A composition for improving the epidermal junction layer and its application

Abstract

The present invention provides a composition for improving the epidermal junction layer and its application. It was found that the combination of a symbiotic fermentation product of Thermus thermophilus and Bacillus sp. and grape seed extract synergistically improves the expression of proteins associated with the epidermal junction layer, increasing the expression of occludin by 316%, heparan sulfate proteoglycan (HSPG) by 64%, and hyaluronic acid receptor protein (CD44) by 69%. Compared with single ingredients, this composition is more effective in improving the health of the epidermal junction layer. It can be used to prepare cosmetics to meet the urgent need for anti-aging, has good application prospects, and also provides a new approach to the development of traditional anti-aging skin care products.

Description

Technical Field

[0001] The present invention relates to the technical field of skin care products, and in particular to a composition for improving the dermal-epidermal junction layer and an application thereof. Background Art

[0002] Delaying skin aging is a dynamic, multi-step process involving different layers of the skin. Currently, in the development of personal care products specifically for anti-wrinkle, most people think of strengthening the skin barrier and protection, and repairing collagen and elastin in the dermis. Few mention improving the health of the dermal-epidermal junction (DEJ). In fact, improving the health of the DEJ is crucial for skin care. Studies have found that the structure of the DEJ in 30-year-old women's skin has changed. This phenomenon is particularly pronounced in the elderly, while such changes are rarely seen in 20-year-old skin, indicating that flattening of the DEJ is a primary characteristic of early skin aging.

[0003] Finding substances that improve and maintain the dermal-epidermal junction (DEJ) can strengthen the basal layer from the inside, enhance the cohesion between the dermis and epidermis, and improving the dermal-epidermal junction (DEJ) can restore the skin's appearance to a plump and smooth state, thereby increasing skin nutrient absorption, increasing the thickness of the epidermis, and improving the epidermis' tolerance to external mechanical forces. This is an important direction for future anti-aging skin care products.

[0004] The skin is composed of the epidermis, dermis, and subcutaneous adipose tissue. The dermis and epidermis form an intermeshing structure at their junction. The dermis, adjacent to the epidermis, rises upward in a papillary manner and embeds between the epidermal spikes. The subcutaneous extensions of the epidermis are called spikes. This intermeshing structure of spikes and papillae forms the basement membrane of the epidermal-epidermal junction. The basement membrane is a complex lattice composed of laminin, type IV collagen, type VII collagen, heparan sulfate glycoproteins, integrins, nidogen, and fibronectin, embedded in a mucopolysaccharide-rich gel matrix. This structure facilitates the exchange of substances between the epidermis and dermis. Flattening of the epidermal-epidermal junction is closely related to the loss of related glycoproteins, such as heparan sulfate proteoglycans (HSPGs) and hyaluronic acid receptor protein (CD44).

[0005] Therefore, there is an urgent need to find a preparation that can contribute to improving the health of the dermal-epidermal junction layer so that it can be used to prepare anti-aging skin care products. Summary of the Invention

[0006] In response to the above problems, the present invention provides a composition for improving the epidermal junction layer and its application. It was found that the compounding of the symbiotic fermentation product of Thermus thermophilus and Bacillus and grape seed extract has a synergistic effect in improving the expression of proteins related to the epidermal junction layer, which can increase the expression of occludin by 316%, increase the expression of heparan sulfate proteoglycan (HSPG) by 64%, and increase the expression of hyaluronic acid receptor protein (CD44) by 69%. Compared with a single raw material, it has a more significant effect in improving the health of the epidermal junction layer of the skin. With this effective ingredient, together with a cosmetically acceptable carrier, cosmetics of various dosage forms are made according to conventional preparation methods, which can meet the urgent need of anti-aging and have good application prospects. It also provides a new idea for the development of traditional anti-aging skin care products.

[0007] After extensive research, the present invention discovered that encapsulated grape seed extract (E-AGSE), a fermentation product of Thermus thermophilus and Bacillus (TIRACLE Pro), or a combination of the two, can target and stimulate the expression of the dermal-epidermal junction, targeting the expression of basement membrane proteoglycans, particularly stimulating the expression of glycoproteins such as HSPGs in the basement membrane, and specifically increasing the expression of heparan sulfate proteoglycans (HSPGs), hyaluronic acid receptor protein (CD44), and occludin, thereby improving skin nourishment and complexion. E-AGSE, TIRACLE Pro, or a combination of the two can be used in the development of skin care products to make cosmetics in various dosage forms that can penetrate deep into the basal layer of the skin, helping the skin regain a bright, elastic, and youthful appearance.

