A cosmetic composition and its application in preparing skin care products

Abstract

The present invention relates to the field of cosmetic technology, and in particular to a cosmetic composition and its application in the preparation of skin care products. The invention provides a composition of a filtrate of a fermentation product of bifidobacterium / lactobacillus / soybean seed extract and a microcurrent tetrapeptide, and the composition has at least one of the following effects: I) anti-oxidation; II) protecting cells; III) regulating the transcription level of related genes; IV) regulating the expression level of related proteins; V) inhibiting aging indicator bacteria. Therefore, the composition can achieve a good anti-aging effect, and there is a synergistic effect between the two components. The composition can be applied to the field of cosmetics, with a wider range of efficacy, especially in the field of beauty and skin care.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a cosmetic composition and application thereof in preparing skin care products. Background Art

[0002] Skin aging is a complex biological process. As we age, our skin gradually loses its elasticity, and wrinkles and sagging appear. The factors that cause skin aging can be divided into two types: endogenous and exogenous. Exogenous aging factors mainly include: ultraviolet radiation, environmental pollution, bad living habits, etc. Among them, ultraviolet rays can induce photoaging, destroy skin collagen and elastic fibers, and are the main culprit of exogenous aging. The main ultraviolet ray that causes photoaging is UVA. Endogenous aging factors mainly include: genetic factors, changes in hormone levels, and abnormal intracellular metabolism. These are often closely related to aging. As we age, the renewal rate of skin cells slows down, and the repair ability decreases, leading to natural aging of the skin.

[0003] As the anti-aging concept continues to gain popularity, consumers' understanding of skin care has gone beyond basic cleansing and moisturizing, and they are pursuing deeper skin nourishment and aging protection. Against this backdrop, the anti-aging cosmetics market has ushered in unprecedented development. Various anti-aging products are common on the market, and the active ingredients they contain range from traditional antioxidants to advanced bioactive peptides, from natural plant extracts to high-tech nanocarriers. Various innovative ingredients emerge in an endless stream, providing a solid scientific basis for anti-aging products.

[0004] However, while the industry is booming, anti-aging cosmetics still face many problems that need to be solved. For example: ① False advertising is common, exaggerating product effects and misleading consumers. ② Quality varies, and some products lack effective quality control, affecting the effect of use. ③ Ingredient safety risks cannot be ignored. Some products may contain harmful or unverified ingredients, endangering consumer health.

[0005] It can be seen that the anti-aging cosmetics market has huge development potential and broad market prospects. Although the existing categories are rich, the quality still needs to be further improved. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a beauty and skin care composition with anti-aging effect and its application in the preparation of skin care products.

[0007] The composition provided by the invention comprises bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide.

[0008] The present invention compounds the filtrate of the fermentation product of bifidobacterium / lactobacillus / soybean seed extract and the microcurrent tetrapeptide. The filtrate of the fermentation product of bifidobacterium / lactobacillus / soybean seed extract is a product obtained by fermenting bifidobacteria and lactic acid bacteria with black beans as the main raw material. It has not been reported to have anti-aging function before. The main component of the microcurrent tetrapeptide is tetrapeptide-1 (Tetrapeptide-1), and the peptide sequence is LEAP. The compounding of the two can have a significant anti-aging effect and it has been verified that there is a synergistic effect between the two components.

[0009] In order to further improve the synergistic effect, the present invention optimizes the mass ratio of the two components in the composition. In an embodiment of the present invention, the mass ratio of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and the microcurrent tetrapeptide in the provided composition is (0.25~100): (0.25~100).

[0010] As a feasibility case, the mass ratio of the filtrate of the fermentation product of bifidobacterium / lactobacillus / soybean seed extract and the microcurrent tetrapeptide is: (0.25~1.25): (0.25~1.25), (0.25~1.25): (0.25~1.5), (0.25~1.25): (0.25~2.5), (0.25~1.25): (0.25~10), (0.25~1.25): (0.25~50), (0.25~1.25): (0.25~100), (0.25~1.25): (1.25~1.5), (0.25~1.25): (1.25~2.5), (0.25~1.25): (1.25~10), (0.25 ~1.25): (1.25~50), (0.25~1.25): (1.25~100), (0.25~1.25): (1.5~2.5), (0.25~1.25): (1.5~10), (0.25~1.25): (1.5~50), (0.25~1.25): (1.5~100), (0.25~1.25): (2.5~10), (0.25~1.25): (2.5~50), (0.25~1.25): (2.5~100), (0.25~1.25): (10~50), (0.25~1.25): (10~100) or (0.25~1.25): (50~100);

[0011] In other cases, the mass ratio of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate to the microcurrent tetrapeptide was (0.25~1.5):(0.25~1.25), (0.25~1.5):(0.25~1.5), (0.25~1.5):(0.25~2.5), (0.25~1.5):(0.25~10), (0.25~1.5):(0.25~50), (0.25~1.5):(0.25~100), (0.25~1.5):(1.25~1.5), (0.25~1.5):(1.25~2.5), (0.25~1.5):(1.25~10), (0.25 ~1.5): (1.25~50), (0.25~1.5): (1.25~100), (0.25~1.5): (1.5~2.5), (0.25~1.5): (1.5~10), (0.25~1.5): (1.5~50), (0.25~1.5): (1.5~100), (0.25~1.5): (2.5~10), (0.25~1.5): (2.5~50), (0.25~1.5): (2.5~100), (0.25~1.5): (10~50), (0.25~1.5): (10~100) or (0.25~1.5): (50~100.

[0012] In other cases, the mass ratio of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate to the microcurrent tetrapeptide was (0.25~2.5):(0.25~1.25), (0.25~2.5):(0.25~1.5), (0.25~2.5):(0.25~2.5), (0.25~2.5):(0.25~10), (0.25~2.5):(0.25~50), (0.25~2.5):(0.25~100), (0.25~2.5):(1.25~1.5), (0.25~2.5):(1.25~2.5), (0.25~2.5):(1.25~10), (0.25 ~2.5): (1.25~50), (0.25~2.5): (1.25~100), (0.25~2.5): (1.5~2.5), (0.25~2.5): (1.5~10), (0.25~2.5): (1.5~50), (0.25~2.5): (1.5~100), (0.25~2.5): (2.5~10), (0.25~2.5): (2.5~50), (0.25~2.5): (2.5~100), (0.25~2.5): (10~50), (0.25~2.5): (10~100) or (0.25~2.5): (50~100.

[0013] In other cases, the mass ratios of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (0.25~10):(0.25~1.25), (0.25~10):(0.25~1.5), (0.25~10):(0.25~2.5), (0.25~10):(0.25~10), (0.25~10):(0.25~50), (0.25~10):(0.25~100), (0.25~10):(1.25~1.5), (0.25~10):(1.25~2.5), (0.25~10):(1.25~10), (0.25~10):(0.25~50). 25~10): (1.25~50), (0.25~10): (1.25~100), (0.25~10): (1.5~2.5), (0.25~10): (1.5~10), (0.25~10): (1.5~50), (0.25~10): (1.5~100), (0.25~10): (2.5~10), (0.25~10): (2.5~50), (0.25~10): (2.5~100), (0.25~10): (10~50), (0.25~10): (10~100) or (0.25~10): (50~100).

