Anti-aging composition and application thereof

Abstract

The invention discloses an anti-aging composition, which is prepared from fisetin and dihydroberberine, or is prepared from the fisetin and the dihydroberberine. The invention also discloses application of the anti-aging composition in preparation of a product for delaying skin fibroblast aging, and discloses an anti-aging preparation containing the anti-aging composition. According to the anti-aging composition provided by the invention, fisetin and dihydroberberine contained in the anti-aging composition show a remarkable synergistic effect in cell anti-aging and function maintenance, so that aging of skin fibroblasts can be remarkably delayed, and healing and repairing of skin are promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of compositions, and particularly relates to an anti-aging composition and its applications. Background Art

[0002] The skin is the largest organ of the human body, with multiple functions such as protection, temperature regulation, and perception of the external environment. As people age, significant changes occur in the structure and function of the skin, manifested as reduced elasticity, increased wrinkles, pigmentation, and weakened wound healing ability. These changes are mainly attributed to the decline in the function of skin fibroblasts. Fibroblasts are the main cell type in the dermis of the skin, responsible for synthesizing and maintaining the matrix components of the skin, such as collagen, elastin, and other extracellular matrix components. The aging of fibroblasts not only leads to the degradation of skin structure but also affects the overall health and appearance of the skin.

[0003] At the cellular level, fibroblast aging is closely related to multiple biological processes, including cell cycle arrest, oxidative stress, inflammatory responses, and extracellular matrix remodeling. Senescent cells usually show increased activity of β-galactosidase (β-gal), upregulated expression of cell cycle inhibitory genes such as CDKN1A (encoding p21) and CDKN2A (encoding p16), and increased secretion of pro-inflammatory cytokines such as IL6, IL8, and IL11. These changes together lead to the functional decline and structural degradation of the skin.

[0004] In recent years, the research on natural compounds in delaying skin aging has received extensive attention. Fisetin and Dihydroberberine are two natural compounds with significant biological activities, showing potential anti-aging effects. Fisetin is a flavonoid compound, well-known for its strong antioxidant and anti-inflammatory properties. It can delay the aging process in multiple cell models by scavenging reactive oxygen species (ROS) and inhibiting inflammatory signaling pathways. In addition, fisetin has also been found to regulate the expression of cell cycle-related proteins, thus promoting cell survival and proliferation.

[0005] Dihydroberberine is the reduced form of berberine and has attracted much attention due to its improved metabolic function and anti-inflammatory effects. Research shows that dihydroberberine can regulate intracellular metabolic pathways, enhance cell viability, and improve the tolerance of cells to oxidative stress. In addition, it also has the ability to regulate the expression of inflammatory factors and may promote skin health by inhibiting inflammatory responses.

[0006] Although fisetin and dihydroberberine each show good biological activities, their synergistic anti-aging effects in skin fibroblasts have not been fully studied. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an anti-aging composition, which contains fisetin and dihydroberberine, and fisetin and dihydroberberine exhibit a significant synergistic effect in cell anti-aging and function maintenance, and can significantly delay the aging of skin fibroblasts and promote the healing and repair of the skin.

[0008] To solve the above technical problems, the present invention provides the following technical solutions:

[0009] In a first aspect of the present invention, there is provided an anti-aging composition, which comprises the following components or consists of the following components:

[0010] (i) Fisetin; and

[0011] (ii) Dihydroberberine.

[0012] In some embodiments of the present invention, in the anti-aging composition, the molar ratio of fisetin to dihydroberberine is (1-3):(1-3), for example, it can be 1:1, 1:2, 1:3, 2:1, 2:3, 3:1, 3:2, etc. Preferably, the molar ratio of fisetin to dihydroberberine is 1:1.

[0013] Fisetin and dihydroberberine are two natural compounds with potential anti-aging effects. Fisetin is a flavonoid compound, which can delay the aging process in a variety of cell models by scavenging reactive oxygen species (ROS) and inhibiting inflammatory signaling pathways. In addition, fisetin can also regulate the expression of cell cycle-related proteins, thereby promoting cell survival and proliferation. Dihydroberberine can regulate intracellular metabolic pathways, enhance cell viability and improve the tolerance of cells to oxidative stress. In addition, it also has the ability to regulate the expression of inflammatory factors.