[0008] The symbiotic fermentation product of Thermus thermophilus and Bacillus is rich in amino acids, peptides, polyphenols and trace elements for maintaining skin nutrition, and is prepared by: (1) subjecting Thermus thermophilus to one or more enrichment fermentations at 65°C and a pH value of 7.8±0.2, allowing it to be in an exponential growth phase; (2) adding Bacillus subtilis and conducting a secondary fermentation at a pH value of 7.0-7.2 and a temperature of 30±2°C, and sterilizing the fermentation broth by centrifugation.

[0009] The grape seed extract preparation method follows the extraction method of Example 8 of U.S. Patent No. 11 / 890,278. Grape seeds free of impurities are first extracted with 600 ml of 100% ethanol at room temperature for 18 hours. The ethanol extract is filtered through Whatman filter paper and then the ethanol is removed by rotary evaporation to obtain a concentrated primary extract. The primary extract is then repeated with the above process, dried in a vacuum drying oven, and further concentrated to a final extract, which is then ground to obtain the grape seed extract.

[0010] In one aspect, the present invention provides a composition for improving the dermal-epidermal junction layer, comprising a symbiotic fermentation product of Thermus thermophilus and Bacillus, and also comprising grape seed extract.

[0011] Furthermore, the content of the symbiotic fermentation product of Thermus thermophilus and Bacillus is 0.005%-1%, and the content of the grape seed extract is 0.002%-1%.

[0012] In another aspect, the present invention provides a use of a mixture for preparing a preparation for improving the dermal-epidermal junction, wherein the mixture comprises a symbiotic fermentation product of Thermus thermophilus and Bacillus, and a grape seed extract.

[0013] Furthermore, the mixture improves the dermal-epidermal junction layer by promoting the expression of perlecan.

[0014] Furthermore, the mixture improves the dermal epidermal junction layer by promoting the expression of any one or more of Occludin, HSPG or CD44.

[0015] Furthermore, the content of the symbiotic fermentation product of Thermus thermophilus and Bacillus is 0.005%-1%, and the content of the grape seed extract is 0.002%-1%.

[0016] On the other hand, the present invention provides a use of a symbiotic fermentation product of Thermus thermophilus and Bacillus in preparing a preparation for improving the dermal-epidermal junction.

[0017] Furthermore, the symbiotic fermentation products of Thermus thermophilus and Bacillus improve the epidermal junction layer by promoting the expression of basement membrane proteoglycan, thereby delaying skin aging.

[0018] Furthermore, the symbiotic fermentation product of Thermus thermophilus and Bacillus improves the dermal epidermal junction layer by promoting the expression of any one or more of Occludin, HSPG or CD44.

[0019] Furthermore, the content of the symbiotic fermentation product of Thermus thermophilus and Bacillus is 0.005%-1%.

[0020] In another aspect, the present invention provides use of a grape seed extract in preparing a preparation for promoting the expression of any one or more of occludin, HSPG, or CD44.

[0021] Furthermore, the content of the grape seed extract is 0.002%-1%.

[0022] In another aspect, the present invention provides a use of a mixture for preparing a preparation for promoting the expression of any one or more of Occludin, HSPG or CD44, wherein the mixture comprises a symbiotic fermentation product of Thermus thermophilus and Bacillus, and a grape seed extract.

[0023] Furthermore, the content of the symbiotic fermentation product of Thermus thermophilus and Bacillus is 0.005%-1%, and the content of the grape seed extract is 0.002%-1%.

[0024] In another aspect, the invention provides a use of a symbiotic fermentation product of Thermus thermophilus and Bacillus in the preparation of a preparation for promoting the expression of any one or more of Occludin, HSPG or CD44.

[0025] Furthermore, the content of the symbiotic fermentation product of Thermus thermophilus and Bacillus is 0.005%-1%, and the content of the grape seed extract is 0.002%-1%.