[0014] In other cases, the mass ratios of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (0.25~50):(0.25~1.25), (0.25~50):(0.25~1.5), (0.25~50):(0.25~2.5), (0.25~50):(0.25~10), (0.25~50):(0.25~50), (0.25~50):(0.25~100), (0.25~50):(1.25~1.5), (0.25~50):(1.25~2.5), (0.25~50):(1.25~10), (0.25~50):(0.25~100). 25~50): (1.25~50), (0.25~50): (1.25~100), (0.25~50): (1.5~2.5), (0.25~50): (1.5~10), (0.25~50): (1.5~50), (0.25~50): (1.5~100), (0.25~50): (2.5~10), (0.25~50): (2.5~50), (0.25~50): (2.5~100), (0.25~50): (10~50), (0.25~50): (10~100) or (0.25~50): (50~100).

[0015] In other cases, the mass ratio of the filtrate of the fermentation product of bifidobacterium / lactobacillus / soybean seed extract to the microcurrent tetrapeptide was (0.25~100):(0.25~1.25), (0.25~100):(0.25~1.5), (0.25~100):(0.25~2.5), (0.25~100):(0.25~10), (0.25~100):(0.25~50), (0.25~100):(0.25~100), (0.25~100):(1.25~1.5), (0.25~100):(1.25~2.5), (0.25~100):(1.25~10), (0.25 ~100): (1.25~50), (0.25~100): (1.25~100), (0.25~100): (1.5~2.5), (0.25~100): (1.5~10), (0.25~100): (1.5~50), (0.25~100): (1.5~100), (0.25~100): (2.5~10), (0.25~100): (2.5~50), (0.25~100): (2.5~100), (0.25~100): (10~50), (0.25~100): (10~100) or (0.25~100): (50~100).

[0016] In other cases, the mass ratio of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate to the microcurrent tetrapeptide was (1.25~1.5):(0.25~1.25), (1.25~1.5):(0.25~1.5), (1.25~1.5):(0.25~2.5), (1.25~1.5):(0.25~10), (1.25~1.5):(0.25~50), (1.25~1.5):(0.25~100), (1.25~1.5):(1.25~1.5), (1.25~1.5):(1.25~2.5), (1.25~1.5):(1.25~10). ~1.5): (1.25~50), (1.25~1.5): (1.25~100), (1.25~1.5): (1.5~2.5), (1.25~1.5): (1.5~10), (1.25~1.5): (1.5~50), (1.25~1.5): (1.5~100), (1.25~1.5): (2.5~10), (1.25~1.5): (2.5~50), (1.25~1.5): (2.5~100), (1.25~1.5): (10~50), (1.25~1.5): (10~100) or (1.25~1.5): (50~100.

[0017] In other cases, the mass ratio of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate to the microcurrent tetrapeptide was (1.25~2.5):(0.25~1.25), (1.25~2.5):(0.25~1.5), (1.25~2.5):(0.25~2.5), (1.25~2.5):(0.25~10), (1.25~2.5):(0.25~50), (1.25~2.5):(0.25~100), (1.25~2.5):(1.25~1.5), (1.25~2.5):(1.25~2.5), (1.25~2.5):(1.25~10), (1.25 ~2.5): (1.25~50), (1.25~2.5): (1.25~100), (1.25~2.5): (1.5~2.5), (1.25~2.5): (1.5~10), (1.25~2.5): (1.5~50), (1.25~2.5): (1.5~100), (1.25~2.5): (2.5~10), (1.25~2.5): (2.5~50), (1.25~2.5): (2.5~100), (1.25~2.5): (10~50), (1.25~2.5): (10~100) or (1.25~2.5): (50~100.

[0018] In other cases, the mass ratios of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (1.25-10):(0.25-1.25), (1.25-10):(0.25-1.5), (1.25-10):(0.25-2.5), (1.25-10):(0.25-10), (1.25-10):(0.25-50), (1.25-10):(0.25-100), (1.25-10):(1.25-1.5), (1.25-10):(1.25-2.5), (1.25-10):(1.25-10). 25~10): (1.25~50), (1.25~10): (1.25~100), (1.25~10): (1.5~2.5), (1.25~10): (1.5~10), (1.25~10): (1.5~50), (1.25~10): (1.5~100), (1.25~10): (2.5~10), (1.25~10): (2.5~50), (1.25~10): (2.5~100), (1.25~10): (10~50), (1.25~10): (10~100) or (1.25~10): (50~100).

[0019] In other cases, the mass ratios of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (1.25~50):(0.25~1.25), (1.25~50):(0.25~1.5), (1.25~50):(0.25~2.5), (1.25~50):(0.25~10), (1.25~50):(0.25~50), (1.25~50):(0.25~100), (1.25~50):(1.25~1.5), (1.25~50):(1.25~2.5), (1.25~50):(1.25~10). 25~50): (1.25~50), (1.25~50): (1.25~100), (1.25~50): (1.5~2.5), (1.25~50): (1.5~10), (1.25~50): (1.5~50), (1.25~50): (1.5~100), (1.25~50): (2.5~10), (1.25~50): (2.5~50), (1.25~50): (2.5~100), (1.25~50): (10~50), (1.25~50): (10~100 or (1.25~50): (50~100).

[0020] In other cases, the mass ratio of the filtrate of the fermentation product of bifidobacterium / lactobacillus / soybean seed extract to the microcurrent tetrapeptide was (1.25~100):(0.25~1.25), (1.25~100):(0.25~1.5), (1.25~100):(0.25~2.5), (1.25~100):(0.25~10), (1.25~100):(0.25~50), (1.25~100):(0.25~100), (1.25~100):(1.25~1.5), (1.25~100):(1.25~2.5), (1.25~100):(1.25~10), (1.25 ~100): (1.25~50), (1.25~100): (1.25~100), (1.25~100): (1.5~2.5), (1.25~100): (1.5~10), (1.25~100): (1.5~50), (1.25~100): (1.5~100), (1.25~100): (2.5~10), (1.25~100): (2.5~50), (1.25~100): (2.5~100), (1.25~100): (10~50), (1.25~100): (10~100) or (1.25~100): (50~100).

[0021] In other cases, the mass ratio of bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate to microcurrent tetrapeptide was (1.5~2.5):(0.25~1.25), (1.5~2.5):(0.25~1.5), (1.5~2.5):(0.25~2.5), (1.5~2.5):(0.25~10), (1.5~2.5):(0.25~50), (1.5~2.5):(0.25~100), (1.5~2.5):(1.25~1.5), (1.5~2.5):(1.25~2.5), (1.5~2.5):(1.25~10), (1.5~2.5):(1.25~1.5). 5~2.5): (1.25~50), (1.5~2.5): (1.25~100), (1.5~2.5): (1.5~2.5), (1.5~2.5): (1.5~10), (1.5~2.5): (1.5~50), (1.5~2.5): (1.5~100), (1.5~2.5): (2.5~10), (1.5~2.5): (2.5~50), (1.5~2.5): (2.5~100), (1.5~2.5): (10~50), (1.5~2.5): (10~100) or (1.5~2.5): (50~100).

[0022] In other cases, the mass ratios of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (1.5~10):(0.25~1.25), (1.5~10):(0.25~1.5), (1.5~10):(0.25~2.5), (1.5~10):(0.25~10), (1.5~10):(0.25~50), (1.5~10):(0.25~100), (1.5~10):(1.25~1.5), (1.5~10):(1.25~2.5), (1.5~10):(1.25~10). .5~10): (1.25~50), (1.5~10): (1.25~100), (1.5~10): (1.5~2.5), (1.5~10): (1.5~10), (1.5~10): (1.5~50), (1.5~10): (1.5~100), (1.5~10): (2.5~10), (1.5~10): (2.5~50), (1.5~10): (2.5~100), (1.5~10): (10~50), (1.5~10): (10~100) or (1.5~10): (50~100).

