An anti-aging composition for sensitive skin, its application, and cosmetics

Abstract

This invention belongs to the field of new materials for daily chemical products, and discloses an anti-aging composition, application, and cosmetic for sensitive skin. The composition includes halophilic bacteria fermentation product and acetyl hexapeptide-8; the weight ratio of the halophilic bacteria fermentation product and acetyl hexapeptide-8 is 1:0.1~10. This composition is non-irritating, suitable for sensitive skin, and has good effects on improving crow's feet, under-eye wrinkles, and firmness, with a very fast improvement speed.

Description

Technical Field

[0001] This invention relates to the field of biology, and more particularly to an anti-aging composition for sensitive skin, its application, and cosmetics. Background Technology

[0002] With the fast pace of modern life and changes in the living environment, the number of people with sensitive skin is increasing daily. As this population expands, the demand for skincare products is no longer limited to basic moisturizing and repair; there are higher-level needs, especially regarding anti-aging. This is primarily because sensitive skin ages earlier and faster than normal skin. Sensitive skin is easily irritated by external stimuli, leading to a weakened skin barrier, redness, and a fragile skin base that damages skin cells, accelerating aging and resulting in fine lines and sagging. Furthermore, aging cells amplify sensitivity signals, making the skin even more fragile, creating a cycle where sensitivity exacerbates aging, and aging is accompanied by sensitivity.

[0003] Most existing patents on anti-aging for sensitive skin claim to be suitable for sensitive skin by incorporating anti-aging active ingredients into a mild formula base. However, they do not actually address both skin sensitivity and aging simultaneously. This cycle continues, with skin sensitivity remaining unresolved, resulting in less effective anti-aging. Therefore, there is still room for improvement in both the mechanism and efficacy.

[0004] Halophilic bacteria are a class of microorganisms adapted to high-salt environments, encompassing multiple groups within the domains of halophilic archaea and bacteria. Extreme halophiles, such as *Halobacter* and *Halococcus*, belong to the phylum Archaea. Their cells contain red pigments and a purple membrane structure; their cell walls lack peptidoglycan, and the rhodopsin in the purple membrane can absorb light energy. They are distributed in environments such as salt lakes, salt pans, preserved products, and the Dead Sea, and can survive at salt concentrations of 15%-32%. They maintain osmotic balance by synthesizing compatible solutes such as glycine and betaine and accumulating potassium chloride; the function of their cytoplasmic proteins depends on high concentrations of sodium chloride.

[0005] There is limited information regarding the application of halophilic bacteria and their fermentation products in cosmetics, such as:

[0006] The patent application, with publication number CN115581656A, is for an emotional awakening iceberg condensate with soothing and repairing effects based on a combination of marine organisms and its preparation method. In this combination, mussel mucin and halophilic bacteria fermentation products mainly provide soothing effects.

[0007] Through ongoing research into the fermentation products of halophilic bacteria, we have discovered new functions and phenomena. Based on this, we propose this invention. Summary of the Invention

[0008] The purpose of this invention is to provide an anti-aging composition for sensitive skin. This composition is non-irritating, suitable for sensitive skin, and has a good effect on improving crow's feet, under-eye wrinkles, and firmness, with a very fast improvement speed.

[0009] The present invention also discloses the application of the composition and cosmetics.

[0010] To achieve the above objectives, this application discloses:

[0011] An anti-aging composition for sensitive skin includes halophilic bacteria fermentation product and acetyl hexapeptide-8;

[0012] The weight ratio of the halophilic bacteria fermentation product to acetyl hexapeptide-8 is 1:0.1~10.

[0013] This composition improves intercellular communication by influencing intercellular junction proteins, thereby achieving firming, anti-wrinkle effects and anti-aging benefits.