[0014] The inventors have found through a large number of studies that when fisetin and dihydroberberine are used in combination, they can exhibit a significant synergistic effect in the anti-aging and function maintenance of fibroblasts, and significantly delay the aging of skin fibroblasts.

[0015] Fibroblasts play a crucial role in the maintenance and repair of skin tissue. With the increase of age, the proliferation ability, migration ability and collagen synthesis function of fibroblasts decrease significantly, which not only affects the appearance of the skin, but also leads to the weakening of skin repair and healing ability. The research of the inventors shows that the composition obtained by compounding fisetin and dihydroberberine can significantly delay the aging process of fibroblasts through multiple pathways in a synergistic manner, specifically as follows:

[0016] (1) In terms of anti-aging effect and regulation of aging gene expression, fisetin and dihydroberberine exhibit a significant synergistic effect.

[0017] During the aging process, the expression of specific aging-related genes (such as CDKN1A and CDKN2A) changes significantly, and these genes are closely related to cell cycle arrest and the aging process. The experimental results of the present invention show that fisetin and berberrubine can significantly inhibit the expression of these aging genes, possibly by regulating the activity of cyclins, promoting the normal progress of the cell cycle, and thus enhancing the proliferation ability of cells. In addition, fisetin and berberrubine also show a significant synergistic effect in inhibiting the expression of the aging marker SA-β-gal, further demonstrating their inhibitory effect on the aging process.

[0018] (2) In terms of the regulation of the expression of inflammation-related genes, fisetin and berberrubine synergistically reduce the expression of inflammatory factors, such as IL6, IL8, and IL11, through different signaling pathways.

[0019] Inflammation is an important feature of the aging process. Chronic low-grade inflammation (inflammaging) not only accelerates the aging process but is also closely related to the occurrence of various age-related diseases. Fisetin may reduce the inflammatory response by inhibiting the NF-κB signaling pathway, while berberrubine may enhance the antioxidant response by activating the Nrf2 / ARE pathway. The combined use of the two may jointly regulate the expression of inflammatory response and aging marker genes through interaction, thereby achieving a more significant anti-aging effect.

[0020] (3) In the regulation of cellular reactive oxygen species (ROS), fisetin and berberrubine show a significant synergistic effect.

[0021] ROS are by-products of cell metabolism, and their excessive accumulation often leads to oxidative stress, thereby accelerating cell aging. The research of the present invention shows that the use of fisetin or berberrubine alone can reduce the ROS level in aging cells, and when the two are used in combination, their antioxidant effect is more significant. This significant antioxidant effect may be related to the synergistic enhancement of the activity of intracellular antioxidant enzymes or the direct scavenging of ROS by the two. This finding provides a theoretical basis for the development of new anti-aging treatment strategies.

[0022] (4) In the regulation of the proliferation and migration of normal human fibroblasts, the synergistic effect of fisetin and berberrubine has also been verified.

[0023] The ability of cell proliferation and migration is crucial for skin healing and tissue regeneration. Through the CCK-8 experiment, it was found that the combined treatment of fisetin and berberrubine significantly promoted cell proliferation, with a proliferation rate reaching 170%. In the cell migration experiment, it was observed that when berberrubine was used alone, the migration ratio was 30%, while after the combined use of fisetin and berberrubine, the migration ratio was significantly increased to 60%. These results indicate that fisetin and berberrubine have a synergistic effect in promoting cell proliferation and migration. This synergistic effect may be related to the complementary effect of the two on the cell signaling pathway. For example, fisetin may promote cell proliferation by regulating the expression of cyclin, while berberrubine may enhance cell migration by affecting cytoskeleton reorganization.

[0024] The second aspect of the present invention provides the application of the anti-aging composition in the preparation of products for delaying the aging of skin fibroblasts. Among them, the products include but are not limited to cosmetics, drugs, and health products.