[0023] In other cases, the mass ratios of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (1.5~50):(0.25~1.25), (1.5~50):(0.25~1.5), (1.5~50):(0.25~2.5), (1.5~50):(0.25~10), (1.5~50):(0.25~50), (1.5~50):(0.25~100), (1.5~50):(1.25~1.5), (1.5~50):(1.25~2.5), (1.5~50):(1.25~10). .5~50): (1.25~50), (1.5~50): (1.25~100), (1.5~50): (1.5~2.5), (1.5~50): (1.5~10), (1.5~50): (1.5~50), (1.5~50): (1.5~100), (1.5~50): (2.5~10), (1.5~50): (2.5~50), (1.5~50): (2.5~100), (1.5~50): (10~50), (1.5~50): (10~100 or (1.5~50): (50~100).

[0024] In other cases, the mass ratios of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (1.5~100):(0.25~1.25), (1.5~100):(0.25~1.5), (1.5~100):(0.25~2.5), (1.5~100):(0.25~10), (1.5~100):(0.25~50), (1.5~100):(0.25~100), (1.5~100):(1.25~1.5), (1.5~100):(1.25~2.5), (1.5~100):(1.25~10). 5~100): (1.25~50), (1.5~100): (1.25~100), (1.5~100): (1.5~2.5), (1.5~100): (1.5~10), (1.5~100): (1.5~50), (1.5~100): (1.5~100), (1.5~100): (2.5~10), (1.5~100): (2.5~50), (1.5~100): (2.5~100), (1.5~100): (10~50), (1.5~100): (10~100) or (1.5~100): (50~100).

[0025] In other cases, the mass ratios of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (2.5~10):(0.25~1.25), (2.5~10):(0.25~1.5), (2.5~10):(0.25~2.5), (2.5~10):(0.25~10), (2.5~10):(0.25~50), (2.5~10):(0.25~100), (2.5~10):(1.25~1.5), (2.5~10):(1.25~2.5), (2.5~10):(1.25~10). .5~10): (1.25~50), (2.5~10): (1.25~100), (2.5~10): (1.5~2.5), (2.5~10): (1.5~10), (2.5~10): (1.5~50), (2.5~10): (1.5~100), (2.5~10): (2.5~10), (2.5~10): (2.5~50), (2.5~10): (2.5~100), (2.5~10): (10~50), (2.5~10): (10~100) or (2.5~10): (50~100).

[0026] In other cases, the mass ratios of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (2.5~50):(0.25~1.25), (2.5~50):(0.25~1.5), (2.5~50):(0.25~2.5), (2.5~50):(0.25~10), (2.5~50):(0.25~50), (2.5~50):(0.25~100), (2.5~50):(1.25~1.5), (2.5~50):(1.25~2.5), (2.5~50):(1.25~10). .5~50): (1.25~50), (2.5~50): (1.25~100), (2.5~50): (1.5~2.5), (2.5~50): (1.5~10), (2.5~50): (1.5~50), (2.5~50): (1.5~100), (2.5~50): (2.5~10), (2.5~50): (2.5~50), (2.5~50): (2.5~100), (2.5~50): (2.5~50), (2.5~50): (10~50), (2.5~50): (10~100 or (2.5~50): (50~100).

[0027] In other cases, the mass ratios of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (2.5~100):(0.25~1.25), (2.5~100):(0.25~1.5), (2.5~100):(0.25~2.5), (2.5~100):(0.25~10), (2.5~100):(0.25~50), (2.5~100):(0.25~100), (2.5~100):(1.25~1.5), (2.5~100):(1.25~2.5), (2.5~100):(1.25~10), (2.5~100):(1.25~10). 5~100): (1.25~50), (2.5~100): (1.25~100), (2.5~100): (1.5~2.5), (2.5~100): (1.5~10), (2.5~100): (1.5~50), (2.5~100): (1.5~100), (2.5~100): (2.5~10), (2.5~100): (2.5~50), (2.5~100): (2.5~100), (2.5~100): (10~50), (2.5~100): (10~100) or (2.5~100): (50~100).

[0028] In other cases, the mass ratios of bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (10~50):(0.25~1.25), (10~50):(0.25~1.5), (10~50):(0.25~2.5), (10~50):(0.25~10), (10~50):(0.25~50), (10~50):(0.25~100), (10~50):(1.25~1.5), (10~50):(1.25~2.5), (10~50):(1.25~10), (10~50): (1.25~50), (10~50): (1.25~100), (10~50): (1.5~2.5), (10~50): (1.5~10), (10~50): (1.5~50), (10~50): (1.5~100), (10~50): (2.5~10), (10~50): (2.5~50), (10~50): (2.5~100), (10~50): (10~50), (10~50): (10~100 or (10~50): (50~100).

[0029] In other cases, the mass ratios of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (10~100):(0.25~1.25), (10~100):(0.25~1.5), (10~100):(0.25~2.5), (10~100):(0.25~10), (10~100):(0.25~50), (10~100):(0.25~100), (10~100):(1.25~1.5), (10~100):(1.25~2.5), (10~100):(1.25~10), (1 0~100): (1.25~50), (10~100): (1.25~100), (10~100): (1.5~2.5), (10~100): (1.5~10), (10~100): (1.5~50), (10~100): (1.5~100), (10~100): (2.5~10), (10~100): (2.5~50), (10~100): (2.5~100), (10~100): (10~50), (10~100): (10~100) or (10~100): (50~100).

[0030] In other cases, the mass ratios of bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide were (50-100):(0.25-1.25), (50-100):(0.25-1.5), (50-100):(0.25-2.5), (50-100):(0.25-10), (50-100):(0.25-50), (50-100):(0.25-100), (50-100):(1.25-1.5), (50-100):(1.25-2.5), (50-100):(1.25-10), (5 0~100): (1.25~50), (50~100): (1.25~100), (50~100): (1.5~2.5), (50~100): (1.5~10), (50~100): (1.5~50), (50~100): (1.5~100), (50~100): (2.5~10), (50~100): (2.5~50), (50~100): (2.5~100), (50~100): (10~50), (50~100): (10~100) or (50~100): (50~100).

[0031] Preferably, the mass ratio of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate to the microcurrent tetrapeptide in the composition is 1:10 to 10:1. For example, the mass ratio of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate to the microcurrent tetrapeptide is 1:10, 1:1 or 10:1.

[0032] In addition to the above ratios, Bifidobacterium / Lactobacillus / soybean seed extract fermentation product filtrate and microcurrent tetrapeptide also have anti-aging effects at other mass ratios, but the effects are not as good as the aforementioned ratios.

[0033] Furthermore, the present invention also provides a skin care product, comprising auxiliary materials and the above-mentioned composition.

[0034] In an embodiment of the present invention, in the skin care product:

[0035] The concentration of the filtrate of the fermentation product of bifidobacterium / lactobacillus / soybean seed extract is 0.25-100 mg / mL, or the mass fraction is 0.025%-10%. Preferably, the concentration of the filtrate of the fermentation product of bifidobacterium / lactobacillus / soybean seed extract in the skin care product is 0.25-2.5 mg / mL, or the mass fraction is 0.025%-0.25%.

[0036] The concentration of the microcurrent tetrapeptide is 0.25-100 mg / mL, or the mass fraction is 0.025%-10%. Preferably, the concentration of the microcurrent tetrapeptide is 0.25-2.5 mg / mL, or the mass fraction is 0.025%-0.25%.