[0014] Gap junctions are intercellular channels that mediate electrical and biochemical coupling through the exchange of ions, second messengers, and small molecule metabolites. A gap junction consists of two connexins, each provided by an adjacent cell. Each connexin is a hexamer composed of six connexins (Cx). Currently, 21 members of the human connexin gene family have been identified, many of which are found in the skin, including Cx26, 30, 30.3, 31, 31.1, 32, 37, 40, 43, and 45. Connexin-mediated intercellular communication in the epidermis is crucial for maintaining epidermal homeostasis.

[0015] By consulting relevant literature, CCL2 was identified as a common target for both skin sensitivity and aging. As a pro-inflammatory mediator, CCL2 can induce skin inflammation, as well as neuropathic pain and itching, triggering symptoms of sensitive skin. In this state, the skin's aging process is accelerated. On the other hand, CCL2 can induce skin aging through the SASP / mTOR signaling pathway. Aging skin, due to collagen loss, becomes thinner, and its ECM support weakens, making it more susceptible to allergic reactions from external stimuli. Thus, in this vicious cycle of skin sensitivity and aging, blocking CCL2 signaling can simultaneously achieve soothing and anti-aging (firming, anti-wrinkle) effects.

[0016] By reviewing relevant literature, NF-κB was identified as a common target for both skin sensitivity and aging. NF-κB responds to oxidative stress, DNA damage, immune activation, and growth regulation signals, regulating cell proliferation, innate and adaptive immunity, inflammatory responses, and apoptosis. The relationship between NF-κB and skin sensitivity is mainly manifested in two aspects: inflammatory responses and barrier damage. Inflammatory responses: NF-κB can promote the secretion of pro-inflammatory cytokines such as IL-6, IL-1β, and TNFα, and pathway activation can induce atopic dermatitis. Barrier damage: NF-κB activation can stimulate TNFα expression, subsequently inducing upregulation of MMPS, and collagen degradation leads to skin barrier damage. The relationship between NF-κB and skin aging is shown in the following: NF-κB activation can induce upregulation of MMPS, and collagen degradation induces skin aging. In chronic inflammation, sustained activation of NF-κB can also induce telomere shortening in muscle stem cells; telomere shortening is a well-known marker of aging.

[0017] Halophilic bacteria are used in industry for the production of polyhydroxyalkanoates (PHAs), extracellular polysaccharides, and seawater desalination. Some strains can degrade pollutants such as petroleum hydrocarbons and thiocyanates. Metabolites such as peptides and biosurfactants possess antibacterial activity. The halophilic bacteria described in this invention were further selected from 37 known microorganisms with intercellular signal transduction functions screened from a diverse high-salt ecological microbial library. In vitro experiments were conducted on the above-mentioned active ingredients: these experiments revealed that the active ingredients can inhibit the production of CCL2 or NF-κB by keratinocytes, simultaneously disrupting pathways leading to skin sensitivity and aging, while also enhancing the gene expression of intercellular communication linkers, thus improving intercellular communication and exerting an anti-aging effect. Furthermore, it was confirmed that acetyl hexapeptide-8 and the fermentation products of halophilic bacteria have an excellent synergistic effect.

[0018] In the above-mentioned sensitive skin anti-aging composition, the weight ratio of the halophilic bacteria fermentation product to acetyl hexapeptide-8 is 1:1 to 2.5.

[0019] Furthermore, this invention also discloses the use of the above-described sensitive skin anti-aging composition in the preparation of cosmetics. More specifically, the sensitive skin anti-aging composition refers to a composition that has the functions of inhibiting CCL2 inflammatory factors, downregulating NF-κB inflammatory factor function, and enhancing Cx26 connective protein gene expression function.

[0020] Finally, the present invention also discloses a cosmetic containing the sensitive skin anti-aging composition as described above.

[0021] In the aforementioned cosmetics, the concentration of the sensitive skin anti-aging composition is 0.01~6wt%.

[0022] In the aforementioned cosmetics, the concentration of the sensitive skin anti-aging composition is 0.1~2wt%.