[0025] Furthermore, the anti-aging composition is used to regulate the expression of aging genes and / or inflammation-related genes in skin fibroblasts.

[0026] Furthermore, the anti-aging composition is used to regulate the level of reactive oxygen species in skin fibroblasts.

[0027] The third aspect of the present invention provides the application of the anti-aging composition in the preparation of products for promoting skin healing and repair. Among them, the products include but are not limited to cosmetics, drugs, and health products.

[0028] Furthermore, the anti-aging composition is used to promote the proliferation and migration of skin fibroblasts.

[0029] The fourth aspect of the present invention provides an anti-aging preparation, which contains the anti-aging composition. Among them, the anti-aging preparation includes but is not limited to cosmetics, drugs, and health products.

[0030] Furthermore, the anti-aging preparation is a drug and a health product, and the dosage forms of the drug and the health product include but are not limited to oral dosage forms.

[0031] Furthermore, the anti-aging preparation is a cosmetic, and the cosmetic can be prepared into various dosage forms, including but not limited to creams, lotions, gels, aqueous solutions, oils, powders, and solids.

[0032] Furthermore, the types of the cosmetics include but are not limited to toners, eye creams, face creams, hand creams, facial cleansers, cleansing foams, cleansing gels, hand sanitizers, makeup remover oils, makeup remover waters, makeup remover gels, liquid soaps, solid soaps, body washes, body lotions, foundations, serums, and masks.

[0033] Furthermore, the addition amount of the anti-aging composition in the cosmetic is 0.05-5 wt%, for example, it can be 0.05 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, 4.0 wt%, 4.5 wt%, 5.0 wt%, etc.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] 1. For the anti-aging composition provided by the present invention, luteolin and berberrubine therein exhibit a significant synergistic effect in cell anti-aging and function maintenance, and can significantly delay the aging of skin fibroblasts and promote the healing and repair of the skin.

[0036] 2. For the anti-aging composition provided by the present invention, the synergistic effect of luteolin and berberrubine provides a new idea for skin anti-aging and healing. Based on their effects in reducing the ROS level, promoting cell proliferation and migration, the combined use of these two compounds may have broad application prospects in skin care products and regenerative medicine. In addition, the low toxicity and natural source of the composition make it more advantageous in clinical applications. Description of the Drawings

[0037] Figure 1 a shows the results of β-Gal staining of cells in different treatment groups before (control) and after (Senescence) UVB irradiation; Figure 1 b is Figure 1 a statistical bar chart of senescent cells in different treatment groups in a; Figure 1 c-d show the expression results of CDKN1A and CDKN2A genes in cells in different treatment groups before (control) and after (Senescence) UVB irradiation;

[0038] Figure 2 show the expression results of inflammation-related genes IL-6, IL-8 and IL-11 in cells in different treatment groups before (control) and after (Senescence) UVB irradiation;

[0039] Figure 3 a shows the photos of reactive oxygen species staining of cells in different treatment groups before and after UVB irradiation; Figure 3 b is Figure 3 a statistical bar chart of the fluorescence intensity of reactive oxygen species staining in a;

[0040] Figure 4 a is a statistical bar graph of the cell proliferation rate of different treatment groups; Figure 4 b is the result of the scratch assay of cells in different treatment groups; Figure 4 c is a statistical bar graph of the cell migration rate of different treatment groups. Detailed implementation manners

[0041] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts belong to the scope of protection of the present invention.

[0042] Next, the present invention will be further described in combination with specific examples and comparative examples.

[0043] In the following detailed implementation manners, *p, **p, and ***p indicate comparison with the control group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0044] Example 1

[0045] This example provides an anti-aging composition composed of fisetin (Fis) and berberrubine (DHB), and their molar ratio is 1:1.

[0046] Comparative Example 1

[0047] The difference between this comparative example and Example 1 is that the anti-aging composition only contains berberrubine.

[0048] Comparative Example 2

[0049] The difference between this comparative example and Example 1 is that the anti-aging composition only contains fisetin.