[0037] In the present invention, the concentration of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate in the skin care product is 0.25mg / mL~1.25mg / mL, 0.25mg / mL~1.5mg / mL, 0.25mg / mL~2.5mg / mL, 0.25mg / mL~10mg / mL, 0.25mg / mL~50mg / mL, 0.25mg / mL~100mg / mL, 1.25mg / mL~1.5mg / mL, 1.25mg / mL~2.5mg / mL, 1.25mg / mL~10mg / mL, 1.25mg / mL to 50mg / mL, 1.25mg / mL to 100mg / mL, 1.5mg / mL to 2.5mg / mL, 1.5mg / mL to 10mg / mL, 1.5mg / mL to 50mg / mL, 1.5mg / mL to 100mg / mL, 2.5mg / mL to 10mg / mL, 2.5mg / mL to 50mg / mL, 2.5mg / mL to 100mg / mL, 10mg / mL to 50mg / mL, 10mg / mL to 100mg / mL or 50mg / mL to 100mg / mL. More specifically, the concentration of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate is: 0.25mg / ml, 1.25mg / ml, 1.5mg / ml, 2.5mg / ml, 10mg / ml, 50mg / ml or 100mg / ml.

[0038] In the present invention, the concentration of the microcurrent tetrapeptide in the skin care product is 0.25mg / mL~1.25mg / mL, 0.25mg / mL~1.5mg / mL, 0.25mg / mL~2.5mg / mL, 0.25mg / mL~10mg / mL, 0.25mg / mL~50mg / mL, 0.25mg / mL~100mg / mL, 1.25mg / mL~1.5mg / mL, 1.25mg / mL~2.5mg / mL, 1.25mg / mL~10mg / mL, 1.25mg / mL ~50mg / mL, 1.25mg / mL~100mg / mL, 1.5mg / mL~2.5mg / mL, 1.5mg / mL~10mg / mL, 1.5mg / mL~50mg / mL, 1.5mg / mL~100mg / mL, 2.5mg / mL~10mg / mL, 2.5mg / mL~50mg / mL, 2.5mg / mL~100mg / mL, 10mg / mL~50mg / mL, 10mg / mL~100mg / mL or 50mg / mL~100mg / mL. More specifically, the concentration of the microcurrent tetrapeptide is: 0.25mg / ml, 1.25mg / ml, 1.5mg / ml, 2.5mg / ml, 10mg / ml, 50mg / ml or 100mg / ml.

[0039] Preferably, in the skin care product, the concentration of the filtrate of the bifidobacterium / lactobacillus / soybean seed extract fermentation product is 0.25-2.5 mg / mL; and the concentration of the microcurrent tetrapeptide is 0.25-2.5 mg / mL.

[0040] In the present invention, the skin care products are cleaning cosmetics, care cosmetics or beauty / modification cosmetics, and their dosage forms include creams, lotions, solutions, sprays, facial masks, essence water, gels or powders.

[0041] In some embodiments, the acceptable excipients in the cosmetics include, but are not limited to, purified water, oils, lipids, esters, waxes, surfactants, moisturizers, thickeners, chelating agents, pigments, fragrances, antioxidants, preservatives, pH adjusters, and the like.

[0042] Furthermore, the present invention also provides use of the above-mentioned composition and / or the above-mentioned skin care product in the preparation of anti-aging products.

[0043] In the embodiment of the present invention, the anti-aging method includes at least one of the following I) to V):

[0044] I) Antioxidant;

[0045] II) protect cells;

[0046] III) regulate the transcription level of related genes;

[0047] IV) regulating the expression levels of related proteins;

[0048] V) Inhibit aging indicator bacteria.

[0049] In a specific embodiment, the anti-oxidation comprises scavenging free radicals, and preferably, the free radical is DPPH.

[0050] In a specific embodiment, the protected cells are fibroblasts; more specifically, the viability and / or activity of fibroblasts are protected under ultraviolet irradiation.

[0051] In a specific embodiment, the related genes are COL1 gene and COL3 gene. More specifically, regulating the transcription level of the related genes includes increasing the transcription level of COL 1 gene and / or COL3 gene;

[0052] In a specific embodiment, the related protein is COL1 protein. More specifically, regulating the expression level of the related protein includes promoting the expression of COL1 protein.

[0053] In a specific embodiment, the aging indicator bacteria is Moraxella osloensis.

[0054] In the present invention, the aging is photoaging of the skin. Specifically, the aging is aging caused by ultraviolet radiation. More specifically, it is aging caused by UVA.

[0055] The present invention also provides an anti-aging method, which comprises applying the composition as described above and / or the skin care product as described above on the human body surface. The method described in the present invention includes a method for therapeutic purposes and a method for non-therapeutic purposes. Preferably, it is an anti-aging method for non-therapeutic purposes. For example, a cosmetic method that does not involve the human body or does not cause trauma.

[0056] The invention provides a composition of a bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate and a microcurrent tetrapeptide, which has at least one of the following effects: I) anti-oxidation; II) cell protection; III) regulating the transcription level of related genes; IV) regulating the expression level of related proteins; V) inhibiting aging indicator bacteria. Therefore, the composition can achieve a good anti-aging effect, and there is a synergistic effect between the two components. The composition can be applied to the field of cosmetics, with a wider range of efficacy, especially in the field of beauty and skin care. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 DPPH free radical scavenging rate of different concentrations of Huanfuyuan;

[0058] Figure 2DPPH free radical scavenging rate of microcurrent tetrapeptide at different concentrations;

[0059] Figure 3 DPPH free radical scavenging rate of different ratios of Huanfuyuan and microcurrent tetrapeptide combinations;

[0060] Figure 4 Cell survival rate of different concentrations of Huanfuyuan;

[0061] Figure 5 Cell survival rate of different concentrations of microcurrent tetrapeptide;

[0062] Figure 6 Cell survival rate of different ratio combinations of Huanfuyuan and microcurrent tetrapeptide;

[0063] Figure 7 The relative mRNA levels of COL1 gene in different concentrations of Huanfuyuan;

[0064] Figure 8 The relative mRNA levels of COL3 gene in different concentrations of Huanfuyuan;

[0065] Fig. 9 Relative mRNA levels of COL1 gene at different concentrations of microcurrent tetrapeptide;

[0066] Fig.10 Relative mRNA levels of COL3 gene at different concentrations of microcurrent tetrapeptide;

[0067] Fig.11 Relative mRNA levels of COL1 gene in different ratio combinations of Huanfuyuan and microcurrent tetrapeptide;

[0068] Fig.12 Relative mRNA levels of COL3 gene in different ratio combinations of Huanfuyuan and microcurrent tetrapeptide;

[0069] Fig.13 The relative expression of COL1 protein in Huanfuyuan, microcurrent tetrapeptide and the combination;

[0070] Fig.14 The relative growth rate of the bacterial flora of Huanfuyuan, microcurrent tetrapeptide and the combination;

[0071] In the figures, ns indicates that there is no significant difference between the groups; There are significant differences between the groups, p < 0.05; There were extremely significant differences between the groups, p<0.01; There were extremely significant differences between the groups, p<0.001; There were extremely significant differences among the groups, p<0.0001. DETAILED DESCRIPTION

[0072] The present invention provides a cosmetic composition and its application in the preparation of skin care products. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0073] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0074] In the present invention, "include", "comprising" and "having" are used interchangeably, and are intended to indicate the inclusiveness of the scheme, meaning that the scheme may have other elements besides the listed elements. At the same time, it should be understood that the descriptions of "include", "comprising" and "having" used in this article also provide "consisting of..." schemes.