[0023] This application has at least the following beneficial effects:

[0024] The composition of the present invention targets intercellular junction proteins to improve signs of aging; the composition can act on common targets that lead to skin sensitivity and aging, precisely improve signs of aging in sensitive skin, and the components have a synergistic effect. Attached Figure Description

[0025] Figure 1 For observation and reference diagrams of test sites in human trials;

[0026] Figure 2A This illustrates the variation of the crow's feet wrinkles from left to right (W0) in Example 1.

[0027] Figure 2B This illustrates the change in under-eye wrinkles from left to right (W0) in Example 1.

[0028] Figure 2C To illustrate the changes in crow's feet wrinkles from left to right (W0) in Example 1;

[0029] Figure 2D To compare the changes in crow's feet wrinkles from left to right (W0) in Example 3;

[0030] Figure 2E To compare the changes in under-eye wrinkles from left to right (W0);

[0031] Figure 2F To compare the changes in crow's feet wrinkles from left to right (W0) in Example 4;

[0032] Note, Figures 2A to 2F The thick lines marked manually are wrinkles identified by the instrument. Detailed Implementation

[0033] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products. Unless otherwise specified, all parts used in the embodiments of the present invention are parts by weight.

[0034] I. Explanation of Raw Material Sources

[0035] Halophilic bacteria fermentation product: Supplier: Lubrizol Specialty Chemicals Manufacturing (Shanghai) Co., Ltd.; Active ingredient content: 0.09~0.11wt%;

[0036] Acetyl hexapeptide-8: Supplier: Lubrizol Specialty Chemicals Manufacturing (Shanghai) Co., Ltd.; Active ingredient content: 0.045~0.055%;

[0037] Acetyl Tetrapeptide-2: Supplier: Dongguan Weiqi Technology Co., Ltd.; Active ingredient content: 0.045~0.055%;

[0038] Palmitoyl tripeptide-5: Supplier: DSM Nutritional Products Limited; Active ingredient content: 0.09~0.13%.

[0039] The substances listed in Table 2 of this invention represent commodities and do not represent the net content of active ingredients.

[0040] II. Examples and Comparative Examples

[0041] Reference Table 1 shows the base material formulations for each case.

[0042] Table 1. Formulation of the base material

[0043]

[0044] The preparation process is as follows:

[0045] Add phase A raw material to the main pot and heat to 70-90℃ until completely dissolved;

[0046] 2. Heat the B phase raw material to 70-90℃ until completely dissolved;

[0047] 3. Add phase B to the main pot of phase A and emulsify and homogenize at 70-90℃ for 1-6 minutes;

[0048] 4. Cool down to below 60℃, then add the dissolved C phase into the main pot;

[0049] 5. Cool down to below 45℃, add the dissolved D phase to the main pot, and stir well;

[0050] 6. Cool down to below 37℃ and filter out the material.

[0051] The variable components for each case can be found in Table 2 below;

[0052] Table 2 Formulation Table (Unit: wt%)

[0053] Acetyl hexapeptide-8 Acetyl tetrapeptide-2 Palmitoyl tripeptide-5 Fermentation products of halophilic bacteria Example 1 2.5 0 0 1 Comparative Example 1 3.5 0 0 0 Comparative Example 2 0 0 0 3.5 Comparative Example 3 0 2.5 0 1 Comparative Example 4 0 0 2.5 1

[0054] III. Human Body Testing

[0055] The human testing methods involved in this invention include: 7-day improvement rate of crow's feet wrinkles, 7-day improvement rate of under-eye wrinkles, 7-day improvement and relief, and 7-day improvement rate of firmness;

[0056] 1. Testing instruments

[0057] The main instruments and equipment involved in the test are shown in Table 3.

[0058] Table 3 Main Instruments

[0059]

[0060] 2. Testing cycle:

[0061] Before using the sample (W0) and week 1 after using the sample (W1).

[0062] 3. Testing process:

[0063] 1) On the day of the visit, the subjects did not use any products on their face, cleaned their face with water, and sat quietly in a constant temperature and humidity room for 30 minutes.