[0026] The encapsulated grape seed extract (E-AGSE), the fermentation product of Thermus thermophilus and Bacillus (TIRACLE Pro), or a composite composition thereof provided by the present invention can be used to prepare skin care products with anti-aging effects.

[0027] Furthermore, the dosage forms of the skin care products provided by the present invention mainly include one or more of facial cream, emulsion, suspension, facial mask, cream, paste, gel, dry powder, wet powder, aqueous solution, essence or spray.

[0028] The skin care products with anti-aging and other effects described in the present invention, in addition to including grape seed extract (E-AGSE), fermentation product of Thermus thermophilus and Bacillus sp. (TIRACLE Pro), or a composite composition of the two, can be appropriately combined with various ingredients commonly used in the field of skin care products and quasi-drugs as needed, within the scope that does not impair the effects of the present invention, such as any one or a mixture of powder components, moisturizers, emulsifiers, light stabilizers, thickeners, solubilizers, metal ion sequestering agents, pigments, pH regulators, skin nutrients, vitamins, preservatives, antioxidants, sunscreens, penetration enhancers, liposomes, skin nutrient components, antioxidant adjuvants, flavors, fragrances, pigments, etc.

[0029] The beneficial effects of the present invention are:

[0030] 1. Provided is a composition for improving the epidermal junction layer to resist aging;

[0031] 2. Discovered new uses for E-AGSE, TIRACLE Pro, or a combination of the two to improve the health of the dermal-epidermal junction (DEJ) and thus combat aging;

[0032] 3. Discovered a new use of E-AGSE, TIRACLE Pro, or a combination of the two to improve the dermal-epidermal junction by promoting the expression of perlecan;

[0033] 4. Discovered new uses of E-AGSE, TIRACLE Pro, or a combination of the two in promoting the expression of any one or more of occludin, HSPG, or CD44;

[0034] 5. It was found that TIRACLE Pro can increase the expression of occludin by 54%, the expression of heparan sulfate proteoglycan (HSPG) by 14%, and the expression of hyaluronic acid receptor protein (CD44) by 6%;

[0035] 6. E-AGSE was found to increase the expression of occludin by 152%, heparan sulfate proteoglycan (HSPG) by 17%, and hyaluronic acid receptor protein (CD44) by 16%.

[0036] 7. It was found that E-AGSE can increase the expression of occludin by 316%, the expression of heparan sulfate proteoglycan (HSPG) by 64%, and the expression of hyaluronic acid receptor protein (CD44) by 69%;

[0037] 8. The provided E-AGSE and TIRACLE Pro compound composition can strengthen the basal layer from the inside, enhance the cohesion between the dermis and epidermis, restore the skin's appearance to a plump and smooth state, thereby increasing the skin's nutrient absorption, increasing the thickness of the epidermis, and improving the health of the dermis-epidermal junction layer, thereby significantly improving the skin's wrinkles and fine lines. DETAILED DESCRIPTION

[0038] The present invention will be further described in detail below with reference to the examples. It should be noted that the examples described below are intended to facilitate understanding of the present invention and do not have any limiting effect on the present invention.

[0039] Example 1 Preparation of symbiotic fermentation products of Thermus thermophilus and Bacillus

[0040] The Thermus thermophilus used in this example has a strain accession number of BAA-163, purchased from the American Type Culture Collection; the Bacillus subtilis is Bacillus subtilis subsp. Subtilis, with a strain accession number of CICC10023, purchased from the China Industrial Microbiology Culture Collection.

[0041] The basic fermentation medium formula is as follows: 10L of culture medium contains 30g peptone, 2g malt extract, 3.5g anhydrous magnesium sulfate, 1.2g potassium dihydrogen phosphate, 20g ammonium sulfate, 0.5g anhydrous ferric chloride, and 2.5g sodium chloride. Water is used to make up to 10L. Different amounts of culture medium can be scaled down or up according to the ratio. The seed solution is inoculated into the culture medium at a rate of 3-10%.

[0042] All embodiments of the present invention use the above culture medium formula as the culture medium for bacterial fermentation. Of course, in actual industrial production, the proportions can be adjusted according to different requirements. These formulas are all common formulas for culturing Thermus thermophilus / Bacillus.