[0075] “And / or” as used herein includes the meanings of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0076] In the present invention, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0077] It should be understood that in various embodiments of the present invention, the size of the serial number does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0078] The numerical ranges and parameters involved in the present invention have been presented in the specific embodiments as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have certain reasonable deviations within a certain range, for example: within ±10%, ±5%, ±1% or ±0.5%.

[0079] Some implementations of the present invention are shown in the embodiments of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases. In fact, any concentration of each component between the two endpoint values ​​shown in the embodiments can achieve a good anti-aging effect. In the case with better effects, the mass ratios of Huanfuyuan and microcurrent tetrapeptide are 1:10, 1:1, and 10:1. The following embodiments also list some cases with poor results in the experiments: for example, using different components (acetyl tetrapeptide-3), using different ratios, etc., but the effects of these attempts are not as good as the effects under the ratios described above, which will not be repeated here.

[0080] In the embodiment of the present invention, the statistical analysis method is to use GraphPad Prism 9 software for statistical analysis and drawing. If the data meet the normal distribution and variance homogeneity, the paired t test is used for the comparison before and after the experiment within the group, and the one-way analysis of variance is used for the comparison of the means between the groups. P < 0.05 indicates that the difference is statistically significant.

[0081] In the embodiments of the present invention, the synergistic analysis adopts the Bliss independence model. The Bliss independence model is used to simulate the effect of a single drug in combination, and is a common and reliable model for calculating drug synergy coefficients in the field. For related literature, see Wei Zhao et al. "A New Bliss Independence Model to Analyze Drug Combination Data" or CI BLISS "The toxicity of poisons applied jointly". The Bliss synergy score is calculated according to the Bliss independence model, and the formula is S BLISS =E A,B -(E A +E B -E A E B ), where S BLISS <0 is antagonistic, S BLISS =0 is additive effect, S BLISS >0 indicates synergistic effect.

[0082] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0083] The main component of the microcurrent tetrapeptide is tetrapeptide-1, the peptide sequence of which is LEAP, which is purchased from Lipotec.

[0084] The bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate, Guozhuang original preparation number 20230032, the registered Chinese name is bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate, INCI name is Bifidobacterium / Lactobacillus / Soybean Seed Extract Ferment Filtrate, also referred to as Huanfuyuan in this article, from Guangzhou Yixian e-commerce. The preparation method mainly includes: soaking black beans and water in a certain proportion, obtaining soybean seed extract after heating and water extraction, and obtaining a fermentation base after sterilization. Transfer to a fermentation tank, add a strain composition (containing bifidobacteria and lactobacillus) for fermentation, and obtain a fermentation filtrate after sterilization and filtration. Finally, after passing the quality inspection, it is packaged and stored in the warehouse.

[0085] The present invention will be further described below in conjunction with embodiments:

[0086] Example

[0087] A DPPH free radical scavenging test to determine the antioxidant effect

[0088] 1. Determine the DPPH free radical scavenging rate of the sample

[0089] (1) Preparation of stock solution: 95% ethanol: Take 95 ml of ethanol in a measuring cylinder and add ultrapure water to 100 ml. 0.03% DPPH stock solution: Weigh 15 mg of DPPH powder into a 50 ml brown volumetric flask, add about 40 ml of 95% ethanol, sonicate to completely dissolve it, dilute to the mark, shake well, and use as DPPH stock solution.

[0090] (2) Preparation of 0.03‰ DPPH working solution: Based on the required amount of DPPH solution, measure 1 / 10 volume of 0.03% DPPH stock solution and dilute it to prepare 0.03‰ DPPH working solution.

[0091] (3) The positive control Trolex was diluted to 0.025 mM with pure water to verify the test system.

[0092] (4) The test substance is diluted with pure water to form samples of multiple concentrations. Referring to Table 1, set up sample tubes (T), sample background (T0), DPPH tubes (C) and solvent background (C0) in a 96-well plate. Three parallel wells should be set up for each sample well (T) of each test concentration of each sample, and three parallel wells should also be set up for the DPPH tube (C).

[0093] (5) Add 50 μl of sample solution of the same concentration and 100 μl of pure water to the sample tube (T) and sample background (T0), and add 150 μl of pure water to the DPPH tube (C) and solvent background (C0).

[0094] (6) Add 50 μl of DPPH ethanol solution to each of the sample tube (T) and DPPH tube (C). Replace the sample background (T0) and solvent background (C0) with 50 μl of 95% ethanol.

[0095] (7) Incubate the mixture at room temperature for 5 min and measure OD517.

[0096] (8) Calculate the DPPH free radical scavenging rate: scavenging rate (%) = (1- )×100%

[0097] (9) In the formula, T is the absorbance of the sample well, i.e., the absorbance of the solution after the sample reacts with DPPH; T0 is the background absorbance of the sample; C is the average of the three absorbances of the DPPH well, i.e., the absorbance of the DPPH solution when no sample is added; and C0 is the background absorbance of the solvent.

[0098] (10) Statistical analysis and graphing: GraphPad Prism 9 software was used for statistical analysis and graphing. If the data met the normal distribution and homogeneity of variance, paired t-test was used for pre- and post-experimental comparison within the group, and one-way analysis of variance was used for comparison of means between groups. P < 0.05 indicated statistically significant differences.

[0099] Table 1 DPPH free radical scavenging rate determination experimental sample solution table

[0100]

[0101] 1.1 Antioxidant effect of Huanfuyuan (DPPH free radical scavenging rate)

[0102] Table 2 DPPH free radical scavenging rate values ​​of different concentrations of Huanfuyuan

[0103]

[0104] Summary: The positive standard 0.025mM Trolex showed a good DPPH free radical scavenging rate, indicating that the experimental system was successful. Different concentrations of Huanfuyuan showed a good DPPH free radical scavenging rate, indicating that Huanfuyuan has a certain antioxidant capacity and anti-aging effect.

[0105] 1.2 Antioxidant effect of microcurrent tetrapeptide (DPPH free radical scavenging rate)

[0106] Table 3 DPPH free radical scavenging rate of microcurrent tetrapeptide at different concentrations

[0107]

[0108] Summary: The positive standard 0.025mM Trolex showed good DPPH free radical scavenging rate, indicating that the experimental system was successful. However, different concentrations of microcurrent tetrapeptide did not show good DPPH free radical scavenging rate, indicating that microcurrent tetrapeptide has no antioxidant ability.

[0109] 1.3 Antioxidant effect of different proportions of Huanfuyuan + microcurrent tetrapeptide (DPPH free radical scavenging rate)

[0110] Table 4 DPPH free radical scavenging rate of different ratios of Huanfuyuan and microcurrent tetrapeptide

[0111]

[0112] Summary: When Huanfuyuan and microcurrent tetrapeptide were combined in different ratios, there was no significant difference in DPPH free radical scavenging rate compared with Huanfuyuan alone. Only when Huanfuyuan and microcurrent tetrapeptide were combined in 10:1, the scavenging rate was slightly higher than that of Huanfuyuan alone. This shows that Huanfuyuan and microcurrent tetrapeptide did not synergistically improve antioxidant capacity in different combinations.

[0113] MTT assay to determine cell viability

[0114] (1) HFF fibroblasts were plated into 96-well plates.

[0115] (2) On the second day, the culture medium was replaced with PBS solution and the cells were subjected to 30 J / cm 2 After UVA irradiation, fresh pre-warmed culture medium was replaced and the test samples were then added to stimulate the cells for 24 h. The final culture medium volume in each well was 200 μl.