[0050] Experimental tests

[0051] 1. Synergistic effect of fisetin and berberrubine in anti-aging effect and regulation of senescence gene expression

[0052] In this experiment, the human skin fibroblast cell line BJ was used, and UVB irradiation (40 mJ / cm 2)The cells were treated for 4 days to induce cellular senescence. Subsequently, berberrubine (98% DHB, 20 μM) of Comparative Example 1, fisetin (98% Fis, 20 μM) of Comparative Example 2, and the composition DHB (10 μM) + Fis (10 μM) of Example 1 were added respectively, and the drugs in each group were dissolved in DMSO; a positive control group and a negative control group were set up. In the positive control group, dasatinib combined with quercetin (D+Q, 1 μM + 20 μM) was added, and an equal proportion of DMSO was added to the negative control group. Then the treatment was continued for 72 hours.

[0053] β-Gal staining (also known as SA-β-Gal staining) is an experimental technique for detecting cellular senescence. This technique is based on the characteristic that the activity of β-galactosidase in senescent cells increases significantly at around pH 6.0. By using X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) as a substrate, under the catalytic action of β-galactosidase, a dark blue product is generated, thereby realizing the detection of senescent cells. The BJ cells treated with drugs were detected for senescence by β-gal staining method, and the percentage of senescence-positive cells was counted. The results are as Figure 1 shown in a~b.

[0054] Please refer to Figure 1 a~b. In the cells treated with UVB irradiation alone, about 80% of the cells showed senescence positivity. However, after adding berberrubine and fisetin respectively, the proportion of senescence-positive cells decreased significantly to about 60%, similar to the D+Q treatment group. Notably, the combined treatment of berberrubine and fisetin could greatly resist cellular senescence, and the proportion of senescence-positive cells was only 20%. These results indicate that in the composition of Example 1, berberrubine and fisetin produced a significant anti-aging synergistic effect. In addition, no obvious cytotoxicity to normal cells was shown in all drug treatment groups.

[0055] The CDKN1A gene inhibits the cell cycle process by encoding the p21 protein, thereby promoting cellular senescence and preventing the proliferation of damaged cells. The p16 ^ INK4a protein accumulates during cellular senescence, thereby inhibiting the cell cycle and serving as one of the markers of cellular senescence. To explore the mechanism of berberrubine and fisetin in resisting cellular senescence, the expression levels of related genes were further detected. The results are as Figure 1 shown in c~d.

[0056] Please refer to Figure 1From c to d, UVB irradiation significantly induced the expression of CDKN1A and CDKN2A genes, increasing by approximately 4-fold. However, the individual treatments with berberrubine and fisetin were able to inhibit the UVB-induced elevation of gene expression. In particular, the combined treatment of berberrubine and fisetin maximally inhibited the upregulation of CDKN1A and CDKN2A gene expression. This finding indicates that berberrubine and fisetin may play their anti-aging roles by regulating the expression of cell cycle inhibitors.

[0057] The above experimental results show that berberrubine and fisetin can not only resist UVB-induced cellular senescence individually, but also exhibit a significant synergistic effect when used in combination.

[0058] 2. Synergistic effect of fisetin and berberrubine in the regulation of inflammation-related gene expression

[0059] Cellular senescence is accompanied by a complex cellular secretion phenomenon called senescence-associated secretory phenotype (SASP). SASP consists of various cytokines, chemokines, proteases, and growth factors and plays an important role in the process of cellular senescence. Although SASP has certain positive effects in tissue repair and tumor suppression, its long-term presence can lead to chronic inflammation, tissue damage, and various age-related diseases. Key inflammatory factors in SASP include interleukin 6 (IL-6), interleukin 8 (IL-8), and interleukin 11 (IL-11). These factors not only act as inflammatory mediators to transmit signals between cells but also play important roles in cellular senescence and the tumor microenvironment.

[0060] In this experiment, the human skin fibroblast cell line BJ was used, and cells were treated with UVB irradiation (40 mJ / cm 2 ) for 7 days to induce cellular senescence. Then, the relative expression levels of IL-6, IL-8, and IL-11 genes were detected by real-time fluorescence quantitative PCR (RT-PCR), and the results are as Figure 2 shown.