[0064] 2) Initial screening: Initial screening is conducted after the informed consent form is signed;

[0065] 3) W0 test: Visio 4D and VISIA-CR (ST1, CP) were used to take pictures in sequence, and then skin firmness (F4 value) and EI value were measured;

[0066] 4) Provide sample usage instructions and on-site sample trials to the enrolled subjects;

[0067] 5) W1 test: A follow-up test will be conducted one week after the sample is used, following the same steps as 1) and 3).

[0068] 6) Organize the data and generate a report.

[0069] Data statistical analysis methods:

[0070] Rate of change = (Wn - W0) / W0 × 100%

[0071] Where W0 represents the value before using the sample, and Wn represents the value after using the sample. The improvement rate is the absolute value of the rate of change when the parameter improves.

[0072] The specific test methods are as follows:

[0073] 7-day improvement rate of crow's feet: After using the sample for 7 days, the three parameters of crow's feet (wrinkle volume, wrinkle perimeter, and wrinkle area) improved compared with the baseline values, suggesting that the sample has the effect of improving crow's feet. The 7-day improvement rate of crow's feet was obtained by using the change rate calculation formula.

[0074] 7-day improvement rate of under-eye wrinkles: After using the sample for 7 days, the three parameters of under-eye wrinkles (wrinkle volume, wrinkle perimeter, and wrinkle area) improved compared with the baseline values, suggesting that the sample has the effect of improving under-eye wrinkles. The 7-day improvement rate of under-eye wrinkles was obtained by using the change rate calculation formula.

[0075] 7-day improvement and soothing effect: After using the sample for 7 days, the a* value and EI value of the skin in the test area decreased compared with the baseline value, suggesting that the sample has a soothing (redness) effect. The 7-day improvement and soothing change rate was obtained by using the change rate calculation formula.

[0076] 7-day skin firmness improvement rate: After using the sample for 7 days, the skin firmness (F4 value) in the test area decreased compared with the baseline value, suggesting that the sample has the effect of improving skin firmness. The 7-day skin firmness change rate was obtained through the change rate calculation formula.

[0077] Reference for observation and testing sites Figure 1 ;

[0078] Note: 1. The analysis of parameters such as firmness (F4 value), a* value, EI value, under-eye wrinkles, and crow's feet is distributed across the left and right halves of the face;

[0079] 2. VISIA-CR: Frontal view, light source: CP, used for skin a* value analysis.

[0080] The test results are shown in Table 4 and the appendix. Figure 2A To be continued Figure 2F ;

[0081] Table 4 Test Results

[0082]

[0083] Note: The abnormality in the circumference data of the 7-day improvement rate of under-eye wrinkles in Comparative Example 2 may indicate that it did not have an actual improvement effect on the circumference of under-eye wrinkles. The slight decrease in data may be due to testing error.

[0084] Results analysis:

[0085] 1. As can be seen from Example 1, the composition exhibits superior soothing and anti-wrinkle properties.

[0086] 2. Based on the test results of Comparative Example 1 and Comparative Example 2, both acetyl hexapeptide-8 and halophilic bacteria fermentation product can exhibit certain degrees of soothing and anti-wrinkle properties when used alone; however, considering the results of Example 1 and Comparative Examples 1 and 2, only when both are used simultaneously can a better synergistic effect be observed.

[0087] 3. As is well known in the art, the three peptides verified in this invention all possess excellent anti-wrinkle properties and very low irritation. For example, acetyl hexapeptide-8, also known as Argireline, belongs to the neurotransmitter inhibitory peptide class and is mainly used as a peptide cosmetic ingredient in the anti-aging field; acetyl tetrapeptide-2 can comprehensively promote the expression of collagen genes of types I, III, IV, VII, and XVII, and can also inhibit the gene expression of matrix metalloproteinase 1 (MMP-1) and matrix metalloproteinase 9 (MMP-9); acetyl tetrapeptide-2 can significantly promote the gene expression and protein synthesis of elastin in the skin, providing strong protection for maintaining skin elasticity; palmitoyl tripeptide-5 is a synthetic tripeptide ingredient widely used in anti-aging skincare products, mainly working by promoting collagen production, repairing the skin barrier, and improving skin elasticity. Its molecular structure has been modified to penetrate the skin more efficiently, achieving anti-wrinkle and firming effects.