[0116] (3) Discard the culture medium and add MTT solution (0.5 mg / mL) to each well and incubate at 37°C for 4 hours. Discard the culture medium and add 150 µl DMSO to dissolve the precipitate. Measure the absorbance at OD 490 nm using a multifunctional microplate reader and calculate the percentage of cell viability using the following formula:

[0117] Cell viability (%) = 100% × (OD sample - ODctl) / ODctl

[0118] Among them, ODsample is the OD value of the sample, and ODctl is the OD value of the control.

[0119] 2.1 Huanfuyuan anti-UVA damage (MTT)

[0120] Table 5 Numerical table of cell survival rates of different concentrations of Huanfuyuan

[0121]

[0122] Summary: After UVA treatment, the cell viability decreased significantly, indicating that UVA stimulation caused damage to the cell survival rate. On this basis, after treatment with different concentrations of Huanfuyuan, the detected cell viability increased significantly compared with UVA treatment alone, indicating that Huanfuyuan has the effect of resisting cell damage caused by UVA and has anti-aging effects.

[0123] 2.2 Microcurrent Tetrapeptide Anti-UVA Damage (MTT)

[0124] Table 6 Numerical table of cell survival rates of different concentrations of microcurrent tetrapeptides

[0125]

[0126] Summary: After UVA treatment, the cell viability decreased significantly, indicating that UVA stimulation caused damage to the cell survival rate. On this basis, after treatment with different concentrations of microcurrent tetrapeptide, the detected cell viability increased to a certain extent compared with UVA treatment alone, indicating that microcurrent tetrapeptide has the effect of resisting cell damage caused by UVA and has anti-aging effects.

[0127] 2.3 Different proportions of Huanfuyuan + microcurrent tetrapeptide synergistically resist UVA damage (MTT)

[0128] Table 7 Cell survival rate values ​​of different ratios of Huanfuyuan and microcurrent tetrapeptide combination

[0129]

[0130] Summary: After UVA treatment, the cell viability decreased significantly, indicating that UVA stimulation caused damage to the cell viability, and the anti-aging model was successfully established; under UVA stimulation, Huanfuyuan, microcurrent tetrapeptide and combination were used to treat and calculate the cell viability. The results showed that when Huanfuyuan: microcurrent tetrapeptide was combined in a 1:1 ratio, the cell viability increased significantly compared with Huanfuyuan and microcurrent tetrapeptide alone, and S BLISS >0, indicating that Huanfuyuan and microcurrent tetrapeptide can synergistically resist the damage of UVA to cell survival rate when combined in a 1:1 ratio, and have a synergistic anti-aging effect.

[0131] Detection and analysis of three anti-aging genes (qPCR)

[0132] (1) HFF fibroblasts grown in 12-well plates were stimulated with UVA to establish the model.

[0133] (2) 24 hours after modeling, cells were collected.

[0134] (3) Use an RNA total extraction kit to extract cellular RNA, and use a reverse transcription kit to reverse transcribe and synthesize cDNA.

[0135] (4) Amplification was performed using SYBR Green qPCR Master Mix fluorescent quantitative PCR instrument, and the specificity of the primers was checked by melting curve.

[0136] (5) The relative expression levels of the target genes (type I collagen COL1 and type III collagen COL3) were calculated using the 2^-ΔΔCt method, with GAPDH as the internal reference gene (primer sequences of GAPDH: F: 5'-ACAACTTTGGTATCGTGGAAGG-3'; R: 5'-GCCATCACGCCACAGTTTC-3')

[0137] 3.1 Anti-aging effect of Huanfuyuan (qPCR)

[0138] Table 8 qPCR experimental design table.

[0139]

[0140] COL1 PCR

[0141] Primers for Collagen I (COL1):

[0142] F:5'- GTGCGATGACTGATCTGTGA-3'

[0143] R:5'- CGGTGGTTTCTTGGTCGGT-3'

[0144] COL1 is type I collagen, which accounts for about 80% of the dermis of the skin. Type I collagen helps the dermis (middle layer of the skin) form fibroblasts, helps new cells grow to provide structure, strength and support for the skin, makes the skin plump and promotes the expression of COL1 protein, which can achieve a certain effect in resisting the formation of wrinkles, thereby achieving anti-aging effects.

[0145] Table 9 Relative mRNA level values ​​of COL1 gene in different concentrations of Huanfuyuan

[0146]

[0147] Summary: After UVA treatment, compared with BC, the relative mRNA level of COL1 gene decreased significantly, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used, and the relative mRNA level of COL1 gene increased significantly, indicating that the positive control of this experiment was effective. Compared with the UVA alone treatment group, after 0.25mg / ml Huanfuyuan treatment, the relative mRNA level of COL1 gene decreased significantly, while after 1.25mg / ml and 2.5mg / ml Huanfuyuan treatment, the relative mRNA level of COL1 gene increased significantly. This result shows that Huanfuyuan concentrations of 1.25mg / ml and 2.5mg / ml can significantly increase the expression of COL1 gene, and has a certain anti-aging effect.

[0148] COL3 PCR

[0149] Primers for Collagen III (COL3):

[0150] F: 5'-TTGAAGGAGGATGTTCCCATCT-3'

[0151] R: 5'-ACAGACACATATTTGGCATGGTT-3

[0152] COL3 is type III collagen, one of the most abundant proteins in the human body. It is composed of three alpha chains that contain many amino acids, mainly glycine, hydroxyproline and proline. COL3 is mainly found in the skin, blood vessel walls, internal organs and muscles. The functions of COL3 are similar to other types of collagen, including providing strength and elasticity to tissues, maintaining the firmness and elasticity of the skin, supporting the structure of blood vessel walls, and maintaining the health of muscles and joints.

[0153] Table 10 Relative mRNA level values ​​of COL3 gene in different concentrations of Huanfuyuan

[0154]

[0155] Summary: After UVA treatment, compared with BC, the relative mRNA level of COL3 gene decreased significantly, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used, and the relative mRNA level of COL3 gene increased significantly, indicating that the positive control of this experiment was effective. Compared with the UVA alone treatment group, after 0.25mg / ml Huanfuyuan treatment, the relative mRNA level of COL3 gene decreased significantly, while after 1.25mg / ml and 2.5mg / ml Huanfuyuan treatment, the relative mRNA level of COL3 gene increased significantly. This result shows that Huanfuyuan concentrations of 1.25mg / ml and 2.5mg / ml can significantly increase the expression of COL3 gene, and has a certain anti-aging effect.

[0156] 3.2 Anti-aging effect of microcurrent tetrapeptide (qPCR)

[0157] Table 11 qPCR experimental design table

[0158]

[0159] COL1 PCR

[0160] Primers for Collagen I (COL1):

[0161] F:5'-CTGGACTCCGACACTCTGGA-3'

[0162] R:5'-CAGGAAAGGTTCTGAAGTGACC-3'

[0163] COL1 is type I collagen, which accounts for about 80% of the dermis of the skin. Type I collagen helps the dermis (middle layer of the skin) form fibroblasts, helps new cells grow to provide structure, strength and support for the skin, makes the skin plump and promotes the expression of COL1 protein, which can achieve a certain effect in resisting the formation of wrinkles, thereby achieving anti-aging effects.