[0061] Please refer to Figure 2 , in senescent cells, the expression levels of IL-6 and IL-8 genes increased by 6-fold respectively, while the expression level of IL-11 gene increased by 12-fold, indicating that SASP was significantly activated.

[0062] On the fourth day of the experiment, berberrubine (98% purity, 20 μM) of Comparative Example 1, fisetin (98% purity, 20 μM) of Comparative Example 2, and the composition DHB (10 μM) + Fis (10 μM) of Example 1 were added to the cells respectively and continuously treated for 72 hours. The relative expression levels of IL-6, IL-8, and IL-11 genes were detected by real-time fluorescence quantitative PCR (RT-PCR), and the results are asFigure 2 as shown

[0063] Figure 2 The results showed that when berberrubine and fisetin were used alone, they could effectively inhibit the overexpression of IL-6, IL-8 and IL-11 genes in senescent cells. When the two were used in combination, the inhibitory effect reached the maximum, and the expression levels of the three genes were close to those of normal cells. This result indicates that fisetin and berberrubine have a synergistic effect in inhibiting the expression of SASP-related genes.

[0064] 3. Synergistic effect of fisetin and berberrubine in regulating cellular reactive oxygen species (ROS)

[0065] Reactive Oxygen Species (ROS) are by-products generated during cell metabolism, including superoxide, hydrogen peroxide and hydroxyl radicals. Although low levels of ROS play an important role in cell signaling and defense mechanisms, excessive ROS can lead to oxidative stress, damaging proteins, lipids and DNA within cells, thereby accelerating the process of cell aging. The excessive accumulation of ROS has been proven to be closely related to a variety of age-related diseases. Therefore, regulating the level of ROS is of great significance for delaying aging.

[0066] In this experiment, the human skin fibroblast cell line BJ was used, and the cells were treated with UVB irradiation (40 mJ / cm 2 ) for 7 days to induce senescence. On the fourth day of senescent cell induction, berberrubine (DHB, 20 μM) of Comparative Example 1, fisetin (Fis, 20 μM) of Comparative Example 2, and the composition DHB (10 μM) + Fis (10 μM) of Example 1 were added to the cells respectively; a positive control group and a negative control group were set up. In the positive control group, dasatinib combined with quercetin (D+Q, 1 μM + 20 μM) was added, and an equal proportion of DMSO was added to the negative control group. The treatment continued for 72 hours. The ROS in the cells was labeled with a ROS green fluorescent probe, and the cell nuclei were stained with a DAPI probe. The ROS level of the cells was observed under a 63X oil immersion lens using a Zeiss LSM-900 laser confocal microscope. The obtained results are as Figure 3 as shown

[0067] Please refer to Figure 3a-b, normal cells only have weak ROS signals, while the ROS signals of senescent cells are significantly enhanced, and the average fluorescence intensity is 6 times higher than that of normal cells. After adding dihydroberberine, fisetin, and dasatinib combined with quercetin (D+Q), the ROS signals of senescent cells were reduced. In particular, when dihydroberberine and fisetin were used in combination, the ROS level was suppressed to the greatest extent, close to the level of normal cells. This shows that fisetin and dihydroberberine have a significant synergistic effect in regulating ROS, which may delay cell senescence by reducing oxidative stress.

[0068] 4. Synergistic effect of fisetin and dihydroberberine in regulating proliferation and migration of normal human fibroblasts

[0069] Normal human fibroblasts play a key role in the maintenance and repair of skin tissue. Their proliferation and migration abilities are essential for wound healing, tissue regeneration, and skin health. Fibroblast proliferation helps fill damaged tissue, while their migration ability ensures that the cells can effectively cover and repair damaged areas. Therefore, regulating the proliferation and migration abilities of fibroblasts may be important for improving skin healing and anti-aging.