[0088] Based on the actual test results of this invention, the combined use of acetyl tetrapeptide-2, palmitoyl tripeptide-5, and halophilic monoclonal fermentation products does not achieve the expected results before the experiment of this invention.

[0089] In summary, the present invention, through the compatibility study of halophilic monoclonal fermentation products and peptides, has demonstrated that it has good soothing, anti-aging and anti-wrinkle effects with acetyl hexapeptide-8, exhibiting good synergy.

[0090] IV. In vitro experimental section

[0091] Experiment 1: Detection of CCL2 (Inflammatory Factor) Levels in RAW264.7 Macrophages Stimulated by LPS

[0092] 1. Experimental Procedure

[0093] 1) Cell seeding: at 1×10 5 Cells were seeded into 24-well plates at a seeding density of cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0094] 2) Solution preparation: Prepare working solutions of different concentrations of the test substance according to the experimental design table in Table 5 below.

[0095] Table 5 Experimental Group Design Table

[0096]

[0097] 3) Induction and drug administration: When the cell deposition rate in the 24-well plates reached 40%–60%, 100 μL of 10× LPS working solution was added to each well according to the experimental design. The wells were shaken left and right to mix the drug. Samples were added simultaneously. The final LPS concentration was 1 μg / mL. Each group had 3 replicates. After drug administration, the wells were placed in an incubator (37℃, 5% CO2) and cultured for another 24 h.

[0098] 4) Sample collection: After incubation, collect the cell culture supernatant into EP tubes (Note: Determine the amount of sample to be collected according to the detection indicators). After collection, freeze the sample in a -80℃ freezer.

[0099] 5) CCL2 content detection: Perform the detection according to the instructions of the Mouse CCL2 ELISA kit.

[0100] 2. CCL2 content test results are shown in Table 6;

[0101] Table 6 Test Results

[0102] Sample Name Acetyl hexapeptide-8 content (wt%) wt% of halophilic bacteria fermentation products CCL2 content (pg / ml) Example 2 0.072 0.028 809.67 Example 3 0.72 0.28 443.01 Comparative Example 5 0.1 0 1333.69 Comparative Example 6 0 0.1 1213.18 Comparative Example 7 1 0 1039.5 Comparative Example 8 0 1 949.32 negative control group - - 1647.04 Positive control group - - 309.69

[0103] As can be seen from the test results in Table 6, the composition of the present invention exhibits a strong synergistic effect in inhibiting inflammatory factor (CCL2), and this synergistic effect is evident even at a composition concentration of 0.1%.

[0104] Experiment 2: NF-κB gene expression level test

[0105] 1. Experimental procedure:

[0106] 1) Cell seeding: HaCaT was seeded at a rate of 6 × 10⁻⁶. 5 The cells were seeded at a density of cells / well in 6-well plates and incubated in an incubator (37°C, 5% CO2) for 24 h.

[0107] 2) Drug administration pretreatment: According to the experimental groups in Table 7 below, the sample groups were added with fresh culture medium containing the corresponding concentration of the sample and cultured at 37 ℃ and 5% CO2 for 2 h.

[0108] Table 7 Experimental Group Design Table

[0109]

[0110] 3) UVB modeling: After pretreatment, HaCaT cells were washed three times with D-Hanks. 1 mL of D-Hanks was added to each well to submerge the cells. The control group was wrapped in tin foil and placed in the dark. UVB modeling was performed on the groups requiring radiation, according to the experimental group requirements.