[0164] Table 12 Relative mRNA levels of COL1 gene in different concentrations of microcurrent tetrapeptide

[0165]

[0166] Summary: After UVA treatment, compared with BC, the relative mRNA level of COL1 gene decreased significantly, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used, and the relative mRNA level of COL1 gene increased significantly, indicating that the positive control of this experiment was effective. Compared with the UVA alone treatment group, after 0.25mg / ml and 1.25mg / ml microcurrent tetrapeptide treatment, the relative mRNA level of COL1 gene increased significantly. This result shows that microcurrent tetrapeptide can significantly increase the expression of COL1 gene in cells at 0.25mg / ml and 1.25mg / ml, and has a certain anti-aging effect.

[0167] COL3 PCR

[0168] Primers for Collagen III (COL3):

[0169] F: 5'-TTGAAGGAGGATGTTCCCATCT-3'

[0170] R: 5'-ACAGACACATATTTGGCATGGTT-3

[0171] COL3 is type III collagen, one of the most abundant proteins in the human body. It is composed of three alpha chains that contain many amino acids, mainly glycine, hydroxyproline and proline. COL3 is mainly found in the skin, blood vessel walls, internal organs and muscles. The functions of COL3 are similar to other types of collagen, including providing strength and elasticity to tissues, maintaining the firmness and elasticity of the skin, supporting the structure of blood vessel walls, and maintaining the health of muscles and joints.

[0172] Table 13 Relative mRNA levels of COL3 gene in different concentrations of microcurrent tetrapeptide

[0173]

[0174] Summary: After UVA treatment, compared with BC, the relative mRNA level of COL3 gene decreased significantly, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used, and the relative mRNA level of COL3 gene increased significantly, indicating that the positive control of this experiment was effective. Compared with the UVA alone treatment group, after 0.25mg / ml and 1.25mg / ml microcurrent tetrapeptide treatment, the relative mRNA level of COL3 gene increased significantly. This result shows that microcurrent tetrapeptide can significantly increase the expression of COL3 gene in cells at 0.25mg / ml and 1.25mg / ml, and has a certain anti-aging effect.

[0175] 3.3 Huanfuyuan + microcurrent tetrapeptide anti-aging synergistic effect (qPCR)

[0176] Table 14 qPCR experimental design table

[0177]

[0178] COL1 PCR

[0179] Primers for Collagen I (COL1):

[0180] F:5'- GTGCGATGACTGATCTGTGA-3'

[0181] R:5'- CGGTGGTTTCTTGGTCGGT-3'

[0182] COL1 is type I collagen, which accounts for about 80% of the dermis of the skin. Type I collagen helps the dermis (middle layer of the skin) form fibroblasts, helps new cells grow to provide structure, strength and support for the skin, makes the skin plump and promotes the expression of COL1 protein, which can achieve a certain effect in resisting the formation of wrinkles, thereby achieving anti-aging effects.

[0183] Table 15 Relative mRNA level values ​​of COL1 gene in different ratios of Huanfuyuan and microcurrent tetrapeptide

[0184]

[0185] Summary: After UVA treatment, the relative mRNA level of COL1 gene decreased significantly compared with BC, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used, and the relative mRNA level of COL1 gene increased significantly, indicating that the positive control of the experiment was effective. When the ratio of Huanfuyuan and microcurrent tetrapeptide combination was 1:1, the gene expression level of COL1 increased significantly compared with that of Huanfuyuan and microcurrent tetrapeptide alone, and S BLISS >0, indicating that Huanfuyuan and microcurrent tetrapeptide have synergistic anti-aging effects, indicating that the combination of Huanfuyuan and microcurrent tetrapeptide has a synergistic effect in anti-aging efficacy.

[0186] COL3 PCR

[0187] Primers for Collagen III (COL3):

[0188] F: 5'-TTGAAGGAGGATGTTCCCATCT-3'

[0189] R: 5'-ACAGACACATATTTGGCATGGTT-3

[0190] COL3 is type III collagen, one of the most abundant proteins in the human body. It is composed of three alpha chains that contain many amino acids, mainly glycine, hydroxyproline and proline. COL3 is mainly found in the skin, blood vessel walls, internal organs and muscles. The functions of COL3 are similar to other types of collagen, including providing strength and elasticity to tissues, maintaining the firmness and elasticity of the skin, supporting the structure of blood vessel walls, and maintaining the health of muscles and joints.

[0191] Table 16 Relative mRNA level values ​​of COL3 gene in different ratios of Huanfuyuan and microcurrent tetrapeptide

[0192]

[0193] Summary: After UVA treatment, the relative mRNA level of COL3 gene decreased significantly compared with BC, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used, and the relative mRNA level of COL3 gene increased significantly, indicating that the positive control of the experiment was effective. When the ratio of Huanfuyuan and microcurrent tetrapeptide combination was 1:1, the gene expression level of COL3 increased significantly compared with that of Huanfuyuan and microcurrent tetrapeptide alone, and S BLISS >0, indicating that Huanfuyuan and microcurrent tetrapeptide have synergistic anti-aging effects, indicating that the combination of Huanfuyuan and microcurrent tetrapeptide has a synergistic effect in anti-aging efficacy.

[0194] Detection and analysis of four anti-aging proteins (WB)

[0195] (1) HFF fibroblasts grown in 12-well plates were stimulated with UVA to establish the model.

[0196] (2) 24 hours after modeling, cells were collected and extracted by adding RIPA cell lysis buffer.

[0197] (3) Protein quantification: Use a protein quantification method (such as the BCA method) to determine the extracted protein concentration.

[0198] (4) SDS-PAGE electrophoresis: Use polyacrylamide gel electrophoresis (SDS-PAGE) to separate protein samples according to molecular weight.

[0199] (5) Transfer: Assemble the gel, membrane and transfer buffer in a transfer apparatus, and use a wet transfer apparatus to transfer the separated proteins from the gel to the PVDF membrane.

[0200] (6) Blocking: Place the membrane in 5% skimmed milk powder and incubate for 1 h.

[0201] (7) Primary antibody incubation: Add the primary antibody (specific antibody) of the target protein to 5% skim milk powder and incubate overnight to allow it to fully bind to the target protein in the membrane.

[0202] (8) Washing: Wash the membrane with buffer TBST to remove unbound primary antibody.

[0203] (9) Secondary antibody incubation: Add the secondary antibody corresponding to the source of the primary antibody (e.g., goat anti-rabbit IgG) to a solution containing 5% skim milk powder and incubate for 2 h to allow it to bind to the primary antibody. The secondary antibody is usually labeled with an enzyme (e.g., horseradish peroxidase-HRP) or a fluorescent dye.

[0204] (10) Washing: Wash the membrane with buffer TBST to remove unbound secondary antibody.

[0205] (11) Signal detection: The secondary antibody-labeled protein was detected using the substrate chemiluminescence ECL method, and the target protein was imaged and stored using the ChemiDoc imaging system.

[0206] (12) Result analysis: ImageJ was used to quantitatively analyze the target protein based on the signal intensity and location.

[0207] 4.1 Anti-aging effect of Huanfuyuan + microcurrent tetrapeptide (1:1) (WB)

[0208] Table 17 WB experimental design table

[0209]

[0210] COL1 WB

[0211] COL1 is type I collagen, which accounts for about 80% of the dermis of the skin. Type I collagen helps the dermis (middle layer of the skin) form fibroblasts, helps new cells grow to provide structure, strength and support for the skin, makes the skin plump and promotes the expression of COL1 protein, which can achieve a certain effect in resisting the formation of wrinkles, thereby achieving anti-aging effects.