[0043] The symbiotic fermentation culture process is as follows:

[0044] 1. Seed culture of Thermus thermophilus

[0045] On a clean bench, 2 ml of the frozen stock solution of Thermus thermophilus BAA-163 was added to a 2.5 L Erlenmeyer flask containing 1 L of culture medium with a pH of 7.8 ± 0.2. The culture was carried out at 150 rpm and 65 ° C for 12 h to obtain 1 L of Thermus thermophilus seed solution in which the bacteria were in the exponential growth phase.

[0046] 2. Add Bacillus, carry out secondary fermentation at a pH of 7.0-7.2 and a fermentation temperature of 30±2°C, and centrifuge the fermentation liquid for sterilization.

[0047] Example 2 Preparation of a composite composition of E-AGSE and TIRACLE Pro

[0048] The fermentation product of Thermus thermophilus and Bacillus sp. (TIRACLEPro) was prepared according to the method provided in Example 1.

[0049] Grape seed extract (E-AGSE) is prepared according to the extraction method of Example 8 of U.S. Patent No. 11 / 890,278. Grape seeds free of impurities are first extracted with 600 ml of 100% ethanol at room temperature for 18 hours. The ethanol extract is filtered through Whatman filter paper and then the ethanol is removed by rotary evaporation to obtain a concentrated primary extract. The above process is repeated for the primary extract, which is then dried in a vacuum drying oven and further concentrated to a final extract, which is then ground to obtain the grape seed extract.

[0050] E-AGSE and TIRACLE Pro are mixed to obtain a compound composition, wherein the content of TIRACLE Pro is 0.005%-1%, and the content of E-AGSE is 0.01%-1%.

[0051] Example 3: Cell experiment with TIRACLE Pro

[0052] Epidermal keratinocytes (NHEKs, purchased from Thermo Fisher Scientific (Cat No. A13401)) were obtained from human neonates; each cell batch tested negative for HIV-1, HIV-2, HCV, and other antigens. Appropriate 6-well plates were selected, and 2 mL of the cell suspension was added to each well of EpiLife medium (Thermo Fisher Scientific; Cat No. MEPI500CA). The cells were cultured for 24 hours under a constant 5% v / v CO2 flow, at a temperature of 36°C to 38°C and a humidity of 50% to 65%, respectively. The culture medium was replaced, and the cells were cultured for another 24 hours under the same experimental conditions to obtain a cell culture fluid.

[0053] The cell culture medium was then taken and the sample solution diluted 1:100 (TIRACLE Pro) with the stock solution of fresh culture medium was added, and cultured for 24 hours at 37°C and 5% CO2. At the same time, a group that did not receive any dose of sample served as a blank control of the experiment.

[0054] Finally, the culture medium was removed and the cells were washed with phosphate-buffered saline (PBS) without test materials. Total RNA was extracted from the tissues and converted into cDNA by reverse transcription. Fluorescence PCR was performed using 2 -△△CT Results were calculated using the same method. GraphPad Prism Program software was used for data processing and graphing. Statistical analysis was performed using the t-test between groups. P < 0.05 indicated significant differences, and P < 0.01 indicated extremely significant differences. All cell culture and assay procedures were performed in the Signal Biosciences Laboratory, which meets the standards of a Biosafety Level 2 (BL-2) cell culture laboratory. The test results are shown in Table 1.

[0055] Table 1. Changes in NHEKs gene expression after TIRACLE Pro (0.5%) treatment

[0056]

[0057] The results showed that a 0.5% TIRACLE Pro solution increased occludin expression by 54%, heparan sulfate proteoglycan (HSPG) expression by 14%, and hyaluronic acid receptor protein (CD44) expression by 6% compared to the blank control. It can be concluded that TIRACLE Pro significantly promotes perlecan gene expression after basic in vitro treatment of keratinocytes.

[0058] This embodiment also uses 0.005%, 0.05%, 0.3%, 0.5%, and 1% TIRACLE Pro solutions to conduct experiments, respectively, to examine the effects of different concentrations of TIRACLE Pro solutions on promoting the expression of occludin, heparan sulfate proteoglycans (HSPG), and hyaluronic acid receptor protein (CD44). The results show that TIRACLE Pro solutions in the range of 0.005%-1% can effectively promote the expression of occludin, heparan sulfate proteoglycans (HSPG), and hyaluronic acid receptor protein (CD44).