[0111] 4) Drug administration: After modeling, D-Hanks gently rinsed the cells once or twice. Fresh culture medium was added to the normal control group, and fresh culture medium containing the corresponding concentration of sample was added to the sample group. The cells were cultured at 37 ℃ and 5% CO2 for 24 h.

[0112] 5) Sample collection: After the culture is completed, cell samples are collected, total RNA is extracted from each experimental group, and cDNA is synthesized;

[0113] 6) Detection: The expression of NF-κB and the internal reference gene was detected using q-PCR technology. The relative RNA expression level of the target gene was calculated using the expression of the internal reference gene as a reference.

[0114] 2. The experimental results are shown in Table 8;

[0115] Table 8 Experimental Results

[0116] Sample Name Acetyl hexapeptide-8 content (wt%) wt% of halophilic bacteria fermentation products Relative expression level of NF-κB gene NF-κB downregulation rate Example 8 0.0072 0.0028 0.609 39% Comparative Example 9 0.01 0 0.699 30% Comparative Example 10 0 0.01 0.670 33% Blank control group - - 0.560 - Model control group - - 1.000 -

[0117] Results analysis: As can be seen from the test results in Table 8, the composition of the present invention exhibits strong synergistic effect in inhibiting inflammatory factors (NF-κB), and this synergistic effect can be observed even at a composition concentration of 0.01%.

[0118] Experiment 3: Assay of Connectin Gene Expression Levels

[0119] 1. Experimental Procedure

[0120] 1) Cell seeding: NHEK cell (normal human epidermal keratinocytes) suspension was plated and cultured in an incubator (37℃, 5% CO2) for 24 hours.

[0121] 2) Drug administration: Prepare samples according to the concentrations set in Table 9 below, and treat each sample for 24 hours.

[0122] Table 9. Enhancement of connexin gene expression by the composition.

[0123] Sample Name Acetyl hexapeptide-8 content (wt%) wt% of halophilic bacteria fermentation products Cx26 linker protein gene expression enhancement rate Example 9 1 1 44% Example 10 1 2 88% Comparative Example 11 1 - NS Comparative Example 12 - 1 21% Comparative Example 13 - 2 53%

[0124] 3) Sample collection: Extract total RNA from each experimental group and synthesize cDNA.

[0125] 4) Detection: The expression of Cx26 and the internal reference gene was detected using q-PCR technology. The relative RNA expression level of the target gene was calculated using the expression of the internal reference gene as a reference.

[0126] 2. Results Analysis: The test results show that acetyl hexapeptide-8 does not enhance the gene expression of connexins, while the fermentation product of halophilic bacteria can enhance the gene expression of connexins. The combination of the two has a synergistic effect, indicating that the composition of the present invention has a synergistic effect in improving the gene expression of connexins.

Claims

1. A sensitive skin anti-aging composition, characterized in that, Including halophilic bacteria fermentation products and acetyl hexapeptide-8; The weight ratio of the halophilic bacteria fermentation product to acetyl hexapeptide-8 is 1:1 to 2.

5. The halophilic bacteria fermentation product was provided by Lubrizol Specialty Chemicals Manufacturing (Shanghai) Co., Ltd.; the content of the active ingredient was 0.09~0.11wt%.

2. Use of the sensitive skin anti-aging composition as described in claim 1 to prepare cosmetics, wherein the sensitive skin anti-aging composition refers to a composition having the function of inhibiting CCL2 inflammatory factors and downregulating... NF-κB A composition that enhances the function of inflammatory factors and the expression of the Cx26 linker protein gene.

3. A cosmetic product, characterized in that, Contains the sensitive skin anti-aging composition as described in claim 1.

4. The cosmetic product according to claim 3, characterized in that, The concentration of the sensitive skin anti-aging composition is 0.01~6wt%.

5. The cosmetic product according to claim 3, characterized in that, The concentration of the sensitive skin anti-aging composition is 0.1~2wt%.

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