[0212] Table 18 Relative expression values ​​of MMP1 protein of Huanfuyuan, microcurrent tetrapeptide and combinations of different proportions

[0213]

[0214] Summary: After UVA treatment, the expression of COL1 protein decreased significantly compared with BC, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used, and the expression of COL1 protein increased significantly, indicating that the positive control of this experiment was effective. Under UVA stimulation, the expression of COL1 protein in all monomers and combinations increased significantly, indicating that all monomers and combinations have anti-aging effects. In addition, the expression level of COL1 protein in the combination of Huanfuyuan and microcurrent tetrapeptide at a ratio of 1:1 was significantly increased compared with that of Huanfuyuan and microcurrent tetrapeptide alone, and S BLISS >0, indicating that Huanfuyuan and microcurrent tetrapeptide have a synergistic anti-aging effect. At the same time, compared with the combination of Huanfuyuan and acetyl tetrapeptide-3 (1:1), the COL1 protein expression level of the combination of Huanfuyuan and microcurrent tetrapeptide (1:1) showed a significant increase, indicating that the combination of Huanfuyuan and microcurrent tetrapeptide significantly increased the expression of COL1 protein compared with the combination of Huanfuyuan and acetyl tetrapeptide-3. In summary, the combination of Huanfuyuan and microcurrent tetrapeptide has a synergistic effect in anti-aging efficacy, and the effect is better than the anti-aging effect of the combination of Huanfuyuan and acetyl tetrapeptide-3.

[0215] Five antibacterial detection analysis

[0216] (1) Take out the glycerol storage tube of the strain from the -80°C ultra-low temperature freezer, streak a single colony on the prepared R2A solid plate, and then place it in a 30°C incubator for 48 h;

[0217] (2) Take the R2A culture plate out of the 30°C incubator and pick a single colony with an inoculation loop in a clean bench. Place the colonies in sterile 12 ml shaking tubes and add 4 ml of R2A liquid culture medium to each tube. Place the tubes in a constant temperature shaker at 220 rpm and culture overnight at 30°C.

[0218] (3) Take the bacterial solution in step 2, prepare a sterile cell shake flask, take a certain volume, inoculate it into fresh sterile R2A liquid culture medium at a ratio of 2%, and then place it in a constant temperature shaker at a speed of 220 rpm and 30°C for 6 h;

[0219] (4) Meanwhile, prepare several sterile 96-well plates, add 30ul of R2A liquid culture medium and other components to the positive control group, and add 30ul of raw materials to the experimental groups respectively, and set aside for use;

[0220] (5) Take 200ul of the bacterial solution from step 3, add it to a sterile 96-well plate, and measure the OD600; then resuspend it in fresh sterile R2A medium and adjust its OD600 reading to between 0.14 and 0.16;

[0221] (6) Mix the resuspended bacterial solution and biolog dye in the ratio (2ul: 170ul = 1: 85) (protect from light), and add 172ul to a 96-well plate;

[0222] (7) Measure the OD590 of each 96-well plate at 0 h, and then place it in a 30°C incubator in the dark;

[0223] (8) Measure OD590 at different time periods and compare and record the data.

[0224] 5.1 Antibacterial experiment of Huanfuyuan + microcurrent tetrapeptide (1:1)

[0225] Table 19 Antibacterial experiment design table

[0226]

[0227] 5.2 Moraxella osloensis

[0228] Table 20 Numerical table of relative growth rates of bacteria of Huanfuyuan, microcurrent tetrapeptide and combination

[0229]

[0230] Conclusion: After culturing Moraxella osloensis for 24 hours, the growth rate of the bacteria treated with the combination of Huanfuyuan and microcurrent tetrapeptide was significantly lower than that of BC. In addition, the growth rate of the bacteria treated with the combination of Huanfuyuan and microcurrent tetrapeptide at a ratio of 1:1 was significantly lower than that of Huanfuyuan and microcurrent tetrapeptide alone, and S BLISS >0, indicating that Huanfuyuan and microcurrent tetrapeptide can synergistically inhibit the growth of Moraxella osloensis, and have certain microecological anti-aging effects. After culturing Moraxella osloensis for 48 hours, the growth rate of the flora treated with Huanfuyuan, microcurrent tetrapeptide and their combination was significantly lower than that of BC. This indicates that Huanfuyuan and microcurrent tetrapeptide can significantly inhibit the growth of Moraxella osloensis. After culturing Moraxella osloensis for 72 hours, the growth rate of the flora treated with Huanfuyuan, microcurrent tetrapeptide and their combination was significantly lower than that of BC. This indicates that Huanfuyuan and microcurrent tetrapeptide can significantly inhibit the growth of Moraxella osloensis. In addition, under the condition of a ratio of 1:1, the growth rate of the flora of the composition of Huanfuyuan and microcurrent tetrapeptide was significantly lower than that of Huanfuyuan and microcurrent tetrapeptide alone, indicating that Huanfuyuan and microcurrent tetrapeptide can synergistically inhibit the growth of Moraxella osloensis, and have good microecological anti-aging effects.

[0231] Six synergy calculations

[0232] The above data were collected and the Bliss synergy score was calculated according to the Bliss independence model. The formula is S BLISS =E A,B -(E A +E B -E A E B ), where S BLISS <0 is antagonistic, S BLISS =0 is additive effect, S BLISS >0 indicates synergistic effect. The E value is the average value of the test sample effect minus the average value of the control group after corresponding UVA treatment. The results are shown in Table 21:

[0233] Table 21

[0234]

[0235] Note: The ratio refers to the mass ratio of the filtrate and the microcurrent tetrapeptide.

[0236] The above results show that in most cases, the two components in the experimental composition have a good synergistic effect. Therefore, the combination of these two components can have a good synergistic anti-aging effect.

[0237] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition comprising a filtrate of a bifidobacterium / lactobacillus / soybean seed extract fermentation product and a microcurrent tetrapeptide; in, The mass ratio of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate to the microcurrent tetrapeptide is 1:10~10:1; The peptide sequence of the microcurrent tetrapeptide is LEAP.

2. The composition according to claim 1, characterized in that The mass ratio of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate to the microcurrent tetrapeptide is 1:10, 1:1 or 10:

1.

3. A skin care product comprising an auxiliary material and the composition according to claim 1 or 2.

4. The skin care product according to claim 3, characterized in that: in: The concentration of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate is 0.25-100 mg / mL, or the mass fraction is 0.025%-10%; The concentration of microcurrent tetrapeptide is 0.25~100 mg / mL, or the mass fraction is 0.025%~10%.

5. The skin care product according to claim 4, characterized in that: in: The concentration of the bifidobacterium / lactobacillus / soybean seed extract fermentation product filtrate is 0.25-2.5 mg / mL, or the mass fraction is 0.025%-0.25%; The concentration of microcurrent tetrapeptide is 0.25~2.5mg / mL, or the mass fraction is 0.025%~0.25%.

6. Use of the composition according to claim 1 or 2 and / or the skin care product according to any one of claims 3 to 5 in the preparation of anti-aging products.

7. The use according to claim 6, characterized in that: The anti-aging treatment includes at least one of the following I) to V): I) Antioxidant; II) protect cells; III) regulate the transcription level of related genes; IV) regulating the expression levels of related proteins; V) Inhibit aging indicator bacteria.

8. The use according to claim 7, characterized in that: The anti-oxidation includes scavenging free radicals; The protective cells are protective fibroblasts; The regulation of the transcription level of the relevant gene includes increasing COL 1 Genes and / or COL3 Transcription levels of genes; The regulating the expression level of related proteins includes promoting the expression of COL1 protein; The aging indicator bacteria is Moraxella osloensis.

9. The use according to any one of claims 6 to 8, characterized in that: The aging is caused by ultraviolet radiation.

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