[0059] Example 4 Cell experiment of E-AGSE

[0060] Epidermal keratinocytes (NHEKs, purchased from Thermo Fisher Scientific (Cat No. A13401)) were obtained from human neonates; each cell batch tested negative for HIV-1, HIV-2, HCV, and other antigens. Appropriate 6-well plates were selected, and 2 mL of the cell suspension was added to each well of EpiLife medium (Thermo Fisher Scientific; Cat No. MEPI500CA). The cells were cultured for 24 hours under a constant flow of 5% v / v CO2, at a temperature of 36°C to 38°C and a humidity of 50% to 65%, respectively. The medium was then replaced, and the cells were cultured for another 24 hours under the same experimental conditions to obtain the cell culture fluid.

[0061] Subsequently, the cell culture medium was taken and the sample solution diluted 1:100 (E-AGSE) with the original solution in fresh culture medium was added, and cultured for 24 hours at 37°C and 5% CO2. At the same time, a group that did not receive any dose of sample served as a blank control of the experiment.

[0062] Subsequently, the culture medium was removed and the cells were washed with phosphate-buffered saline (PBS) without the test materials. Total RNA was extracted from the tissues and converted to cDNA by reverse transcription. Quantitative fluorescence PCR (qPCR) was performed to calculate the relative gene fold expression changes for each treatment. Statistical analysis was performed using a two-tailed ANOVA Student's t-test. All of the above cell culture and detection procedures were performed in the Signal Biosciences Laboratory, which meets the standards of a Biosafety Level 2 (BL-2) cell culture laboratory. The results are shown in Table 2.

[0063] Table 2. Changes in NHEKs gene expression after E-AGSE (0.01%) treatment

[0064]

[0065] The results showed that a 0.01% E-AGSE solution increased occludin expression by 152%, heparan sulfate proteoglycan (HSPG) expression by 17%, and hyaluronic acid receptor protein (CD44) expression by 16% compared to the blank control. It can be concluded that E-AGSE significantly promotes perlecan gene expression after basic in vitro treatment of keratinocytes.

[0066] This embodiment also uses 0.002%, 0.01%, 0.02%, 0.1%, 0.2%, and 1% E-AGSE solutions to conduct experiments, respectively, to examine the effects of different concentrations of E-AGSE solutions on promoting the expression of occludin, heparan sulfate proteoglycans (HSPG), and hyaluronic acid receptor protein (CD44). The results show that E-AGSE solutions in the range of 0.002%-1% can effectively promote the expression of occludin, heparan sulfate proteoglycans (HSPG), and hyaluronic acid receptor protein (CD44).

[0067] Example 5: Cell experiment of TIRACLE Pro+E-AGSE

[0068] Epidermal keratinocytes (NHEKs, purchased from Thermo Fisher Scientific (Cat No. A13401)) were obtained from human neonates; each cell batch tested negative for HIV-1, HIV-2, HCV, and other antigens. Appropriate 6-well plates were selected, and 2 mL of the cell suspension was added to each well of EpiLife medium (Thermo Fisher Scientific; Cat No. MEPI500CA). The cells were cultured for 24 hours under a constant flow of 5% v / v CO2, at a temperature of 36°C to 38°C and a humidity of 50% to 65%, respectively. The medium was then replaced, and the cells were cultured for another 24 hours under the same experimental conditions to obtain the cell culture fluid.

[0069] The cell culture medium was then taken and the sample solution diluted 1:100 (E-AGSE + TIRACLE Pro) with the original solution in fresh culture medium was added, and cultured for 24 hours at 37°C and 5% CO2. At the same time, a group that did not receive any dose of sample served as a blank control for the experiment.

[0070] Subsequently, the culture medium was removed and the cells were washed with phosphate-buffered saline (PBS) without the test materials. Total RNA was extracted from the tissue and converted into cDNA by reverse transcription. Quantitative PCR (qPCR) was performed using specific gene primers to calculate the relative gene fold expression changes for each treatment. Statistical analysis was performed using a two-tailed ANOVA Student's t-test. All of the above cell culture and detection procedures were performed in the Signal Bioscience Laboratory, which meets the standards of a biosafety level 2 (BL-2) cell culture room. The test results are shown in Table 3.

