody, cover with a membrane, and incubate at 37 °C for 2 h. Discard the liquid, add 300 μL of washing buffer (PBS containing 0.05% Tween-20) to each well, let stand for 1 min, discard, and repeat the washing 3 times.

[0070] (4) Add detection antibody: Add 100 μL of biotinylated detection antibody working solution to each well, cover with membrane, and incubate at 37 °C for 1 h. Wash 3 times.

[0071] (5) Add enzyme conjugate: Add 100 μL of streptavidin-HRP working solution to each well, cover with a membrane, and incubate at 37 °C in the dark for 30 min. Wash 3 times.

[0072] (6) Color development and termination: Add 100 μL of TMB substrate solution to each well and incubate at 37 °C in the dark for 15 min. Add 100 μL of 2 M H2SO4 to each well to terminate the reaction.

[0073] (7) Reading and calculation: Within 30 min, the OD value was measured at a wavelength of 450 nm using a microplate reader (BioTek, USA). The standard curve was fitted using four-parameter logistic regression (4PL) with the standard concentration as the x-axis and the OD value as the y-axis (R²≥0.99) to calculate the MMP concentration in the sample.

[0074] Figure 4 This is a schematic diagram illustrating the results of MMP activity assays in HaCaT cells using the compositions of each embodiment. Figure 4 As shown, MMP activity in the H2O2-induced aging model group was significantly higher than that in the blank control group, indicating accelerated extracellular matrix degradation. After intervention with the compositions of Examples 1 to 8 of this invention, MMP activity was downregulated to varying degrees. Among them, the treatment in Example 4 showed the most significant effect, followed by the treatments in Examples 3 and 5, further confirming the positive effect of this composition in inhibiting matrix metalloproteinase activity and reducing collagen degradation.

[0075] Example 11: Detection of aging-related β-galactosidase (SA-β-gal) activity

[0076] HaCaT cells were used at a rate of 1×10 4 Cells were seeded at a density of 1 / well in 24-well plates and cultured to 80% confluence before being processed into groups. Cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. After rinsing with PBS, SA-β-gal staining working solution (containing 1 mg / mL X-Gal, 40 mM citrate / sodium phosphate buffer pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2) was added, and the cells were incubated at 37°C in the dark for 16 h. Five fields of view (200×) were randomly selected under an inverted optical microscope to count the blue-green positive cells and the total cell count, and the percentage of SA-β-gal positive cells was calculated.

[0077] Figure 5 This is a schematic diagram illustrating the results of the treatment of HaCaT cells with the compositions of each embodiment to detect β-galactosidase activity. Figure 5As shown, the percentage of SA-β-gal positive cells in the H2O2-induced aging model group was significantly higher than that in the blank control group, indicating a significant increase in the degree of cellular senescence. After intervention with the compositions of each embodiment of the present invention, the percentage of SA-β-gal positive cells all showed a significant decrease. Among them, the treatment effect of Example 4 was the most significant, followed by the treatment effects of Examples 3 and 5. This result fully demonstrates that in a 1000g tea oil system, when the amount of psoralen is 0.99g to 1.99g and the amount of acetyl hexapeptide-8 is 0.01g to 0.1g, the composition has excellent anti-cellular senescence activity.

[0078] In summary, the compositions based on 1000 grams of tea oil provided in Examples 1 to 8 of this invention have all been proven to have multi-pathway anti-skin aging effects. Based on the test results of Examples 9 to 11, it can be determined that in a 1000-gram tea oil system, when the amount of psoralen is 0.99 to 1.99 grams and the amount of acetyl hexapeptide-8 is 0.01 to 0.1 grams, the composition can produce a synergistic effect in inhibiting the TGF-β / Smad signaling pathway, promoting extracellular matrix synthesis, and clearing senescent cells. This preferred scope covers Examples 2 to 5, among which Example 4, due to its optimal performance on multiple key efficacy indicators, is established as the preferred embodiment of this invention.

[0079] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A TGF-β / Smad signaling pathway inhibitor, characterized in that, The components include the following parts by weight: 1000 parts tea oil, 0.09~9.8 parts psoralen, and 0.01~0.2 parts acetyl hexapeptide-8.

2. The TGF-β / Smad signaling pathway inhibitor according to claim 1, characterized in that, The components by weight also include: 1000 parts of tea oil, 0.99~1.99 parts of psoralen, and 0.01~0.1 parts of acetyl hexapeptide-8.

3. The PI3K-AKT signaling pathway inhibitor according to claim 1, characterized in that, The components by weight also include: 1000 parts tea oil, 1.99 parts psoralen, and 0.01 parts acetyl hexapeptide-8.

4. A method for preparing a TGF-β / Smad signaling pathway inhibitor as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Weigh each component according to its mass fraction; (2) Heat the tea oil slowly; (3) Add psoralen and acetyl hexapeptide-8 to the tea oil under stirring conditions, and stir until completely dissolved; (4) Allow the system to stand and cool to obtain the inhibitor.

5. The method according to claim 4, characterized in that, In step (2), the heating temperature is 40°C.

6. The method according to claim 4, characterized in that, In step (3), the stirring speed is 200 rpm.

7. The method according to claim 4, characterized in that, In step (4), the cooling temperature is room temperature.

8. A cosmetic product, characterized in that, It contains a TGF-β / Smad signaling pathway inhibitor as described in any one of claims 1 to 3 as an active ingredient.

9. The cosmetic product according to claim 8, characterized in that, The cosmetics are in the form of skin care oil, lotion, cream, serum, freeze-dried powder, or skin care water.

10. A method for evaluating the anti-aging efficacy of the cosmetic as described in claim 8 or 9, characterized in that, The method includes one or more of the following: Method 1: By establishing an H2O2-induced HaCaT cell senescence model, the expression levels of TGF-β1 and / or Smad2 mRNA were detected by qPCR. Method 2: By establishing an H2O2-induced HaCaT cell senescence model, the expression level of COL1a1b mRNA was detected by qPCR. Method 3: By establishing an H2O2-induced HaCaT cell senescence model, the activity of matrix metalloproteinases (MMPs) was detected using the ELISA method; Method 4: By establishing an H2O2-induced HaCaT cell senescence model, the activity of senescence-related β-galactosidase SA-β-gal was detected by staining.

Images