[0070] In this experiment, human skin fibroblast cell line BJ was used, and the cells were treated with dihydroberberine (DHB, 20 μM) of Comparative Example 1, fisetin (Fis, 20 μM) of Comparative Example 2, and the composition DHB (10 μM) + Fis (10 μM) of Example 1 for 24 hours, and DMSO in equal proportion was used as a negative control. CCK-8 (cell counting kit-8) was used to detect the proliferation ability of the cells. The results are shown in Figure 4 As shown in a.

[0071] Figure 4 The results of a showed that the use of dihydroberberine or fisetin alone had no significant effect on cell proliferation. However, when dihydroberberine and fisetin were used in combination, cell proliferation increased significantly, with the proliferation rate reaching 170%, indicating that the two had a synergistic effect in promoting fibroblast proliferation.

[0072] Next, the cell migration ability was evaluated by scratch test. The specific method is as follows: On the cell culture dish, a straight line (scratch) was drawn in the monolayer of cells with a sterile pipette tip, and then gently rinsed with PBS to remove the detached cells. After the cells were treated, photos were taken at 0 hours and 24 hours to record the change in the width of the scratch. The cell migration ability was evaluated by calculating the proportion of scratch closure. The results are shown in Figure 2. Figure 4 As shown in b~c.

[0073] See also Figure 4From b to c, the cell migration ratio of the DMSO treatment group was 10%. When berberrubine was used alone, the migration ratio increased to 30%, showing a certain promoting effect, while fisetin alone did not significantly affect migration. However, under the combined treatment of berberrubine and fisetin, the cell migration ability was significantly enhanced, and the migration ratio reached 60%. This further demonstrated the synergistic effect of fisetin and berberrubine in promoting fibroblast migration. Therefore, fisetin and berberrubine showed a significant synergistic effect in promoting the proliferation and migration of normal human fibroblasts, which may provide a new strategy for skin repair and anti-aging treatment.

[0074] In summary, for the composition provided by the present invention, fisetin and berberrubine therein have a synergistic effect in regulating the anti-aging of skin fibroblasts, providing a new idea for skin anti-aging and healing. Based on their effects in reducing the ROS level, promoting cell proliferation and migration, the combined use of these two compounds may have broad application prospects in skin care products and regenerative medicine. In addition, in the composition provided by the present invention, both fisetin and berberrubine are of natural origin and have low toxicity, which makes it more advantageous in clinical applications.

[0075] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

1. An anti-aging composition, characterized in that The anti-aging composition comprises or consists of the following components: (i) fisetin; and (ii) Dihydroberberine.

2. An anti-aging composition according to claim 1, characterized in that: In the anti-aging composition, the molar ratio of fisetin to dihydroberberine is (1-3):(1-3).

3. An anti-aging composition according to claim 2, characterized in that: In the anti-aging composition, the molar ratio of fisetin to dihydroberberine is 1:

1.

4. Use of the anti-aging composition according to any one of claims 1 to 3 in the preparation of a product for delaying the aging of skin fibroblasts; wherein: The products include cosmetics, medicines and health products.

5. The use according to claim 4, characterized in that: The anti-aging composition is used to regulate the expression of aging genes and / or inflammation-related genes in skin fibroblasts; and / or, The anti-aging composition is used for regulating the level of reactive oxygen species in skin fibroblasts.

6. Use of the anti-aging composition according to any one of claims 1 to 3 in the preparation of products that promote skin healing and repair; wherein: The products include cosmetics, medicines and health products.

7. The use according to claim 6, characterized in that: The anti-aging composition is used for promoting the proliferation and migration of skin fibroblasts.

8. An anti-aging preparation, characterized in that The anti-aging preparation comprises the anti-aging composition according to any one of claims 1 to 3; wherein the anti-aging preparation comprises cosmetics, medicines and health products.

9. An anti-aging preparation according to claim 8, characterized in that: The anti-aging preparation is a medicine and a health product, and the dosage form of the medicine and the health product includes an oral dosage form.

10. An anti-aging preparation according to claim 8, characterized in that: The anti-aging preparation is a cosmetic, and the dosage form of the cosmetic includes cream, lotion, gel, water, oil, powder and solid; and / or, the anti-aging composition is added in an amount of 0.05-5wt% in the cosmetic.

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