[0071] Table 3. Changes in NHEKs gene expression after E-AGSE (0.02%) + TIRACLE Pro (0.5%) treatment

[0072]

[0073] The results showed that the E-AGSE (0.02%) + TIRACLE Pro (0.5%) solution increased occludin expression by 316%, heparan sulfate proteoglycan (HSPG) expression by 64%, and hyaluronan receptor protein (CD44) expression by 69% compared to the blank control. Compared to E-AGSE alone (the E-AGSE content in Example 4 was 0.01%), occludin expression increased by more than 2-fold, heparan sulfate proteoglycan (HSPG) expression increased by nearly 4-fold, and hyaluronan receptor protein (CD44) expression increased by more than 4-fold. Compared to TIRACLE Pro alone, occludin expression increased by nearly 6-fold, heparan sulfate proteoglycan (HSPG) expression increased by more than 4-fold, and hyaluronan receptor protein (CD44) expression increased by more than 11-fold. In summary, it can be concluded that E-AGSE+TIRACLE Pro produces significant gene marker expression ability after in vitro basic treatment of human keratinocytes.

[0074] Based on these data, it can be concluded that E-AGSE and TIRACLE Pro produce significant effects in the expression of gene markers in the dermal-epidermal junction (DEJ), and the E-AGSE+TIRACLE Pro combination can produce higher significant gene marker expression ability and synergistic enhancement effects after in vitro basic treatment of human keratinocytes.

[0075] This embodiment also used TIRACLE Pro solutions with contents of 0.005%, 0.05%, 0.3%, 0.5%, and 1% and E-AGSE solutions with contents of 0.002%, 0.01%, 0.02%, 0.1%, 0.2%, and 1% to conduct experiments in combination to examine the effects of different concentrations of E-AGSE + TIRACLE Pro solutions on promoting the expression of occludin, heparan sulfate proteoglycans (HSPG), and hyaluronic acid receptor protein (CD44). The results showed that when the TIRACLE Pro content was 0.005%-1% and the E-AGSE content was 0.002%-1%, they could effectively promote the expression of occludin, heparan sulfate proteoglycans (HSPG), and hyaluronic acid receptor protein (CD44), and the synergistic effect was significant compared with TIRACLE Pro solution or E-AGSE solution.

[0076] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by the claims.

Claims

1. A composition for improving the epidermal junction layer, characterized in that: The invention comprises a symbiotic fermentation product of Thermus thermophilus and Bacillus, and also comprises grape seed extract; the strain accession number of the Thermus thermophilus is BAA-163, and the strain accession number of the Bacillus is CICC10023. The grape extract is prepared by: selecting grape seeds without any impurities, extracting them with 600 ml of 100% ethanol at room temperature for 18 hours, filtering the ethanol extract through Whatman filter paper, and then removing the ethanol through a rotary evaporator to obtain a concentrated primary extract; repeating the above operation on the primary extract, and then drying it in a vacuum drying oven, further concentrating it into a final extract, and grinding it to obtain the grape seed extract.

2. The composition according to claim 1, wherein The content of the symbiotic fermentation product of Thermus thermophilus and Bacillus is 0.005%-1%, and the content of grape seed extract is 0.002%-1%.

3. Use of a composition in preparing a preparation for improving the dermal-epidermal junction layer, characterized in that: The composition is as described in any one of claims 1-2.

4. The use according to claim 3, characterized in that The composition improves the dermal-epidermal junction layer by promoting the expression of basement membrane proteoglycan, thereby achieving the effect of delaying skin aging.

5. The use according to claim 4, characterized in that The composition improves the dermal epidermal junction by promoting the expression of any one or more of occludin, heparan sulfate proteoglycan or hyaluronic acid receptor protein.

6. Use of a composition for preparing a preparation for promoting the expression of any one or more of occludin, heparan sulfate proteoglycan or hyaluronic acid receptor protein, characterized in that: The composition is as described in any one of claims 1-2.

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